WO2005047531A1 - Assay for pkc-beta activation of phosphorylation of myosin heavy chain polypeptide - Google Patents

Assay for pkc-beta activation of phosphorylation of myosin heavy chain polypeptide Download PDF

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Publication number
WO2005047531A1
WO2005047531A1 PCT/AU2004/001573 AU2004001573W WO2005047531A1 WO 2005047531 A1 WO2005047531 A1 WO 2005047531A1 AU 2004001573 W AU2004001573 W AU 2004001573W WO 2005047531 A1 WO2005047531 A1 WO 2005047531A1
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Prior art keywords
seq
pkc
polypeptide
peptide
heavy chain
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PCT/AU2004/001573
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French (fr)
Inventor
Trevor Biden
Russell Ludewyke
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Garvan Institute Of Medical Research
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Priority claimed from AU2003906287A external-priority patent/AU2003906287A0/en
Application filed by Garvan Institute Of Medical Research filed Critical Garvan Institute Of Medical Research
Publication of WO2005047531A1 publication Critical patent/WO2005047531A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase

Definitions

  • derived from shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
  • Protein kinase C isozymes are single polypeptide chains ranging from about 592 to about 737 amino acids in length.
  • the isozymes contain a regulatory domain and a catalytic domain connected by a linker peptide.
  • the regulatory and catalytic domains can be further subdivided into constant and variable regions.
  • the catalytic domain of protein kinase C is very similar to that seen in other protein kinases while the regulatory domain is unique to the PKC isozymes.
  • the PKC isozymes are approximately 40-80% identical at the amino acid level. However, the identity of any one isozyme between different species is generally greater than about 80%.
  • PKC- ⁇ activity has been shown to be upregulated in hyperglycemic patients and animals.
  • PKC- ⁇ activity has been shown to be increased in diabetic subjects, and models of diabetes, especially in diabetic subjects that have been exposed to glucose (King et al, Growth Factors in Health and Disease, eds. Westmark, Betscholtz and Hokfelt, Exerpta Medica, Amsterdam, pp. 303-317, 1990).
  • the increase in activity of PKC- ⁇ is thought to be caused by an increase in the concentration of diaglycerol (DAG) as a result of increased glucose levels in a subject.
  • DAG diaglycerol
  • the PKC- ⁇ is a PKC- ⁇ l or PKC- ⁇ ll, for example, the PKC- ⁇ comprises an amino acid sequence at least 80% identical to an amino acid sequence set forth in SEQ ID NO: 169 or SEQ ID NO: 171.
  • the peptide, polypeptide or protein comprising the PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide is in the sample being assayed and the ligand is an antibody that specifically binds to a phosphorylated PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide.
  • the level of PKC- ⁇ activity is determined by performing a process comprising: (i) contacting a sample comprising the peptide, polypeptide or protein comprising a PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide with an antibody that specifically binds to a phosphorylated PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody-antigen complex to form; and
  • the antibody is additionally capable of selectively binding to a mimetic of a phosphorylated PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide.
  • the mimetic of a phosphorylated PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide is a peptide, polypeptide or protein comprising an amino acid sequence set forth in SEQ ID NO: 178 or SEQ ID NO: 179.
  • the level of labelled peptide, polypeptide or protein comprising a phosphorylated PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide bound to the antibody is inversely proportional to the level of unlabelled peptide, polypeptide or protein comprising a phosphorylated PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide in the standard.
  • Figure 1 is a copy of a photographic representation of a dot blot showing that a polyclonal antibody raised against a peptide comprising the amino acid sequence set forth in SEQ ID NO: 10 is capable of specifically binding to the phosphorylated form of the peptide (P-Ser peptide).
  • the antibody did not recognize an unphosphorylated peptide (Ser peptide, i.e. SEQ ID NO: 9).
  • DNP-BSA dinitrophenylated bovine serum albumin
  • PKC isozymes may phosphorylate the PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide, albeit to a significantly reduced degree compared to a PKC- ⁇ isozyme. Accordingly, a PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide is preferentially phosphorylated by a PKC- ⁇ enzyme, albeit not necessarily exclusively.
  • amino acid sequence of human myosin heavy chain IIA is set forth in SEQ ID NO: 2
  • amino acid sequence of rat myosin heavy chain IIA is set forth in SEQ ID NO: 4
  • amino acid sequence of mouse myosin heavy chain IIA is set forth in SEQ ID NO: 6
  • amino acid sequence of chicken myosin heavy chain IIA is set forth in SEQ ID NO: 8.
  • a phosphatase inhibitor may be used when screening for a phosphorylated antigen specific antibody to inhibit the removal of a phosphate group from the antigen of interest.
  • the assay used to determine the amount or level of a protein in a quantitative assay.
  • the present invention provides a method for determining the level of PKC- ⁇ activity comprising: (i) contacting a sample comprising a peptide, polypeptide or protein comprising a PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide with a first antibody that binds to said peptide, polypeptide or protein at a site other than the PKC- ⁇ phosphorylation site for a time and under conditions sufficient for the antibody to bind to the peptide, polypeptide or protein;
  • the competitor may be added at the same time as or after contacting the sample. Following washing to remove unbound protein (e.g., competitor) the level of detectable label is detected.
  • the level of label detected is inversely proportional to the level of the peptide, polypeptide or protein comprising a phosphorylated PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide in the sample. Using a standard curve the level of the peptide, polypeptide or protein comprising a phosphorylated PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide in the sample may be quantified.
  • a suitable standard will be apparent to the skilled artisan and/or is described herein.
  • Evanescent biosensors are also preferred as they do not require the pre-treatment of a biological sample prior to detection of a protein of interest.
  • An evanescent biosensor generally relies upon light of a predetermined wavelength interacting with a fluorescent molecule, such as for example, a fluorescent antibody attached near the probe's surface, to emit fluorescence at a different wavelength upon binding of the phosphorylation site of a myosin heavy chain polypeptide to the antibody or ligand.
  • Methods using immunofluorescence are preferable, as they are quantitative or at least semi-quantitative.
  • Methods of quantitating the degree of fluorescence of a stained cell are known in the art and described, for example, in Immunohistochemistry (Cuello, 1984 John Wiley and Sons, ASIN 0471900524).
  • PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide (such as, for example myosin heavy chain IIA), and, following washing, eluting those peptides that remain bound to the ligand or antibody.
  • Said ligand or antibody is bound to another molecule to allow for ease of purification, such as, for example, a molecule selected from the group consisting of protein A, protein G, agarose or Sepharose, biotin, glutathione S- transferase (GST), and a FLAG epitope.
  • the PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide is isolated simply through centrifugation, or through binding to another molecule, eg. streptavidin, or binding of a specific antibody, eg. anti-FLAG antibodies, or anti-GST antibodies.
  • Methods using a ligand or antibody covalently bound to an affinity matrix are particularly preferred.
  • the method of the present invention comprises dissociating the peptide, polypeptide or protein comprising a PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide from the antibody or ligand used to capture said polypeptide.
  • Methods of dissociating proteins are known in the art, and described, for example, in Scopes (In: Protein Purification: Principles and Practice, Third Edition, Springer Verlag, 1994).
  • proteins are dissociated by changing the pH of a sample (e.g. with glycine (pH3) or triethanolamine (pHl l)), increasing the salt concentration of a sample with an ionic detergent (e.g. with 5M lithium chloride), treating a sample with an ionic detergent (e.g. SDS), treating a sample with a dissociating agent (e.g. urea), treating a sample with a chaotropic agent (e.g. thiocyanate) or a combination of these methods.
  • a ionic detergent e.
  • the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the gene from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
  • Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichia pastoris, S. cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PH05 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.
  • Preferred mammalian cells for expression of the nucleic acid fragments include epithelial cells, fibroblasts, kidney cells, T cells, or erythroid cells, including a cell line selected from the group consisting of COS, CHO, murine 10T, MEF, NIH3T3, MDA- MB-231, MDCK, HeLa, K562, HEK 293 and 293T.
  • neoplastic cells such as, for example, leukemic/leukemia cells, is also contemplated herein.
  • the amount of hydrolysed Pi in solution may be determined using a malachite green method essentially as described in Kodoma et al, J. Biochem., 99(5): 1465-1472, 1986. Essentially, malachite green/molybdate is incubated with a solution comprising hydrolysed Pi under conditions sufficient to allow interaction. Interaction of hydrolysed Pi, malachite green and molybdate cause the solution to change colour, with the degree of change indicative of the amount of phosphate. This may be quantitated using a standard curve, and determining absorbance at 650 nm. As will be apparent to the skilled artisan, an assay using malachite greenmolybdate does not require labelled ATP.
  • the binding between the phosphorylation site and the ligand is determined by performing a process comprising: (i) contacting a peptide, polypeptide or protein comprising a PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide with a PKC- ⁇ in a vesicle under conditions sufficient for the PKC- ⁇ to phosphorylate said PKC- ⁇ phosphorylation site; and (ii) determining the level of phosphorylation of the PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide, wherein the level of phosphorylation of the PKC- ⁇ phosphorylation site of a myosin heavy chain is indicative of a PKC- ⁇ activity.
  • the cell or subject is a wild-type cell or subject.
  • wild-type shall be taken to mean a cell or subject that is a naturally occurring cell or subject, i.e. a cell or subject that has not been modified at the genetic level or treated with a compound to modify that cell or subject.
  • the cell or subject has not been modified at the genetic level or treated with a compound that modulates the activity of PKC- ⁇ in said cell or subject.
  • the cell or subject has been modified at the genetic level.
  • the cell or subject has been modified at the genetic level to induce a diabetes-type phenotype.
  • a cell or animal subject is administered with a compound that induces a diabetes-type phenotype.
  • Compounds that induce a diabetes-type phenotype are known in the art and include, for example, alloxan (as described in Kodoma et al, Diabetes Res. clin. Pract., 20(3): 189-189, 2003) and streptozotocin (as described in Sandier et al, Diabetes 4: 78-83, 1983 and Ar'Rajab et al, Eur. Surg. Res. 22(5): 270- 275, 1990) (available from Sigma, St. Louis, MO, USA).
  • the animal subject is administered with a compound that induces an asthma type phenotype.
  • a compound that induces an asthma type phenotype for example, an animal subject may be made sensitive to ovalbumin, and any subsequent exposure to ovalbumin in an aerosol form induces an allergic asthma-type response.
  • said subject may develop aberrant modified animal subjects that suffer from a colon cancer-type phenotype are known in the art and include, for example, AN3 1 KAL 1 MutL homologue 1 (MCH1) deficient mice, insulin like growth factor 1 deficient mice, p21 (WAFl/cipl) deficient mice and DNA methyl transferase 1 deficient mice.
  • MCH1 AN3 1 KAL 1 MutL homologue 1
  • WAFl/cipl DNA methyl transferase 1 deficient mice.
  • a biological sample is treated prior to use in the assay of the present invention.
  • the kit comprises recombinant or isolated a PKC- ⁇ enzyme.
  • the kit comprises a PKC- ⁇ l enzyme.
  • the kit comprises a PKC- ⁇ ll enzyme.
  • the kit comprises a PKC- ⁇ l and a PKC- ⁇ ll enzyme.
  • Conditions associated with aberrant PKC- ⁇ activity are known in the art.
  • conditions associated with decreased PKC- ⁇ activity include, hypoglycemia, melanoma, inflammatory and hyperplastic skin disorders (e.g. psoriasis, cutaneous T- cell lymphoma, cutaneous dermatitis and allergic contact dermatitis) and immunodeficiency.
  • Conditions associated with increased PKC- ⁇ activity include, for example, hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, colon cancer, breast cancer, leukemia, hepatocarcinoma, gastric cancer, asthma and hypertension.
  • a subject has not been previously diagnosed with a condition associated with PKC- ⁇ activity.
  • the diagnostic/prognostic method of the invention may be used to determine a condition that is associated with PKC- ⁇ activity.
  • the present invention also encompasses the use of an antibody or ligand that specifically binds to an amino acid that is phosphorylated by PKC- ⁇ in the manufacture of a diagnostic or prognostic for the diagnosis or prognosis of a condition that is associated with aberrant PKC- ⁇ activity.
  • the condition associated with aberrant PKC- ⁇ activity is selected from the group consisting of hypoglycaemia, hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, hypertension, colorectal cancer and immunodeficiency.
  • the polypeptide is myosin heavy chain IIA. More preferably, the polypeptide comprises an amino acid sequence that is at least about 80% homologous to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6 or 8. Even more preferably, the polypeptide is phosphorylated at a serine residue that corresponds to the serine at position 1916 of SEQ ID NO: 2 or 6, position 1917 of SEQ ID NO: 4 or SEQ ID NO: 1915 of SEQ ID NO: 8.
  • a modulator of PKC- ⁇ activity partially or completely inhibits PKC- ⁇ enzyme activity (i.e. PKC- ⁇ phosphorylation of a PKC- ⁇ phosphorylation site of a myosin heavy chain polypeptide.
  • a modulator of PKC- ⁇ activity is a peptide that comprises a phosphorylation site of a myosin heavy chain polypeptide.
  • the peptide comprises the sequence set forth in SEQ ID NO: 172.
  • a peptide comprising the amino acid sequence set forth in SEQ ID NO: 177 is capable of suppressing histamine secretion in RBL-2h3 cells.
  • the invention provides a process for determining a compound that modulates PKC- ⁇ activity, said process comprising:
  • the agent is provided with an indication as to its use, for example, as determined using a method described herein.
  • the present invention provides a process for determining a candidate compound for the treatment of a condition associated with aberrant PKC- ⁇ activity said method comprising:
  • Formulation of a pharmaceutical compound will vary according to the route of administration selected (e.g., solution, emulsion, capsule).
  • An appropriate composition comprising the identified modulator to be administered can be prepared in a physiologically acceptable vehicle or carrier.
  • suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils, for instance.
  • Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers and the like (See, generally, Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., Pa., 1985).
  • the agent can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, nebulizer or pressurized aerosol dispenser).
  • RBL-2H3 cells express non-muscle myosin IIA. These cells were incubated in the presence of a phorbolester and/or an antigen which cause an increase in phosphorylation of the myosin IIA polypeptide.
  • Rat basophilic mast cells (RBL-2H3) are maintained and used as adherent monolayers in RPMI-1640 medium supplemented with 2mM glutamine, 25mM Hepes, 10% fetal calf serum.
  • the RBL-2H3 cell line is activated via crosslinking of the Fc ⁇ Rl receptors on its surface.
  • the DNP-specific monclonal IgE added to the cells attaches to the Fc ⁇ Rl receptors, and primes the cells to be activated through this receptor.
  • a reporter assay based on co-overexpression of a C-terminal fragment of human myosin heavy chain IIA tagged to GFP (Straussman R et al, 2001, J Cell Sci 114:3047-3057).
  • a myosin reporter comprising a C-terminal fragment of human myosin heavy chain IIA tagged to GFP (essentially as described in Straussman et al, JCell Sci 114:30A1-3051, 2001) is used.
  • Fluorescence intensity is examined in cells overexpressing the MHC-reporter (identified by GFP fluorescence) to determine effects of the co-expressed PKC constracts.
  • stimulated cells are lysed and the GFP-tagged polypeptide is immunoprecipitated with a mixture of mouse mono-clonal antibodies (clones 7.1 and 13.1; Roche).
  • Phosphorylation due to co-expressed PKC constructs is then determined by immunoblotting using the antibody described in Example 1 essentially as described supra.
  • EXAMPLE 8 Determining PKC- ⁇ activity in a rat model of diabetes
  • SBP systolic blood pressure
  • An antibody capable of being used in a two site ELISA assay i.e. capable of being used in an ELISA that accurately predicts the amount of PKC- ⁇ phosphorylated myosin heavy chain are selected for use in an ELISA assay for determining the amount of phosphorylated myosin heavy chain IIA, and as a consequence PKC- ⁇ activity in a sample.
  • Any myosin heavy chain IIA present in the sample binds to the antibody, and the sample then migrates (by capillary action) up the test strip, passing over the monoclonal antibody that specifically detects PKC- ⁇ phosphorylated myosin heavy chain IIA (as described in Example 10) that has been conjugated to the test strip in a specific localised area (i.e. a line). Any PKC- ⁇ phosphorylated myosin heavy chain IIA in the sample is then captured by this antibody.
  • Example 10 A commercial antibody to non-muscle MHC (Biomedical Technologies Inc), which recognises predominantly MHC2A is pre-absorbed onto a micro-titre plate. Cell extracts are then added to the wells, and the signal detected using the antisera described in Example 1 coupled to HRP. The ELISA assay is performed essentially as described in Example 10.
  • the level of phosphorylated myosin heavy chain polypeptide determined in a test sample is then compared to the standard curve and the total phosphorylated myosin heavy chain polypeptide estimated.
  • CMNREVSDLKNKLRR (SEQ ID NO: 173) in which the serine phosphorylated by PKC- ⁇ is replaced with an aspartic acid thereby mimicking the negative charge of the phosphorylated serine residue.
  • RBL-2H3 cells were maintained as monolayer cultures and harvested by trypsin treatment. Cells were transferred to 96-well culture plates at a density of 4.9 X 10 cells per well in 0.4ml Earle's modified Eagle's medium supplemented with 15%(v/v) fetal bovine serum. For those experiments involving antigenic activation, the cells were sensitized by the addition of 75 ng/ml dinitrophenol (DNP)-specific IgE and incubated overnight at 37°C with 5% CO 2 . To measure the secretory response, cultures were also incubated overnight with [ 3 H] 5-hydroxytryptamine ([ 3 H] 5HT), (0.5 ⁇ Ci/ml), which becomes incorporated into the histamine containing granules.
  • DNP dinitrophenol
  • the cells were washed twice in a calcium-free, potassium glutamate buffer: 139mM glutamic acid, 5mM ATP, 5mM glucose: 7mM magnesium acetate; ImM EGTA and
  • EXAMPLE 19 Determining a compound that modulates PKC- ⁇ activity
  • the PKC- ⁇ phosphorylation site of myosin heavy chain IIA comprises a serine residue adjacent to the serine that is phosphorylated. This may lead to low levels of inadvertent phosphorylation of the PKC- ⁇ phosphorylation site by another PKC isozyme.
  • a mutagenesis protocol is used to determine a peptide with higher specificity for PKC- ⁇ .
  • Lipids (5 mg/ml in chloroform:methanol (19:1 v/v)), are dried under nitrogen and sonicated into 100 mM MOPS (pH 7.5) 1% (v/v) Triton X-100 until clear, before addition to the assay buffer.
  • Reactions are stopped by addition of 10 ⁇ l cold ATP (150 mM). After vortexing 40 ⁇ l of reaction medium is withdrawn and spotted onto P81 paper squares. These are dropped into H 3 PO 4 (10ml cone, acid in 11ml), and then washed 5 times for 5 min in 3 % (v/v) H 3 PO , and then once in 100% EtOH. After drying radioactivity is determined by Cerenkov counting. Control incubations are undertaken under similar conditions but using Histone HIIIS as a generic PKC substrate, and a myosin heavy chain phosphorylation site mutant (MNREVSALKNKLRR, SEQ ID NO: 180) as a negative control. Also used as a control is a peptide comprising the amino acid sequence set forth in SEQ ID NO: 130.

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Abstract

The present invention provides a method for determining protein kinase C-ß (PKC-ß) activity in a sample. In particular, the method of the invention detects the interaction of a PKC-ß with a substrate peptide derived from myosin heavy chain IIA that comprises a PKC-ß phosphorylation site. In one embodiment, the method comprises determining the level of phosphorylation of the substrate peptide using an antibody that specifically binds to the peptide following phosphorylation by PKC-ß. Alternatively, the method comprises determining the level of phosphate incorporated into the peptide by a PKC-ß. Also provided is a method for diagnosing a disorder associated with aberrant PKC-ß activity and a method for determining a compound that modulates PKC-ß activity. The present invention additionally provides a method for modulating PKC-ß activity using a peptide inhibitor. Peptides are also provided that are useful in the performance of the methods of invention.

Description

ASSAY FOR PKC-BETA ACTIVATION BY PHOSPHORYLATION OF MYOSIN HEAVY CHAIN POLYPEPTIDE
Field of the Invention
The present invention relates to novel assays for determining the activity of protein kinase C-β (PKC-β) activity. In particular, the assay of the present invention determines PKC-β activity by detecting the interaction of PKC-β with a substrate peptide from a myosin heavy chain comprising a PKC-β phosphorylation site and/or detecting phosphorylation of a PKC-β phosphorylation site of a myosin heavy chain polypeptide.
Background of the Invention 1. General
This specification contains nucleotide and amino acid sequence information prepared using Patentln Version 3.1, presented herein after the claims. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1, <210>2, <210>3, etc). The length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence, are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the term "SEQ ID NO:", followed "by the sequence identifier (e.g. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as <400>1).
The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine andN represents any nucleotide residue.
As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
Unless specifically stated otherwise, each feature described herein with regard to a specific embodiment of the invention, shall be taken to apply mutatis mutandis to each and every other embodiment of the invention.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, histology and immunology. Such procedures are described, for example, in the following texts that are incorporated by reference: 1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III; 2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text; 3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; 5. Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; 6. Perbal, B., A Practical Guide to Molecular Cloning (1984); 7. Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R.L. (1976). Biochem. Biophys. Res. Commun. 73336-342 8. Merrifield, R.B. (1963). J. Am. Chem. Soc. 85, 2149-2154. 9. Barany, G. and Merrifield, R.B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York. 10. Wunsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Mϋler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart. ll. Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer- Verlag, Heidelberg. 12. Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg. 13. Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474.
Description of the related art
The phosphorylation of serine and threonine residues in proteins is essential to the normal regulation of a multitude of biological mechanisms including the modulation of membrane structure, receptor desensitization, transcriptional control, cell signalling, exocytosis, cell growth, differentiation and mediation of immune responses. A particularly important family of serine/threonine kinases are the protein kinase C (PKC) family of enzymes. Presently, there are at least ten known isozymes of PKC that differ in their tissue distribution, enzymatic specificity, and regulation (Nishizuka Annu. Rev. Biochem. 58: 31-44 (1989); Nishizuka Science 258: 607-614 (1992)).
Protein kinase C isozymes are single polypeptide chains ranging from about 592 to about 737 amino acids in length. The isozymes contain a regulatory domain and a catalytic domain connected by a linker peptide. The regulatory and catalytic domains can be further subdivided into constant and variable regions. The catalytic domain of protein kinase C is very similar to that seen in other protein kinases while the regulatory domain is unique to the PKC isozymes. The PKC isozymes are approximately 40-80% identical at the amino acid level. However, the identity of any one isozyme between different species is generally greater than about 80%.
Protein kinase C enzymes are allosterically regulated by a number of factors, including membrane phospholipids, calcium, and certain membrane lipids, such as, for example, diacylglycerols that are liberated in response to the activities of phospholipases (Bell and Burns, J Biol Chem. 266: A661-A66A (1991); Nishizuka, Science 258: 607-614 (1992). The protein kinase C isozymes, α, β-I, β-II and γ, require membrane phospholipid, calcium and diacylglycerol/phorbol esters for complete activation. The δ, ε, η, and θ forms of PKC are calcium-independent in their mode of activation. The ζ and λ forms of PKC are independent of both calcium and diacylglycerol and are believed to require only membrane phospholipid for their activation.
The β isozymes of PKC (i.e. PKC-β-I and PKC-β-II) are of particular interest as aberrant activity of these proteins have been found to be associated with a number of human conditions, such as, for example, diabetes and complications of diabetes, hypertension and immunodeficiency.
PKC-β activity has been shown to be upregulated in hyperglycemic patients and animals. In particular, PKC-β activity has been shown to be increased in diabetic subjects, and models of diabetes, especially in diabetic subjects that have been exposed to glucose (King et al, Growth Factors in Health and Disease, eds. Westmark, Betscholtz and Hokfelt, Exerpta Medica, Amsterdam, pp. 303-317, 1990). The increase in activity of PKC-β is thought to be caused by an increase in the concentration of diaglycerol (DAG) as a result of increased glucose levels in a subject.
The observation that PKC-β activity is increased in hyperglycemic subjects is of particular interest as PKC-β has been shown to modulate smooth muscle growth and contraction, endothelial cell permeability, signal transduction of hormones and growth factors and cardiomyocyte contractility (Inogucchi et al, Proc. Natl. Acad. Sci. USA, 89: 11059-11065, 1992 and references cited therein). All of these processes have been shown to be abnormal in both diabetic subjects and animal models of diabetes. In fact, studies have shown that increased PKC-β activity is associated with diabetes induced damage to heart muscle and aorta (Inogucchi et al, Proc. Natl. Acad. Sci. USA, 89: 11059-11065, 1992).
Furthermore, increased PKC-β activity has been shown to be associated with a variety of symptoms of diabetes, including, formation of advanced glycation end products, hypertension and activation of the renin-angiotensin system (Weiss et al, Nephrol Dial. Transplant. 49: 87-93, 2000; and Kreisberg et al, Kidney Int. Suppl. 51: S3-S11, 1995). Hypertension and activation of the renin-angiotensin system have in turn been shown to contribute to the development and progression of diabetic renal disease (Fioretto et al, N. Engl. J. Med. 339: 69-75, 1993; and Parving et al, Br. Med. J. 294: 1443-1447, 1987).
Inhibition of PKC-β has been shown to attenuate the structural and functional manifestations of diabetic renal injury, for example suppression of PKC-β activity reduced both diffuse and nodular glomerulosclerosis and tubulointerstitial pathology in the m(Ren-2)27 rat model of diabetes (Kelly et al, Diabetes, 52: 512-518). Furthermore, inhibition of PKC-β also suppressed overexpression of the profibrotic growth factor TGF-β. PKC-β activity has also been shown to ameliorate the development and progression of diabetic retinopathy, and retinal vascular leakage in both human and animal subjects (Aiello, Surv. Opthamol, 47: s263-s279, 2002).
PKC-β has also been implicated in the immune response, with PKC-β null mice developing an immunodeficiency characterized by impaired humoral immune responses and reduced cellular responses of B-cells. Furthermore, PKC-β has been shown to be associated with Bruton tyrosine kinase in antigen receptor-mediated signal transduction (Leitges et al, Science 273: 788-791, 1996).
Mice deficient in PKC-β fail to activate NfkB and promote cell survival of B-cells and mast cells upon B cell receptor signalling. Furthermore, B-cells in PKC-β deficient mice are unable to recruit the kinases IKKA and IKKB in addition to other proteins required for B-cell receptor signalling (Su et al., Nature Immunol, 3: 780-786, 2002).
Increased expression and/or activity of PKC-β has also been observed in various forms of cancer. For example, (Gδkmen-Polar et al, Cancer Research, 61: 1375-1381, 2001) showed that PKC-βll expression is enhanced in both developing and established colon cancer. Furthermore, Murray et al, J. Cell Biol, 145: 699-711, 1999, demonstrated that increased PKC-βll expression caused increased hyperproliferation in colonic epithelium and an increased susceptibility to carcinogen-induced preneoplastic lesions.
Notwithstanding that a variety of disorders have been shown to be associated with aberrant PKC-β activity, there is still no rapid, reliable assay used to determine PKC-β activity, especially for high-throughput analysis of analytes. Rather, researchers have traditionally relied upon assays utilizing cell lines that express a single PKC-β isozyme or type of PKC isozyme (e.g. PKC-βl and PKC-βll expressing cell). The disadvantage of such assays is that they are time consuming and expensive. Furthermore, often several experiments are required to determine whether or not the observed phenotype is, in fact, PKC-β specific.
PKC activation assays performed in cell free systems depend upon individually assaying the ability of each PKC isozyme to phosphorylate a target protein. Accordingly, while potentially saving the cost of performing these assays in cells or animals, these assays fail to resolve the requirement of testing each PKC isozyme.
Accordingly, there remains a clear need in the art for a rapid and reliable assay that specifically determines PKC-β activity. Such an assay would be of particular benefit in diagnosing disorders associated with aberrant PKC-β activity and in the identification of new compounds that specifically modulate PKC-β activity.
Summary of the Invention
In work leading up to the present invention, the inventors identified a specific serine residue ("serine 1917" or "S1917") in human myosin heavy chain IIA that is phosphorylated by PKC-β. Furthermore, the inventors produced a polyclonal antibody that is capable of distinguishing between myosin heavy chain IIA that is phosphorylated at serine 1917 and myosin heavy chain IIA that is not phosphorylated at serine 1917. Using this antibody, the inventors have shown that modulators of PKC-β expression and/or activity also modulate the amount of phosphorylated myosin heavy chain IIA in a sample. These findings have provided the means for producing a novel assay that specifically determines the activity of PKC-β. As will be apparent to the skilled artisan this assay may also be used to diagnose and/or prognose a disorder associated with aberrant PKC-β activity. Additionally, this assay may be used in assays to identify a modulator of PKC-β activity.
Accordingly, the present invention provides a method for determining protein kinase C- β (PKC-β) activity in a sample comprising contacting a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a variant thereof with a ligand that specifically binds to an amino acid residue within said phosphorylation site or a sample comprising said ligand for a time and under conditions sufficient for binding to occur and then detecting the binding wherein the binding is indicative of PKC-β activity. Preferably, the PKC-β is a PKC-βl or PKC-βll, for example, the PKC-β comprises an amino acid sequence at least 80% identical to an amino acid sequence set forth in SEQ ID NO: 169 or SEQ ID NO: 171.
In one embodiment, the peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 142. It is preferable, that the variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 142 to 167.
In another embodiment, the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide is a myosin heavy chain IIA polypeptide or a fragment thereof comprising an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 142. For example, the peptide, polypeptide or protein comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.
Preferably, the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or variant thereof is capable of being phosphorylated by PKC-β at a site equivalent to amino acid position 9 of SEQ ID NO: 9.
In a preferred embodiment, the peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide is in the sample being assayed and the ligand is an antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide.
In one embodiment, the level of PKC-β activity is determined by performing a process comprising: (i) contacting a sample comprising the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with an antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody-antigen complex to form; and
(ii) determining the level of said complex, wherein the level of said complex is indicative of PKC-β activity.
Preferably, the antibody specifically binds to a peptide, polypeptide or protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO
10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO 18, SEQ ID NO
20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO 28, SEQ ID NO
30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO 38, SEQ ID NO
40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO 48, SEQ ID NO
50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO 58, SEQ ID NO
60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO 68, SEQ ID NO
70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO 78, SEQ ID NO
80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO 88, SEQ ID NO
90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO 98, SEQ ID NO
100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID
NO: 110, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119,
SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139 and SEQ ID NO: 141. For example, the antibody specifically binds to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10.
In another embodiment, the antibody is capable of specifically binding a myosin heavy chain polypeptide that is phosphorylated at the serine residue at an amino acid equivalent to amino acid position 1916 of SEQ ID NO: 2 or SEQ ID NO: 6 or amino acid position 1917 of SEQ ID NO: 4 or amino acid position 1915 of SEQ ID NO: 8.
In an additional embodiment, the antibody is additionally capable of selectively binding to a mimetic of a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide. For example, the mimetic of a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide is a peptide, polypeptide or protein comprising an amino acid sequence set forth in SEQ ID NO: 178 or SEQ ID NO: 179.
In one embodiment, the antibody is selected from the group consisting of a monoclonal antibody, a fragment of a monoclonal antibody, a recombinant antibody and a polyclonal antibody. Preferably, the antibody is a polyclonal antibody. In ane embodiment, the level of the complex is determined by performing a process comprising contacting the complex with another antibody capable of binding to said complex for a time and under conditions sufficient for another antibody-antigen complex to form and detecting the level of the other complex, wherein the level of said other complex is indicative of PKC-β activity. Preferably, the antibody and/or the other antibody is labelled with a detectable marker, for example, an enzyme label, a radiolabel and a fluorescent label.
In one embodiment, determining the level of the complex comprises comparing the level of the complex determined in the sample to the level determined using a standard comprising a known level of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide, wherein a similar level of the complex determined in the sample and the standard indicates the level of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide present in the sample.
Preferably, the standard is produced by performing a process comprising: (i) contacting a known level of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide with the antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody-antigen complex to form; and (ii) determining the level of the complex, wherein the level of the complex determined is indicative of the known level of the peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide.
In one embodiment, the method for determining the level of PKC-β activation comprises:
(i) contacting a sample comprising a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with a first antibody that binds to said peptide, polypeptide or protein at a site other than the PKC-β phosphorylation site for a time and under conditions sufficient for the antibody to bind to the peptide, polypeptide or protein; (ii) contacting the bound peptide, polypeptide or protein at (i) with a second antibody that specifically binds to the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody- antigen complex to form, wherein either the first antibody or the second antibody specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide ; and
(ii) determining the level of said complex, wherein the level of said complex is indicative of PKC-β activity.
In one embodiment, the first antibody specifically binds to a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide.
In an alternativel embodiment, the second antibody specifically binds to a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide.
Preferably, the first antibody is/was previously immobilized on a solid support, matrix or resin.
Preferably, the level of the complex is determined by performing a process comprising comparing the level of complex determined in the sample with the level of complex determined in a standard, wherein the level of the complex determined in the sample and in the standard indicates the level of phosphorylated peptide, polypeptide or protein in the sample and PKC-β activity.
In one embodiment, the standard is produced by performing a process comprising:
(i) contacting a first antibody that binds to a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide at a site other than the PKC-β phosphorylation site with (a) a known amount of a peptide, polypeptide or protein comprising an unphosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof and (b) a known amount of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof for a time and under conditions sufficient for the antibody to bind to the peptide, polypeptide or protein; (ii) contacting the bound peptide, polypeptide or protein at (ii) with a second antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody-antigen complex to form; and (iii) determining the level of complex relative to the total amount of level of peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof, thereby determining the level of complex that is indicative of the known amount of the peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide.
In one embodiment, the peptide, polypeptide or protein comprising an unphosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof and the peptide, polypeptide or protein comprising an phosphorylated
PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof are labelled with a detectable marker. Preferably, the detectable marker is a fluorescent marker.
In another embodiment of the invention, the level of PKC-β activity is determined by performing a process comprising:
(i) contacting a sample comprising a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with an antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody-antigen complex to form and contacting said antibody with a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof labelled with a detectable marker; and
(ii) determining the level of detectable marker bound to the antibody, wherein the level of detectable marker bound to the antibody is indicative of the level of PKC-β activity.
As will be apparent to the skilled artisan, it is preferable that, the level of detectable marker bound to the antibody is inversely proportional to the level of PKC-β activity. Preferably, the antibody is/was previously immobilized on a solid support, matrix or resin.
In one embodiment, the method additionally comprises washing the sample to remove any non-specifically bound or unbound protein prior to determining the level of detectable marker bound to the antibody.
In a preferred embodiment, the level of detectable marker bound to the antibody is determined using a process comprising comparing the level of detectable marker bound to the antibody in the sample to a standard, wherein a comparable level of detectable marker bound to the antibody in the sample and the standard indicates the level of peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide in the sample and PKC-β activity.
Preferably, the standard is produced using a method comprising:
(i) contacting the antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide with (a) a known amount a labelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide; and (b) a known amount of an unlabelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof for a time and under conditions sufficient for an antibody-antigen complex to form; and
(ii) determining the level of detectable marker bound to the antibody, wherein the level of labelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide bound to the antibody is indicative of the level of unlabelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide in the standard.
Preferably, the level of labelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide bound to the antibody is inversely proportional to the level of unlabelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide in the standard.
In a preferred embodiment, the label is a fluorescent label.
In yet another embodiment of the invention, the ligand is a PKC-β enzyme in an extract or cellular sample being assayed. In accordance with this embodiment, the binding between the phosphorylation site and the ligand is determined by performing a process comprising: (i) contacting a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with a PKC-β or a sample comprising or suspected of comprising a PKC-β under conditions sufficient for the PKC-β to phosphorylate said PKC-β phosphorylation site; and (ii) determining the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide, wherein the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide is indicative of a PKC-β activity.
Preferably, the peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO; 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138 and SEQ ID NO: 140. Ina preferred embodiment of the invention, the peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide comprises an amino acid set forth in SEQ ID NO: 130.
In one embodiment, the peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide is contacted with the PKC-β or sample comprising or suspected of comprising a PKC-β in the presence of a labelled phosphate. For example, the label is selected from the group consisting of a radioactive label, a fluorescent label, an enzymatic label and a colorimetric label. In a preferred example, the labelled phosphate is γ32P-ATP.
As will be apparent to the skilled artisan, the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide is determined by determining the level of labelled phosphate incorporated into the PKC-β phosphorylation site of a myosin heavy chain polypeptide.
Preferably, the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide is determined by performing a process comprising comparing the level of detectable marker bound to the antibody in the sample to a standard.
In one embodiment, the standard is produced by performing a process comprising: (i) incubating a known amount of the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide in the presence of a known amount of a PKC-β for a time and under conditions sufficient for phosphorylation of the PKC-β phosphorylation site to occur; and (ii) determining the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide, wherein the level of phosphorylation in the standard is indicative of the activity of the PKC-β in the sample.
In a still further embodiment of the invention, the ligand is a compound that specifically binds to a phosphorylated amino acid. In accordance with this embodiment, the level of PKC-β activity in a sample is determined by performing a process comprising: (i) isolating a peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide from the sample; (ii) contacting the isolated peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide with a compound that binds to a phosphorylated amino acid for a time and under conditions sufficient for binding to occur; and (iii) determining the level of bound compound wherein the level of bound compound is indicative of PKC-β activity.
Preferably, the peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide is isolated by performing a process comprising contacting a sample comprising the peptide, polypeptide or protein with an antibody or ligand that specifically binds said peptide, polypeptide or protein and isolating antibody with the peptide, polypeptide or protein bound thereto. For example, the antibody is/was previously immobilized on a solid support, matrix or resin.
Preferably, the compound that binds to a phosphopeptide fluoresces when in contact with a phosphopeptide.
In another embodiment, the compound that specifically binds to the phosphorylated amino acid is an antibody that specifically binds to a phosphorylated serine.
In accordance with each of the embodiments described herein it is preferred that the method is performed using a sample comprising a cell or an extract thereof or a mixture thereof. For example, the cell or cell extract is or has been isolated from a subject. The cell may be a wild-type cell or a cell that has been modified at the genetic level to induce a diabetes-like phenotype, or a cell that has been contacted with a compound to induce a diabetes-like phenotype.
In another embodiment, the method is performed using a sample from an animal subject. For example, the animal subject is is a wild-type animal subject, or an animal subject that has been modified at the genetic level to induce a diabetes-type phenotype or an animal that has been administered a compound to induce a diabetes-type phenotype. In a preferred embdoiment the the method is performed using a sample from a human subject. For example, the sample is from a subject suspected of suffering from a condition associated with aberrant PKC-β activity.
In one embodiment, the aberrant PC-β activity is enhanced PKC-β activity and the condition is selected from the group consisting of asthma, hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, hypertension, cancer, colorectal cancer and immunodeficiency.
In an alternative embodiment, the aberrant PC-β activity is reduced PKC-β activity and the condition is selected from the group consisting of hypoglycemia, melanoma, an inflammatory skin disorder and a hyperplastic skin disorder.
In a further embodiment of the invention, the method is performed using a sample from a cell free system.
The present mention clearly contemplates additionally comprising providing the sample.
The present invention additionally provides a method for diagnosing or prognosing a condition associated with aberrant PKC-β activity in a subject comprising: (i) determining the level of PKC-β activity in a sample from the subject using the method described herein; and (ii) determining the level of PKC-β activity in a control sample using the method described herein, wherein increased or decreased PKC-β activity at (i) compared to (ii) indicates that the subject suffers from a condition associated with aberrant PKC-β activity.
In one embodiment, increased PKC-β activity is determined at (i) compared to (ii) and the condition is selected from the group consisting of hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, hypertension, cancer, colorectal cancer and immunodeficiency. In an alternative embodiment, decreased PKC-β activity is determined at (i) compared to (ii) and the condition is selected from the group consisting of hypoglycemia, melanoma, an inflammatory skin disorder and a hyperplastic skin disorder.
In one embodiment, wherein the subject is in need of treatment. Alternatively, or in addition, the subject has received or is receiving treatment.
The invention additionally provides for the use of an antibody that specifically binds to a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide in the manufacture of a diagnostic or prognostic for the diagnosis or prognosis of a condition selected from the group consisting of hypoglycaemia, hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, aberrant vitamin D function, hypertension, immunodeficiency, melanoma, an inflammatory skin disorder and a hyperplastic skin disorder.
Preferably, the antibody specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO 12, SEQ
ID NO: 14, SEQ ID NO: 16, SEQ ID NO 18, SEQ ID NO: 20, SEQ ID NO 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO 28, SEQ ID NO: 30, SEQ ID NO 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO 38, SEQ ID NO: 40, SEQ ID NO 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO 48, SEQ ID NO: 50, SEQ ID NO 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO 58, SEQ ID NO: 60, SEQ ID NO 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO 68, SEQ ID NO: 70, SEQ ID NO 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO 78, SEQ ID NO: 80, SEQ ID NO 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO 88, SEQ ID NO: 90, SEQ ID NO 92, SEQ
ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139 and SEQ ID NO: 141.
More preferably, the antibody specifically binds to the amino acid sequence set forth in SEQ ID NO: 10. In another embodiment, the present invention provides a method for determining a compound that modulates PKC-β activity comprising:
(i) determining the level of PKC-β activity in a sample in the presence of a candidate compound by performing the method described herein; and (ii) determining the level of PKC-β activity in a sample in the absence of the candidate compound by performing the method described herein, wherein an increase or decrease in PKC-β activity at (i) compared to (ii) indicates that the candidate compound is a modulator of PKC-β activity.
In one embodiment, the modulator increases PKC-β activity. Alternatively, the modulator decreases PKC-β activity.
The present invention additionally provides a process for determining a compound that modulates PKC-β activity, said process comprising: (i) determining the level of PKC-β activity in a sample in the presence of a candidate compound by performing the method described herein; and
(ii) determining the level of PKC-β activity in a sample in the absence of a candidate compound by performing the method described herein, wherein an increase or decrease in PKC-β activity at (i) compared to (ii) indicates that the candidate compound is a modulator of PKC-β activity.
(iii) optionally, determining the structure of the candidate compound;
(iv) optionally, providing the name or structure of the candidate compound; and
(v) optionally, providing the candidate compound.
Also provided is a process for determining a candidate compound for the treatment of a condition associated with aberrant PKC-β activity comprising: (i) determining the level of PKC-β activity in a sample in the presence of a candidate compound by performing the method described herein; and (ii) determining the level of PKC-β activity in a sample in the absence of a candidate compound by performing the method described herein, wherein an increase or decrease in PKC-β activity at (i) compared to (ii) indicates that the candidate compound is a modulator of PKC-β activity and is a candidate compound for the treatment of a condition associated with aberrant PKC-β activity; (iii) optionally, determining the structure of the candidate compound;
(iv) optionally, providing the name or structure of the candidate compound; and (v) optionally, providing, the candidate compound
Also provided is a process for manufacturing a medicament for the treatment of a condition associated with aberrant PKC-β activity comprising: (i) determining a candidate compound for the treatment of a condition associated with aberrant PKC- activity using a process comprising:
(a) determining the level of PKC-β activity in a sample in the presence of a candidate compound by performing the method described herein; and
(b) determining the level of PKC-β activity in a sample in the absence of a candidate compound by performing the method described herein, wherein an increase or decrease in PKC-β activity at (i) compared to (ii) indicates that the candidate compound is a modulator of PKC-β activity and is a candidate compound for the treatment of a condition associated with aberrant PKC-β activity; (ii) optionally, isolating the candidate compound;
(iii) optionally, providing the name or structure of the candidate compound; (iv) optionally, providing the candidate compound; and
(v) using the candidate compound in the manufacture of a medicament for the treatment of a condition associated with aberrant PKC-β activity.
The invention also provides a kit for determining PKC-β activity comprising an antibody capable of specifically binding to a peptide, polypeptide or protein comprising phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide and a detection means.
Preferably, the antibody specifically binds to a peptide, polypeptide or protein comprising the amino acid sequence set forth in SEQ ID NO: 10.
Preferably, the kit additionally comprises a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a variant thereof. For exaple, the peptide, polypeptide or protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 167.
In one embodiment, the kit additionally comprises a PKC-β. The invention also provides a method for modulating PKC-β activity in a cell or a subject comprising administering an effective amount of a peptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 172, 174 or 177 to said cell or subject.
Also provided is a method of the treatment of a condition in a subject associated with aberrant PKC-β activity comprising administering to the subject an effective amount of a peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 172, 174 or 177.
Preferably, the condition is selected from the group consisting of hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, hypertension, cancer, colon cancer and immunodeficiency. For example, the condition is associated with histamine secretion, e.g., the condition is asthma.
The invention also provides for the use of a of a peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 172, 174 or 177 in the manufacture of a medicament for the treatment of a condition associated with aberrant PKC-β activity.
Preferably, the condition is selected from the group consisting of hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, hypertension, cancer, colon cancer and immunodeficiency. For example, the condition is associated with histamine secretion, e.g., the condition is asthma.
The invention also provides an isolated peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 172, 174 or 177, in addition to a composition comprising a peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 172, 174 or 177.
Also provided is an isolated peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 167.
The invention also contemplates a peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 167 when used to determine the level of PKC-β activity in a sample in addition to an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 when used to determine the level of PKC-β activity in a sample.
Brief Description of the Drawings Figure 1 is a copy of a photographic representation of a dot blot showing that a polyclonal antibody raised against a peptide comprising the amino acid sequence set forth in SEQ ID NO: 10 is capable of specifically binding to the phosphorylated form of the peptide (P-Ser peptide). The antibody did not recognize an unphosphorylated peptide (Ser peptide, i.e. SEQ ID NO: 9).
Figure 2 is a copy of a photographic representation of a Western blot showing that the polyclonal antibody is capable of detecting a protein in a cell extract derived from RBL-2H3 cells, rat mucosal mast cells, that is approximately the same molecular weight as myosin heavy chain IIA. Furthermore, the amount of phosphorylated protein detected was increased in cells that had been treated with PMA, a known inducer of PKC-β activity, indicating that the antibody specifically bound phosphorylated myosin heavy chain.
Figure 3 is a copy of a photographic representation of a Western blot showing that myosin heavy chain IIA is phosphorylated by PKC-β. RBL-2H3 cells were incubated in the presence of an antigen (Antigen) or phorbolester and an antigen (P+A) both of which induce PKC activity. Cells were also incubated in the presence or absence of Go6967 (an inhibitor of PKC-α, PKC-βl and PKC-βll) as indicated at the base of the figure. As shown, assays detecting phosphorylated myosin heavy chain IIA (S-1917) bound to less protein in cells incubated in the presence of Go6967 (Go) compared to cells only incubated with phosphorylation and/or antigen. This reduction in detected protein was shown not to be due to reduced expression of myosin heavy chain as shown by the use of an antibody to detect total myosin heavy chain expression (Myosin HC). Furthermore, densitometric analysis of results showed the degree to which the inhibitor of PKC activity was capable of reducing the phosphorylation of myosin heavy chain IIA. As shown at the left hand side of the Figure, Go6967 (Go) had no detectable effect on myosin heavy chain IIA phosphorylation in the absence of antigen or phorbolester and antigen (known inducers of PKC-β).
Figure 4 is a graphical representation showing the level of phosphorylation of a peptide comprising and amino acid sequence set forth in SEQ ID NO: 130 by PKC-α or PKC-β. The amount of peptide phosphorylated per minute per 100 units of the relevant enzyme is represented on the Y axis. The concentration (μM) of the peptide is indicated on the Y-axis. The level of peptide phosphorylated with PKC-α is indicated by the circles and PKC-β by the squares.
Figure 5A is a copy of a photographic representation showing phosphorylation of myosin heavy chain IIA in a kidney isolated from a (mRen27)-2 rat that has been treated with streptozotocin (i.e. a diabetic rat). Rat kidney endothelium was incubated with a polyclonal antibody that specifically binds PKC-β phosphorylated myosin heavy chain IIA and the bound polyclonal antibody detected with an anti-rabbit antibody bound to a fluorescent label. No staining was observed in the kidney endothelium of (mRen27)-2 rats that were not treated with streptozotocin.
Figure 5B is a copy of a photographic representation showing phosphorylation of myosin heavy chain IIA in cardiac endothelium from a diabetic rat. Rat cardiac endothelium was incubated with the previously described polyclonal antibody and the bound polyclonal antibody detected with an Alexa Fluor 568 labelled anti-rabbit antibody (Molecular Probes). No phosphorylated myosin heavy chain IIA was observed in the cardiac endothelium of (mRen27)-2 rats that were not treated with streptozotocin.
Figure 5C is a copy of a photographic representation showing the degree of phosphorylation of myosin heavy chain IIA in a pancreas isolated from a non-diabetic control rat. A section of a pancreas isolated from a control rat was incubated with the previously described polyclonal antibody. Pancreatic islet cells are stained, indicating PKC-β activity in these cells. This was not observed in diabetic rats as the islet cells are destroyed (not shown)..
Figure 6 is a representation of a CLUSTAL alignment showing the region surrounding the PKC-β phosphorylation site of myosin IIA of human, rat, mouse and chicken. Note the high degree of conservation in the sequence surrounding the phosphorylation site (underlined). The bold region represents a peptide comprising the amino acid sequence set forth in SEQ ID NO: 10, wherein the underlined serine is phosphorylated. Residues that are conserved between all species are indicated by asterisks (*). Conservation of the groups STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY or FYW is indicated by ":". Conservation of the groups CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, HFY is indicated by ".". Gaps are indicated by dashes "-".
Figure 7A is a graphical representation showing the effect of peptides that mimic a PKC-β phosphorylation site of a myosin heavy chain polypeptide have on histamine secretion. RBL-2H3 cells were permeabilized and incubated with a peptide that corresponded to the PKC-β phosphorylation site of myosin heavy chain IIA (SS), a peptide that corresponds to a mutant form of a PKC-β phosphorylation site of myosin heavy chain IIA that could not be phosphorylated (SA), a peptide that mimics a phosphorylated PKC-β phosphorylation site of a myosin heavy chain IIA (SD) or with no peptide (as indicated at the base of the figure). Histamine secretion was induced by the addition of dinitrophenylated bovine serum albumin (DNP-BSA). Cells incubated in the presence of the SA peptide showed reduced histamine secretion compared to induced cells incubated without a peptide, and induced cells treated with either SS or SD peptides.
Figure 7B is a graphical representation showing the effect of peptides that mimic a PKC-β phosphorylation site of a myosin heavy chain polypeptide have on histamine secretion. RBL-2H3 cells were permeabilized and incubated with a peptide that corresponded to the PKC-β phosphorylation site of myosin heavy chain IIA (SS), a peptide that corresponded to a mutant form of a PKC-β phosphorylation site of myosin heavy chain IIA that could not be phosphorylated (SA), a peptide that mimicked a phosphorylated PKC-β phosphorylation site of a myosin heavy chain IIA (SD) or with no peptide (as indicated at the base of the figure). Histamine secretion was induced by the addition of "GTPγS (GTPgS)". Cells incubated in the presence of the SA peptide showed reduced histamine secretion compared to induced cells incubated without a peptide, and induced cells treated with either SS or SD peptides.
Detailed Description of the Preferred Embodiments The present invention provides a method for determining protein kinase C-β (PKC-β) activity in a sample comprising contacting a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with a ligand that specifically binds to an amino acid residue within said phosphorylation site or a sample comprising said ligand for a time and under conditions sufficient for binding to occur and then detecting the binding wherein the binding is indicative of PKC-β activity. As used herein the term "protein kinase C-β" or "PKC-β" shall be taken to include all isozymes of PKC-β, such as, for example, PKC-βl and PKC-βll. The term "PKC-β" shall be understood to include any peptide, polypeptide or protein having at least about 80% identity to the amino acid sequence set forth in SEQ ID NO: 169 or 171 or functional fragment thereof. Preferably, the percentage identity to SEQ ID NO: 169 or 171 or functional fragment thereof is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
As used herein the term "functional fragment" of a PKC-β shall be understood to mean any fragment of a PKC-β polypeptide that is capable of phosphorylating a PKC-β phosphorylation site of a myosin heavy chain IIA polypeptide when incubated with a peptide, polypeptide or protein comprising said PKC-β phosphorylation site under conditions sufficient for phosphorylation to occur (e.g., a peptide comprising the sequence set forth in SEQ ID NO: 130).
As will be apparent from the preceding discussion, "PKC-β" activity shall be understood to mean that a protein is capable of covalently attaching a phosphate group to another protein, and, in particular, a PKC-β phosphorylation site of a myosin IIA polypeptide.
As used herein the term "a PKC-β phosphorylation site of a myosin heavy chain polypeptide" shall be taken to mean an amino acid residue of the heavy chain of a myosin heavy chain polypeptide that is phosphorylated by a PKC-β isozyme including sufficient flanking amino acid residues to permit phosphorylation by PKC-β to occur. Preferably, the phosphorylation site will comprise about 1 to 3 or about 3 to 5 or about 5 to 7 or about 7 to 9 or more flanking in residues N-terminal and/or C-terminal to the site of phosphorylation. Preferably, the myosin heavy chain is specifically phosphorylated by a PKC-β isozyme, i.e., other PKC isozymes phosphorylate the PKC- β phosphorylation site of a myosin heavy chain polypeptide at a level significantly lower than that at which the PKC-β isozyme phosphorylates the site. For example, a PKC-β isozyme phosphorylates the PKC-β phosphorylation site of a myosin heavy chain polypeptide at least about 2 time more efficiently than another PKC isozyme, more preferably, at least about 3 times more efficiently, more preferably at least about 4 times more efficiently, more preferably at least about 5 times more efficiently. By "level of efficiency" is meant that a PKC-β isozyme phosphorylates more PKC-β phosphorylation site of a myosin heavy chain polypeptide than another PKC isozyme. Accordingly, a PKC-β isozyme phosphorylates at least about 2 times or at least about three times or at least about four times or at least about five times more PKC-β phosphorylation site of a myosin heavy chain polypeptide than another PKC isozyme.
As will be apparent from the preceding description other PKC isozymes may phosphorylate the PKC-β phosphorylation site of a myosin heavy chain polypeptide, albeit to a significantly reduced degree compared to a PKC-β isozyme. Accordingly, a PKC-β phosphorylation site of a myosin heavy chain polypeptide is preferentially phosphorylated by a PKC-β enzyme, albeit not necessarily exclusively.
As discussed herein a PKC-β phosphorylation site of a myosin heavy chain polypeptide is derived from a region of a myosin heavy chain polypeptide or a peptide, polypeptide or protein comprising the region that comprises an amino acid that is capable of being phosphorylated by a PKC-β. Accordingly, the amino acid may be either phosphorylated or unphosphorylated.
By "myosin heavy chain polypeptide" is meant a component of a myosin hexamer, wherein the myosin heavy chain has a molecular weight of at least about 200 kDa, and wherein the C-terminus of the protein forms an α-helix and the N-terminus of the protein forms a globular head that comprises an actin binding site and an ATP binding site. Preferably, a myosin heavy chain polypeptide is a myosin II polypeptide. Myosins II are involved is a wide variety of cellular processes, such as, for example muscle contraction (both smooth and skeletal muscle), cytokenesis and polarized cell locomotion.
In an even more preferred embodiment, the myosin II polypeptide is a non-muscle myosin IIA polypeptide. Preferably, the myosin heavy chain polypeptide comprises and amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6 or 8. Preferably, the percentage identity to SEQ ID NO: 2, 4, 6 or 8 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
For the purposes of nomenclature, the amino acid sequence of human myosin heavy chain IIA is set forth in SEQ ID NO: 2, the amino acid sequence of rat myosin heavy chain IIA is set forth in SEQ ID NO: 4, the amino acid sequence of mouse myosin heavy chain IIA is set forth in SEQ ID NO: 6 and the amino acid sequence of chicken myosin heavy chain IIA is set forth in SEQ ID NO: 8.
Preferably, the myosin heavy chain polypeptide is human non-muscle myosin IIA (SEQ ID NO: 2). However, the invention shall not be limited to human non-muscle myosin IIA. As shown in Figure 5, the region of human, rat, mouse and chicken non- muscle myosin IIA comprising the PKC-β phosphorylation site identified by the inventors is highly conserved. Accordingly, the present invention encompasses a PKC- β phosphorylation site in any non-muscle myosin IIA polypeptide.
In another preferred embodiment of the invention, the myosin heavy chain polypeptide is non-muscle myosin IIA that has been phosphorylated by PKC-β. Preferably, the non-muscle myosin IIA comprises an amino acid sequence at least about 80% identical to the sequence set forth in SEQ ID NO: 2, 4 or 6, and is phosphorylated at the serine residue at position 1916 of SEQ ID NO: 2 or 6, position 1917 of SEQ ID NO: 4 or position 1915 of SEQ ID NO: 8.
In another embodiment, the myosin II polypeptide is encoded by a nucleic acid that comprises a nucleotide sequence at least about 80% identical to the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5 or 7. Preferably, the percentage identity to SEQ ID NO: 1, 3, 5 or 7 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
For the purposes of nomenclature, the nucleotide sequence of human myosin heavy chain IIA is set forth in SEQ ID NO: 1, the nucleotide sequence of rat myosin heavy chain IIA is set forth in SEQ ID NO: 3, the nucleotide sequence of mouse myosin heavy chain IIA is set forth in SEQ ID NO: 5 and the nucleotide sequence of chicken myosin heavy chain IIA is set forth in SEQ ID NO: 7.
In determining whether or not two amino acid sequences fall within the defined percentage identity limits supra, those skilled in the art will be aware that it is possible to conduct a side-by-side comparison of the amino acid sequences. In such comparisons or alignments, differences will arise in the positioning of non-identical residues depending upon the algorithm used to perform the alignment. In the present context, references to percentage identities and similarities between two or more amino acid sequences shall be taken to refer to the number of identical and similar residues respectively, between said sequences as determined using any standard algorithm known to those skilled in the art. In particular, amino acid identities and similarities are calculated using software of the Computer Genetics Group, Inc., University Research Park, Maddison, Wisconsin, United States of America, eg., using the GAP program of Devereaux et al, Nucl Acids Res. 12, 387-395, 1984, which utilizes the algorithm of Needleman and Wunsch, J Mol. Biol. 48, 443-453, 1970. Alternatively, the CLUSTAL W algorithm of Thompson et al, Nucl. Acids Res. 22, 4673-4680, 1994, is used to obtain an alignment of multiple sequences, wherein it is necessary or desirable to maximise the number of identical/similar residues and to minimise the number and/or length of sequence gaps in the alignment. Amino acid sequence alignments can also be performed using a variety of other commercially available sequence analysis programs, such as, for example, the BLAST program available atNCBI.
As used herein the term "NCBI" shall be taken to mean the database of the National Center for Biotechnology Information at the National Library of Medicine at the National Institutes of Health of the Government of the United States of America, Bethesda, MD, 20894.
In determining whether or not two nucleotide sequences fall within a particular percentage identity limitation recited herein, those skilled in the art will be aware that it is necessary to conduct a side-by-side comparison or multiple alignment of sequences. In such comparisons or alignments, differences may arise in the positioning of non- identical residues, depending upon the algorithm used to perform the alignment. In the present context, reference to a percentage identity between two or more nucleotide sequences shall be taken to refer to the number of identical residues between said sequences as determined using any standard algorithm known to those skilled in the art. For example, nucleotide sequences may be aligned and their identity calculated using the BESTFIT program or other appropriate program of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America (Devereaux et al, Nucl. Acids Res. 12, 387-395, 1984).
In one embodiment of the invention, a peptide, polypeptide or protein comprising
PKC-β phosphorylation site of a myosin heavy chain polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 9 to 141. In a particularly preferred embodiment, the PKC-β phosphorylation site of a myosin heavy chain polypeptide is phosphorylated at an amino acid corresponding to the serine residue at position 9 of the sequence set forth in SEQ ID NO: 9 (i.e. SEQ ID NO: 10). In this regard, the present invention contemplates a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide, such as, for example, a peptide, polypeptide or protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO
10, SEQ ID NO 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO 78, SEQ ID NO 80, SEQ ID NO 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO 88, SEQ ID NO 90, SEQ ID NO 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO 98, SEQ ID NO 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139 and SEQ ID NO: 141.
A peptide, polypeptide or protein comprising an unphosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138 and SEQ ID NO: 140.
In a preferred embodiment, a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 130 that is capable of being phosphorylated at a position corresponding -to amino acid position 7 of said sequence, i.e., the peptide, polypeptide or protein comprises an amino acid sequence set forth in SEQ ID NO: 131.
In one embodiment, the PKC-β phosphorylation site of a myosin heavy chain polypeptide is a myosin heavy chain IIA polypeptide or a fragment thereof comprising an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 141 or a variant thereof.
As used herein, the term variant shall be taken to refer to a peptide or polypeptide or protein comprising an amino acid sequence sufficiently similar to an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 141 to allow for a PKC-β to bind and phosphorylate the peptide, polypeptide or protein. Preferably the level of sequence identity shall be at least about 80% or 85% or 90% or 95% or 98%. For example, the amino acid sequence of a variant is set forth in any one of SEQ ID Nos: 142 to 167.
As used herein the term "ligand" shall be taken in its broadest context to include any chemical compound, polynucleotide, peptide, protein, lipid, carbohydrate, small molecule, natural product, polymer, etc. that is capable of selectively binding, whether covalently or not, to one or more specific sites on a target molecule comprising a PKC- β phosphorylation site of a myosin heavy chain. The ligand may bind to its target via any means including hydrophobic interactions, hydrogen bonding, electrostatic interactions, van der Waals interactions, pi stacking, covalent bonding, or magnetic interactions amongst others. It is particularly preferred that a ligand is able to specifically bind to a PKC-β phosphorylation site of a myosin heavy chain polypeptide that has been phosphorylated by a PKC-β.
Antibody-based assays In one embodiment, the PKC-β phosphorylation site of a myosin heavy chain polypeptide is in an extract or cellular sample or sample being assayed. Preferably, the level of phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide in the extract or cellular sample or sample being assayed is detected using an antibody.
As used herein the term "antibody" refers to an intact monoclonal or polyclonal antibodies, an immunoglobulin (IgA, IgD, IgG, IgM, IgE) fraction or mixture thereof, a humanized antibody, or a recombinant single chain antibody, as well as fragments thereof, such as, for example a Fab, a F(ab)2 or a Fv fragment.
The present inventors have produced a polyclonal antibody that is capable of specifically binding to a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide. Accordingly, in a preferred embodiment, the antibody is a polyclonal antibody.
In a further preferred embodiment, the antibody is capable of specifically binding to a PKC-β phosphorylation site of a myosin heavy chain polypeptide that has been phosphorylated by a PKC-β. As used herein, the term "specifically binding to" and equivalents, for example, "specific binding" shall be taken to mean that the antibody or ligand binds to a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide producing a signal at a level that is significantly above background (i.e., a high-signal-to-noise ratio). The degree of binding is measured, for example using a labelled antibody and/or a labelled secondary antibody and detecting the label following binding of the antibody, e.g., using a Western Blot. Appropriate methods for determining the ability of an antibody to preferentially bind to a phosphorylated polypeptide are known in the art and/or described herein, and include for example, a Western blot, an ELISA or the use of a biosensor.
Preferably, an antibody that specifically binds to a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide does not bind to a non-PKC-β phosphorylation site of a myosin heavy chain polypeptide at a level that facilitates detection of the bound antibody, e.g., using an assay described herein.
In a preferred embodiment, the antibody specifically binds to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 9 wherein the serine at position 9 is phosphorylated. Accordingly, it is preferable that the antibody specifically binds to a peptide, polypeptide or protein comprising the amino acid sequence set forth in SEQ ID NO: 10.
In another preferred embodiment, the antibody preferentially, specifically or selectively binds to a peptide, polypeptide or protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO 12, SEQ ID NO: 14, SEQ
IDNO: 16, SEQ ID NO : 18, SEQ ID NO: 20, SEQ ID NO 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO : 28, SEQ ID NO: 30, SEQ ID NO 32, SEQ ID NO: 34, SEQ IIDD NNOO:: 3366,, SSEEQQ IIDD NNOO: 38, SEQ ID NO: 40, SEQ ID NO 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO : 48, SEQ ID NO: 50, SEQ ID NO 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO : 58, SEQ ID NO: 60, SEQ ID NO 62, SEQ ID NO: 64, SEQ IDNO: 66, SEQ ID NO : 68, SEQ ID NO: 70, SEQ ID NO 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO : 78, SEQ ID NO: 80, SEQ ID NO 82, SEQ ID NO: 84, SEQ IIDD NNOO:: 8866,, SSEEQQ IIDD NNOO: 88, SEQ ID NO: 90, SEQ ID NO 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO : 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139 and SEQ ID NO: 141. For example, the preferable or selective binding is compared to the binding to a peptide, polypeptide or protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45 SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55 SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65 SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75 SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85 SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95 SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124; SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO 134, SEQ ID NO: 136, SEQ ID NO: 138 and SEQ ID NO: 140. . For example, the preferential binding is determined using a Western Blot, dot blot or other immunoassay.
It is further preferred that an antibody useful in performing the method of the invention is capable of binding to a myosin heavy chain polypeptide that has been phosphorylated by a PKC-β. Preferably, the antibody is capable of phosphorylating the myosin heavy chain polypeptide at an amino acid equivalent to amino acid position 1916 of SEQ ID NO: 2 or SEQ ID NO: 6 or amino acid position 1917 of SEQ ID NO: 4 or amino acid position 1915 of SEQ ID NO: 8.
In yet another embodiment, an antibody capable of binding to a peptide, polypeptide or protein comprising phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide is also capable of binding to a mimetic of said site. As used herein, the term "mimetic" shall be taken to mean a compound that is capable of forming a secondary structure that is sufficiently similar to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide so as to bind an antibody that specifically binds said site. For example, a mimetic peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 131 or 132. Other suitable mimetic peptides will be apparent from the description herein and include, for example, a peptide, polypeptide or protein comprising an amino acid sequence set forth in SEQ ID NO: 9 wherein the serine at position 9 is replaced with aspartic acid or alanine. By substituting the serine at the corresponding position in a peptide, polypeptide or protein comprising a sequence set forth in any one of SEQ ID NOs: 9 to 167 a suitable mimetic is produced.
Antibodies referred to herein are obtained from a commercial source, or alternatively, produced by conventional means. Commercial sources will be known to those skilled in the art, for example, Eurogentec (Seraing, Belgium) produce monoclonal and polyclonal antibodies capable of specifically detecting a phosphorylated antigen.
High titer antibodies are preferred, as these are more useful commercially in kits for analytical, diagnostic and/or therapeutic applications. By "high titer" is meant a titer of at least about 1:103 or 1:104 or 1:105. Methods of determining the titer of an antibody will be apparent to the skilled artisan. For example, the titer of an antibody in purified antiserum may be determined using an ELISA assay to determine the amount of IgG in a sample. Typically an anti-IgG antibody or Protein G is used in such an assay. The amount detected in a sample is compared to a control sample of a known amount of purified and/or recombinant IgG. Alternatively, a kit for determining antibody may be used, e.g. the Easy TITER kit from Pierce (Rockford, IL, USA).
Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art, and are described, for example in, Harlow and Lane (In: Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In one such technique, an immunogen comprising the antigenic polypeptide is initially injected into any one of a wide variety of animals (e.g., mice, rats, rabbits, sheep, humans, dogs, pigs, chickens and goats). The immunogen is derived from a natural source, produced by recombinant expression means, or artificially generated, such as by chemical synthesis (e.g., BOC chemistry or FMOC chemistry). In this step, the polypeptides or fragments thereof of this invention may serve as the immunogen. Preferably, the polypeptide is a phosphorylated polypeptide. The polypeptide may be isolated in a phosphorylated state, or, alternatively, may be phosphorylated using a recombinant or isolated kinase, such as, for example, PKC-β (available from Oxford Biomed, Oxford, MI, 48371, USA), or produced using synthetic means.
A peptide, polypeptide or protein is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen and optionally a carrier for the protein is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and blood collected from said the animals periodically. Optionally, the immunogen is injected in the presence of an adjuvant, such as, for example Freund's complete or incomplete adjuvant, lysolecithin and dinitrophenol to enhance the immune response to the immunogen. Monoclonal or polyclonal antibodies specific for the polypeptide may then be purified from the blood isolated from an animal by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support. Preferably, the polypeptide used to affinity purify an antibody is a phosphorylated polypeptide.
Preferably, a two step purification process is used to produce the antibody. For example, an antibody is contacted to a peptide, polypeptide or protein that comprises a non-phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide. Unbound antibodies are then collected. These antibodies are then contacted to a peptide, polypeptide or protein that comprises a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide. Bound antibodies are then collected and are specific to the phosphorylated site. As will be apparent the purification steps need not be performed in any order.
Accordingly, only an antibody capable of binding a phosphorylated antigen is purified. In accordance with this method, a phosphatase inhibitor may be used when screening for a phosphorylated antigen specific antibody to inhibit the removal of a phosphate group from the antigen of interest.
Monoclonal antibodies specific for the antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol <J:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described supra. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngenic with the immunized animal. A variety of fusion techniques may be employed, for example, the spleen cells and myeloma cells may be combined with a nonionic detergent or electrofused and then grown in a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and growth media in which the cells have been grown is tested for the presence of binding activity against the polypeptide (immunogen). Hybridomas having high reactivity and specificity are preferred.
Monoclonal antibodies are isolated from the supematants of growing hybridoma colonies using methods, such as, for example, affinity purification as described supra. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies are then harvested from the ascites fluid or the blood of such an animal subject. Contaminants are removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and/or extraction. The PKC-β phosphorylation site of a myosin heavy chain polypeptide of this invention may be used in the purification process in, for example, an affinity chromatography step. As described supra a monoclonal antibody that specifically binds to a phosphorylated antigen is preferred. It is preferable that an immunogen used in the production of an antibody is one which is sufficiently antigenic to stimulate the production of antibodies that will bind to the immunogen and is preferably, a high titer antibody. In one embodiment, an immunogen may be an entire protein.
In another embodiment, an immunogen may consist of a peptide representing a fragment of a polypeptide. Preferably, a phosphorylated fragment of a polypeptide, i.e., a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide. Preferably an antibody raised to such an immunogen also recognizes the full-length protein from which the immunogen was derived, such as, for example, in its native state or having native conformation. Even more preferably, an antibody raised to such an immunogen recognizes the full-length protein when it is phosphorylated (and not when it is unphosphorylated).
Alternatively, or in addition, an antibody raised against a peptide immunogen will recognise the full-length protein from which the immunogen was derived when the protein is denatured. By "denatured" is meant that conformational epitopes of the protein are disrupted under conditions that retain linear B cell epitopes of the protein. As will be known to a skilled artisan linear epitopes and conformational epitopes may overlap.
As discussed supra antibody fragments are contemplated by the present invention. The term "antibody fragment" refers to a portion of a full-length antibody, generally the antigen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments.
Papain digestion of an antibody produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment.
Pepsin treatment yields an F(ab')2 fragment that has two antigen binding fragments that are capable of cross-linking antigen, and a residual other fragment (which is termed pFc1). Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. As used herein, "functional fragment" with respect to antibodies, refers to Fv, F(ab) and F(ab')2 fragments.
An "Fv" fragment is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a non-covalent association (VH -VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH -VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen.
A Fab fragment [also designated as F(ab)] also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.
"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269- 315 (1994).
In a particularly preferred embodiment of the present invention, an antibody is capable of binding to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10.
As exemplified herein, an antibody that specifically recognizes a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO: 10 may be a polyclonal antibody. Methods for determining binding of an antibody
In one embodiment of the present invention the level of PKC-β activity is determined by performing a process comprising:
(i) contacting a sample comprising a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with an antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody-antigen complex to form; and (ii) determining the level of said complex, wherein the level of said complex is indicative of PKC-β activity.
In accordance with this embodiment, the amount of a phosphorylation site bound to an antibody, i.e., the level of the complex is determined using an assay known to the skilled artisan, and preferably, an immunoassay.
The level of a protein or phosphorylated protein or an antibody bound thereto may be determined using a variety of techniques known to the skilled artisan such as, for example, a technique selected from the group consisting of, immunohistochemistry, immunofluorescence, an immunoblot, a Western blot, a dot blot, an enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay, fluorescence resonance energy transfer (FRET), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry, eg LC MS/MS), biosensor technology, evanescent fiber-optics technology or protein chip technology.
In one embodiment the assay used to determine the amount or level of a protein is a semi-quantitative assay.
In another embodiment the assay used to determine the amount or level of a protein in a quantitative assay.
Immunoassays
Preferably, the level of antibody bound to a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain (i.e., PKC-β activity) is determined using an immunoassay. Preferably using an assay selected from the group consisting of, immunohistochemistry, immunofluorescence, ELISA, Western blotting, RIA, a biosensor assay, a protein chip assay, a mass spectrometry assay, a fluorescence resonance energy transfer assay and an immunostaining assay (e.g. immunofluorescence).
For example, a standard solid-phase ELISA format is useful in determining the level of an enzyme labelled ligand or antibody.
In one form such an assay involves immobilizing a biological sample onto a solid matrix, such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g. a glass slide).
An antibody that specifically binds a peptide, polypeptide or protein comprising a phosphorylation site of a myosin heavy chain polypeptide is brought into direct contact with the cell, and forms a direct bond with any of its target protein present in said sample. This antibody is generally labelled with a detectable reporter molecule, such as for example, an enzyme (e.g. horseradish peroxidase (HRP)), alkaline phosphatase (AP) or β-galactosidase. Alternatively, a second labelled antibody can be used that binds to the first antibody. Following washing to remove any unbound antibody the detectable marker is detected by the addition of a substrate, such as for example hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indol-beta-D- galaotopyranoside (x-gal). A suitable substrate will depend on the label used and will be apparent to the skilled artisan. For example, in the case of β-galactosidase a suitable substrate is x-gal.
In accordance with the preceding description the present invention clearly contemplates determining the level of the complex by performing a process comprising contacting the complex with another antibody capable of binding to said complex for a time and under conditions sufficient for another antibody-antigen complex to form and detecting the level of the other complex, wherein the level of said complex is indicative of PKC- activity.
The level of the peptide, polypeptide or protein comprising a phosphorylation site of a myosin heavy chain polypeptide may then be determined, for example using a standard curve that has been produced using known quantities of the peptide, polypeptide or protein comprising a phosphorylation site of a myosin heavy chain polypeptide (e.g. peptide, polypeptide or protein comprising a phosphorylation site of a myosin heavy chain polypeptide ) or another suitable standard, such as for example, described herein.
In the case of a fluorescent label, a fluorescence linked immunosorbent assay (FLIS A) is useful for determining the level of a labeled ligand or antibody in a sample. A FLISA is performed essentially as described supra for the ELISA assay, however, a substrate is not required to detect the bound labeled ligand or antibody. Rather, following washing to remove any unbound ligand/antibody the sample is exposed to a light source of the appropriate wavelength to stimulate the fluorescent label and the level of fluorescence emitted by each sample determined. A FLISA is also known as an immunofluorescence assay (IF A). The present invention contemplates such an assay.
As will be apparent to the skilled artisan, other detection methods based on an immunosorbent assay are useful in the performance of the present invention. For example, an immunosorbent method based on the description supra using a radiolabel for detection, or a gold label (e.g. colloidal gold) for detection, or a liposome, for example, encapsulating NAD+ for detection (e.g., as described in Kumada et al, Journal of Chemical Engineering of Japan, 34: 943-947, 2001) or an acridinium linked immunosorbent assay.
In another form, an immunoassay, e.g., an ELISA or FLISA comprises immobilizing an antibody or ligand that specifically binds a phosphorylation site of a myosin heavy chain polypeptide on a solid matrix, such as, for example, a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate dipstick or a glass support. A sample is then brought into physical relation with said antibody, and said phosphorylation site of a myosin heavy chain polypeptide is bound or ' captured \ The bound protein is then detected using a labeled antibody. For example, an antibody that binds to a distinct epitope on the peptide, polypeptide or protein comprising a phosphorylation site of a myosin heavy chain polypeptide. Alternatively, a third labeled antibody can be used that binds the second (detecting) antibody.
In accordance with this embodiment, the present invention provides a method for determining the level of PKC-β activity comprising: (i) contacting a sample comprising a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with a first antibody that binds to said peptide, polypeptide or protein at a site other than the PKC-β phosphorylation site for a time and under conditions sufficient for the antibody to bind to the peptide, polypeptide or protein;
(ii) contacting the bound peptide, polypeptide or protein at (i) with a second antibody that specifically binds to the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody-antigen complex to form; and
(iii) deteπriining the level of said complex, wherein the level of said complex is indicative of PKC-β activity.
In one form of the assay the first antibody specifically binds to a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide. However, it is also contemplated that the second antibody specifically binds to a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide.
To facilitate the detection of the peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide, either the first antibody or the second is/was previously immobilized on a solid support, matrix or resin.
To facilitate determining or quantifying the level of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide in a sample, the level detected with an assay described supra is compared to a standard. For example, a standard is produced using a method comprising:
(i) contacting a first antibody that binds to a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide at a site other than the PKC-β phosphorylation site with (a) a known amount of a labelled unphosphorylated peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof and (b) a known amount of a labelled phosphorylated peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof for a time and under conditions sufficient for the antibody to bind to the peptide, polypeptide or protein; (ii) contacting the bound peptide, polypeptide or protein at (ii) with a second antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody-antigen complex to form; and (iii) determining the level of complex relative to the level of the detectable label, wherein a comparable level of the level of complex determined in the sample and the level of the complex determined in the standard indicates that a similar level of phosphorylated peptide, polypeptide or protein is present in both samples.
In accordance with this embodiment, the labelled peptide, polypeptide or protein comprising a phosphorylated or unphosphorylated PKC-β phosphorylation site may be produced using synthetic and/or recombinant means, e.g., as described herein. A suitable label will be apparent to the skilled artisan and includes, for example, a label described supra or an epitope, e.g., a FLAG epitope or a HA epitope to facilitate detection with an antibody.
Such an assay enables the user to determine the level of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site relative to the total level of peptide, polypeptide or protein comprising an unphosphorylated PKC-β phosphorylation site in an assay. By mixing various known concentrations of a phosphorylated and an unphosphorylated peptide, polypeptide or protein comprising a PKC-β phosphorylation site a standard curve is produced that enables the skilled artisan to determine or estimate the level of peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site in a sample.
In one embodiment, the method for producing the standard additionally comprises mixing the known amount/s of the labelled peptide, polypeptide or protein comprising a unphosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof and labelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof.
In another embodiment, the method additionally comprises providing the labelled unphosphorylated peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof and/or labelled phosphorylated peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof. For example, by using a method described herein.
The present invention additionally contemplates a standard produced using the method described supra. Alternatively, the present invention encompasses a standard comprising (a) a known amount of a labelled unphosphorylated peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof and (b) a known amount of a labelled phosphorylated peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof.
In a preferred embodiment, the present invention contemplates a competitive immunoassay, such as, for example, a competitive ELISA or FLISA. In accordance with this embodiment, an antibody capable of specifically binding peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide is, for example, immobilised on a solid support. A sample is then brought into contact with said antibody in the presence of a known amount of a labeled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide (e.g., a recombinant or synthetic peptide), i.e. a competitor. The competitor may be added at the same time as or after contacting the sample. Following washing to remove unbound protein (e.g., competitor) the level of detectable label is detected. The level of label detected is inversely proportional to the level of the peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide in the sample. Using a standard curve the level of the peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide in the sample may be quantified. A suitable standard will be apparent to the skilled artisan and/or is described herein.
In accordance with the foregoing, the present invention clearly contemplates a method for determining the level of PKC-β comprising:
(i) contacting a sample comprising a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with an antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody-antigen complex to form and contacting said antibody with a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof labelled with a detectable marker; and (ii) determining the level of detectable marker bound to the antibody, wherein the level of detectable marker bound to the antibody is inversely proportional to the level of PKC-β activity.
In one embodiment, the antibody is/was previously immobilized on a solid support, matrix or resin. Such immobilisation facilitates determining the level of PKC-β activity in a sample.
In one embodiment, the method additionally comprises washing the sample to remove any non-specifically bound or unbound protein prior to determining the level of detectable marker bound to the antibody.
As discussed supra the present embodiment is suitable for determining the level of PKC-β activity in a sample, for example, by comparing the level of detectable marker bound to the antibody in the sample to a standard.
In one embodiment, the standard is produced using a process comprising: (i) contacting the antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide with (a) a known amount a labelled phosphorylated peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide and (b) a known amount of a unlabelled phosphorylated peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof for a time and under conditions sufficient for an antibody-antigen complex to form; and (ii) determining the level of detectable marker bound to the antibody, wherein a comparable level of detectable marker in the sample and the standard indicates that a similar level of phosphorylated peptide, polypeptide or protein is present in both samples.
The use of both labelled and unlabelled peptides, polypeptides or proteins causes competition between the peptides, polypeptides or proteins for binding to the antibody. By determining the level of bound labelled peptide, polypeptide or protein relative to total labelled peptide, polypeptide or protein and total labelled and unlabelled peptide, polypeptide or protein a quantitative measure is produced.
Preferably, a number of different concentrations of unlabelled peptide, polypeptide or protein is used in the presence of a constant amount of labelled peptide, polypeptide or protein to determine the change in the level of labelled peptide, polypeptide or protein detected. This facilitates determining or producing a standard curve useful for determining the level of peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide in a sample.
In accordance with this embodiment, the labelled or unlabelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site may be produced using synthetic and/or recombinant means, e.g., as described herein. A suitable label will be apparent to the skilled artisan and includes, for example, a label described supra or an epitope, e.g., a FLAG epitope or a HA epitope to facilitate detection with an antibody.
In one embodiment, the method for producing the standard additionally comprises mixing the known amount/s of the labelled and unlabelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof.
In another embodiment, the method additionally comprises providing the labelled and/or unlabelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof. For example, by using a method described herein.
The present invention additionally contemplates a standard produced using the method described supra. Alternatively, the present invention encompasses a standard comprising (a) a known amount a labelled phosphorylated peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide and (b) a known amount of a unlabelled phosphorylated peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof. In another embodiment, the presence of an enhanced level of a phosphorylation site of a myosin heavy chain polypeptide is detected using a radioimmunoassay (RIA). The basic principle of the assay is the use of a radiolabeled antibody or antigen to detect antibody-antigen interactions. An antibody or ligand that specifically binds to a phosphorylation site of a myosin heavy chain polypeptide is bound to a solid support and a sample brought into direct contact with said antibody. In order to detect the amount of bound antigen, an isolated and/or recombinant form of the antigen is radiolabeled and brought into contact with the same antibody. Following washing the amount of bound radioactivity is detected. As any antigen in the biological sample inhibits binding of the radiolabeled antigen the amount of radioactivity detected is inversely proportional to the amount of antigen in the sample. Such an assay may be quantitated by using a standard curve using increasing known concentrations of the isolated antigen.
In a particularly preferred embodiment, Western blotting is used to determine the level of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide in a sample. In such an assay protein from a sample is separated using sodium doedecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using a technique known in the art and described, for example, in Scopes (In: Protein Purification: Principles and Practice, Third Edition, Springer Verlag, 1994). Separated proteins are then transferred to a solid support, such as, for example, a membrane (e.g., a PVDF membrane), using methods known in the art, for example, electrotransfer. This membrane is then blocked and probed with a labeled antibody or ligand that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide. Alternatively, a labeled secondary, or even tertiary, antibody or ligand is used to detect the binding of a specific primary antibody. The level of label is then determined using an assay appropriate for the label used. An appropriate assay will be apparent to the skilled artisan.
For example, the level of a phosphorylation site of a myosin heavy chain polypeptide may be determined using methods known in the art, such as, for example, densitometry. In one embodiment, the intensity of a protein band or spot is normalized against the total amount of protein loaded on a SDS-PAGE gel using methods known in the art. Alternatively, an amount of a phosphorylation site of a myosin heavy chain polypeptide detected is normalized against the amount of a control/reference protein. Such control proteins are known in the art, and include, actin, glyceraldehyde 3 -phosphate dehydrogenase (GAPDH), β2 microglobulin, hydroxy-methylbilane synthase, hypoxanthine phosphoribosyl-transferase 1 (HPRT), ribosomal protein L13c, succinate dehydrogenase complex subunit A and TATA box binding protein (TBP). Preferably, the control protein is a myosin heavy chain polypeptide, more preferably, a myosin IIA polypeptide. Following normalization, a relative amount of a phosphorylation site of a myosin heavy chain polypeptide in a biological sample is determined.
In another embodiment, the level of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide is normalized with reference to the total amount or level of the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of^a myosin heavy chain polypeptide, for example using parallel samples. By "parallel samples" is meant two samples derived from the same initial sample and, as a consequence, comprising approximately equal levels of proteins.
The detection of the phosphorylation status of a phosphorylation site of a myosin heavy chain polypeptide using a method such as, for example, mass spectrometry, matrix- assisted laser desorption/ionization time of flight (MALDI-TOF), electrospray ionisation (ESI), protein chip, biosensor technology, or fluorescence resonance energy transfer, is clearly contemplated in the present invention.
For example, a biosensor device generally employs an electrode surface in combination with current or impedance measuring elements to be integrated into a device in combination with the assay substrate (such as that described in U.S. Patent No. 5,567,301). An antibody that specifically binds to a protein of interest is preferably incorporated onto the surface of a biosensor device and a biological sample contacted to said device. A change in the detected current or impedance by the biosensor device indicates protein binding to said antibody. Some forms of biosensors known in the art also rely on surface plasmon resonance to detect protein interactions, whereby a change in the surface plasmon resonance surface of reflection is indicative of a protein binding to a ligand or antibody (U.S. Patent No. 5,485,277 and 5,492,840).
Biosensors are of particular use in high throughput analysis due to the ease of adapting such systems to micro- or nano-scales. Furthermore, such systems are conveniently adapted to incorporate several detection reagents, allowing for multiplexing of diagnostic reagents in a single biosensor unit. This permits the simultaneous detection of several proteins or peptides in a small amount of body fluids.
Evanescent biosensors are also preferred as they do not require the pre-treatment of a biological sample prior to detection of a protein of interest. An evanescent biosensor generally relies upon light of a predetermined wavelength interacting with a fluorescent molecule, such as for example, a fluorescent antibody attached near the probe's surface, to emit fluorescence at a different wavelength upon binding of the phosphorylation site of a myosin heavy chain polypeptide to the antibody or ligand.
Micro- or nano-cantilever biosensors are also preferred as they do not require the use of a detectable label. A cantilever biosensor utilizes a ligand or antibody capable of specifically detecting the analyte of interest that is bound to the surface of a deflectable arm of a micro- or nano-cantilever. Upon binding of the analyte of interest (e.g. a protein) the deflectable arm of the cantilever is deflected in a vertical direction (i.e. upwards or downwards). The change in the deflection of the deflectable arm is then detected by any of a variety of methods, such as, for example, atomic force microscopy, a change in oscillation of the deflectable arm or a change in pizoresistivity. Exemplary micro-cantilever sensors are described in USSN 20030010097.
Alternatively, a biosensor that utilizes a lipid membrane is used. Such a biosensor uses a lipid membrane that incorporates a lipid bilayer that comprises an ion channel or ionophore, wherein the lipid bilayer is tethered to a metal electrode (such biosensors are described in AU 623,747, US 5,234,566 and USSN 20030143726). One form of such a biosensor involves two receptors or antibodies that bind to each other being incorporated into a lipid bilayer. One of these receptors/antibodies is bound to an ion channel or ionophore that spans the outer half of the membrane, and this membrane/antibody is also capable of binding to the analyte of interest. The second receptor/antibody is tethered to a membrane molecule (i.e. not the ionophore or ion channel). When the receptors/antibodies are not bound to each other, the ion channel aligns with another half membrane spanning ionophore (i.e. an ionophore that spans the inner half of the membrane) thereby facilitating detectable ion transmission across the membrane. However, when the two receptors/antibodies bind each other, the outer membrane ionophore is displaced thereby disrupting membrane conductivity. The analyte of interest competes with the second receptor/antibody for the binding site on the first receptor/antibody. The presence of the analyte breaks the bond between the two receptors/antibodies and allows the half membrane ionophores to align and provide an ion conductive path.
To determine the level of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide using a biosensor device a standard is used that is essentially the same as that used for a sandwich ELISA.
Alternatively, increasing known amounts of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide are used to determine the level of said peptide, polypeptide or protein in a sample. In this regard, it may also be desirable to detect the total level of the a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide in the sample to facilitate normalisation.
To produce protein chips, the proteins, peptides, polypeptides, antibodies or ligands that are able to bind specific antibodies or proteins of interest are bound to a solid support such as for example glass, polycarbonate, polytetrafluoroethylene, polystyrene, silicon oxide, metal or silicon nitride. This immobilization is either direct (e.g. by covalent linkage, such as, for example, Schiff s base formation, disulfide linkage, or amide or urea bond formation) or indirect. Methods of generating a protein chip are known in the art and are described in for example U.S. Patent Application No. 20020136821, 20020192654, 20020102617 and U.S. Patent No. 6,391,625. In order to bind a protein to a solid support it is often necessary to treat the solid support so as to create chemically reactive groups on the surface, such as, for example, with an aldehyde-containing silane reagent. Alternatively, an antibody or ligand may be captured on a microfabricated polyacrylamide gel pad and accelerated into the gel using microelectrophoresis as described in, Arenkov et al. Anal. Biochem. 278:123- 131, 2000.
A protein chip may comprise only one protein, ligand or antibody, and be used to screen one or more patient samples for the presence of one polypeptide of interest. Such a chip may also be used to simultaneously screen an array of patient samples for a polypeptide of interest. Preferably, a protein sample to be analysed using a protein chip is attached to a reporter molecule, such as, for example, a fluorescent molecule, a radioactive molecule, an enzyme, or an antibody that is detectable using methods known in the art. Accordingly, by contacting a protein chip with a labeled sample and subsequent washing to remove any unbound proteins the presence of a bound protein is detected using methods known in the art, such as, for example using a DNA microarray reader.
Alternatively, biomolecular interaction analysis-mass spectrometry (BIA-MS) is used to rapidly detect and characterise a protein present in complex biological samples at the low- to sub-finole level (Nelson et al Electrophoresis 21: 1155-1163, 2000). One technique useful in the analysis of a protein chip is surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) technology to characterise a protein bound to the protein chip. Alternatively, the protein chip is analysed using ESI as described in U.S. Patent Application 20020139751.
A suitable standard for use with a protein chip is that for use with a biosensor.
As will be apparent from the preceding discussion, it is particularly preferred to employ a detection system that is antibody or ligand based as such assays are amenable to the detection of phosphorylated peptides, polypeptides and/or proteins. Immunoassay formats are even more particularly preferred.
In an alternative embodiment, the binding of an antibody to a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide is determined within a cell, using a method known in the art, such as, for example, immunohistochemistry or immunofluorescence.
For example, a cell or tissue section that is to be analyzed to determine PKC-β activity is fixed to stabilize and protect both the cell and the proteins contained within the cell. Preferably, the method of fixation does not disrupt or destroy the antigenicity of a phosphorylation site of a myosin heavy chain polypeptide (and, in particular, the phosphorylation of the phosphorylation site of a myosin heavy chain polypeptide), thus rendering it undetectable. Methods for fixing a cell are known in the art and include, for example, treatment with paraformaldehyde, treatment with alcohol, treatment with acetone, treatment with methanol, treatment with Bouin's fixative and treatment with glutaraldehyde. Following fixation, a cell is incubated with a ligand or antibody capable of binding a phosphorylation site of a myosin heavy chain polypeptide, preferably, a phosphorylated PKC-β site of a myosin heavy chain polypeptide. The ligand or antibody may be labeled with a detectable marker, such as, for example, a fluorescent label (e.g. FITC or Texas Red), a fluorescent semiconductor nanocrystal (as described in US 6,306,610) or an enzyme (e.g. horseradish peroxidase (HRP)), alkaline phosphatase (AP) or β-galactosidase. Alternatively, a second labeled antibody that binds to the first antibody can be used to detect the first antibody. Following washing to remove any unbound antibody, the level of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide is detected using the relevant detection means. Means for detecting a fluorescent label will vary depending upon the type of label used and will be apparent to the skilled artisan.
Optionally, a method of detecting a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide using immunofluorescence or immunohistochemistry will comprise additional steps such as, for example, cell permeabilization (using, for example, n-octyl-BD-glucopyranoside, deoxycholate, a non-ionic detergent such as Triton X-100 NP-40, low concentrations of ionic detergents, such as, for example SDS or saponin) and or antigen retrieval (using, for example, heat).
Methods using immunofluorescence are preferable, as they are quantitative or at least semi-quantitative. Methods of quantitating the degree of fluorescence of a stained cell are known in the art and described, for example, in Immunohistochemistry (Cuello, 1984 John Wiley and Sons, ASIN 0471900524).
Phosphorylation-based assays
In another embodiment of the invention, the ligand is a PKC-β enzyme in an extract or cellular sample or sample being assayed and the peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide is not phosphorylated at an amino acid that is a PKC-β phosphorylation site.
In accordance with this embodiment, the present invention provides a method of determining PKC-β activity comprising detecting the binding between a PKC-β and a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide wherein the interaction between the PKC-β enzyme and the PKC-β phosphorylation site causes phosphorylation of said phosphorylation site. In accordance with this embodiment, the binding between the peptide, polypeptide or protein and the PKC-β is determined by detecting the level of phosphate associated with the phosphorylation site by a method comprising: (i) isolating the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide; (ii) determining the level of phosphate bound to the PKC-β phosphorylation site of a myosin heavy chain polypeptide, wherein the level of phosphate bound to the PKC-β phosphorylation site of a myosin heavy chain polypeptide is indicative of PKC-β activity.
In accordance with this embodiment, the peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide is isolated from a sample that is being assayed. Such samples are described herein.
Methods for isolating a polypeptide are known in the art and described, for example, in Scopes (In: Protein Purification: Principles and Practice, Third Edition, Springer Verlag, 1994).
For example, a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide is isolated from a cellular source using an affinity purification method. Methods of affinity purification typically involve contacting a cellular sample with a ligand or antibody that specifically binds a
PKC-β phosphorylation site of a myosin heavy chain polypeptide (such as, for example myosin heavy chain IIA), and, following washing, eluting those peptides that remain bound to the ligand or antibody. Said ligand or antibody is bound to another molecule to allow for ease of purification, such as, for example, a molecule selected from the group consisting of protein A, protein G, agarose or Sepharose, biotin, glutathione S- transferase (GST), and a FLAG epitope. Accordingly, the PKC-β phosphorylation site of a myosin heavy chain polypeptide is isolated simply through centrifugation, or through binding to another molecule, eg. streptavidin, or binding of a specific antibody, eg. anti-FLAG antibodies, or anti-GST antibodies. Methods using a ligand or antibody covalently bound to an affinity matrix are particularly preferred.
In one embodiment, the peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide is a recombinant polypeptide expressed as a fusion with a tag protein, produced using a method known in the art and/or described herein. The expression of such a fusion protein facilitates rapid isolation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide.
Preferably, the peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide is isolated by contacting a sample with an antibody or ligand that specifically binds said PKC-β phosphorylation site of a myosin heavy chain polypeptide and isolating the PKC-β phosphorylation site of a myosin heavy chain polypeptide bound to the antibody or ligand.
In one embodiment, the method of the present invention comprises dissociating the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide from the antibody or ligand used to capture said polypeptide. Methods of dissociating proteins are known in the art, and described, for example, in Scopes (In: Protein Purification: Principles and Practice, Third Edition, Springer Verlag, 1994). For example, proteins are dissociated by changing the pH of a sample (e.g. with glycine (pH3) or triethanolamine (pHl l)), increasing the salt concentration of a sample with an ionic detergent (e.g. with 5M lithium chloride), treating a sample with an ionic detergent (e.g. SDS), treating a sample with a dissociating agent (e.g. urea), treating a sample with a chaotropic agent (e.g. thiocyanate) or a combination of these methods.
Preferably, the proteins are dissociated in such a way to facilitate the isolation of the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide. For example, a PKC-β phosphorylation site of a myosin heavy chain polypeptide may be captured by an antibody that specifically binds to said polypeptide that is bound to a Sepharose column. Following capture and washing to remove any unbound proteins, the PKC-β phosphorylation site is eluted using a method known in the art and/or described herein and collected for further analysis.
In one embodiment of the present invention, the level of phosphate bound to a PKC-β phosphorylation site of a myosin heavy chain polypeptide is determined by contacting the isolated polypeptide with a marker that binds to a phosphorylated amino acid residue and determining the amount of phosphorylated amino acids bound to the marker. In one embodiment, the marker is a fluorescent marker, such as, for example Pro-Q Diamond dye (as described in Martin et al, Proteomics, 3(7): 1244-1245, 2003). Such a dye specifically binds to phosphate residues that are incorporated into a peptide, polypeptide or protein. Accordingly, following "capture" of a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide using a specific antibody or ligand, the sample is stained with, for example, Pro-Q Diamond dye, and the level of phosphate in the sample determined. As will be apparent to the skilled artisan, an antibody or ligand may also be phosphorylated, and, as such, it may be necessary to also determine the amount of phosphate associated with said antibody or ligand. The use of a dye such as Pro-Q Diamond dye is particularly useful for high-throughput screening of PKC-β activity, as it does not require optimization unlike an antibody based assay. Furthermore, the sensitivity of such an assay means that it is particularly amenable to micro- type formats, thereby requiring smaller amounts of sample for analysis.
As will be apparent to the skilled artisan, the level of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide detected suing an assay of the present embodiment may be normalised using a standard comprising one or more known quantities of peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide. Such quantification facilitates comparing results between different assays.
In another embodiment of the present invention a marker specifically recognizes phosphoserine. Preferably, the marker is a ligand or antibody that is capable of specifically binding to a phosphoserine. Phosphoserine antibodies are available from a variety of sources, such as, for example Research Diagnostics Inc., Pleasant Hill Road Flanders NJ 07836 USA. Such a phosphoserine specific antibody is labeled with a detectable marker, such as, for example, a fluorescent marker, a radioactive marker, a colorimetric marker or an enzymatic marker. Alternatively, the anti-phosphoserine antibody is detected using a labeled secondary antibody or ligand that binds to the phosphoserine antibody.
Methods of determining antibody binding to a PKC-β phosphorylation site of a myosin heavy chain polypeptide described supra (and, in particular immunoassay formats) are to be taken to apply mutatis mutandis to this embodiment of the invention. Accordingly, in one embodiment, the level of phosphate bound to a PKC-β phosphorylation site is determined by contacting the isolated polypeptide with an antibody or ligand that binds phosphoserine for a time and under conditions sufficient for an antibody-antigen complex to form and determining the level of said complex wherein the level of said complex is indicative of the level of phosphate bound to a PKC-β phosphorylation site.
Methods for detecting the level of protein bound to an antibody or ligand as described herein-above are to be taken to apply mutatis mutandis to this embodiment of the invention.
Another embodiment of the present invention provides a method for determining PKC- β activity comprising detecting the interaction between a PKC-β and a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide. In accordance with this embodiment the binding between the phosphorylation site and the ligand (i.e., PKC-β activity) is determined by performing a process comprising:
(i) contacting a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with a PKC-β or a sample comprising or suspected of comprising a PKC-β under conditions sufficient for the PKC-β to phosphorylate said PKC-β phosphorylation site; and (ii) determining the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide, wherein the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide is indicative of a PKC-β activity.
In accordance with this embodiment, a ligand that is capable of binding an amino acid residue within a PKC-β phosphorylation site of a myosin heavy chain polypeptide is a PKC-β enzyme. Preferably, the PKC-β enzyme is in the extract or cellular sample that is being assayed.
It is preferable, that a PKC-β phosphorylation site of a myosin heavy chain polypeptide is not phosphorylated prior to performing the method of the present embodiment, thereby facilitating phosphorylation by a PKC-β in a sample. As will be apparent to a skilled artisan, it is preferable that the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide is isolated prior to performing the present invention. The peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide may be isolated from a natural source, such as, for example a cell, or alternatively may be produced by synthetic or recombinant means.
Methods for isolating a protein from a cellular source are known in the art and described, for example, in Scopes (hv_ Protein Purification: Principles and Practice, Third Edition, Springer Verlag, 1994). For example, a peptide, polypeptide or protein comprising a PKC-β phosphorylation site is isolated using affinity purification using the methods described supra.
In another embodiment, a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide is produced by recombinant means. For example, a myosin IIA polypeptide is expressed in a cell using an expression construct comprising at least a promoter and any relevant regulatory element to express the recombinant protein in said cell. Preferably, the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide is expressed as a fusion protein with another polypeptide that facilitates purification of the polypeptide of interest. Such "tags" include, but are not limited to influenza vims hemagglutinin (HA), Simian Viras 5 (V5, polyhistidine (e.g. 6xHis), c-myc, FLAG, epitope tags as described by Slootstra et al. Mol Divers 2(3):156-16A, 1997, GST (glutathione-S-transferase), MBP (maltose binding protein), GAL4, β-galactosidase, or a fluorescent protein, such as, for example green fluorescent protein, GFP, red shifted green fluorescent protein (RFP) or cyan shifted green fluorescent protein (CFP) (as will be apparent from the foregoing, a labelled peptide, polypeptide or protein is also useful for producing, for example, a standard of the invention). Alternatively, the PKC-β phosphorylation site of a myosin heavy chain polypeptide is labeled with a protein that directly associates with another known protein, such as for example, biotin, streptavidin or Strep-Tag, an 8 amino acid streptavidin binding peptide (available from Sigma-Genosys, Sydney, Australia).
The term "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (i.e. upstream activating sequences, transcription factor binding sites, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue specific manner. In the present context, the term "promoter" is also used to describe a recombinant, synthetic or fusion molecule, or derivative which confers, activates or enhances the expression of a nucleic acid molecule to which it is operably linked, and which encodes the peptide or protein. Preferred promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter the spatial expression and/or temporal expression of said nucleic acid molecule.
Placing a nucleic acid molecule under the regulatory control of, i.e., "in operable connection with", a promoter sequence means positioning said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the coding sequence that they control. To constract heterologous promoter/structural gene combinations, it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the gene from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
Typical promoters suitable for expression in viruses of bacterial cells and bacterial cells such as for example a bacterial cell selected from the group comprising E. coli, Staphylococcus sp, Corynebacterium sp., Salmonella sp., Bacillus sp., and Pseudomonas sp., include, but are not limited to, the lacz promoter, the Ipp promoter, temperature-sensitive λL or promoters, T7 promoter, T3 promoter, SP6 promoter or semi-artificial promoters such as the IPTG-inducible tac promoter or lacUV5 promoter. A number of other gene constract systems for expressing the nucleic acid fragment of the invention in bacterial cells are well-known in the art and are described for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987)and (Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001). Expression of a recombinant PKC-β phosphorylation site of a myosin heavy chain polypeptide in bacterial cells is preferred, as these cells do not express an endogenous PKC-β isozyme. Accordingly, a recombinant PKC-β phosphorylation site of a myosin heavy chain polypeptide produced in bacterial cells is unlikely to be phosphorylated and, as such, can be used directly in the method of the present invention.
Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichia pastoris, S. cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PH05 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.
Typical promoters suitable for expression in a virus of a mammalian cell, or in a mammalian cell, mammalian tissue or intact mammal include, for example a promoter selected from the group consisting of, retroviral LTR elements, the SV40 early promoter, the SV40 late promoter, the cytomegalovirus (CMV) promoter, the CMV IE (cytomegalo viras immediate early) promoter, the EFlα promoter (from human elongation factor lα), the EM7 promoter, the UbC promoter (from human ubiquitin C).
Preferred mammalian cells for expression of the nucleic acid fragments include epithelial cells, fibroblasts, kidney cells, T cells, or erythroid cells, including a cell line selected from the group consisting of COS, CHO, murine 10T, MEF, NIH3T3, MDA- MB-231, MDCK, HeLa, K562, HEK 293 and 293T. The use of neoplastic cells, such as, for example, leukemic/leukemia cells, is also contemplated herein.
Methods of producing expression constructs are known in the art and are described, for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) or Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).
Following production of a suitable gene construct, said constract is introduced into the relevant cell. Methods of introducing the gene constructs into a cell or organism for expression are known to those skilled in the art and are described for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) and (Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001). The method chosen to introduce the gene construct in depends upon the cell type in which the gene constract is to be expressed. Means for introducing recombinant DNA into cells include, but are not limited to electroporation or chemical transformation into cells previously treated to allow for said transformation, PEG mediated transformation, microinjection, transfection mediated by DEAE-dextran, transfection mediated by calcium phosphate, transfection mediated by liposomes such as by using Lipofectamine (Invitrogen) and/or cellfectin (Invifrogen), transduction by Adenoviuses, Herpesvirases, Togavirases or Retroviruses and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agacetus Inc., WI,USA).
Following introduction of the gene constract into a cell and expression of the recombinant peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide, the protein is purified by a method known in the art, such as for example affinity purification.
A recombinant polypeptide that is produced in a cell that comprises a PKC-β isozyme may be phosphorylated. Accordingly, it may be necessary to dephosphorylate the recombinant peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide following purification. Methods of dephosphorylating a protein are known in the art and described, for example, in Seki et al, Arch. Biochem. Biophys. 316(2): 673-679, 1995. For example, a sample is treated with a phosphatase, such as, for example, alkaline phosphatase or lambda phosphatase in order to hydrolize phosphate group bound to the PKC-β phosphorylation site of a myosin heavy chain polypeptide.
In a preferred embodiment, a recombinant polypeptide is treated with a phosphatase under conditions sufficient for the phosphatase to dephosphorylate said recombinant polypeptide. Preferably, the phosphatase is one that is capable of dephosphorylating a PKC phosphorylation site, such as, for example, protein phosphatase 1, protein phosphatase 2 A, calcineurin (calmodulin dependent protein phosphatase, CaMPP), particularly CaMPP- 1, CaMPP-2 and/or CaMPP-3. As will be apparent to the skilled artisan combinations of these enzymes increase the rate and/or amount of dephosphorylation of a polypeptide.
Recombinant or isolated forms of useful phosphatases may be obtained from commercial sources. For example, protein phosphatase 1 and protein phosphatase 2A are available from Upstate Biotechnology, Inc. (Lake Placid, NY, USA). Alternatively, phosphatases may be isolated using methods known in the art, for example, CaMPP- 1, CaMPP-2 and CaMPP-3 are isolated using methods described in Seki et al, Arch. Biochem. Biophys. 316(2): 673-679, 1995.
Alternatively, should a phosphorylated protein be desired, for example, for the production of a standard describe supra the recombinant or synthetic protein is contacted with a PKC-β for a time and under conditions sufficient for phosphorylation to occur. For example, recombinant PKC-β-I and/or recombinant PKC-β-II are available from Oxford Biomedical Research (Oxford, MI, USA).
In another embodiment, a short peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide (e.g. a peptide, polypeptide or protein comprising any one of SEQ ID NOs: 9 to 120) is produced synthetically. Synthetic peptides are prepared using known techniques of solid phase, liquid phase, or peptide condensation, or any combination thereof, and can include natural and/or unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc (Nα-amino protected Nα-t-butyloxycarbonyl) amino acid resin with the deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield, J Am. Chem. Soc, 55:2149-2154, 1963, or the base-labile Nα-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids described by Carpino and Han, J Org. Chem., 57:3403-3409, 1972. Both Fmoc and Boc Nα-amino protected amino acids can be obtained from various commercial sources, such as, for example, Fluka, Bachem, Advanced Chemtech, Sigma, Cambridge Research Biochemical, Bachem, or Peninsula Labs.
Synthetic peptides may also be produced using techniques known in the art and described, for example, in Stewart and Young (In: Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, 111. (1984) and/or Fields and Noble (Int. J. Pept. Protein Res., 55:161-214, 1990), or using automated synthesizers. Accordingly, peptides of the invention may comprise D-amino acids, a combination of D- and L- amino acids, and various unnatural amino acids (e.g., β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids, etc) to convey special properties. Synthetic amino acids include omithine for lysine, fluorophenylalanine for phenylalanine, and norleucine for leucine or isoleucine. Furthermore, phospho-amino acids may also be used to generate a phosphorylated synthetic polypeptide. Following isolation or production of an unphosphorylated peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide, the polypeptide is used to determine the level of PKC-β activity in a sample.
In one embodiment, the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide is bound or adsorbed to a solid support, such as, for example, a microtiter plate.
In one embodiment, a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide is incubated with a sample comprising a PKC-β and a labeled phosphate. In accordance with this embodiment, the labeled phosphate is labeled with a detectable marker, such as, for example, a radioactive label, a fluorescent label, an enzymatic label or a colorimetric label.
In a preferred embodiment, a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide is incubated with a sample comprising a PKC-β and γ^P-ATP. Accordingly, phosphorylation of the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide by PKC-β results in incorporation of the 32P into the PKC-β phosphorylation site of a myosin heavy chain polypeptide. Phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide is then detected using methods known in the art, such as, for example scintillation. Such a method is of particular use as it is not only highly reproducible but it is also quantitative. Accordingly, the amount of radioactive phosphate incorporated into a PKC-β phosphorylation site of a myosin heavy chain polypeptide is indicative of the amount of PKC-β activity in that sample.
Alternative labels for ATP are clearly contemplated by the present invention, such as, for example, the pyrene labeled ATP (Pyr-ATP) described by Hiratsuka in Biophys J. 72: 843-849, 1997 or any of the fluorescently labelled ATP available from MoBiTec, Germany.
Alternatively, following incubation of the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide, the sample is washed to remove any unbound phosphate. The sample is then incubated with a phosphatase, such as, for example, alkaline phosphatase or lambda protein phosphatase under conditions sufficient to allow complete hydrolysis of any phosphates within the PKC-β phosphorylation site of a myosin heavy chain polypeptide. A compound such as calcium molybdate is then added to the solution under conditions sufficient to allow the reduction of calcium molybdate to heteropolymolybdenum in the presence of free phosphate (e.g. by adding l-amino-2-naρthol-4-sulfonic acid), heteropolymolybdenum is a blue colour, and the intensity of the blue color produced is proportional to the amount of phosphate released from the PKC-β phosphorylation site of a myosin heavy chain polypeptide. The amount of heteropolymolybdenum in a sample is determined using, for example, light spectrometry.
As stated supra, the method of the present invention may be performed in a cell or in a cell free system or in a cell lysate.
In one embodiment, the method of the present embodiment of the invention comprises determining the level of phosphorylation of the PKCrβ phosphorylation site of a myosin heavy chain polypeptide a process comprising comparing the level of detectable marker bound to the antibody in the sample to a standard.
For example, the standard is produced using a method comprising:
(i) incubating a known amount of the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide in the presence of a known amount of a PKC-β under conditions sufficient for phosphorylation of the PKC-β phosphorylation site to occur; and (ii) determining the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide, wherein a comparable level of phosphorylation in the sample and the standard indicates that a similar level PKC-β is present in both samples.
Assays for detection of myosin related activity
In another embodiment of the present invention, a ligand is a PKC-β polypeptide or enzyme and the binding of the PKC-β to a phosphorylation site of a myosin heavy chain polypeptide is determined by detecting a change in myosin activity. Accordingly, in one embodiment the binding between the peptide, polypeptide or protein comprising a phosphorylation site and the ligand is determined by performing a process comprising: (i) contacting a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with a PKC-β enzyme under conditions sufficient for the PKC-β enzyme to phosphorylate said PKC-β phosphorylation site; and (ii) determining a change in the level of activity of the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide wherein the level of activity of the peptide, polypeptide or protein is indicative of PKC- β activity.
In one embodiment, the myosin heavy chain polypeptide is a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 140.
In another embodiment, the myosin heavy chain polypeptide is a non-muscle myosin heavy chain polypeptide or a fragment thereof, and even more preferably a myosin heavy chain IIA polypeptide or a fragment thereof. More preferably, the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 2, 4, 6 or 8 or a fragment thereof.
In one embodiment the myosin heavy chain polypeptide comprises an amino acid sequence set forth in SEQ ID NOs: 2, 4, 6 or 8 wherein the polypeptide is capable of being phosphorylated by a PKC-β at a position corresponding to amino acid position 1916 of SEQ ID NO: 2 or 6, amino acid position 1917 of SEQ ID NO: 4 or amino acid position 1915 of SEQ ID NO: 8.
In one embodiment, an assay of phosphorylated myosin activity determines the ATPase activity of a myosin heavy chain polypeptide. Methods for determining myosin ATPase activity are known in the art and described, for example, in Kuznicki and Dom, Journal of Biological Chemistry, 259(14): 9302-9307. Essentially, such an assay determines the rate of release or hydrolysis of labelled Pi from ATP (e.g., hydrolysis of 32Pi from [γ32P] ATP). Preferably, this assay is performed in the presence and in the absence of actin, more preferably F-actin, as the binding of myosin to actin modulates ATPase activity. Isolated myosin ± actin are incubated in the presence of [γ32P] ATP (or any other suitably labelled ATP) under conditions to facilitate hydrolysis of the ATP. Proteins and ATP are then removed from solution, e.g. by centrifugation, and the level of γ32P in solution determined, e.g. by scintillation.
Alternatively, the amount of hydrolysed Pi in solution may be determined using a malachite green method essentially as described in Kodoma et al, J. Biochem., 99(5): 1465-1472, 1986. Essentially, malachite green/molybdate is incubated with a solution comprising hydrolysed Pi under conditions sufficient to allow interaction. Interaction of hydrolysed Pi, malachite green and molybdate cause the solution to change colour, with the degree of change indicative of the amount of phosphate. This may be quantitated using a standard curve, and determining absorbance at 650 nm. As will be apparent to the skilled artisan, an assay using malachite greenmolybdate does not require labelled ATP.
In another embodiment, an assay of phosphorylated myosin activity determines the interaction of a myosin heavy chain polypeptide with an actin polypeptide. Such an interaction may be determined by colocalising the myosin heavy chain polypeptide and the actin polypeptide using immunofluorescence or immunohistochemistry. Immunohistochemistry and/or immunofluorescent methods are known in the art and/or described herein.
Alternatively, interaction of a phosphorylated myosin heavy chain polypeptide and an actin polypeptide may be determined using immuno-affinity purification and/or coimmunoprecipitation experiments, essentially as described herein and in Scopes (In: Protein Purification: Principles and Practice, Third Edition, Springer Verlag, 1994).
Biological samples
In one embodiment, the method of the present invention is performed in a cell free system. Accordingly, in one embodiment the present invention provides a method of determining PKC-β activity comprising detecting the interaction between a PKC-β and a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide. In accordance with this embodiment the binding between the phosphorylation site and the ligand is determined by performing a process comprising: (i) contacting a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with a PKC-β in a vesicle under conditions sufficient for the PKC-β to phosphorylate said PKC-β phosphorylation site; and (ii) determining the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide, wherein the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain is indicative of a PKC-β activity.
In one embodiment, a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide and a PKC-β is incubated with at least a molecule that activates the PKC-β (e.g. diaglyarol and/or calcium) and a source of phosphate (e.g. ATP).
Such a cell free system enables the skilled artisan to rapidly assess the phosphorylation of a PKC-β phosphorylation site of a myosin heavy chain polypeptide. For example, a cell free system enables the skilled artisan to rapidly screen a large number of compounds (using methods known in the art and/or described herein) to determine those that are capable of specifically modulating the ability of a PKC-β enzyme to modulate a PKC-β phosphorylation site of a myosin heavy chain polypeptide.
In a preferred embodiment, the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide is in an extract or cellular sample being assayed. An extract or cellular sample may be any sample that comprises a PKC-β phosphorylation site of a myosin heavy chain polypeptide and/or a PKC-β.
In one embodiment, the extract is derived from a cell. In accordance with this embodiment, the phosphorylation site of a myosin heavy chain polypeptide may be endogenously expressed in a cellular sample, or alternatively, may be expressed by recombinant means. Methods of expressing a polypeptide by recombinant means are known in the art.
Preferably, the cellular sample is a mammalian cellular sample. Any cell type may be used in a screen for PKC-β activity, provided that the cell expresses a peptide, polypeptide or protein comprising a PKC-β myosin heavy chain polypeptide of the present invention and/or a PKC-β isozyme. Preferred cell include COS, CHO, murine 10T, MEF, NIH3T3, MDA-MB-231, MDCK, HeLa, K562, HEK 293 and 293T. In a particularly preferred embodiment, the cells are RBL-2H3 cells. Alternatively, the cell is derived from a subject. In this regard, any cell that expresses a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a PKC-β isozyme is preferred. Preferably, the cell is derived from a tissue or body fluid that is associated with a disorder caused by or associated with aberrant PKC-β activity. Preferably, the tissue or body fluid is selected from the group consisting of pancreas, heart, skeletal muscle, liver, retina, colon, skin, stomach, lung, whole blood, serum and plasma.
More preferably, the cell is a PBMC, a white blood cell or a mast cell. .
In one embodiment, a subject is any organism that expresses a PKC-β. In one embodiment, a subject is an animal subject, preferably a model animal subject. Preferably, the animal subject is selected from the group consisting of a rat, a mouse, chicken, a guinea pig, a rabbit, a bovine species, a sheep, and a zebrafϊsh.
In another embodiment, the subject is a human. In accordance with this embodiment, it is preferable that the human subject is suspected to be suffering from a disorder associated with PKC-β activity. Preferably, the subject is suspected to be suffering from a disorder associated with increased activity of a PKC-β enzyme, such as, for example, hyperglycemia, diabetes, diabetic nephropathy, diabetic retinopathy, macular edema, colon cancer, breast cancer, leukemia, hepatocarcinoma, gastric cancer, asthma or hypertension. In another embodiment, the human subject suffers from a condition associated with decreased PKC-β activity, such as, for example, hypoglycemia, melanoma, inflammatory and hyperplastic skin disorders (e.g. psoriasis, cutaneous T- cell lymphoma, cutaneous dermatitis and allergic contract dermatitis) or immunodeficiency.
In one embodiment, the cell or subject is a wild-type cell or subject. As used herein the term "wild-type" shall be taken to mean a cell or subject that is a naturally occurring cell or subject, i.e. a cell or subject that has not been modified at the genetic level or treated with a compound to modify that cell or subject. In particular, the cell or subject has not been modified at the genetic level or treated with a compound that modulates the activity of PKC-β in said cell or subject. In another embodiment, the cell or subject has been modified at the genetic level. Preferably, the cell or subject has been modified at the genetic level to induce a diabetes-type phenotype. As used herein, the term "diabetes-type phenotype" shall be taken to mean that a cell or subject is incapable of producing wild-type levels of insulin and/or is insulin resistant in at least one tissue compared to a wild-type cell or subject. In the case of an animal subject, that subject may also, or alternatively, display complications of diabetes, such as, for example, diabetic nephropathy, heart disease, diabetic neuropathy and/or diabetic retinopathy. As PKC-β activity and/or expression has been shown to be associated with diabetes or complications of diabetes, such genetically modified cells or subjects represent attractive models in which to study PKC-β activity and, in particular, to determine modulators of PKC-β activity that are useful in the treatment of such disorders.
Genetically modified animal models that suffer from a diabetes-type phenotype are known in the art and include, for example, NOD mice, mice deficient in insulin receptor substrate 1 and/or insulin receptor substrate 2 (as described by Lausten et al, Genes Dev., 16: 3213-3222, 2002), ob/ob mice and db/db mice. Preferably, a genetically modified animal subject that has a diabetes-type phenotype is a rat that expresses a transgene encoding a renin gene (preferably, the mouse Ren-2 gene), i.e. the (mRen-2)27 rat model.
In another embodiment, a cell or animal subject is administered with a compound that induces a diabetes-type phenotype. Compounds that induce a diabetes-type phenotype are known in the art and include, for example, alloxan (as described in Kodoma et al, Diabetes Res. clin. Pract., 20(3): 189-189, 2003) and streptozotocin (as described in Sandier et al, Diabetes 4: 78-83, 1983 and Ar'Rajab et al, Eur. Surg. Res. 22(5): 270- 275, 1990) (available from Sigma, St. Louis, MO, USA).
In a preferred embodiment, a cell or animal subject that is genetically modified to induce a diabetes-type phenotype is administered a compound that induces a diabetes- type phenotype. For example, a (mRen-2)27 rat is administered streptozotocin to induce structural and functional changes associated with human diabetic nephropathy. Alternatively, administration of streptozocin to hairless mice induces pathophysiological changes in the skin and stratum corneum similar to those observed in humans suffering from diabetes mellitus. In another embodiment, the cell or animal subject has been modified at the genetic level to induce an asthma-type phenotype. As used herein, the term "asthma-type phenotype" shall be taken to mean that a subject displays aberrant expansion of lung T- helper cells that produce TH2 cytokines as a response to mast cells activated by IgE.
Genetically modified animal subjects that suffer from an asthma-type phenotype are known in the art, and include, for example, prostaglandin D2 deficient mice, C3a anaphylatoxin receptor deficient mice or T-box 1 deficient mice.
In another embodiment, the animal subject is administered with a compound that induces an asthma type phenotype. For example, an animal subject may be made sensitive to ovalbumin, and any subsequent exposure to ovalbumin in an aerosol form induces an allergic asthma-type response.
In a further embodiment, the cell or animal subject has been modified at the genetic level to induce a colon cancer type phenotype. As used herein the term "colon cancer type phenotype" shall be taken to mean that the phenotype of the animal is similar to that of a disease or disorder such as, for example, hereditary non-polyposis colorectal cancer, familial adenomatous polyposis or ulcerative colitis. For example, a cell of the colonic epithelium or a cell derived from colonic epithelium is hyperproliferative. In the case of an animal subject, said subject may develop aberrant modified animal subjects that suffer from a colon cancer-type phenotype are known in the art and include, for example, AN3 1 KAL 1 MutL homologue 1 (MCH1) deficient mice, insulin like growth factor 1 deficient mice, p21 (WAFl/cipl) deficient mice and DNA methyl transferase 1 deficient mice.
In a still further embodiment, the cell or animal subject is administered with a compound that induces a colon cancer type phenotype. Compounds that induce a colon cancer type phenotype are known in the art and include, for example, azoxy methane (as described in Gokmen-Polar et al, Cancer Research, 61: 1375- 1381 , 2001 ).
In yet another embodiment, a biological sample is treated prior to use in the assay of the present invention.
In one embodiment, a biological sample is treated to lyse a cell in said sample. Such methods include the use of detergents, enzymes, repeatedly freezing and thawing said cells, sonication or mechanical shearing (e.g. vortexing) of said cells in the presence of glass beads, amongst others.
In another embodiment, a biological sample is treated to denature a protein present in said sample. Methods of denaturing a protein include heating a sample, treatment with 2-mercaptoethanol, or treatment with detergents and other compounds such as, for example, guanidinium or urea.
In yet another embodiment, a biological sample is treated to concentrate a protein is said sample. Methods of concentrating proteins include precipitation, freeze drying, use of funnel tube gels (TerBush and Novick, Journal of Biomolecular Techniques, 10(3); 1999), ultrafiltration or dialysis.
In a still further embodiment, a biological sample is a cell-free sample comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide. Preferably, said cell-free sample also comprises a PKC-β, e.g., a PKC-β-I and/or a P-β-II, such that the PKC-β phosphorylation site of a myosin heavy chain polypeptide is phosphorylated. Such a cell free sample may also comprise buffering solutions, and a phosphate source, such as, for example ATP, in order to facilitate phosphorylation of the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide. In one embodiment, such a cell free sample is a vesicle.
Kits
Another aspect of the present invention provides a kit for determining PKC-β activity. In one embodiment, said kit comprises an antibody or ligand that specifically binds to a PKC-β phosphorylation site of a myosin heavy chain polypeptide. Preferably, the antibody or ligand is capable of specifically binding a PKC-β phosphorylation site of a myosin heavy chain polypeptide comprising the sequence set forth in SEQ ID NO: 9 to 167. Preferably, the peptide is phosphorylated. More preferably, the antibody or ligand is capable of binding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10.
In another embodiment, the antibody or ligand is capable of binding a PKC-β phosphorylation site of a myosin heavy chain polypeptide comprising a PKC-β phosphorylated or unphosphorylated phosphorylation site of a myosin heavy chain polypeptide that is a polypeptide at least about 80% homologous to the polypeptide set forth in SEQ ID NO: 2, 4, 6 or 8. Preferably, the polypeptide is phosphorylated at position 1916 of SEQ ID NO: 2 or 6, position 1917 of SEQ ID NO: 4 or SEQ ID NO: 1915 of SEQ ID NO: 8.
In another embodiment the kit for determining PKC-β activity comprises a phosphorylated or non-phosphorylated peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide. Preferably, the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide is a polypeptide comprising the sequence set forth in any one of SEQ ID NOs: 9 to l20.
In another embodiment, the kit comprises a phosphorylated or non-phosphorylated peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide that is a polypeptide at least about 80% homologous to the polypeptide set forth in SEQ ID NO: 2, 4, 6 or 8. Preferably, the polypeptide is phosphorylated at the serine at position 1916 of SEQ ID NO: 2 or 6, position 1917 of SEQ ID NO: 4 or SEQ ID NO: 1915 of SEQ ID NO: 8.
In a still further embodiment of the present invention, the kit comprises recombinant or isolated a PKC-β enzyme. In one embodiment, the kit comprises a PKC-βl enzyme. In another embodiment, the kit comprises a PKC-βll enzyme. In a further embodiment, the kit comprises a PKC-βl and a PKC-βll enzyme.
Diagnosis/prognosis of a condition associated with aberrant PKC-β activity A further aspect of the present invention provides a method of diagnosing or prognosing a condition associated with aberrant PKC-β activity in a subject comprising determining phosphorylation of a PKC-β phosphorylation site of a myosin heavy chain polypeptide in a sample isolated from said subject, wherein a modulated level of phosphorylation is indicative of a disorder associated with aberrant PKC-β activity.
In a preferred embodiment, the present invention provides a method for diagnosing or prognosing a condition associated with aberrant PKC-β activity in a subject comprising:
(i) determining the level of PKC-β activity in a sample from the subject using a method described herein; and (ii) determining the level of PKC-β activity in a control sample using a method described herein, wherein increased or decreased PKC-β activity at (i) compared to (ii) indicates that the subject suffers from a condition associated with aberrant PKC-β activity.
Preferably, the subject is a subject suspected of suffering from a condition associated with aberrant PKC-β activity.
Alternatively, the subject has already been diagnosis with a disorder associated with aberrant PKC-β activity using the method of the present invention or any other method, and the method of the present invention is used to monitor the PKC-β activity in a cell derived from said subject. In one embodiment, the subject has received or is receiving treatment, and the method of diagnosis/prognosis is used to monitor the effectiveness of said treatment.
In one embodiment, the subject suffers or is suspected of suffering from a disorder that is associated with increased expression or activity of a PKC-β enzyme.
In another embodiment, the subject suffers from a disorder selected from the group consisting of hypoglycaemia, hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, hypertension colorectal cancer and immunodeficiency.
One embodiment of the present invention provides a method for diagnosing or prognosing a condition associated with aberrant PKC-β activity in a subject comprising: (i) determining the level of phosphorylation of a PKC-β phosphorylation site of a myosin heavy chain polypeptide in a test sample from said subject; and (ii) comparing the level of phosphorylation at (i) with the level of phosphorylation in a reference sample from a healthy or normal subject; wherein a level of the nucleic acid at (ii) that is enhanced or reduced in the test sample relative to the sample from the normal or healthy subject is indicative of a disorder associated with aberrant PKC-β activity in said subject. In an alternative embodiment, the present invention provides a method for diagnosing or prognosing a condition associated with aberrant PKC-β activity in a subject comprising:
(i) determining the level of PKC-β activity in a sample from the subject using a method described herein; and
(ii) determining the level of PKC-β activity in a control sample using a method described herein, wherein increased or decreased PKC-β activity at (i) compared to (ii) indicates that the subject suffers from a condition associated with aberrant PKC-β activity.
Conditions associated with aberrant PKC-β activity are known in the art. For example, conditions associated with decreased PKC-β activity include, hypoglycemia, melanoma, inflammatory and hyperplastic skin disorders (e.g. psoriasis, cutaneous T- cell lymphoma, cutaneous dermatitis and allergic contact dermatitis) and immunodeficiency. Conditions associated with increased PKC-β activity include, for example, hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, colon cancer, breast cancer, leukemia, hepatocarcinoma, gastric cancer, asthma and hypertension.
In one embodiment, the diagnostic method of the present invention is used to determine a subject in need of treatment with a modulator of PKC-β activity.
In one embodiment, the subject has been previously diagnosed as suffering from a condition associated with aberrant PKC-β activity.
For example, a subject suffering from a condition associated with aberrant PKC-β activity known in the art and/or described supra, is tested using a diagnostic PKC-β assay described herein to determine whether or not PKC-β activity in said subject is aberrant. Those subjects that have aberrant PKC-β activity may then be treated with a modulator of PKC-β activity to increase or decrease PKC-β activity as appropriate.
For example, a biological sample derived from a subject suffering from asthma (e.g. a blood sample in which the white blood cells have been lysed) is used in an assay described herein to determine the level of PKC-β activity in said sample. A subject suffering from asthma that also has an increased PKC-β activity should be treated with an appropriate amount of an inhibitor of PKC-β activity, e.g. LY333531 (Eli Lily and Co., Indianapolis, IN, USA), to inhibit PKC-β activity.
As will be apparent to the skilled artisan, a subject receiving treatment for a disorder associated with aberrant PKC-β activity may also be monitored using the method of the present invention. Biological samples derived from the subject while receiving treatment for a disorder associated with aberrant PKC-β activity are analysed to determine PKC-β activity. Furthermore, the subject may also be monitored for various aspects of the condition, for example a subject suffering from asthma may also be monitored for forced expiration volume in one second (FEV1), a measure of lung function. By using such assays, a subject may be monitored to determine the efficacy of a treatment of a condition with aberrant PKC-β activity.
Alternatively, a subject suffering from diabetes may be monitored for aberrant PKC-β activity, in order to determine the risk of said subject developing complications of diabetes, such as, for example diabetic retinopathy, diabetic nephropathy and/or macular edema. For example, a blood sample isolated from the subject may be assayed to determine the level of PKC-β activity. An increase in PKC-β phosphorylation of a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide indicates that the subject may require treatment. For example, the subject may require treatment for aberrant PKC-β activity, eg. with a PKC-β inhibitor.
Alternatively, or in addition, the subject may require treatment for a complication of diabetes. As will be apparent to the skilled artisan the method of the present invention may be used to continually monitor the level of PKC-β activity in a subject.
In another embodiment, a subject has not been previously diagnosed with a condition associated with PKC-β activity. In accordance with this embodiment, the diagnostic/prognostic method of the invention may be used to determine a condition that is associated with PKC-β activity.
In accordance with this aspect of the invention, it is preferred that the aberrant PKC-β activity is increased PKC-β activity. Increased PKC-β activity may be a result of increased expression of PKC-βl or PKC-βll or both PKC-βl and PKC-βll.
Alternatively, increased PKC-β activity may be a result of a mutation in the nucleic acid that encodes a PKC-β enzyme, that results in a change in the amino acid composition of the encoding protein, wherein the resulting amino acid change causes an increase in PKC-β activity. Alternatively, aberrant PKC-β activity may be caused by a post-translational modification to a PKC-β enzyme, such as, for example, phosphorylation of the enzyme or glycosylation of the enzyme, wherein the modification results in aberrant activity of the enzyme.
Methods of determining the activity of a PKC-β enzyme are described herein-above are to be taken to apply mutatis mutandis to this aspect of the invention.
Test samples and reference samples
Preferably, a test sample in which a phosphorylation of a PKC-β phosphorylation site of a myosin heavy chain polypeptide is detected is a sample that expresses or comprises a PKC-β enzyme. Preferably, a sample is derived from a tissue selected from the group consisting of pancreas, heart, skeletal muscle, liver, colon, skin, stomach, lung and retina. Cell preparations or protein preparation derived from such tissues or cells are not to be excluded. The sample can be prepared on a solid matrix for histological analyses, or alternatively, in a suitable solution such as, for example, an extraction buffer or suspension buffer, and the present invention clearly extends to the testing of biological solutions thus prepared.
In another embodiment, the sample comprises a body fluid or cell selected from the group consisting of whole blood, plasma, serum, a PBMC, a white blood cell, buffy coat fraction and a mast cell.
In one embodiment a test or reference sample is obtained previously from a patient.
In another embodiment, a sample is treated prior to use in a diagnostic or prognostic assay. A sample may be treated to lyse a cell in the sample (e.g. by using a detergent), to denature a protein in a sample or to concentrate the amount of protein in a sample.
As will be apparent, the diagnostic and prognostic methods provided by the present invention require a degree of quantification to determine the amount of a protein that is diagnostic or prognostic of a condition associated with aberrant PKC-β activity. Such quantification can be determined by the inclusion of appropriate reference samples in the assays described herein. In one embodiment, the reference sample comprises a cell or tissue (or derivative thereof) from the same subject taken at a time point when the individual was healthy. In another embodiment, the reference sample comprises a cell or tissue (or derivative thereof) from a healthy or normal individual. In accordance with both of these embodiments, the reference sample and the test sample are both processed, albeit not necessarily at the same time, and data obtained for both samples are compared.
Alternatively, a reference sample is not included in an assay, rather the reference sample is derived from an established data set that has been previously generated. Accordingly, in one embodiment, a reference sample comprises data from a sample population study of healthy individuals, such as, for example, statistically significant data for the healthy range of the amount of phosphorylation of a PKC-β phosphorylation site of a myosin heavy chain polypeptide. Data derived from processing, analysing or assaying a test sample is then compared to data obtained for the sample population.
Data obtained from a sufficiently large number of reference samples so as to be representative of a population allows the generation of a data set for determining the average level of a particular parameter. Accordingly, the level of phosphorylation of a PKC-β phosphorylation site of a myosin heavy chain polypeptide can be determined for any population of individuals, and for any sample derived from said individual, for subsequent comparison to levels of the expression product determined for a sample being assayed. Where such normalized data sets are relied upon, internal controls are preferably included in each assay conducted to control for variation.
As used herein the term "healthy individual" shall be taken to mean an individual who is not known to suffer from a condition associated with aberrant PKC-β activity. This form of information is derived from clinical data on the individual. Preferably, the individual does not suffer from a condition selected from the group consisting of hypoglycaemia, hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, aberrant vitamin D function, hypertension and immunodeficiency. Preferably, the individual does not have any indication that they may be developing any of these conditions. For example, early indications that a subject may suffer from diabetes include, frequent urination, excessive thirst, extreme hunger, unusual weight loss, increased fatigue, irritability and blurred vision. As used herein the term "normal individual" shall be taken to mean an individual having a normal level of PKC-β phosphorylation of a PKC-β phosphorylation site of a myosin heavy chain polypeptide in a sample derived from the individual.
As will be apparent to the skilled artisan, the present invention also provides a method of treatment of an individual comprising performing the diagnostic or prognostic method described herein.
In one embodiment, the method of treatment comprises the additional step of treating the subject with an appropriate therapeutic composition. Such a composition may comprise a PKC-β inhibitor, such as, for example LY333531. Alternatively, a therapeutic composition may directly treat a disorder associated with aberrant PKC-β activity. For example, a therapeutic composition may comprise insulin in order to treat diabetes induced hyperglycemia; or thiazides, beta-blockers, ACE inhibitors, calcium- channel blockers, alpha-blockers or angiotensin-II receptor antagonists for the treatment of increased blood pressure associated with diabetes. Patients suffering from diabetic retinopathy or macular edema may undergo laser surgery or photocoagulation, or if they additionally suffer from a vitreous haemorrhage the subject may require vitrectomy surgery. Subjects suffering from diabetic nephropathy may require treatment with an ACE inhibitor or an angiotensin-II receptor inhibitor.
The present invention also encompasses the use of an antibody or ligand that specifically binds to an amino acid that is phosphorylated by PKC-β in the manufacture of a diagnostic or prognostic for the diagnosis or prognosis of a condition that is associated with aberrant PKC-β activity. More preferably, the condition associated with aberrant PKC-β activity is selected from the group consisting of hypoglycaemia, hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, hypertension, colorectal cancer and immunodeficiency.
Preferably, the amino acid that is phosphorylated by PKC-β is an amino acid within a PKC-β phosphorylation site of a myosin heavy chain polypeptide.
In one embodiment, the antibody or ligand specifically binds to a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9 to 120. In a particularly preferred embodiment, the antibody is a polyclonal antibody capable of specifically binding to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10.
In another embodiment, the antibody specifically binds to a myosin heavy chain IIA polypeptide. Preferably, the myosin IIA polypeptide comprises an amino acid sequence at least about 80% homologous to the sequence set forth in SEQ ID NO: 2, 4, 6 or 8. More preferably, the polypeptide is phosphorylated at the serine residue at position 1916 of SEQ ID NO: 2 or 6, position 1917 of SEQ ID NO: 4 or SEQ ID NO: 1915 of SEQ ID NO: 8.
Determining a modulator of PKC-β activity
A still further aspect of the present invention provides a method of determining a modulator of PKC-β activity comprising: (i) determining the level of phosphorylation of a PKC-β phosphorylation site of a myosin heavy chain polypeptide in the presence of a candidate compound; and (ii) determining the level of phosphorylation of a PKC-β phosphorylation site of a myosin heavy chain polypeptide in the absence of a candidate compound, wherein a difference in the level of said phosphorylation at (i) and (ii) indicates that the candidate compound is a modulator of PKC-β activity.
In one embodiment, the method comprises: (i) determining the level of PKC-β activity using a method described herein in a sample in the presence of a candidate compound; and (ii) determining the level of PKC-β activity using a method described herein in a sample in the absence of a candidate compound, wherein an increase or decrease in PKC-β activity at (i) compared to (ii) indicates that the candidate compound is a modulator of PKC-β activity.
In a preferred embodiment, the PKC-β phosphorylation site of a myosin heavy chain polypeptide is a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9 to 120. More preferably, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 10.
In another embodiment, the polypeptide is myosin heavy chain IIA. More preferably, the polypeptide comprises an amino acid sequence that is at least about 80% homologous to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6 or 8. Even more preferably, the polypeptide is phosphorylated at a serine residue that corresponds to the serine at position 1916 of SEQ ID NO: 2 or 6, position 1917 of SEQ ID NO: 4 or SEQ ID NO: 1915 of SEQ ID NO: 8.
In one embodiment of the present invention, a modulator of PKC-β activity enhances PKC-β enzyme activity (i.e. enhances PKC-β phosphorylation of a PKC-β phosphorylation site of a myosin heavy chain polypeptide.
In another embodiment of the present invention, a modulator of PKC-β activity partially or completely inhibits PKC-β enzyme activity (i.e. PKC-β phosphorylation of a PKC-β phosphorylation site of a myosin heavy chain polypeptide.
Methods of determining PKC-β activity described herein shall be taken to apply mutatis mutandis to this aspect of the invention. Accordingly, the level of PKC-β activity is determined in the presence and absence of a putative modulator, and the effect of that modulator is assessed by comparing the PKC-β phosphorylation of a myosin heavy chain polypeptide in each sample.
For example, PKC-β activity may be determined by direct means, such as, for example, by determining real time association or dissociation constants in the presence and absence of the candidate, or modified binding of an antibody that recognizes a conformational epitope of the complex. Biosensors used essentially as described herein, in the presence or absence of the candidate compound or antibody, are particularly suited to such applications.
Alternatively, the association, dissociation, or structure of the complex may be determined by indirect means, such as, for example, using an ELISA, or other known assay format for detecting protein-protein interactions. Such indirect means generally use a reporter system to detect formation or dissociation of the protein complex.
Standard solid-phase ELISA assay formats are particularly useful for identifying antagonists of the protein-protein interaction. In accordance with this embodiment, a peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide may be immobilized on a solid matrix, such as, for example an array of polymeric pins or a glass support. The PKC-β in solution is brought into physical relation with the immobilized protein to phosphorylate the PKC-β phosphorylation site of a myosin heavy chain polypeptide. The PKC-β containing solution may also comprise a putative modulator of PKC-β activity, or, in the case of a control, may comprise no modulator. As a positive control, a PKC-β containing solution may comprise a known PKC-β modulator, such as, for example LY333531 (Eli Lilly and Co., IN, USA) or the Protein Kinase CβC2-4 inhibitor available from Calbiochem, La Jolla, CA, USA. The phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide is detected with an antibody or ligand essentially as described herein above. The antibody is generally labeled with a fluorescent molecule or conjugated to an enzyme (e.g. horseradish peroxidase), or alternatively, a second labeled antibody can be used that binds to the first antibody. This facilitates determining the level of phosphorylation of the peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide. A compound that enhances or suppresses the level of phosphorylation is considered a modulator of PKC-β activity. It will be apparent to the skilled person that the assay format described herein is amenable to high throughput screening of samples, such as, for example, using a microarray of bound peptides or fusion proteins.
Cell free assays are also useful for high throughput analysis of putative modulators of PKC-β activity. For example, a PKC-β phosphorylation site of a myosin heavy chain polypeptide is incubated in a vesicle comprising a PKC-β enzyme and γ^p under conditions to allow phosphorylation of the PKC-β phosphorylation site. Optionally, the vesicle additionally comprises a putative modulator of PKC-β activity. The level of PKC-β activity in a sample is determined by detecting the amount of 32P that has been incorporated into a PKC-β phosphorylation site of a myosin heavy chain polypeptide. A modulator of PKC-β activity will increase or decrease the amount of phosphorylation of the PKC-β phosphorylation site in a sample.
In one embodiment of the present invention, a modulator is a small molecule (e.g. a chemical compound, agonist, antagonist, allosteric modulator, competitive inhibitor, or non-competitive inhibitor of PKC-β activity).
In one embodiment, the compound administered comprises nucleic acid, For example, the nucleic acid is an antagonist of PKC-β expression or a protein that is required for PKC-β activity (eg. RACKl and/or Anexin I), such as, for example, an antisense nucleic acid, peptide nucleic acid (PNA), ribozyme, or interfering RNA, which is complementary, in whole or in part, to a target molecule comprising a sense strand, and can hybridize with the target molecule. When introduced into a cell using suitable methods, such a nucleic acid inhibits the expression of the PKC-β or protein required for PKC-β activity encoded by the sense strand. Antisense nucleic acid, ribozymes (eg. Cech et /øUSSN 4,987,071; Cech et al, USSN 5,116,742; Barrel and Szostak, Science 261, 1411-1418, 1993), nucleic acid capable of forming a triple helix (eg. Helene, Anticancer Drug Res. 6, 569-584, 1991), PNAs (Hyrup et al, Bioorganic & Med. Chem. 4, 5-23, 1996; O^Keefe et al, Proc. Natl Acad. Sci. USA 93, 14670-14675, 1996), interfering RNAs (Elbashir et al, Nature 411, 494-498, 2001; Sharp, Genes Devel 15, 485-490, 2001; Lipardi et al, Cell 107, 297-307, 2001; Nishikura, Cell 107, 415-418, 2001) or small interfering RNAs (siRNA) may be produced by standard techniques known to the skilled artisan, based upon the sequences disclosed herein.
Accordingly, the antisense nucleic acid, ribozyme, PNA, interfering RNA or siRNA comprises a sequence that is complementary to at least about 15-20 contiguous nucleotides of a sequence having at least about 80% identity to SEQ ID NO: 169 or SEQ ID NO: 171 (i.e. it is complementary to PKC-βl or PKC-βll-encoding mRNA) and can hybridize thereto. For example, such antagonistic nucleic acid can be complementary to a target nucleic acid having the sequence of SEQ ID NOs: 123 or 124 or a portion thereof sufficient to allow hybridization. Longer molecules, comprising a sequence that is complementary to at least about 25, or 30, or 35, or 40, or 45, or 50 contiguous nucleotides of mRNA encoding a PKC-β or a protein that is required for PKC-β activity are also encompassed by the present invention.
In one embodiment, a constract comprising an antisense nucleic acid, ribozyme, PNA, interfering RNA or siRNA, can be introduced into a suitable cell to inhibit expression of PKC-β or a protein that is required for PKC-β activity. In another embodiment, such a construct can be introduced into some or all of the cells of a mammal. The antisense nucleic acid, ribozyme, PNA, or interfering RNA, inhibits expression of the target gene. Accordingly, PKC-β activity is inhibited
The use of antibodies that can inhibit one or more functions characteristic of a PKC-β protein, such as a binding activity, a signalling activity, and/or a phosphorylation activity, is also encompassed by the present invention. In one embodiment, antibodies of the present invention can inhibit binding of a PKC-β to a PKC-β phosphorylation site. In one embodiment, an antibody inhibits the binding of a PKC-β to a PKC-β phosphorylation site of a myosin heavy chain polypeptide.
In preferred embodiment, a modulator of PKC-β activity is a peptide that comprises a phosphorylation site of a myosin heavy chain polypeptide. In a particularly preferred embodiment, the peptide comprises the sequence set forth in SEQ ID NO: 172.
As exemplified herein, a peptide comprising the amino acid sequence set forth in SEQ ID NO: 172 is capable of suppressing histamine secretion in RBL-2H3 cells.
In an alternate particularly preferred embodiment, a modulator of PKC-β activity is a peptide that comprises a mutated phosphorylation site of a myosin heavy chain polypeptide. Preferably, the peptide comprises an amino acid sequence set forth in SEQ ID NO: 174 or 177.
As exemplified herein, a peptide comprising the amino acid sequence set forth in SEQ ID NO: 177 is capable of suppressing histamine secretion in RBL-2h3 cells.
Furthermore, the use of ligands, such as, for example, peptides that modulate interaction of a PKC-β with a PKC-β phosphorylation site is also encompassed by the present invention. For example a peptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9 to 167 may be used to suppress a PKC-β activity in a cell, by competing for PKC-β phosphorylation with the endogenous PKC-β phosphorylation sites within said cell. Preferably, such a peptide is expressed within the cell, although the peptide may also be introduced into the cell using methods known in the art.
As will be apparent to the skilled artisan the present invention encompasses peptides that modulate the interaction of a PKC-β with another peptide, polypeptide or protein, wherein that interaction is essential for a PKC-β activity.
The modulators identified using the methods described herein are useful for the therapeutic or prophylactic treatment of a disorder selected from the group consisting of hypoglycaemia, hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, colon cancer, breast cancer, hepatocarcinoma, gastric cancer, melanoma, leukemia, inflammatory and hyperplastic skin disorders (e.g. psoriasis, cutaneous T-cell lymphoma, cutaneous dermatitis and allergic contact dermatitis, asthma and immunodeficiency.
As exemplified herein a peptide that inhibits PKC-β phosphorylation of a myosin heavy chain polypeptide is capable of suppressing histamine secretion in a cellular model of asthma. Accordingly, such a peptide is particularly useful in the production of a compound for the treatment of asthma.
A further aspect of the present invention provides a method of manufacturing a compound that modulates PKC-β activity comprising: (i) determining a modulator of PKC-β activity; and (ii) using the modulator in the manufacture of a compound that modulates PKC-β activity.
In one embodiment, the method comprises the additional step of isolating the modulator of PKC-β activity. Alternatively, a modulator is identified and is produced for use in the manufacture of a compound that modulates PKC-β activity.
In a preferred embodiment, the modulator is a peptide that comprises an amino acid sequence set forth in SEQ ID NO: 174.
In one embodiment,, the invention provides a process for determining a compound that modulates PKC-β activity, said process comprising:
(i) determining the level of PKC-β activity in a sample in the absence of a candidate compound by performing the method of the invention;
(ii) determining the level of PKC-β activity in a sample in the presence of the candidate agent by performing the process of any one of the invention, wherein a difference in the level of PKC-β activity at (i) compared to (ii) indicates that the candidate compound modulates PKC-β activity; (iii) optionally, determining the structure of the candidate compound;
(iv) optionally, providing the name or structure of the candidate compound; and
(v) providing, the candidate compound.
In one example, the candidate compound is provided with an indication as to its use, for example, as determined using a method described herein. Naturally, for compounds that are known albeit not previously tested for their function using a screen provided by the present invention, determination of the structure of the compound is implicit in step (i) supra. This is because the skilled artisan will be aware of the name and/or structure of the compound at the time of performing the screen.
As used herein, the term "providing the compound" shall be taken to include any chemical or recombinant synthetic means for producing said agent or alternatively, the provision of an agent that has been previously synthesized by any person or means. For example, a peptidyl compound is synthesized using is produced synthetically as described herein.
In another embodiment, a peptidyl agent is produced using recombinant means. For example, an oligonucleotide or other nucleic acid (eg., a nucleic acid encoding a dominant negative inhibitor of the protein of interest) is placed in operable connection with a promoter. Methods for producing such expression constracts, introducing an expression constract into a cell and expressing and/or purifying the expressed peptide, polypeptide or protein are known in the art and described supra.
Alternatively, the peptide, polypeptide or protein is expressed using a cell free system, such as, for example, the TNT system available from Promega. Such an in vitro translation system is useful for screening a peptide library by, for example, ribosome display, covalent display or mRNA display.
Methods for producing antibodies, preferably a monoclonal antibody, or a fragment or recombinant fragment thereof are described supra.
In a preferred embodiment, the compound or modulator or the name or structure of the compound or modulator is provided with an indication as to its use e.g., as determined by a screen described herein.
The present inventors have also determined compounds useful for treating a condition associated with aberrant PKC-β activity. Accordingly, the present invention additionally provides a process for determining a candidate compound for the treatment of a condition associated with aberrant PKC-β activity comprising: (i) determining the level of PKC-β activity in a sample in the absence of a candidate compound by performing the method of the invention; (ii) determining the level of PKC-β activity in a sample in the presence of the candidate compound by performing the process of any one of the invention, wherein a difference in the level of PKC-β activity at (i) compared to (ii) indicates that the candidate compound modulates PKC-β activity and is a candidate compound for the treatment of a disorder associated with PKC-β activity,
Conditions associated with aberrant PKC-β activity are known in the art and/or described herein.
In one example, the agent is provided with an indication as to its use, for example, as determined using a method described herein.
The present invention additionally provides a process for determining a candidate compound for the treatment of a condition associated with aberrant PKC-β activity : (i) determining the level of PKC-β activity in a sample in the absence of a candidate compound by performing the method of the invention; (ii) determining the level of PKC-β activity in a sample in the presence of the candidate compound by performing the process of any one of the invention, wherein a difference in the level of PKC-β activity at (i) compared to (ii) indicates that the candidate compound modulates PKC-β activity and is a candidate compound for the treatment of a disorder associated with PKC-β activity; (iii) optionally, determining the structure of the candidate agent; and (iv) providing the candidate compound or the name or structure of the candidate compound.
In one example, the agent is provided with an indication as to its use, for example, as determined using a method described herein.
Furthermore, the present invention provides a process for determining a candidate compound for the treatment of a condition associated with aberrant PKC-β activity said method comprising:
(i) determining the level of PKC-β activity in a sample in the absence of a candidate compound by performing the method of the invention;
(ii) determining the level of PKC-β activity in a sample in the presence of the candidate compound by performing the process of any one of the invention, wherein a difference in the level of PKC-β activity at (i) compared to (ii) indicates that the candidate compound modulates PKC-β activity and is a candidate compound for the treatment of a disorder associated with PKC-β activity; (iii) optionally, determining the structure of the candidate compound; (iv) optionally, providing the name or structure of the candidate compound; and (v) providing the candidate compound.
Suitable agents are known in the art and/or described supra.
The present invention additionally provides a process for manufacturing a medicament for the treatment of a condition associated with aberrant PKC-β activity :
(i) determining the level of PKC-β activity in a sample in the absence of a candidate compound by performing the method of the invention;
(ii) determining the level of PKC-β activity in a sample in the presence of the candidate compound by performing the process of any one of the invention, wherein a difference in the level of PKC-β activity at (i) compared to (ii) indicates that the candidate agent modulates PKC-β activity and is a candidate compound for the treatment of a disorder associated with PKC-β activity;
(iii) optionally, determining the structure of the candidate compound;
(iv) optionally, providing the name or structure of the candidate compound; and (v) optionally, providing the candidate compound; and
(vi) using the candidate compound in the manufacture of a medicament for the treatment of a condition associated with aberrant PKC-β activity.
Preferably the condition is diabetes or asthma.
A still further aspect of the present invention provides a method of treating a disorder associated with aberrant PKC-β activity comprising administering an amount of a compound effective to modulate PKC-β activity in a cell.
In one embodiment, a disorder associated with reduced PKC-β activity is treated with a modulator capable of enhancing PKC-β activity. Disorders associated with reduced PKC-β activity are known in the art and include, for example conditions associated with decreased PKC-β activity include, hypoglycemia, melanoma, inflammatory and hyperplastic skin disorders (e.g. psoriasis, cutaneous T-cell lymphoma, cutaneous dermatitis and allergic contact dermatitis) and immunodeficiency. In another embodiment, a disorder associated with increased PKC-β activity is treated with a modulator capable of suppressing PKC-β activity. Disorders associated with increased PKC-β activity are known in the art and include hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, colon cancer, breast cancer, leukemia, hepatocarcinoma, gastric cancer, asthma and hypertension.
In a particularly preferred embodiment, the compound administered is capable of modulating histamine secretion.
Preferably the compound is a peptide. In a particularly preferred embodiment, the compound administered comprises a peptide comprising the amino acid sequence set forth in SEQ ID NOs: 172, 174 or 177. Preferably, the peptide is an antagonist of PKC- β activity.
One or more modulatory compounds can be administered to a subject by an appropriate route, either alone or in combination with another compound. An effective amount of a compound is an amount sufficient to achieve the desired therapeutic or prophylactic effect, under the conditions of administration, such as an amount sufficient for inhibition or promotion of PKC-β activity.
Alternatively, nucleic acid encoding a modulator of PKC-β activity such as, for example, nucleic acid encoding a peptide comprising a sequence set forth in SEQ ID NOs: 172, 174 or 177, is introduced to a subject in need of treatment and expressed therein operably under control of a suitable promoter sequence.
Such nucleic acids may be delivered to a cell or tissue requiring treatment using a gene delivery vehicle. For example, a nucleic acid encoding a PKC-β modulatory peptide or polypeptide may be contained in an adeno-associated vector (e.g. as disclosed in US Patent No. 5,739,941 and 5,622,850), an attenuated adenovirus vector (USSN 5,935,935), a lentiviral vector (USSN 5,994,136), a retroviral vector (USSN 5,817,491) or a synthetic (non-viral) vector (USSN 4,394,448 and 5,676,954).
Alternatively, a peptide or nucleic acid may be delivered using a liposome.
In another embodiment, a therapeutic peptide is delivered to a subject (e.g. to the lungs of a subject with asthma by inhalation). Preferably, the peptide is capable of transfecting a cell of that subject. Peptide delivery methods are known in the art and described, for example, in Schwartz and Zhang, Current Opinions in Moelcular Therapetuics, 2(2), 2000. For example, a modulatory peptide or protein may be conjugated to a cell-permeable peptide selected from the group consisting of, Drosophila antennapedia, HIV TAT, Herpes virus Vp22, Transportan, Dipahytic, MGP, HA1, LARLY Pepl, penetratin and Loligomer.
A variety of routes of administration are possible but not necessarily limited to oral, dietary, topical, parenteral (e.g. intravenous, intra-arterial, intra-musular, subcutaneous injection) and inhalation routes of administration, depending on the compound and/or condition to be treated.
Formulation of a compound to be administered will depend upon the route of administration selected (e.g. solution, emulsion, capsule). An appropriate composition or medicament comprising the compound can be prepared in a physiological carrier or vehicle (see, generally Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., Pa., 1985).
Where the compound is a peptide or protein, the compound may be administered via in vivo expression of the recombinant protein or peptide. In one embodiment, nucleic acid encoding the proteinpeptide can be incorporated into a retroviral, adenoviral or other suitable vector for delivery.
Alternatively, or in addition the nucleic acid encoding the protein/peptide can be introduced into a host cell capable of expressing said protein/peptide for delivery. In accordance with this embodiment, the cells can be implanted (either alone or in a barrier device), injected or introduced by any other method known in the art.
Preferably, the compound is a pharmaceutical compound.
Formulation of a pharmaceutical compound will vary according to the route of administration selected (e.g., solution, emulsion, capsule). An appropriate composition comprising the identified modulator to be administered can be prepared in a physiologically acceptable vehicle or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils, for instance. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers and the like (See, generally, Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., Pa., 1985). For inhalation, the agent can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, nebulizer or pressurized aerosol dispenser).
Furthermore, where the agent is a protein or peptide, the agent can be administered via in vivo expression of the recombinant protein. In vivo expression can be accomplished via somatic cell expression according to suitable methods (see, e.g. U.S. Pat. No. 5,399,346). In this embodiment, nucleic acid encoding the protein can be incorporated into a retroviral, adenoviral or other suitable vector (preferably, a replication deficient infectious vector) for delivery, or can be introduced into a transfected or transformed host cell capable of expressing the protein for delivery. In the latter embodiment, the cells can be implanted (alone or in a barrier device), injected or otherwise introduced in an amount effective to express the protein in a therapeutically effective amount.
As will be apparent to a skilled artisan, a compound that is active in vivo is particular preferred. A compound that is active in a human subject is even more preferred. Accordingly, when manufacturing a compound that is a modulator of PKC-β activity it is preferable to ensure that any components added to the modulator of PKC-β activity to not inhibit or modify the activity of said modulator.
The present invention is described further in the following non-limiting examples.
EXAMPLE 1 A polyclonal antibody that specifically recognizes a phosphorylated PKC-β site of a myosin heavy chain polypeptide.
A rabbit polyclonal antibody was raised against a peptide comprising the amino acid sequence set forth in SEQ ID NO: 9 that comprised a phosphoserine at position 9 (i.e. SEQ ID NO: 10) of the peptide using methods known in the art. Briefly, a peptide consisting of an amino acid sequence as set forth in SEQ ID NO: 10 was produced and conjugated to a carrier protein. A peptide consisting of the amino acid sequence set forth in SEQ ID NO: 9 was also produced (i.e. an unphosphorylated peptide). The phosphorylated peptide was then injected into SPF rabbits, with booster injections performed as required. Rabbits were periodically bled, and an indirect ELISA assay used to detect antibodies capable of specifically binding the phosphorylated peptide.
The phosphorylated peptide (SEQ ID NO: 10) was adsorbed to a microtiter plate and the plate blocked with phosphate buffered saline with 5% milk powder. Serum isolated from immunized rabbits was then added to the plate. Following washing, to remove any unbound proteins, the amount of protein bound to the peptide was determined using an anti-rabbit antibody conjugated to a detectable marker. Following identification of serum capable of binding the phosphorylated peptide (SEQ ID NO: 10) this method was repeated with the unphosphorylated peptide (SEQ ID NO: 9). Serum that only binds to the phosphorylated peptide was selected for further analysis.
Antiserum that has the highest titer of anti-phosphorylated peptide is selected for purification. The antiserum is passed through an affinity column to which the nonphosphorylated peptide (SEQ ID NO: 9) is attached. This removes antibodies that bind to epitopes outside the phosphorylation domain. ELISA analysis of the flow- through using both nonphosphorylated and phosphorylated peptides as antigens confirms that the flow through is now enriched for antibodies against the phosphorylated peptide.
To remove any remaining antibodies that may recognize the unphosphorylated site, the flow through is affinity purified on a peptide column containing the phosphorylated peptide (SEQ ID NO: 10). The flow through is discarded and the antibodies that attach to the column are eluted and assayed against the two peptides. ELISA results indicate that this additional affinity column produces a more discriminating antibody that is phosphospecific.
As shown in Figure 1, the polyclonal antibody was only capable of binding to a peptide comprising the amino acid sequence set forth in SEQ ID NO: 10. Furthermore, the level of antibody detected was also observed to reflect the amount of phosphorylated peptide present in a sample (as shown in the dot-blot shown in Figure 1).
Cellular extracts derived from RBL-2H3 cells were also assayed to determine the PKC- β activity in these cells. Protein samples were derived from cells that were stimulated with phorbol 12-myristate 13-acetate (PMA) and from unstimulated samples. Protein concentration of cellular samples was determined by the Bradford assay, using BSA as a standard. Samples containing 10 μg of protein were diluted to 30 μl in loading buffer, denatured for 5 min at 95°C, and separated by electrophoresis in 12.5% SDS-PAGE gels. After electroblotting onto Hybond transfer membranes (Amersham Pharmacia Biotech, Budkinghamshire, U.K.) gel loading and transfer efficiency were assessed by staining the blot with 0.1% Ponceau's Solution (Sigma Chemical Co.). Blots were blocked overnight at 4°C in 5% wt vol non-fat dry milk before incubation with the primary antibody for 60 min at room temperature. The rabbit polyclonal antibody described herein was used to determine the level of PKC-β phosphorylation of myosin heavy chain IIA. After the blot was incubated with horseradish peroxidase- conjugated anti-rabbit secondary antibody (Amersham), antibody binding was visualised by enhanced chemiluminescence detection reagents. The bands of the resulting autoradiographs were compared for optical density. Relative quantities were compared normalised to control values, arbitrarily assigned as 100%.
As seen in Figure 2, the PKC inducing compound PMA was capable of increasing the amount of phosphorylated myosin heavy chain IIA compared to unstimulated cells, suggesting that this protein is phosphorylated by a PKC enzyme.
EXAMPLE 2 PKC phosphorylates myosin heavy chain IIA
RBL-2H3 cells express non-muscle myosin IIA. These cells were incubated in the presence of a phorbolester and/or an antigen which cause an increase in phosphorylation of the myosin IIA polypeptide.
As shown in Figure 3 cells incubated in the presence of antigen for 2.5 minutes showed a dramatic increase in the level of protein detected with the polyclonal antibody described in Example 1. Furthermore, when these cell were incubated in the presence of Go6976 (an inhibitor of PKCα, PKCβl and PKCβll) the amount of protein detected by the monoclonal antibody was reduced. The amount of protein detected by an antibody that detects myosin heavy chain was unchanged, suggesting that the changes in the amount of protein detected by the previously described antibody was attributable to the changed phosphorylation status of myosin heavy chain IIA, and not changes in the level of expression of myosin heavy chain IIA.
Also reduced was the level of histamine secretion by the cells. Histamine secretion is caused by activation with phorbolester and/or antigen and is associated with PKC- activation. The inhibition of secretion by Go6967 indicates the suppression of PKC- activity.
Similar results were also obtained in cells that are incubated in the presence of both antigen and phorbolester (an activator of PKC). In this case, the administration of Go6976 resulted in less phosphorylated myosin heavy chain polypeptide being detected and approximately 43% less histamine being secreted.
Accordingly, these results clearly demonstrate that the region of myosin heavy chain IIA that comprises the sequence set forth in SEQ ID NO: 9 is a site for PKC-α or PKC- β phosphorylation, and that the phosphorylation may be detected with the polyclonal antibody described in Example 1. Furthermore, by normalizing results attained with the polyclonal antibody against a control protein (for example, myosin heavy chain) the degree of inhibition or modulation of PKC-β activity in a sample may be determined.
EXAMPLE 3 Constitutively active PKC-β phosphorylates myosin heavy chain IIA independent of phorbolester and/or antigen activation
Recombinant vectors expressing either a constitutively active PKC-β or a constitutively active PKC-α are produced essentially as described in Maramutsu et al, Mol. Cell Biol. 9(2): 831-836, 1989. Essentially, a cDNA encoding PKC-β lacking amino acids 6 to 59 or a cDNA encoding the N-terminal 253 amino acids of PKC-α fused to the N-terminal 17 amino acids of the cyclic- AMP-dependent protein kinase catalytic subunit are cloned into an expression vector under the control of the SR alpha promoter (as described in Takebe et al., Mol. Cell Biol, 8(1): A66-A12, 1988).
HEK-293 cells are the transfected with either of the recombinant expression vectors using standard calcium-phosphate methods. Cells are then grown at 37°C with 5% CO2 for 1 to 2 days (i.e., without exposure to phorbolester and/or antigen, which are both stimuli of PKC activity), before being lysed with sample buffer comprising 8M urea. Samples are then incubated on ice for 1 hour and cell debris precipitated by centrifugation. Protein concentration of the resulting cellular samples is then determined by the Bradford assay, using BSA as a standard. Samples containing 10 μg of protein are diluted to 30 μl in loading buffer, denatured for 5 min at 95°C, and separated by electrophoresis in 12.5% SDS-PAGE gels. After electroblotting onto Hybond transfer membranes (Amersham Pharmacia Biotech, Budkinghamshire, U.K.) gel loading and transfer efficiency are assessed by staining the blot with 0.1% Ponceau's Solution (Sigma Chemical Co.). Blots are blocked overnight at 4°C in 5% wt/vol non-fat dry milk before incubation with the rabbit polyclonal antibody described in Example 1 for 60 min. to determine the amount of PKC-β phosphorylation of myosin heavy chain IIA. The blot is then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (Amersham), and antibody binding visualised by enhanced chemiluminescence detection reagents. The bands of the resulting autoradiographs are compared for optical density. Relative quantities are compared normalised to control values (i.e. the amount of myosin heavy chain IIA phosphorylation detected in cells transfected with an empty expression vector), arbitrarily assigned as 100%.
Results indicating increased phosphorylation of myosin heavy chain IIA in the cells overexpression PKC-β compared to those overexpressing PKC-α indicate that the phosphorylation of myosin heavy chain IIA is PKC-β specific.
EXAMPLE 4 PKC-β specific phosphorylation of myosin heavy chain IIA
The compound 6-(3,4-dihydroxystyryl)-4-hydroxy-2-pyrone (hispidin) has been previously shown to be capable of specifically inhibiting the activity of PKC-β isozymes (Gonindard et al, Cell. Biol Toxicol, 13: 141-153, 1997 and Wadworth SJ and Goldfme, Infect Immun. 70:4650-60, 2002). To determine the particular PKC isozyme/s that phosphorylate myosin heavy chain IIA this compound is used to inhibit PKC-β in cells and the level of phosphorylation of myosin heavy chain IIA is determined.
Rat basophilic mast cells (RBL-2H3) are maintained and used as adherent monolayers in RPMI-1640 medium supplemented with 2mM glutamine, 25mM Hepes, 10% fetal calf serum. The RBL-2H3 cell line is activated via crosslinking of the FcεRl receptors on its surface. The DNP-specific monclonal IgE added to the cells attaches to the FcεRl receptors, and primes the cells to be activated through this receptor.
Activation experiments are carried out by adding lOOng/ml DNP-BSA to the cells, which binds to the IgE attached to the FcεRl receptors leading to receptor signalling and subsequent degranulation. After the designated time, the reaction is stopped by placing the samples on ice. Cells are also incubated in the presence of a concentration of hispidin ranging from 0.5 μM to lOμM. Controls are incubated in the absence of hispidin.
For Western blots, 25 μg protein from treated or untreated cells is separated using SDS PAGE and transferred to PVDF. The PVDF membrane was blocked for 14 hours (overnight) at 4°C with 5% Skim Milk in PBS with the addition of 0.1% Tween-20 (PBS-T). Excess skim milk is removed by three 10 minute rinses in PBS-T. The primary antibody (described in Example 1), diluted in 2% skim milk is incubated with the membrane for 90 minutes at room temperature with gentle rocking. Excess unbound primary antibody is removed by three 10 minute rinses in PBS-T (PBS + Tween 20). The secondary antibody is also diluted in 2% skim milk, and was incubated with the membrane for 60 minutes at room temperature with gentle rocking. Excess unbound secondary antibody is removed by three 10 minute rinses in PBS-T. The proteins are detected using an Enhanced Chemiluminescent detection kit following the manufacturer's instructions (NEN, Perkin Elmer). Results showing reduced phosphorylation in the presence of hispidin compared to control samples demonstrates that PKC-β preferentially phosphorylates myosin heavy chai IIA (i.e., at a site corresponding to position 1916 of SEQ ID NO: 2 or position 1917 of SEQ ID NO: 4).
Immuno-cytochemical techniques are used to confirm the specificity of hispidin by monitoring translocation (activation) of PKCβ and PKCα in response to phorbol ester or antigen. RBL cells are pre-incubated with concentrations of hispidin found to inhibit phosphorylation of myosin heavy chain IIA.
For these studies, cells are plated on coverslips. Cells are stimulated with phorbol ester or antigen, in the presence or absence of hispidin and then washed six times with PBS, fixed, permeabilized with PBS containing 0.1% Triton X-100, and blocked with 1% bovine serum albumin. The permeabilized cells are then incubated at 37°C for 1 h with rabbit polyclonal antibodies (1:100) to human PKCα, PKCBI, or PKCBII (Santa Cruz). Then the cells are then washed 10 times with PBS, the secondary antibody, anti-rabbit immunoglobulin G conjugated to FITC is added at 1:100, and the cells are incubated for 30 min at 25°C. After this incubation the cells are washed 10 times with PBS and mounted for confocal microscopy.
Results showing the inhibition of translocation of PKC-βl and/or PKC-βll and not PKC-α to the cellular membrane indicates the specificity of hispidin for PKC-β. EXAMPLE 5 PKC-β has a higher affinity for myosin heavy chain IIA than PKC-α
A short peptide corresponding to the sequence around the phosphorylation site of myosin heavy chain (MNREVSSLKNKLRR, SEQ ID NO: 130) was produced synthetically was incubated with 100 μM [γ-32P]ATP (100 - 200 c.p.m./pmol), and 10 μl recombinant PKCα, or PKC-βl (Calbiochem, diluted approx. 1/100 in MOPS/Triton = 1 unit/μl). Lipids (5 mg/ml in chloroformmethanol (19:1 v/v)), were dried under nitrogen and sonicated into 100 mM MOPS (pH 7.5) 1% (v/v) Triton X-100 until clear, before addition to the assay buffer. In addition of PKC isoforms and [γ-32P]ATP, the final assay medium (50 μl) contained 24 mM MOPS (pH 7.5), 0.04% (v/v) Triton X- 100, 5 mM Mg(CH2COO)2, 1 mM CaCl2, 125 μg/ml phosphatidylserine and 2.5 μg/ml dioctanoylglycerol. Incubations were performed at 30°C for lOmin. Reactions were started by addition of 10 μl of ATP-Mg Acetate (ATP 500 μM) containing [γ-32P]ATP. Reactions were stopped by addition of 10 μl cold ATP (150 mM). After vortexing 40μl of reaction medium was withdrawn and spotted onto P81 paper squares. These were dropped into H3PO (10ml cone, acid in 11ml), and then washed 5 times for 5 min in 3 % (v/v) H3PO , and then once in 100% EtOH. After drying radioactivity was determined by Cerenkov counting. Control incubations were undertaken under similar conditions but using Histone HIIIS as a generic PKC substrate, and a myosin heavy chain phosphorylation site mutant (MNREVSALKNKLRR, SEQ ID NO: 180) as a negative control.
As shown in Figure 4 PKC-βl phosphorylates the myosin heavy chain polypeptide at a significantly greater level than PKC-α (approximately 4 to 5 times more peptide is phosphorylated by PKC-β than PKC-β). EXAMPLE 6 Knockdown of PKC isozymes
siRNA molecules specifically targeting PKC-α, PKC-βl, PKC-βll, PKC-δ, PKC-ε, PKC-η, PKC-i, PKC-Θ and PKC-ζ are obtained from Ambion (Austin, TX, USA). Rat basophilic mast cells (RBL-2H3) are maintained and used as adherent monolayers in RPMI-1640 medium supplemented with 2mM glutamine, 25mM Hepes, 10% fetal calf serum.
Cells are transfected using siRNA duplexes specific for a PKC isozyme at a final concentration of lOOnM using the Silencer siRNA transfection kit from Ambion.
The RBL-2H3 cell line is activated via crosslinking of the FcεRl receptors on its surface. The DNP-specific monclonal IgE added to the cells attaches to the FcεRl receptors, and primes the cells to be activated through this receptor.
Activation experiments are carried out by adding lOOng/ml DNP-BSA to the cells, which binds to the IgE attached to the FcεRl receptors leading to receptor signalling and subsequent degranulation. After the designated time, the reaction is stopped by placing the samples on ice.
For Western blots, 25μg protein from treated or untreated cells is separated using SDS PAGE and transferred to PVDF. The PVDF membrane was blocked for 14 hours (overnight) at 4°C with 5% Skim Milk in PBS with the addition of 0.1% Tween-20 (PBS-T). Excess skim milk is removed by three 10 minute rinses in PBS-T. The primary antibody (described in Example 1), diluted in 2% skim milk is incubated with the membrane for 90 minutes at room temperature with gentle rocking. Excess unbound primary antibody is removed by three 10 minute rinses in PBS-T (PBS + Tween 20). The secondary antibody is also diluted in 2% skim milk, and was incubated with the membrane for 60 minutes at room temperature with gentle rocking. Excess unbound secondary antibody is removed by three 10 minute rinses in PBS-T. The proteins are detected using an Enhanced Chemiluminescent detection kit following the manufacturer's instructions (NEN, Perkin Elmer). Results showing reduced phosphorylation in the presence of PKC-β specific siRNA compared to control samples and/or samples transfected with siRNA specific to other PKC isozymes demonstrate that PKC-β preferentially phosphorylates myosin heavy chai IIA (i.e., at a site corresponding to position 1916 of SEQ ID NO: 2 or position 1917 of SEQ ID NO: 4).
EXAMPLE 7 Overexpression of inactive dominant negative PKC-β to determine the kinase that phosphorylates myosin heavy chain IIA
Overexpression of a kinase-dead PKC is known to act in an isoform-specific dominant- negative fashion (e.g., see Carpenter et al, J Biol Chem 276: 5368-5374, 2001). Accordingly, the PKC-β specific phosphorylation of myosin heavy chain IIA is demonstrated by its inhibition following overexpression of kinase-dead PKC βl and/or PKC-βll.
We will therefore use a reporter assay based on co-overexpression of a C-terminal fragment of human myosin heavy chain IIA tagged to GFP (Straussman R et al, 2001, J Cell Sci 114:3047-3057). To determine the level of phosphorylation a myosin reporter comprising a C-terminal fragment of human myosin heavy chain IIA tagged to GFP (essentially as described in Straussman et al, JCell Sci 114:30A1-3051, 2001) is used.
RBL cells are plated on coverslips on the morning of the experiment. After 4 hours they are transfected in serum free medium with 2μg DNA (1:1 myosin reporter :PKC β mutant construct) in a 1:4 ratio with Lipofectamine 2000. After 2 hours cells are washed, and returned to tissue culture for a further 24-48 hours.
Cells are then stimulated with phorbol ester or antigen as described supra. For imaging studies cells are washed with PBS, fixed, permeabilized with PBS containing 0.1% Triton X-100, and blocked with 1% bovine serum albumin. The permeabilized cells are then incubated at 37°C for 1 h with the antibody described in Example 1 (1:100). Then the cells will be washed 10 times with PBS, the secondary antibody, anti-rabbit immunoglobulin G conjugated to FITC, will be added at 1:100, and the cells are incubated for 30 min at 25°C. After this incubation the cells are washed 10 times with PBS and mounted for con&cal microscopy. Fluorescence intensity is examined in cells overexpressing the MHC-reporter (identified by GFP fluorescence) to determine effects of the co-expressed PKC constracts. Alternatively, stimulated cells are lysed and the GFP-tagged polypeptide is immunoprecipitated with a mixture of mouse mono-clonal antibodies (clones 7.1 and 13.1; Roche). Phosphorylation due to co-expressed PKC constructs is then determined by immunoblotting using the antibody described in Example 1 essentially as described supra.
EXAMPLE 8 Determining PKC-β activity in a rat model of diabetes
Animals. Six week-old female, heterozygous (mRen-2)27 rats are randomized to receive either 55 mg/kg streptozotocin (STZ) (Sigma, St. Louis, MO) diluted in 0.1 mol/1 citrate buffer (pH 4.5) (diabetic) or citrate buffer alone (non-diabetic) by tail-vein injection after an overnight fast. A subset of diabetic Ren-2 rats are treated with a PKC β specific inhibitor 10 mg kg-1 d-1, milled into rat food for 6 months after STZ or vehicle. Treatment commences within 24 h of STZ injection. Each week, rats are weighed and blood glucose is determined using an AMES glucometer (Bayer Diagnostics, Melbourne, Australia). Every 4 weeks, systolic blood pressure (SBP) is recorded in preheated conscious rats by tail cuff plethysmography (21). Diabetic rats receive a daily injection of insulin (2-4 units intraperitoneally; Ultratard, Novo Nordisk, Bagsraerd, Denmark) to promote weight gain and reduce mortality.
Tissue preparation. Rats are anesthetized (Nembutal 60 mg/kg body wt intraperitoneally; Boehringer-Ingelheim, North Ryde, Australia), and the abdominal aorta is cannulated with an 18-G needle. Perfusion-exsanguination commenced at SBP (180-220 mmHg) via the abdominal aorta with 0.1 mol/1 PBS (pH 7.4; 20-50 ml) to remove circulating blood, and the inferior vena cava adjacent to the renal vein is simultaneously severed allowing free flow of the perfusate. After clearance of circulating blood, 4% paraformaldehyde in 0.1 mol/1 phosphate buffer (pH 7.4) is perfused for another 5 min (100-200 ml of fixative) to fix the tissues. Kidneys are removed from the animal, decapsulated, sliced transversely, and either frozen- embedded in OCT (Tissue-Tek; Miles, Elkhart, IN) for immunofluorescence or paraffin-embedded for light microscopic evaluation.
Immunofluorescence. Four-micron frozen sections are postfixed in Acetor for 5 min at 4°C. Following washing sections are incubated for 30 min with normal goat serum (NGS) diluted 1:10 with 0.1 mol/1 PBS at pH 7.4. Sections are then incubated overnight at 4°C with the rabbit polyclonal antibody described in Example 1. Sections incubated with 1:10 NGS instead of the primary antiserum as the negative control. After thorough washing with PBS (3 x 5 min changes), the sections are incubated with goat anti-rabbit Alexa Fluor 568 (Molecular Probes), diluted 1:200 with PBS for 1 h at room temperature. Sections are rinsed with PBS (2 x 5 min), rinsed in tap water for 5 min, and mounted for microscope viewing.
As shown in Figure 5A myosin IIA was phosphorylated in the kidney epithelium of diabetic rats, while this was not observed in control animals (not shown). This suggests a large increase in PKC-β activity in the kidney epithelium as a result of a diabetic phenotype. This observation was also reflected in the cardiac endothelium of diabetic rats (Figure 5B). Accordingly, these results suggest that diabetes increases the activity of PKC-β in a variety of tissues throughout the body.
Western blot analysis. Protein concentration of whole kidney samples is determined by the Bradford assay, using BSA as a standard. Samples containing 10 μg of protein are diluted to 30 μl in loading buffer, denatured for 5 min at 95°C, and separated by electrophoresis in 12.5% SDS-PAGE gels. After electroblotting onto Hybond transfer membranes (Amersham Pharmacia Biotech, Budkinghamshire, U.K.) gel loading and transfer efficiency are assessed by staining the blot with 0.1% Ponceau's Solution (Sigma Chemical Co.). Blots are blocked overnight at 4°C in 5% wt/vol non-fat dry milk before incubation with the primary antibody for 60 min at room temperature. The rabbit polyclonal antibody described in Example 1 is used to determine the amount of PKC-β phosphorylation of myosin heavy chain IIA. After the blot is incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (Amersham), antibody binding is visualized by enhanced chemiluminescence detection reagents. The bands of the resulting autoradiographs are compared for optical density. Relative quantities are compared normalized to control values, arbitrarily assigned as 100%.
EXAMPLE 9 A cell free system for determining phosphorylation of a PKC-β phosphorylation site of a myosin heavy chain polypeptide. The ability of PKC β, βll, α, γ, δ and ζ (available from Research Diagnostics Inc., NJ, USA) to phosphorylate the peptide described in Example 1 is assessed. Vesicles consisting of 120 μg/mL phosphatidylserine (Avanti Polar Lipids) and sufficient diacylglycerol (Avanti Polar Lipids) to activate the specific enzyme to maximum activity in 20 mM HEPES buffer (Sigma, St. Louis, MO), pH 7.5 μM calcium chloride (Sigma, St. Louis, MO) for assaying the α, βl, βll and γ enzymes only, 1 mM EGTA for all enzymes, 10 mM magnesium chloride (Sigma, St. Louis, MO) and 30 μM (γ"32P) ATP (DuPont). The peptide substrate is also incorporated into the vesicle. The assay is started by addition of protein kinase C enzyme. The assay is then incubated at 30°C for 10 min and stopped by adding 0.5 mL of cold trichloroacetic acid (Amresco) followed by 100 μg/mL of 1 mg/mL bovine serum albumin (Sigma, St. Louis, MO). The precipitate is collected by vacuum filtration on glass fiber filters and quantified by counting using a β scintillation counter. EXAMPLE 10 Preparation of a monoclonal antibody that recognizes a myosin heavy chain IIA phosphorylated by PKC-β
A monoclonal antibody that specifically binds the phosphorylated peptide described in Example 1 (i.e. a peptide comprising an amino acid sequence set forth in SEQ ID NO:
10) is produced using methods known in the art. Briefly, peptide antigens and peptide probes are synthesised essentially using the methods described in Bodanszky, M.
(1984) Principles of Peptide Synthesis, Springer- Verlag, Heidelberg and Bodanszky,
M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg. A phosphoamino acid (i.e. phosphoserine) is incorporated into the phosphorylated peptide essentially as described in Czemik et al, Methods Enzymol.
201: 264-283, 1991. A non-phosphorylated peptide is also produced.
Peptides are purified using HPLC and purity assessed by amino acid analysis.
Female BalB/c mice are immunized with a purified form of the phosphorylated peptide. Initially mice are sensitised by intraperitoneal injection of Hunter's Titermax adjuvant (CytRx Corp., Norcross, GA,). Three boosts of the peptide are administered at 2, 5.5 and 6.5 months post initial sensitisation. The first of these boosts is a subcutaneous injection while the remaining are administered by intraperitoneal injection. The final boost is administered 3 days prior to fusion.
The splenocytes of one of the immunized BALB/c mice is fused to X63-Ag8.653 mouse myeloma cells using PEG 1500. Following exposure to the PEG 1500 cells are incubated at 37°C for 1 hour in heat inactivated foetal bovine serum. Fused cells are then transferred to RPMI 1640 medium and incubated overnight at 37°C with 10% CO2. The following day cells are plated using RPMI 1640 media that has been supplemented with macrophage culture supematants.
Two weeks after fusion, hybridoma cells are screened for antibody production by solid phase ELISA assay. Standard microtitre plates are coated with the synthetic phosphorylated peptide in a carbonate based buffer. Plates are then blocked with BSA, washed and then the test samples (i.e. supernatant from the fused cells) is added, in addition to control samples, (i.e. supernatant from an unfused cell). Antigen-antibody binding is detected by incubating the plates with goat-anti-mouse HRP conjugate (Jackson ImmunoResearch Laboratories) and ABTS peroxidase substrate system (Vector Laboratories, Burlingame, Ca 94010, USA). Absorbance is read on an automatic plate reader at a wavelength of 405 nm.
Hybridoma cells that produce antibodies that bind the phosphorylated peptide are also screened for their ability to bind the unphosphorylated peptide using a solid phase ELISA (essentially as described supra, however, the unphosphorylated peptide is used in place of the phosphorylated peptide). Cells expressing antibodies capable of selectively binding the phosphorylated peptide are selected.
Any colonies that are identified as positive by these screens continue to be grown and screened for several further weeks. Stable colonies are then isolated and stored at - 80°C.
Positive stable hybridomas are then cloned by growing in culture for a short period of time and diluting the cells to a final concentration of 0.1 cells/well of a 96 well tissue culture plate. These clones are then screened using the previously described assays. This procedure is then repeated in order to ensure the purity of the clone. Four different dilutions, 5 cells/well, 2 cells/well, 1 cell/well, 0.5 cells/well of the primary clone are prepared in 96-wells microtiter plates to start the secondary cloning. Cells are diluted in IMDM tissue culture media containing the following additives: 20% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml of penicillin, 100 μg/ml of streptomycin, 1% GMS-S, 0.075% NaHCO3. To determine clones that secrete anti-phosposerine 1917 myosin heavy chain IIA antibody, supematants from individual wells of the 0.2 cells/well microtiter plate are withdrawn after two weeks of growth and tested for the presence of antibody by ELISA assay as described above.
Furthermore, antibodies are used to screen RBL-2H3 cells that have been induced with PMA and/or incubated in the presence of a PKC-β specific inhibitor. Protein is isolated from the cells after induction and separated using SDS-PAGE before transfer to a PVDF membrane. Membranes are blocked, and then screened with the previously described monoclonal antibodies to ensure that they are capable of only detecting a protein of approximately the same molecular weight as myosin heavy chain IIA in the cells incubated with PMA. Those antibodies capable of specifically detecting phosphorylated myosin heavy chain IIA are selected.
All positive clones are then adapted and expanded in RPMI media containing the following additives: 10% FBS, 2 mM L-glutamine, 100 units/ml of penicillin, 100 .μg/ml of streptomycin, 1% GMS-S, 0.075% NaHCO3, and 0.013 mg/ml of oxalaacetic acid. A specific antibody is purified by Protein A affinity chromatography from the supernatant of cell culture.
The titer of the antibodies produced using this method are determined using the Easy Titer kit available from Pierce (Rockford, II, USA). This kit utilises beads that specifically bind mouse antibodies, and following binding of such an antibody these beads aggregate and no longer absorb light to the same degree as unassociated beads. Accordingly, the amount of an antibody in the supernatant of a hybridoma is assessed by comparing the OD measurement obtained from this sample to the amount detected in a standard, such as for example mouse IgG.
EXAMPLE 11 A two-site ELISA to determine PKC-β activity in a sample. A monoclonal antibody that binds to both phosphorylated and non-phosphorylated myosin heavy chain IIA essentially is produced essentially as described in Example 10. However, entire recombinant myosin heavy chain IIA is used as an immunogen. Furthermore, rather than selecting against antibodies that cannot bind to non- phosphorylated myosin heavy chain IIA, antibodies are selected that can bind to both forms of myosin, but can not bind to the region of myosin heavy chain IIA represented by SEQ ID NO: 9 or SEQ ID NO: 10. Accordingly, the antibody binds to a different epitope to the antibody described in Example 1.
Monoclonal antibodies are additionally screened to determine those useful in two site immunoassay. Monoclonal antibodies are absorbed to a microtitre plate at 20°C for 16 hours. Plates are then washed and blocked for 1 hour. Recombinant phosphorylated myosin heavy chain IIA is serially diluted, added to wells of the microtitre plate and incubated for 1 hour.
The monoclonal antibody is capable of specifically detecting PKC-β phosphorylated myosin heavy chain IIA described in Example 10 is conjugated to horseradish peroxidase (HRP) using a HRP conjugation kit (Alpha Diagnostics International, Inc., San Antonio, TX, USA).
Following washing of the microtitre plates, the HRP conjugated monoclonal antibody is added to each well of the plate and incubated. Plates are then washed and ABTS (Sigma Aldrich, Sydney, Australia) is added to each well. Reactions are stopped after approximately 20 minutes and absorbance values measured at 415 nm.
The amount of absorbance detected in negative control wells (no myosin added) is subtracted from the absorbance of each other well to determine the amount of antibody bound to phosphorylated myosin heavy chain polypeptide.
All unlabelled monoclonal antibodies are tested to determine their efficacy as capture antibodies. Antibodies are tested to determine their ability to accurately predict the amount of phosphorylated myosin heavy chain polypeptide in a sample.
An antibody capable of being used in a two site ELISA assay (i.e. capable of being used in an ELISA that accurately predicts the amount of PKC-β phosphorylated myosin heavy chain are selected for use in an ELISA assay for determining the amount of phosphorylated myosin heavy chain IIA, and as a consequence PKC-β activity in a sample.
EXAMPLE 12 A rapid assay to determine PKC-β activity in a sample.
An assay using immunochromatography to determine the presence of a significant amount of PKC-β phosphorylated myosin heavy chain IIA, and as a consequence, PKC-β activity is of particular use, for example,, as a point-of-care diagnostic tool, as it requires little technical knowledge to accurately perform the assay.
A biological sample, e.g. a blood or tissue sample isolated from a subject, is treated to lyse cells in the sample. The lysate is then added to a test strip that comprises a monoclonal antibody capable of binding both phosphorylated and non-phosphorylated myosin heavy chain IIA (as described in Example 11), that has been previously conjugated to visible colloidal gold (available from structure Probe Inc., West Chester, PA, USA). Any myosin heavy chain IIA present in the sample binds to the antibody, and the sample then migrates (by capillary action) up the test strip, passing over the monoclonal antibody that specifically detects PKC-β phosphorylated myosin heavy chain IIA (as described in Example 10) that has been conjugated to the test strip in a specific localised area (i.e. a line). Any PKC-β phosphorylated myosin heavy chain IIA in the sample is then captured by this antibody.
A significant concentration of PKC-β phosphorylated myosin heavy chain IIA captured by the second antibody causes a visible line to form on the test strip. Accordingly, the formation of this line indicates that a sample comprises a significant amount of PKC-β activity. The amount of PKC-β phosphorylated myosin heavy chain IIA required to form a visible line may be varied by altering the amount of the secondary antibody bound to the test strip.
As a positive control an anti-human antibody is also conjugated to the test strip at a separate location to the secondary monoclonal antibody. A visible line forms at this location when there is sufficient myosin heavy chain IIA bound to the labelled antibody in a sample to bind the anti-human antibody.
EXAMPLE 13 A quantitative sandwich ELISA for determining PKC-β activity
A commercial antibody to non-muscle MHC (Biomedical Technologies Inc), which recognises predominantly MHC2A is pre-absorbed onto a micro-titre plate. Cell extracts are then added to the wells, and the signal detected using the antisera described in Example 1 coupled to HRP. The ELISA assay is performed essentially as described in Example 10.
A standard curve is produced using GFP labelled myosin heavy chain polypeptide. Standards comprise recombinant GFP-tagged myosin heavy chain IIA produced by cloning the coding region of human myosin heavy chain IIA (SEQ ID NO: 1) into the pcDNA3.1/NT-GFP vector (Invitrogen) and transfecting into COS cells. The GFP labelled protein is then affinity purified from the cells using an antibody to the GFP protein (Invitrogen).
Phosphorylated labelled myosin heavy chain polypeptide is then produced by incubating the isolated protein in the presence of a mixture of recombinant PKC-βl and PKC-βll (Calbiochem) essentially as described in Example 5. Unphosphorylated recombinant protein is produced using recombinant protein phosphatase 1 and protein phosphatase 2A available from Upstate Biotechnology, Inc. (Lake Placid, NY, USA) .
Mixtures comprising various known concentrations of labelled phosphorylated and unphosphorylated myosin heavy chain polypeptide are then produced. The level of phosphorylated myosin heavy chain polypeptide is then determined in each mixture using the two site ELISA described supra and this level compared to the known level of phosphorylated and total myosin heavy chain in the mixture. This allows for correlation of the level of phosphorylated myosin heavy chain polypeptide with the total level of phosphorylated myosin heavy chain polypeptide in a sample.
The level of phosphorylated myosin heavy chain polypeptide determined in a test sample is then compared to the standard curve and the total phosphorylated myosin heavy chain polypeptide estimated.
EXAMPLE 14 A competitive ELISA to determine PKC-β activity Micro-titre plates are pre-absorbed with anti-rabbit IgG, and then with the anti-phospho MHC antisera. Cell extracts are then added to the sample with a FITC labelled peptide comprising the sequence (MNREVSDLKNKLRR, SEQ ID NO: 173) or phosphomimetic peptide comprising the amino acid sequence (MNREVSELKNKLRR, SEQ ID NO: 181). The FITC labelled peptides are produced by labelling a synthetic peptide with FITC using the Fluorotag FITC kit available from Sigma Aldrich essentially according to manufacurer's instructions.
Following washing to remove unbound antigen the level of phosphorylated heavy chain is determined by detecting the level of labelled peptide bound and comparing this to a standard curve.
The standard curve is produced by combining known levels of the labelled peptide with known levels of the unlabelled peptide (or mimetic). The level of detected label is then correlated with the total level of labelled peptide in the mixture, thereby providing a measure of total phosphorylated myosin heavy chain polypeptide in the sample.
The level of labelled peptide bound to the antibody in an assay using a test sample is then correlated to the standard curve to determine the level of PKC-β activity in the sample.
EXAMPLE 15 PKC-β activity in diabetic subjects
A 96-well plate is coated with 10 μg/ml poly-L -lysine for 30 min at 37 °C. After two washes with sterile PBS, white blood cells are plated into each well and fixed with 8% formaldehyde in PBS for 20 min at room temperature, followed by three washes with PBS/Triton. Endogenous peroxidase is quenched with 1% H2O2 and 0.1% sodium azide in PBS/Triton for 20 min, washed three times in PBS/Triton, blocked with 10% fetal calf serum in PBS/Triton for 1 h and incubated overnight with various dilutions of primary antibody described in Example 1 in PBS/Triton containing 5% BSA at 4 °C.
Cells are then washed three times with PBS/Triton for 5 min and incubated with secondary antibody (peroxidase-conjugated goat anti-rabbit antibody, dilution 1:500) in PBS/Triton with 5% BSA for 1 h at room temperature and washed three times with
PBS/Triton for 5 min and twice with PBS. Subsequently the cells are incubated with 50 μl of a solution containing 0.4 mg/ml OPD, 11.8 mg/ml Na2HPO4-2H2O, 7.3 mg/ml citric acid and 0.015% H2O2 for 15 min at room temperature in the dark. The reaction is stopped with 25 μl of 1 M H2SO4, the ,4490/650 measured.]
Detection of an enhanced level of phosphorylated myosin heavy chain IIA in the subjects demonstrates increased PKC-β activity and shows the utility of the assay for the diagnosis of a subject suffering from a disorder associated with aberrant PKC-β activity, namely diabetes. EXAMPLE 16 Phosphorylation of myosin heavy chain IIA modulates histamine secretion
To determine the effect of PKC-β phosphorylation of myosin heavy chain IIA several peptides were produced that corresponded to the PKC-β phosphorylation site of myosin IIA in various forms.
The peptides produced comprised the following amino acid sequences: CMNREVSSLKNKLRR (SEQ ID NO: 172) corresponds to the non-phosphorylated PKC-β phosphorylation site of myosin IIA.
CMNREVSDLKNKLRR (SEQ ID NO: 173) in which the serine phosphorylated by PKC-β is replaced with an aspartic acid thereby mimicking the negative charge of the phosphorylated serine residue.
CMNREVSALKNKLRR (SEQ ID NO: 174) in which the serine phosphorylated by PKC-β is replaced with an alanine, thereby rendering this peptide incapable of being phosphorylated.
These peptides were then used to determine the effect of PKC-β phosphorylation of myosin heavy chain IIA on secretion from rat basophilic leukemia cells, RBL-2H3 cells.
8.1 Cell culture and secretion
RBL-2H3 cells were maintained as monolayer cultures and harvested by trypsin treatment. Cells were transferred to 96-well culture plates at a density of 4.9 X 10 cells per well in 0.4ml Earle's modified Eagle's medium supplemented with 15%(v/v) fetal bovine serum. For those experiments involving antigenic activation, the cells were sensitized by the addition of 75 ng/ml dinitrophenol (DNP)-specific IgE and incubated overnight at 37°C with 5% CO2. To measure the secretory response, cultures were also incubated overnight with [3H] 5-hydroxytryptamine ([3H] 5HT), (0.5μCi/ml), which becomes incorporated into the histamine containing granules.
8.2 Permeabilization and Stimulation of cells.
The cells were washed twice in a calcium-free, potassium glutamate buffer: 139mM glutamic acid, 5mM ATP, 5mM glucose: 7mM magnesium acetate; ImM EGTA and
20mM PIPES (pH 7.2). To permeabilize streptolysin O (SLO), 0.2 units/ml was added to aliquots of the buffer at 37°C ± 25mg/mL peptide (final concentration = 500μg/mL).
Cells were pre-permeabilized at 28°C for 30min (for cells treated with GTPγS) or 8min
(for cells treated with DNP-BSA). CaCl2 (0.856mM) was added as required so the free calcium concentration was IμM which is optimal for antigenic activation in permeabilized RBL-2H3 cells. After 20 min at 37°C the reaction was stopped by placing the plates on ice and an aliquot of the supernatant taken for the determination of the tritium content using a liquid scintillation counter. The release of [3H] 5HT into the medium was expressed as a percentage of the total cellular content of the amine as determined from lysed, imstimulated cultures or after the remaining amine within the cells had been released by lysis in l%(v/v) Triton X-100.
8.3 Results
Results of experiments using cells stimulated with DNP-BSA (which stimulates the IgE receptor thereby inducing histamine secretion) are shown in Figure 7A, and results of experiments using cells stimulated with GTPγS (guanosine-5'-Q-(3-thiotriphosphate, which bypasses receptor-mediated events in permeabilized cells and directly triggers downstream signalling) are shown in Figure 7B. Secretory responses in cells treated with GTPγS are higher in permeabilized cells. Secretion due to either stimulus was totally abolished by the peptide comprising the sequence set forth in SEQ ID NO: 174, which is incapable of being phosphorylated (Figure 7). Addition of the peptide comprising the sequence set forth in SEQ ID NO: 173, which mimics the negative charge of the PKC-β phosphorylated ser-1917 residue of human myosin heavy chain IIA, tended to increase secretion. The native peptide (SEQ ID NO: 172) was without significant effect (Figure 6).
These experiments demonstrate that ser 1917 phosphorylation of MHC is necessary for secretion, and that compounds preventing phosphorylation at that site, or interfering with its downstream consequences, are beneficial in treatment of asthma. EXAMPLE 17 Unconjugated cell permeable peptides modulate histamine secretion
To introduce the peptides described in Example 16 into unpermeabilized RBL-2H3 cells, a synthetic peptide comprising the sequence:
KETWWETWWTEWSQPKKRKV (SEQ ID NO: 175) (Pepl) is produced. Pep 1 is a peptide carrier that is particularly useful for the delivery of biologically active peptides to cells, as there is no requirement that Pepl be conjugated to the peptide of interest (Morris et al, Nature Biotechnology, 19: 1173 - 1176. Accordingly, Pepl does not interfere (eg by altering the structure of the peptide of interest) with the biological activity of the peptide of interest.
Pepl is incubated with each of the peptides described in Example 16 in PBS at 37°C for 30 minutes.
RBL-2H3 cells are maintained as monolayer cultures and harvested by trypsin treatment. Cells are transferred to 96-well culture plates at a density of 4.9 X 104 cells per well in 0.4ml Earle's modified Eagle's medium supplemented with 15%(v/v) fetal bovine serum. Cells are then grown to 75% confluence and overlayed with the Pepl/peptide complex described supra and incubated for 30 minutes at 37°C. Additional Earle's modified Eagle's medium supplemented with 15%(v/v) fetal bovine serum is then added to the culture plates and the plates incubated at 37°C for an additional 30 minutes. Cells are then extensively washed with PBS. . Following transfection of the peptides those cells used in antigenic activation are sensitized by the addition of 75 ng/ml dinitrophenol (DNP)-specific IgE and incubated overnight at 37°C with 5% CO2. To measure the secretory response, cultures are also incubated overnight with [3H] 5-hydroxytryptamine ([3H] 5HT), (0.5μCi/ml), which becomes incorporated into the histamine containing granules.
Supernatant from the cell cultures is then collected to determine of the tritium content using a liquid scintillation counter. The release of [3H] 5HT into the medium is expressed as a percentage of the total cellular content of the amine as determined from lysed, unstimulated cultures or after the remaining amine within the cells has been released by lysis in l%(v/v) Triton X-100.
EXAMPLE 18 Conjugated cell permeable peptides modulate histamine secretion
To introduce the peptides described in Example 16 into unpermeabilized RBL-2H3 cells, a penetratin peptide comprising the sequence:
RRMKWKK (SEQ ID NO: 176)
was conjugated to the peptides comprising the sequences set forth in SEQ ID NOs: 126 and 127. Accordingly, synthetic peptides comprising the following amino acid sequences were produced.
MNREVSALKNKLRRMKWKK (SEQ ID NO: 177) in which the serine phosphorylated by PKC-β is replaced with an alanine, thereby rendering this peptide incapable of being phosphorylated.
MNREVSDLKNKLRRMKWKK (SEQ ID NO: 178) in which the serine phosphorylated by PKC-β is replaced with an aspartic acid thereby mimicking the negative charge of the phosphorylated serine residue.
Each of these peptides are suspended in phosphate-buffered saline at a concentration of approximately lμM. RBL-2H3 cells are maintained as monolayer cultures and harvested by trypsin treatment. Cells are transferred to 96-well culture plates at a density of 4.9 X 104 cells per well in 0.4ml Earle's modified Eagle's medium supplemented with 15%(v/v) fetal bovine serum. Cells are then grown to approximately 50% confluence and the media replaced with serum free Earle's modified Eagle's medium. A penetratin conjugated peptide solution (described supra) is then added to the media and the cells incubated for 30 minutes at 37°C. Cells are then extensively washed with PBS and Earle's modified Eagle's medium supplemented with 15%(v/v) fetal bovine serum added. Cells are then incubated at 37°C with 5% CO2.
Following transfection of the peptides those cells used in antigenic activation are sensitized by the addition of 75 ng/ml dinitrophenol (DNP)-specific IgE and incubated overnight at 37°C with 5% CO2. To measure the secretory response, cultures are also incubated overnight with [3H] 5-hydroxytryptamine ([3H] 5HT), (0.5μCi/ml), which becomes incorporated into the histamine containing granules.
Supernatant from the cell cultures is then collected to determine of the tritium content using a liquid scintillation counter. The release of [3H] 5HT into the medium is expressed as a percentage of the total cellular content of the amine as determined from lysed, unstimulated cultures or after the remaining amine within the cells has been released by lysis in l%(v/v) Triton X-100.
EXAMPLE 19 Determining a compound that modulates PKC-β activity
A short peptide comprising the amino acid sequence MNREVSSLKNKLRR (SEQ ID NO: 130) is incubated in the presence of various concentrations of a test compound (from, for example, the Pharmacopeia small molecule library, Pharmacopeia Drug Discovery Inc., NJ, USA) and 100 μM [γ-32P]ATP (100 - 200 c.p.m./pmol), and 10 μl recombinant PKC-βl and/or PKC-βll isoforms (Calbiochem, diluted approx. 1/100 in MOPS/Triton = 1 unit/μl). Lipids (5 mg/ml in chloroform:methanol (19:1 v/v)), are dried under nitrogen and sonicated into 100 mM MOPS (pH 7.5) 1% (v/v) Triton X- 100 until clear, before addition to the assay buffer. In addition of PKC isoforms and [γ- 3 P]ATP, the final assay medium (50 μl) contained 24 mM MOPS (pH 7.5), 0.04% (v/v) Triton X-100, 5 mM Mg(CH2COO) , 1 mM CaCl , 125 μg/ml phosphatidylserine and 2.5 g/ml dioctanoylglycerol. Incubations are performed at 30°C for lOmin. Reactions are started by addition of 10 μl of ATP-MgAcetate (ATP 500 μM) containing [γ-32P]ATP. Reactions are stopped by addition of 10 μl cold ATP (150 mM). After vortexing 40 μl of reaction medium was withdrawn and spotted onto P81 paper squares. These are dropped into H3PO4 (10ml cone, acid in 11), and then washed 5 times for 5 min in 3 % (v/v) H3PO4, and then once in 100% EtOH. After drying radioactivity is determined by Cerenkov counting. Control incubations are undertaken under similar conditions but using Histone HIIIS as a generic PKC substrate, or a phosphorylation site mutant peptide comprising the sequence MNREVSALKNKLRR (SEQ ID NO: 180).
A compound that reduces or enhances the level of radioactivity detected modulates the level of PKC-β activity. Molecules that suppress PKC-β activity are of particular interest for the treatment of, for example, diabetes and/or asthma and are selected for further analysis.
EXAMPLE 20 Mutagenic screening of a PKC-β phosphorylation site
The PKC-β phosphorylation site of myosin heavy chain IIA comprises a serine residue adjacent to the serine that is phosphorylated. This may lead to low levels of inadvertent phosphorylation of the PKC-β phosphorylation site by another PKC isozyme. To determine a peptide with higher specificity for PKC-β a mutagenesis protocol is used.
Peptides comprising an amino acid sequence set forth in any one of SEQ ID NOs: 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164 or 168 are synthetically produced.
Each peptide is incubated with 100 μM [γ-32P]ATP (100 - 200 c.ρ.m./pmol), and 10 μl recombinant PKCα or βl (Calbiochem, diluted approx. 1/100 in MOPS/Triton = 1 unit/μl). Lipids (5 mg/ml in chloroform:methanol (19:1 v/v)), are dried under nitrogen and sonicated into 100 mM MOPS (pH 7.5) 1% (v/v) Triton X-100 until clear, before addition to the assay buffer. In addition to PKC isoforms and [γ-3 P]ATP, the final assay medium (50 μl) contains 24 mM MOPS (pH 7.5), 0.04% (v/v) Triton X-100, 5 mM Mg(CH2COO)2, 1 mM CaCl2, 125 μg/ml phosphatidylserine and 2.5 μg/ml dioctanoylglycerol. Incubations are performed at 30°C for lOmin. Reactions are started by addition of 10 μl of ATP-Mg Acetate (ATP 500 μM) containing [γ-32P]ATP. Reactions are stopped by addition of 10 μl cold ATP (150 mM). After vortexing 40 μl of reaction medium is withdrawn and spotted onto P81 paper squares. These are dropped into H3PO4 (10ml cone, acid in 11ml), and then washed 5 times for 5 min in 3 % (v/v) H3PO , and then once in 100% EtOH. After drying radioactivity is determined by Cerenkov counting. Control incubations are undertaken under similar conditions but using Histone HIIIS as a generic PKC substrate, and a myosin heavy chain phosphorylation site mutant (MNREVSALKNKLRR, SEQ ID NO: 180) as a negative control. Also used as a control is a peptide comprising the amino acid sequence set forth in SEQ ID NO: 130.
By determining the level of phosphorylation by either PKCα or PKCβ a peptide that is phosphorylated more efficiently by PKCβ is determined. Peptides that are phosphorylated by PKC-β the then peptide comprising the amino acid sequence set forth in SEQ ID NO: 130 and less efficiently than PKC-α are selected for use in the assay described in Example 19.

Claims

We claim:
1. A method for determining protein kinase C-β (PKC-β) activity in a sample comprising contacting a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a variant thereof with a ligand that specifically binds to an amino acid residue within said phosphorylation site or a sample comprising said ligand for a time and under conditions sufficient for binding to occur and then detecting the binding wherein the binding is indicative of PKC-β activity.
The method according to claim 1 wherein the PKC-β is a PKC-βl or PKC-βll.
3. The method according to claim 1 or claim 2 wherein the PKC-β comprises an amino acid sequence at least 80% identical to an amino acid sequence set forth in SEQ ID NO: 169 or SEQ ID NO: 171.
4. The method according to claim 1 wherein the peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 142.
5. The method according to claim 1 wherein the variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 142 to 167.
6. The method according to claim 1 wherein the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide is a myosin heavy chain IIA polypeptide or a fragment thereof comprising an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 142.
7. The method according to claim 6 wherein the peptide, polypeptide or protein comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.
8. The method according to any one of claims 1 or 4 to 6 wherein the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or variant thereof is capable of being phosphorylated by PKC-β at a site equivalent to amino acid position 9 of SEQ ID NO: 9.
9. The method according to claim 1 wherein the peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide is in the sample being assayed and the ligand is an antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide.
10. The method according to claim 9 wherein the level of PKC-β activity is determined by performing a process comprising: (i) contacting a sample comprising the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with an antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody-antigen complex to form; and (ii) determining the level of said complex, wherein the level of said complex is indicative of PKC-β activity.
11. The method according to claim 9 or 10 wherein the antibody specifically binds to a peptide, polypeptide or protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139 and SEQ ID NO: 141.
12. The method according to any one of claims 9 to 11 wherein the antibody specifically binds to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10.
13. The method according to any one of claims 9 to 12 wherein the antibody is capable of specifically binding a myosin heavy chain polypeptide that is phosphorylated at the serine residue at an amino acid equivalent to amino acid position 1916 of SEQ ID NO: 2 or SEQ ID NO: 6 or amino acid position 1917 of SEQ ID NO: 4 or amino acid position 1915 of SEQ ID NO: 8.
14. The method according to any one of claim 9 to 13 wherein the antibody is additionally capable of selectively binding to a mimetic of a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide.
15. The method according to claim 14 wherein the mimetic of a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide is a peptide, polypeptide or protein comprising an amino acid sequence set forth in SEQ ID NO: 178 or SEQ ID NO: 179.
16. The method according to any one of claims 9 to 14 wherein the antibody is selected from the group consisting of a monoclonal antibody, a fragment of a monoclonal antibody, a recombinant antibody and a polyclonal antibody.
17. The method according to claim 16 wherein the antibody is a polyclonal antibody.
18. The method according to claim 10 wherein the level of the complex is determined by performing a process comprising contacting the complex with another antibody capable of binding to said complex for a time and under conditions sufficient for another antibody-antigen complex to form and detecting the level of the other complex, wherein the level of said other complex is indicative of PKC-β activity.
19. The method according to claim 9 or 18 wherein the antibody and/or the other antibody is labelled with a detectable marker.
20. The method according to claim 19 wherein the detectable marker is selected from the group consisting of an enzyme label, a radiolabel and a fluorescent label.
21. The method according to claim 10 wherein determining the level of the complex comprises comparing the level of the complex determined in the sample to the level determined using a standard comprising a known level of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide, wherein a similar level of the complex determined in the sample and the standard indicates the level of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide present in the sample.
22. The method according to claim 20 wherein the standard is produced by performing a process comprising: (i) contacting a known level of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide with the antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody-antigen complex to form; and (ii) determining the level of the complex, wherein the level of the complex determined is indicative of the known level of the peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide.
23. The method according to claim 9 or 10 comprising: (i) contacting a sample comprising a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with a first antibody that binds to said peptide, polypeptide or protein at a site other than the PKC-β phosphorylation site for a time and under conditions sufficient for the antibody to bind to the peptide, polypeptide or protein; (ii) contacting the bound peptide, polypeptide or protein at (i) with a second antibody that specifically binds to the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody- antigen complex to form, wherein either the first antibody or the second antibody specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide ; and (ii) determining the level of said complex, wherein the level of said complex is indicative of PKC-β activity.
24. The method according to claim 23 wherein the first antibody specifically binds to a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide.
25. The method according to claim 23 wherein the second antibody specifically binds to a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide.
26. The method according to any one of claims 23 to 25 wherein the first antibody is/was previously immobilized on a solid support, matrix or resin.
27. The method according to claim 23 wherein the level of the complex is determined by performing a process comprising comparing the level of complex determined in the sample with the level of complex determined in a standard, wherein the level of the complex determined in the sample and in the standard indicates the level of phosphorylated peptide, polypeptide or protein in the sample and PKC-β activity.
28. The method according to claim 27 wherein the standard is produced by performing a process comprising: (i) contacting a first antibody that binds to a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide at a site other than the PKC-β phosphorylation site with (a) a known amount of a peptide, polypeptide or protein comprising an unphosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof and (b) a known amount of a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof for a time and under conditions sufficient for the antibody to bind to the peptide, polypeptide or protein; (ii) contacting the bound peptide, polypeptide or protein at (ii) with a second antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody-antigen complex to form; and (iii) determining the level of complex relative to the total amount of level of peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof, thereby determining the level of complex that is indicative of the known amount of the peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide.
29. The method according to claim 28 wherein the peptide, polypeptide or protein comprising an unphosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof and the peptide, polypeptide or protein comprising an phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof are labelled with a detectable marker.
30. The method according to claim 29 wherein the detectable marker is a fluorescent marker.
31. The method according to claim 9 or 10 wherein the level of PKC-β activity is determined by performing a process comprising: (i) contacting a sample comprising a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with an antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide for a time and under conditions sufficient for an antibody-antigen complex to form and contacting said antibody with a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof labelled with a detectable marker; and (ii) determining the level of detectable marker bound to the antibody, wherein the level of detectable marker bound to the antibody is indicative of the level of PKC-β activity.
32. The method according to claim 31 wherein the level of detectable marker bound to the antibody is inversely proportional to the level of PKC-β activity.
33. The method according to claim 31 wherein the antibody is/was previously immobilized on a solid support, matrix or resin.
34. The method according to claim 31 or claim 33 additionally comprising washing the sample to remove any non-specifically bound or unbound protein prior to determining the level of detectable marker bound to the antibody.
35. The method according to claim 31 wherein the level of detectable marker bound to the antibody is determined using a process comprising comparing the level of detectable marker bound to the antibody in the sample to a standard, wherein a comparable level of detectable marker bound to the antibody in the sample and the standard indicates the level of peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide in the sample and PKC-β activity.
36. The method according to claim 35 wherein the standard is produced using a method comprising: (i) contacting the antibody that specifically binds to a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide with (a) a known amount a labelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide; and (b) a known amount of an unlabelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide or a mimetic thereof for a time and under conditions sufficient for an antibody-antigen complex to form; and (ii) determining the level of detectable marker bound to the antibody, wherein the level of labelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide bound to the antibody is indicative of the level of unlabelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide in the standard.
37. The method according to claim 36 wherein the level of labelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide bound to the antibody is inversely proportional to the level of unlabelled peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide in the standard.
38. The method according to claim 36 or 37 wherein the label is a fluorescent label.
39. The method according to claim 1 wherein the ligand is a PKC-β enzyme in an extract or cellular sample being assayed.
40. The method according to claim 39 wherein the binding between the phosphorylation site and the ligand is determined by performing a process comprising: (i) contacting a peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide with a PKC-β or a sample comprising or suspected of comprising a PKC-β under conditions sufficient for the PKC-β to phosphorylate said PKC-β phosphorylation site; and (ii) determining the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide, wherein the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide is indicative of a PKC-β activity.
41. The method according to claim 40 wherein the peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138 and SEQ ID NO: 140.
42. The method according to claim 41 or 42 wherein the peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide comprises an amino acid set forth in SEQ ID NO: 130.
43. The method according to claim 40 wherein the peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide is contacted with the PKC-β or sample comprising or suspected of comprising a PKC-β in the presence of a labelled phosphate.
44. The method according to claim 43 wherein the label is selected from the group consisting of a radioactive label, a fluorescent label, an enzymatic label and a colorimetric label.
45. The method according to claim 43 or 44 wherein the labelled phosphate is γ P- ATP.
46. The method according to any one of claims 42 to 45 wherein the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide is determined by determining the level of labelled phosphate incorporated into the PKC-β phosphorylation site of a myosin heavy chain polypeptide.
47. The method according to claim 39 wherein the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide is determined by performing a process comprising comparing the level of detectable marker bound to the antibody in the sample to a standard.
48. The method according to claim 47 wherein the standard is produced by performing a process comprising: (i) incubating a known amount of the peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide in the presence of a known amount of a PKC-β for a time and under conditions sufficient for phosphorylation of the PKC-β phosphorylation site to occur; and (ii) determining the level of phosphorylation of the PKC-β phosphorylation site of a myosin heavy chain polypeptide, wherein the level of phosphorylation in the standard is indicative of the activity of the PKC-β in the sample.
49. The method according to claim 1 wherein the ligand is a compound that specifically binds to a phosphorylated amino acid.
50. The method according to claim 49 wherein the level of PKC-β in a sample is determined by performing a process comprising: (i) isolating a peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide from the sample; (ii) contacting the isolated peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide with a compound that binds to a phosphorylated amino acid for a time and under conditions sufficient for binding to occur; and (iii) determining the level of bound compound wherein the level of bound compound is indicative of PKC-β activity.
51. The method of claim 50 wherein the peptide, polypeptide or protein comprising the PKC-β phosphorylation site of a myosin heavy chain polypeptide is isolated by performing a process comprising contacting a sample comprising the peptide, polypeptide or protein with an antibody or ligand that specifically binds said peptide, polypeptide or protein and isolating antibody with the peptide, polypeptide or protein bound thereto.
52. The method according to claim 51 wherein the antibody is/was previously immobilized on a solid support, matrix or resin.
53. The method according to claim 50 wherein the compound that binds to a phosphopeptide fluoresces when in contact with a phosphopeptide.
54. The method according to claim 50 wherein the compound that specifically binds to the phosphorylated amino acid is an antibody that specifically binds to a phosphorylated serine.
55. The method according to claims 1 wherein the method is performed using a sample comprising a cell or an extract thereof or a mixture thereof.
56. The method according to claim 55 wherein the cell or cell extract is or has been isolated from a subject.
57. The method according to claim 55 or claim 56 wherein the cell is a wild-type cell or the cell extract is from a wild-type cell.
58. The method according to claim 55 or 56 wherein the cell has been modified at the genetic level to induce a diabetes-like phenotype.
59. The method according to claim 55 or 56 wherein the cell has been contacted with a compound to induce a diabetes-like phenotype.
60. The method according to claim 1 wherein the method is performed using a sample from an animal subject.
61. The method according to claim 60 wherein the animal subject is a wild-type animal.
62. The method according to claim 60 wherein the animal subject has been modified at the genetic level to induce a diabetes-type phenotype.
63. The method according to claim 60 wherein the animal has been administered a compound to induce a diabetes-type phenotype.
64. The method according to claim 1 wherein the method is performed using a sample from a human subject.
65. The method according to claim 64 wherein the sample is from a subject suspected of suffering from a condition associated with aberrant PKC-β activity.
66. The method according to claim 65 wherein the aberrant PC-β activity is enhanced PKC-β activity and the condition is selected from the group consisting of asthma, hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, hypertension, cancer, colorectal cancer and immunodeficiency.
67. The method according to claim 65 wherein the aberrant PC-β activity is reduced PKC-β activity and the condition is selected from the group consisting of hypoglycemia, melanoma, an inflammatory skin disorder and a hyperplastic skin disorder.
68. The method according to claim 1 wherein the method is performed using a sample from a cell free system.
69. The method according to any one of claims 1 or 55 to 68 additionally comprising providing the sample.
70. A method for diagnosing or prognosing a condition associated with aberrant PKC-β activity in a subject comprising: (i) determining the level of PKC-β activity in a sample from the subject using the method according to any one of claims 1 to 69; and (ii) determining the level of PKC-β activity in a control sample using the method according to any one of claims 1 to 69, wherein increased or decreased PKC-β activity at (i) compared to (ii) indicates that the subject suffers from a condition associated with aberrant PKC-β activity.
71. The method according to claim 70 wherein increased PKC-β activity is determined at (i) compared to (ii) and the condition is selected from the group consisting of hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, hypertension, cancer, colorectal cancer and immunodeficiency.
72. The method according to claim 58 wherein decreased PKC-β activity is determined at (i) compared to (ii) and the condition is selected from the group consisting of hypoglycemia, melanoma, an inflammatory skin disorder and a hyperplastic skin disorder.
73. The method according to claim 70 wherein the subject is in need of treatment.
74. The method according to claim 70 wherein the subject has received or is receiving treatment.
75. Use of an antibody that specifically binds to a peptide, polypeptide or protein comprising a phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide in the manufacture of a diagnostic or prognostic for the diagnosis or prognosis of a condition selected from the group consisting of hypoglycaemia, hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, aberrant vitamin D function, hypertension, immunodeficiency, melanoma, an inflammatory skin disorder and a hyperplastic skin disorder.
76. The use according to claim 63 wherein the antibody specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139 and SEQ ID NO: 141.
77. The use according to claim 75 or 76 wherein the antibody specifically binds to the amino acid sequence set forth in SEQ ID NO: 10.
78. A method for determining a compound that modulates PKC-β activity comprising: (i) determining the level of PKC-β activity in a sample in the presence of a candidate compound by performing the method according to any one of claims 1 to 69 in a sample ; and (ii) determining the level of PKC-β activity in a sample in the absence of the candidate compound by performing the method according to any one of claims 1 to 69 in a sample, wherein an increase or decrease in PKC-β activity at (i) compared to (ii) indicates that the candidate compound is a modulator of PKC-β activity.
79. The method according to claim 78 wherein the modulator increases PKC-β activity.
80. The method according to claim 78 wherein the modulator decreases PKC-β activity.
81. A process for determining a compound that modulates PKC-β activity, said process comprising: (i) determining the level of PKC-β activity in a sample in the presence of a candidate compound by performing the method according to any one of claims 1 to 69; and (ii) determining the level of PKC-β activity in a sample in the absence of a candidate compound by performing the method according to any one of claims 1 to 69, wherein an increase or decrease in PKC-β activity at (i) compared to (ii) indicates that the candidate compound is a modulator of PKC-β activity, (iii) optionally, determining the structure of the candidate compound; (iv) optionally, providing the name or structure of the candidate compound; and (v) optionally, providing the candidate compound.
82. A process for determining a candidate compound for the treatment of a condition associated with aberrant PKC-β activity comprising: (i) determining the level of PKC-β activity in a sample in the presence of a candidate compound by performing the method according to any one of claims 1 to 69; and (ii) determining the level of PKC-β activity in a sample in the absence of a candidate compound by performing the method according to any one of claims 1 to 69, wherein an increase or decrease in PKC-β activity at (i) compared to (ii) indicates that the candidate compound is a modulator of PKC-β activity and is a candidate compound for the treatment of a condition associated with aberrant PKC-β activity; (iii) optionally, determining the structure of the candidate compound; (iv) optionally, providing the name or structure of the candidate compound; and (v) optionally, providing, the candidate compound
83. A process for manufacturing a medicament for the treatment of a condition associated with aberrant PKC-β activity comprising: (i) determining a candidate compound for the treatment of a condition associated with aberrant PKC- activity using a process comprising: (a) determining the level of PKC-β activity in a sample in the presence of a candidate compound by performing the method according to any one of claims 1 to 69; and (b) determining the level of PKC-β activity in a sample in the absence of a candidate compound by performing the method according to any one of claims 1 to 69, wherein an increase or decrease in PKC-β activity at (i) compared to (ii) indicates that the candidate compound is a modulator of PKC-β activity and is a candidate compound for the treatment of a condition associated with aberrant PKC-β activity; (ii) optionally, isolating the candidate compound; (iii) optionally, providing the name or structure of the candidate compound; (iv) optionally, providing the candidate compound; and (v) using the candidate compound in the manufacture of a medicament for the treatment of a condition associated with aberrant PKC-β activity.
84. A kit for determining PKC-β activity comprising an antibody capable of specifically binding to a peptide, polypeptide or protein comprising phosphorylated PKC-β phosphorylation site of a myosin heavy chain polypeptide and a detection means.
85. The kit according to claim 84 wherein the antibody specifically binds to a peptide, polypeptide or protein comprising the amino acid sequence set forth in SEQ ID NO: 10.
86. The kit according to claim 84 or 85 additionally comprising peptide, polypeptide or protein comprising a PKC-β phosphorylation site of a myosin heavy chain polypeptide or a variant thereof.
87. The kit according to claim 86 wherein the peptide, polypeptide or protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 167.
88. The kit according to any one of claims 84 to 87 additionally comprising a PKC- β.
89. A method for modulating PKC-β activity in a cell or a subject comprising administering an effective amount of a peptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 172, 174 or 177 to said cell or subject.
90. A method of the treatment of a condition in a subject associated with aberrant PKC-β activity comprising administering to the subject an effective amount of a peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 172, 174 or 177.
91. The method according to claim 90, wherein the condition is selected from the group consisting of hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, hypertension, cancer, colon cancer and immunodeficiency.
92. The method according to claim 90 wherein the condition is associated with histamine secretion.
93. The method according to claim 92 wherein the condition is asthma.
94. Use of a of a peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 172, 174 or 177 in the manufacture of a medicament for the treatment of a condition associated with aberrant PKC-β activity.
95. The use according to claim 94, wherein the condition is selected from the group consisting of hyperglycemia, diabetes, diabetic retinopathy, diabetic nephropathy, macular edema, hypertension, cancer, colon cancer and immunodeficiency.
96. The use according to claim 94 wherein the condition is associated with histamine secretion.
97. The use according to claim 96 wherein the condition is asthma.
98. An isolated peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 172, 174 or 177 .
99. A composition comprising a peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 172, 174 or 177.
100. An isolated peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 167.
101. A peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 167 when used to determine the level of PKC-β activity in a sample. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 when used to determine the level of PKC-β activity in a sample.
PCT/AU2004/001573 2003-11-14 2004-11-15 Assay for pkc-beta activation of phosphorylation of myosin heavy chain polypeptide WO2005047531A1 (en)

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KR101229807B1 (en) 2009-06-26 2013-02-05 광주과학기술원 Novel Drug Target against Hypersensitivity Immune or Inflammatory Disease

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