Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases

Definitions

• the invention relates to the use of the human Protein Kinase C ⁇ (PKC-eta) isoform, in methods for identification of pharmaceutically useful agents, in particular agents useful for the treatment of type 2 diabetes.
• PKC-eta Protein Kinase C ⁇
• Insulin primarily regulates the direction of metabolism, shifting many processes toward the storage of substrates and away from their degradation [1,2]. Insulin acts to increase the transport of glucose and amino acids as well as key minerals such as potassium, magnesium, and phosphate from the blood into cells. It also regulates a variety of enzymatic reactions within the cells, all of which have a common overall direction, namely the synthesis of large molecules from small units.
• a deficiency in the action of insulin causes severe impairment in (i) the storage of glucose in the form of glycogen and the oxidation of glucose for energy; (ii) the synthesis and storage of fat from fatty acids and their precursors and the completion of fatty-acid oxidation; and (iii) the synthesis of proteins from amino acids.
• Type 1 is insulin-dependent diabetes mellitus (IDDM), for which insulin injection is required; it was formerly referred to as juvenile onset diabetes. In this type, insulin is not secreted by the pancreas and hence must be taken by injection.
• IDDM insulin-dependent diabetes mellitus
• Type 2 diabetes non-insulin-dependent diabetes mellitus (NIDDM)
• NIDDM non-insulin-dependent diabetes mellitus
• Type 2 diabetes is characterized clinically by hyperglycemia and insulin resistance and is commonly associated with obesity.
• Type 2 diabetes is a heterogeneous group of disorders in which hyperglycemia results from both an impaired insulin secretory response to glucose and decreased insulin effectiveness in stimulating glucose uptake by skeletal muscle and in restraining hepatic glucose production (insulin resistance).
• insulin resistance insulin resistance
• patients Before diabetes develops, patients generally lose the early insulin secretory response to glucose and may secrete relatively large amounts of pro-insulin.
• fasting plasma insulin levels may be normal or even increased in type 2 diabetes patients, glucose-stimulated insulin secretion is clearly decreased. The decreased insulin levels reduce insulin-mediated glucose uptake and fail to restrain hepatic glucose production.
• Glucose homeostasis depends upon a balance between glucose production by the liver and glucose utilization by insulin-dependent tissues, such as fat and muscle, and insulin-independent tissues, such as brain and kidney.
• insulin-dependent tissues such as fat and muscle
• insulin-independent tissues such as brain and kidney.
• type 2 diabetes the entry of glucose into fat and muscle is reduced and glucose production in the liver is increased, due to insulin resistance in the tissues.
• RTKs The receptor tyrosine kinases
• the ligands for RTKs are peptide/protein hormones including nerve growth factor (NGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin. Binding of a ligand to an RTK stimulates the receptor's intrinsic protein- tyrosine kinase activity, which subsequently stimulates a signal-transduction cascade leading to changes in cellular physiology and patterns of gene expression.
• RTK signaling pathways have a wide spectrum of functions including regulation of cell proliferation and differentiation, promotion of cell survival, and modulation of cellular metabolism.
• Protein phosphorylation is one of the most versatile posttranslational modifications used in eukaryotic cells and plays a crucial role in the continuous remodeling of different transcriptional regulators.
• the protein kinase C (PKC) family of serine-threonine kinases encompasses mne different genes encoding isozymes that have been shown to transduce a myriad of signals mediated by phospholipid hydrolysis as a consequence of the activation of G protein-coupled receptors, tyrosine kinase receptors, and non-receptor tyrosine kinases [6].
• Lipid signaling molecules can be derived from free fatty acids, and include diacylglycerol, which activates isozymes of the protein kinase C (PKC) family, and ceramide, which has several effectors including PKCs and a protein phosphatase.
• PKC protein kinase C
• lipid availability can increase flux through the hexosamine biosynthesis pathway which can also lead to activation of PKC as well as protein glycosylation and modulation of gene expression.
• the mechanisms giving rise to decreased insulin signaling include serine/threonine phosphorylation of insulin receptor substrate- 1, but also direct inhibition of components such as PKB.
• lipids can inhibit glucose disposal by causing interference with insulin signal transduction, and most likely by more than one pathway depending on the prevalent species of fatty acids.
• Fig. 1 is a graph depicting the effect of PKC-eta over-expression in adipocytes
• FIG. 2 is a graph depicting the effect of PKC-eta on glucose uptake, when over- expressed in (A) non-differentiated L6 muscle cells; and (B) differentiated adipocytes 3T3-L1. Control cells (ctrl) were transfected with an empty plasmid vector. Grey staples indicate non-stimulated cells and white staples indicate insulin-stimulated cells.
• Figure 3 is a graph depicting the effect of PKC-eta over-expression on insulin receptor tyrosine-phosphorylation in CHO-IR cells.
• this invention provides a method for identifying an agent useful for the treatment of a medical condition relating to insulin resistance, said method comprising the steps: (i) contacting a candidate agent with a mammalian PKC-eta polypeptide; and (ii) determining whether said candidate agent decreases or inhibits the biological activities of the polypeptide, such decrease or inhibition being indicative for an agent useful for the treatment of a medical condition relating to insulin resistance.
• the method for identifying an agent useful for the treatment of a medical condition relating to insulin resistance comprises the steps: (i) contacting a candidate agent with a nucleic acid molecule encoding a mammalian PKC-eta polypeptide; and (ii) determining whether said candidate agent decreases or inhibits the expression of the said nucleic acid molecule, such decrease or inhibition being indicative for an agent useful for the treatment of a medical condition relating to insulin resistance.
• the invention features a method for identifying an agent that increases glucose uptake by a cell, the method comprising: (i) contacting a cell with a candidate agent that inhibits an activity of a mammalian (e.g., human) PKC-eta polypeptide; (ii) measuring glucose uptake by the cell in the presence of the candidate agent; and (iii) determining whether the candidate agent increases glucose uptake by the cell.
• the candidate agent can optionally bind to the mammalian PKC-eta polypeptide.
• the invention features a method for identifying an agent that increases glucose uptake by a cell, the method comprising: (i) contacting a cell with a candidate agent that inhibits expression of a nucleic acid encoding a mammalian (e.g., human) PKC-eta polypeptide; (ii) measuring glucose uptake by the cell in the presence of the candidate agent; and (iii) determining whether the candidate agent increases glucose uptake by the cell.
• the candidate agent can optionally bind to the mammalian PKC-eta nucleic acid.
• the medical condition relating to insulin resistance could in particular be associated with reduced glucose uptake and could more specifically be type 2 diabetes.
• the mammalian PKC-eta polypeptide is optionally a human PKC-eta polypeptide.
• a human PKC-eta polypeptide comprises, consists essentially of, or consists of, an amino acid sequence of SEQ T-D NO:3; SEQ ID NO:12; or SEQ LD NO:13.
• Candidate agents that can be used in the methods described herein include, for example, polypeptides, peptides, antibodies or antibody fragments, non-peptide compounds, carbohydrates, small molecules, lipids, single or double stranded DNA, single or double stranded RNA, antisense nucleic acid molecules, and ribozymes.
• An identification method can additionally include a step of comparing glucose uptake by a cell in the presence of the candidate agent with glucose uptake by a cell in the absence of the candidate agent.
• appropriate host cells can be transformed with a vector having a reporter gene under the control of the PKC-eta polypeptide.
• the expression of the reporter gene can be measured in the presence or absence of an agent with known activity (i.e. a standard agent) or putative activity (i.e. a "test, agent” or “candidate agent”).
• a change in the level of expression of the reporter gene in the presence of the test agent is compared with that effected by the standard agent. In this way, active agents are identified and their relative potency in this assay determined.
• a transfection assay can be a particularly useful screening assay for identifying an effective agent.
• a nucleic acid containing a gene such as a reporter gene that is operably linked to a PKC-eta promoter, or an active fragment thereof, is transfected into the desired cell type.
• a test level of reporter gene expression is assayed in the presence of a candidate agent and compared to a control level of expression.
• An effective agent is identified as an agent that results in a test level of expression that is different than a control level of reporter gene expression, which is the level of expression determined in the absence of the agent.
• reporter gene means a gene encoding a gene product that can be identified using simple, inexpensive methods or reagents and that can be operably linked to PKC-eta or an active fragment thereof.
• Reporter genes such as, for example, a luciferase, ⁇ -galactosidase, alkaline phosphatase, or green fluorescent protein reporter gene, can be used to determine transcriptional activity in screening assays according to the invention (see, for example, Goeddel (ed.), Methods Enzymol., Vol. 185, San Diego: Academic Press, Inc. (1990); see also Sambrook, supra).
• the invention provides a method for stimulating glucose uptake in a subject (e.g., a human host), comprising administering to a subject in need of such treatment an amount of an inhibitor (e.g., a selective inhibitor) of PKC-eta effective to increase glucose uptake in the subject.
• a subject e.g., a human host
• an inhibitor e.g., a selective inhibitor
• the subject may have a condition relating to insulin resistance and/or a condition relating to reduced glucose uptake, such as type 2 diabetes.
• the subject has been diagnosed as having such a particular condition.
• the method can include an additional step of detecting an increase in glucose uptake in the subject after administering the inhibitor to the subject.
• the invention further includes a method, which can be carried out in vivo, ex vivo, or in vitro (i.e., outside the body of the host) for increasing glucose uptake in a mammalian cell (e.g., a muscle cell or an adipocyte), the method comprising contacting a mammalian cell with an amount of an inhibitor (e.g., a selective inhibitor) of PKC-eta effective to increase glucose uptake by the cell.
• an inhibitor e.g., a selective inhibitor
• the method can include an additional step of detecting an increase in glucose uptake by the cell after the contacting of the cell with the inhibitor.
• this invention provides a method for the treatment of a medical condition relating to reduced glucose uptake, in particular type 2 diabetes, comprising administering to a subject an effective amount of an agent identified by the method according to the invention for identification of agents useful for such treatment.
• a method of treatment includes: (i) selecting an individual diagnosed as having a medical condition characterized by insulin resistance; and (ii) administering to the individual an amount of an inhibitor of PKC-eta effective to reduce insulin resistance in the individual.
• the method can include an additional step of detecting a reduction in insulin resistance in the individual after the administration of the inhibitor.
• the medical condition can be characterized by reduced glucose uptake and the method can include detecting a reduction in blood glucose levels in the individual after the administration of the inhibitor.
• An exemplary condition that can be treated by such as method is diabetes, e.g., type II diabetes.
• the invention features an isolated nucleic acid molecule containing a nucleotide sequence that is at least 60% identical to the nucleotide sequence shown in SEQ LD NO:6. More preferably, the nucleotide sequence is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identical to the nucleotide sequence shown in SEQ LD NO:6. In the case of a nucleotide sequence that is longer than or equivalent in length to the reference sequence, SEQ LD NO:6, the comparison is made with the full length of the reference sequence.
• nucleotide sequence is shorter that the reference sequence, SEQ LD NO:6, the comparison is made to a segment of the reference sequence of the same length (excluding any loop required by the homology calculation).
• the nucleotide sequence encodes a polypeptide having PKC-eta activity (e.g., serine- threonine kinase activity).
• the nucleotide sequence is identical to SEQ ID NO:6.
• the invention features an isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that is at least about 60% identical to a sequence shown as SEQ ID NO: 13 or a fragment thereof.
• the amino acid sequence is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical to SEQ LD NO: 13 and has a PKC-eta activity (e.g., serine-threonine kinase activity).
• the amino acid sequence can be identical to the sequence of SEQ LD NO: 13.
• nucleic acid molecule comprising a nucleotide sequence that hybridizes under high stringency conditions to the nucleotide sequence of SEQ ID NO:6, the complete complement of SEQ LD NO:6, or a segment thereof as described herein.
• High stringency conditions refers to hybridization in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C.
• the nucleotide sequence encodes a polypeptide having PKC-eta activity (e.g., serine-threonine kinase activity).
• nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising at least 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or more contiguous amino acid residues of SEQ ID NO: 13.
• the polypeptide comprises an immunogenic fragment of at least 20 amino acids of SEQ LD NO: 13.
• the nucleotide sequence encodes a polypeptide having PKC-eta activity (e.g., serine- threonine kinase activity).
• an "isolated nucleic acid” is a nucleic acid, the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three genes.
• the term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene,
• GappedBLAST is utilized as described in Altschul et al (Nucleic Acids Res. 25:3389-3402, 1997).
• BLAST and GappedBLAST programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention.
• the invention features a substantially pure polypeptide containing an amino acid sequence shown as SEQ ID NO: 13.
• the invention also includes a polypeptide, or fragment thereof, that differs from the corresponding sequence shown as SEQ LD NO: 13. The differences are, preferably, differences or changes at a non-essential residue or a conservative substitution.
• the polypeptide includes an amino acid sequence at least about 60% identical to a sequence shown as SEQ ID NO: 13, or a fragment thereof.
• the amino acid sequence is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical to SEQ LD NO: 13 and has a PKC-eta activity (e.g., serine-threonine kinase activity).
• the amino acid sequence can be identical to SEQ ID NO: 13.
• Preferred polypeptide fragments of the invention are at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, or more, of the length of the sequence shown as SEQ ID NO:13 and have a PKC-eta activity (e.g., serine-threonine kinase activity).
• the fragment can be merely an immunogenic fragment, e.g., a fragment that can be used to raise monoclonal and/or polyclonal antibodies that specifically bind to a polypeptide of the sequence of SEQ LD NO: 13.
• the invention encompasses polypeptides carrying modifications such as substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the biological activities of a PKC-eta.
• a substantially pure polypeptide comprising at least 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or more contiguous amino acid residues of SEQ ID NO: 13.
• the polypeptide comprises an immunogenic fragment of at least 20 amino acids of SEQ ID NO: 13.
• the polypeptide has PKC-eta activity (e.g., serine-threonine kinase activity).
• substantially pure as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules.
• the substantially pure polypeptide is at least 75%, 80, 85, 95, or 99% pure by dry weight. Purity can be measured by any appropriate standard method known in the art, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
• the present invention also relates to vectors comprising the nucleic acid molecules of the invention, as well as host cells transformed with such vectors.
• Any of the nucleic acid molecules of the invention may be joined to a vector, which generally includes a selectable marker and an origin of replication for propagation in a host cell.
• vectors for the expression of a polypeptide are preferred.
• Such expression vectors include DNA encoding a polypeptide described herein, operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect gene.
• regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences, which control transcription and translation.
• Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding a polypeptide.
• a promoter nucleotide sequence is operably linked to a PKC-eta DNA sequence if the promoter nucleotide sequence directs the transcription of the PKC-eta sequence.
• the vector may be any vector, which conveniently may be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
• the vector may be an autonomously replicating vector, e.g., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication (examples of such a vector are a plasmid, phage, cosmid, mini-chromosome or virus).
• the vector may be one which, when introduced in a host cell, is integrated in the host cell genome and replicated together with the chromosome(s) into which it has been integrated. Examples of suitable vectors are a bacterial expression vector and a yeast expression vector.
• the vector of the invention may carry any of the DNA molecules of the invention as defined above.
• a suitable host cell can be a prokaryotic cell, a unicellular eukaryotic cell, or a cell derived from a multicellular organism.
• the host cell can thus, e.g., be a bacterial cell such as an E. coli cell, a cell from a yeast such as Saccharomyces cervisiae or Pichiapastoris, an insect cell, or a mammalian cell, such as HEK293, CHO or an equivalent.
• the methods employed to effect introduction of the vector into the host cell are standard methods well known to a person familiar with recombinant DNA methods. Included in the invention is a process for production of a polypeptide described herein, which comprises culturing a host cell as described above under conditions whereby said polypeptide is produced, and optionally recovering said polypeptide, using standard biochemical procedures.
• a further aspect of the invention is a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with at least a part of the nucleic acid molecule according to the invention, said part having a sequence shown in SEQ ID NO:6.
• the invention also provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to at least a part of the nucleic acid molecule according to the invention, said part having a sequence shown in SEQ ID NO:6. Fragments of the nucleic acid molecules described herein, as well as polynucleotides capable of hybridizing to such nucleic acid molecules may be used as a probe or as primers in a polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• Such probes may be used, e.g., to detect the presence of nucleic acids coding for PKC-eta in in vitro assays, as well as in Southern and Northern blots. Cell types expressing PKC-eta may also be identified by the use of such probes. Such procedures are well known, and the skilled artisan will be able to choose a probe of a length suitable to the particular application. For PCR, 5' and 3' primers corresponding to the termini of a desired PKC-eta nucleic acid molecule are employed to isolate and amplify that sequence using conventional techniques.
• polypeptides of the present invention may also be used to raise polyclonal and monoclonal antibodies, which are useful in diagnostic assays for detecting PKC-eta polypeptide expression.
• Such antibodies may be prepared by conventional techniques. See, for example, Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980).
• standard protocols and “standard procedures”, when used in the context of molecular biology techniques, are to be understood as protocols and procedures found in an ordinary laboratory manual such as: Current Protocols in Molecular Biology, editors F. Ausubel et al, John Wiley and Sons, Inc. 1994, or Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989.
• EXAMPLE 1 Identification of kin-13 and PKC-eta.
• RNAi double-stranded RNA
• the Caenorhabditis elegans PKC gene kin-13 was identified by RNAi to suppress the insulin receptor loss of function phenotype daf-2, which is the dauer state, a sort of hibernation in the worm.
• the kin-13 protein (GenBank accession No. P34885;
• SEQ ID NO: 1 was used in a TBLASTN search
• PKC-eta (PKC ⁇ ; GenBank accession No.
• the kin-13 protein was determined to be -58% identical with the human
• PKCepsilon isoform and 55% identical with the eta isoform.
• the PKC-eta gene was cloned using sequence information from the public databases.
• cDNA from human lung was used (Invitrogen; Cat. # D8090-1) with the primers BEKA234 (SEQ ID NO: 4) and BEKA235 (SEQ ID NO: 5).
• PCR was performed using the Advantage 2 PCR Enzyme System (Clontech;
• Plasmid DNA from 5 clones was obtained by using the QIAprep Spin Miniprep Kit (QIAGEN; Cat. # 27104). Sequencing was performed using the ABI PrismBigDye Terminator Cycle Sequencing Ready Reaction Kit, on Applied Biosystems Model ABI 377 XL/96 DNA sequencing system.
• a correct clone was constructed by a three-fragment ligation where the 3'-end from clone #3 was joined to the 5'-end from clone #10, by the use of an internal BglR site. 1 ⁇ g of clone #3 was digested with Notl andHmdIII, 1.3 ⁇ g of clone #3 was digested with Notl andRg/II, and 1.2 ⁇ g of clone #10 was digested with BglR and H dIII. Half of the digestions were loaded on a 1.2% E-Gel.
• a band of approximately 3800 bp was cut out from the N tl- H dlLT digestion, and a band of approximately 650 bp (fragment B) was cut out from the Notl-Bg LI digestion, and a band of approximately 1500 bp (fragment C) was cut out from the Hwdlll-Bglll digestion.
• the fragments were purified using QIAquick Gel Extraction Kit, (Qiagen; Cat # 28704) and subsequent elution in 50 ⁇ l ⁇ 2 O.
• Ligation was performed using Ready-To-Go T4 D ⁇ A ligase (Amersham Pharmacia Biotech; Cat # 27-0361-01). 2 ⁇ l of fragment A was mixed with 6 ⁇ l of fragment B and 6 ⁇ l of fragment C. The ligation mix was transformed into One Shot chemically competent Top 10 E. coli cells (Invitrogen), and plasmid D ⁇ A was obtained as above.
• the resulting plasmid was designated pMB1549, and the insert (SEQ LD ⁇ OS: 6 and 7) was verified by control digestions using the restriction enzymes BamH ⁇ and H fl, which give unique patterns for clone #3, clone #10 and pMB1549.
• PKC-eta for mammalian expression was made by using GatewayTM Cloning Technology (Life Technologies). Gateway compatible primers were designed and PCR was performed using pMB1549 as a template. The PCR was performed in 50 ⁇ l using Taq D ⁇ A Polymerase, Roche (Cat. # 1 435 094) and 1 ⁇ l each of the primers
• BEKA243 (SEQ LD NO: 8) and BEKA244 (SEQ ID NO: 9).
• the Perkin Elmer Gene Amp PCR system 9700 was used with the following program: 95°C, 5 min; (95°C 30 s, 55°C 30 s, 72°C 2 min) x 20; and 72°C, 7 min; followed by cooling to 4°C. 10 ⁇ l of the reaction was loaded on a 1.2% E-Gel, and a fragment of approximately 2000bp was cut out from the gel, and purified using QIAquick Gel Extraction Kit (Qiagen; Cat # 28704).
• PCR fragment was cloned into pDONR201 (Life Technologies; Cat. # 11798-014), according to the manufacturer's instructions.
• the resulting entry clone was designated pBV39, and the insert was confirmed by sequencing.
• amino acids 1 to 226 represents the GST domain
• amino acids 237 to 918 represent human PKC-eta.
• Another clone comprising PKC-eta fused to a histidine tag (SEQ LD NO: 12) was prepared according to known methods.
• SEQ J-D NO: 13 represents the native PKC-eta polypeptide encoded by the gene cloned according to the above Example.
• a Multiple Tissue Expression Array (CLONTECH; Cat. #775) was used in a hybridization experiment with a 269 bp gene specific probe.
• EXAMPLE 4 Reporter assay Inducible reporter vectors that contain the Photinus pyralis (firefly) luciferase reporter gene, driven by a basic promoter element (TATA box), as well as inducible cis- enhancer elements (direct repeats from the promoter regions of various genes), were prepared or purchased.
• the reporter vectors are designed for the in vivo readouts of signal transduction pathways, since the enhancers are convergent points of many signal transduction pathways.
• a plasmid expressing the gene of interest is cotransfected into mammalian cells with a cis-reporter plasmid, increased luciferase expression indicates either direct or indirect transcriptional activation.
• a vector designated pGluREx3 -Luciferase ((gtgCACGTGtgaCAGCTGcaa)x3) was prepared using the pTAL promoter vector (Clontech; cat. #6252).
• the pGluREx3 vector is designed to monitor effects on glucose response (Portois L., et al. (1999) J. Biol. Chem. 274: 8181-8190).
• the vector pCRE-Luciferase designed to monitor the activation of cAMP binding protein (CREB) and cAMP-mediated signal transduction pathways, was purchased from Stratagene (cat. #219075).
• a vector designated pGRE- Luciferase designed to monitor the activation of Glucocorticoid response elements (GRE) mediated signal transduction pathways, was purchased from Clontech (Cat. No. K2049-1).
• Mouse adipocytes (differentiated 3T3-L1 cells) were transiently transfected with the response element construct of interest, in combination with PKC-eta or a backbone (control) plasmid construct, using LipofectAmineTM2000 (Life Technologies). After 48 hrs, the cells were lysed using a lysis buffer (Tris-EDTA + 0.25% Triton-Xl 00) for 10 min at room temperature, and the luciferase activity was measured using a luciferase activity assay (BioThema).
• a lysis buffer Tris-EDTA + 0.25% Triton-Xl 00
• Fig. 1 The results indicate that overexpression of PKC-eta in mouse adipocytes resulted in a 70% decrease of the activity of the GLUx3-Luciferase reporter, indicating a decrease in glucose responsiveness in the cells. Overexpression of PKC-eta in mouse adipocytes also leads to decreased activity of the reporters pCRE and pGRE.
• EXAMPLE 5 Glucose uptake assay
• Glucose uptake was measured as described by Hundal et al. (1994) Biochem. J. 297: 289-295. Briefly, after incubation with hormones for 45 minutes, if not otherwise stated, cell monolayers were rinsed with glucose free PBS. Glucose uptake was quantified by incubating the cells in the presence of 1 ⁇ Ci/ml 3 H-2-deoxy-glucose in PBS for 8 min. Non-specific uptake was determined by quantifying cell-associated radioactivity in the presence of 10 ⁇ M cytochalasin B. Uptake of 2-deoxy-glucose was terminated by rapidly aspirating the medium, followed by three successive washes of cell monolayers with ice cold PBS. The cells were lysed in 0.5 M NaOH, followed by liquid scintillation counting. Rates of transport were normalized for protein content in each well.
• FIGs. 2A and 2B indicate that over-expression of PKC-eta in muscle cells (non-differentiated L6) and adipocytes (differentiated 3T3-L1 cells) decreased the rate of glucose-uptake in an insulin-dependent manner.
• the results confirm the results from the reporter assay (Example 4) and further indicate that one effect of PKC-eta over-expression is reduction of glucose uptake.
• EXAMPLE 6 Down-regulation of insulin receptor tyrosine phosphorylation in CHO- hTR cells overexpressing PKC-eta.
• Tyrosine phosphorylation of the insulin receptor (LR) and insulin receptor substrates (LRS) are essential early steps in insulin signaling.
• the effect of PKC-eta over-expression on insulin receptor phosphorylation was studied.
• a DELFIA assay Dissociation Enhanced Lanthanide Fluoro-Immuno Assay; PerkinElmer Life Sciences was used to measure phosphorylated tyrosines on the IR in lysates from cells infected with an adenovirus containing the PKC-eta gene (Fig.3).
• CHO-hTR Choinese hamster ovary cells over expressing the human LR
• Alpha MEM fetal bovine serum
• FBS fetal bovine serum
• T75 flasks T75 flasks
• the cells were infected with adenovirus at a MOI 7 giving approximately 95% of the cell population overexpressing the PKC-eta gene.
• adenovirus containing GFP was used as a control of the infection. After infection the cells were incubated for 72 hours prior to determination of insulin receptor phosphorylation.
• Confluent CHO-hTR cells plated in T75 flasks (Costar), were infected with adenovirus. The virus stock was diluted in cell medium and incubated with the cells for 6 hours. The medium was subsequently replaced with fresh medium. 48 hours after infection the cells were plated in 12-well cell culture plates. Prior to insulin stimulation the cells were starved for at least 6 hours and then stimulated with 0, 25, or 100 nM insulin for 5 minutes at 37°C. Insulin stimulation was terminated by washing the cells with cold PBS followed by shock freezing with liquid nitrogen (LN 2 ). The cells were thawed, lysed and scraped with cold lysis buffer and transferred to Eppendorf tubes. The lysates were frozen in LN 2 and moved to -70°C.
• LN 2 liquid nitrogen
• DELFIA A Clear FluroNunc 96-well plate (Nunc) was coated overnight at room temperature with goat anti-rabbit IgG lO ⁇ g/ml in PBS/MgCl 2 (Dia-Service #55641). All other incubations were performed 1-2 hours (except enhancement) under agitation (Delfia Shaker) at room temperature. The coated wells were washed 2 times with 300 ⁇ l Delfia Wash Buffer (WB) followed by blocking with Superblock (Ready to use, Pierce). All washing steps were performed with a Delfia Platewasher from Wallac with at least 300 ⁇ l/well.
• WB Delfia Wash Buffer
• the wells coated with anti-rabbit IgG antibodies were incubated with a capturing anti-IR- ⁇ -chain antibody diluted in Delfia Assay Buffer to 1 ⁇ g/ml (Santa Cruz #sc-711) followed by four washes.
• Insulin stimulated and untreated control cell lysates were thawed and centrifuged at 14,000 rpm, 4°C, for 10 minutes. After centrifugation, 100 ⁇ l of the supernatants were added to the wells and lysis buffer was used as blank. In the remaining cell lysates protein concentration were measured with a kit from Pierce (Protein assay Reagent Kit, # 23227).
• biotin-labeled antibodies to phophorylated tyrosine diluted to 0.2 ng/ml in assay buffer (Santa Cruz #sc-7020B) was added.
• the wells were washed 3-4 times before incubation with eu-labeled streptavidin diluted 1/1000 in assay buffer (PerkinElmer Instruments #1244-360).
• the wells were washed six times and the fluorescence was developed by 5- 10 minutes incubation with acidic enhancement solution lOO ⁇ l/well.
• the europium was released from its polycarboxylate-based chelate and immediately a new highly fluorescent hydrophobic chelate was formed that was measured with a time-resolved fluorometer (Wallac VICTOR).
• the europium-chelates were excited at 340 nm and the emission was measured at 615 nm after a delay of 400 ⁇ s.
• the fluorescence of each sample is proportional to tyrosine phosphorylation.
• the counts from each well were normalized to protein concentration, and the average counts from wells treated the same was calculated. The significance of the effect was evaluated with a student T-test. P-values less than 0.05 or preferably 0.005 were considered significant.

Priority Applications (1)

Applications Claiming Priority (2)

Publications (1)

Family

ID=20288884

Family Applications (1)

Country Status (3)

Cited By (3)

Citations (1)

• 2002
  • 2002-09-04 SE SE0202608A patent/SE0202608D0/sv unknown
• 2003
  • 2003-09-03 AU AU2003256209A patent/AU2003256209A1/en not_active Abandoned
  • 2003-09-03 WO PCT/SE2003/001361 patent/WO2004022592A1/en not_active Application Discontinuation

Patent Citations (1)

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events