EP1274859A1 - Specific peptide substrate for human yak1, yak3a, and yak3b and yeast yak1 protein kinases - Google Patents
Specific peptide substrate for human yak1, yak3a, and yak3b and yeast yak1 protein kinasesInfo
- Publication number
- EP1274859A1 EP1274859A1 EP00976987A EP00976987A EP1274859A1 EP 1274859 A1 EP1274859 A1 EP 1274859A1 EP 00976987 A EP00976987 A EP 00976987A EP 00976987 A EP00976987 A EP 00976987A EP 1274859 A1 EP1274859 A1 EP 1274859A1
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- European Patent Office
- Prior art keywords
- ser
- arg
- gly
- leu
- yeast
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- 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
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- 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
- C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01037—Protein kinase (2.7.1.37)
Definitions
- This invention relates to a newly identified peptide substrate for the se ⁇ ne/threonme protein kmase family members, human YAKl, human YAK3a and hYAK3b, and yeast YAKl. More particularly, the peptide of the present invention is derived from the carboxyl terminal end of bovine myehn basic protein (MBP). It is a 15 ammo acid peptide with Ser at position 164 of the bovme MBP sequence as the major site of phosphorylation by hYAKl, hYAK3a and hYAK3b, and yeast YAKl, hereinafter referred to as Serl64. The sequence of Serl64 is:
- the invention also relates to a method of screening for inhibitors of human YAKl and
- this invention relates to using the Ser 164 peptide to search for cellular substrates and/or cellular proteins that interact with the YAK family members of STPK's.
- PSTK se ⁇ ne/threonme kinases
- CDKs cychn-dependent kinases
- cychns cychn-dependent kinases
- cyclm E regulatory proteins
- the complexes of CDKs and cychns are subject to inhibition by low molecular weight proteins such as pl6 (Serrano et al, Nature 1993: 366, 704), which binds to and inhibits CDK4.
- the yeast YAKl is a PSTK with sequence homology to CDKs. It was o ⁇ gmally identified in S cerevisiae as a mediator of cell cycle arrest caused by mactivation of the cAMP-dependent protein kmase PKA (Garrett et al, Mol Cell Biol. 1991: 11, 4045-4052) YAKl kmase activity is low in cycling yeast but increases dramatically when the cells are arrested p ⁇ or to the S-G2 transition. Increased expression of YAKl causes growth arrest m yeast cells deficient in PKA. Therefore, YAKl can act as a cell cycle suppressor in yeast. Frequently, in diseases such as osteoporosis and osteoarth ⁇ tis, patients have established lesions of bone or cartilage, respectively.
- hYAKl a novel human homolog of yeast YAKl, termed hYAKl, which is expressed in osteoblasts, chondrocytes, cardiac and skeletal muscle, and at lower levels, in placenta and pancreas was desc ⁇ bed (CREASY et al., EP Publication Number 98301124.8, published February 16, 1998, the disclosure of which is incorporated herein by reference in its entirety).
- the sequence of hYAKl shares homology with predicted PSTK's from C elegans, S pombe and S cerevisiae and has motifs associated with known protein kinases Inhibitors of hYAKl are expected to stimulate proliferation of cells in which it is expressed
- hYAK3b Another novel human homolog of yeast YAKl, termed hYAK3b, which is expressed in testis, skeletal muscle, and m hematopoietic cells that can undergo erythroid differentiation (XIE, and CREASY, U.S. Patent Number 5,965,420, granted October 12, 1999, and U.S. Application Se ⁇ al Number 09/359,257, filed July 22, 1999, the disclosures of which are incorporated herein by reference in their entireties).
- the sequence of hYAK3b shares 79% ammo acid identity with hYAKl in its kmase domain, and shares homology with predicted PSTK's from C elegans, S pombe and S cerevisiae.
- hYAKl and hYAK3b are members of a novel subfamily of protein kinases with unique structural and enzymatic features. This family had been termed Dyrk (for dual specificity YAK- related kinases) by Dr. Joost and his co-workers. In their publications, hYAKl and hYAK3b were termed Dyrk2 and Dyrk3, respectively (Becker and Joost, Prog. Nucl. Acid Res., 1999, 62, 1-17). Summary of the Invention
- the invention relates to a peptide substrate for hYAKl, hYAK3a and hYAK3b and yeast YAKl .
- This peptide which has the sequence LGGRDSRSGSPMARR(SEQ ID NO:9), is derived from the carboxyl terminal end of bovine MBP (residues 155-169), and in it the Ser residue at position 164 (underlined) was identified as the major phosphoacceptor residue for hYAKl Hereinafter this peptide is referred to as Ser 164 This same residue was identified as the major phosphoacceptor site also for hYAK3a and 3b and yeast YAKl PSTKs
- One aspect of the invention relates to methods for using the Ser 164 peptide as a substrate to screen for inhibitors of the kmase activity of hYAKl, hYAK3a, hYAK3b, yeast YAKl, and other members of this family of protem kinases.
- Such uses include the treatment of bone loss including osteoporosis; inflammatory diseases such as Adult Respiratory Disease Syndrome (ARDS), Rheumatoid arth ⁇ tis, Osteoarth ⁇ tis, Inflammatory Bowel Disease (IBD), pso ⁇ asis, dermatitis, asthma, allergies; infections such as bacte ⁇ al, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; HIV-associated cachexia and other immunodeficiency disorders; septic shock; pain; injury; cancers; anorexia; bulimia; Parkinson's disease; cardiovascular disease including restenosis, atherosclerosis, acute heart failure, myocardial infarction; hypotension, hypertension; u ⁇ nary retention; angina pecto ⁇ s; ulcers; benign prostatic hypertrophy; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, deli ⁇ um.
- ARDS Adult Respiratory Disease Syndrome
- IBD Inflammatory Bowel Disease
- the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with hYAKl, hYAK3a and 3b, and yeast YAKl imbalance with the identified compounds.
- Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with mapprop ⁇ ate hYAKl, hYAK3a and 3b, and yeast YAKl activity or levels.
- Still another aspect of the invention is the use of the SI 64 peptide sequence to identify cellular proteins that are phosphorylated by or interact with hYAKl, hYAK3a and 3b, and yeast YAKl .
- Figure 1 shows the determination of hYAKl -catalyzed phosphorylation sites on bovme MBP.
- Figure 2 shows the activity of hYAKl, hYAK3b, and yeast YAKl against bovine MBP and the SI 64 peptide.
- Figure 3 shows the results of steady state two substrate analysis to determine the kinetic constants of the kmase reaction of purified recombinant hYAKl using the Serl64 as substrate.
- hYAKl refers, among others, generally to a polypeptide having the amino acid sequence set forth m SEQ ID NO:4 or an allehc variant thereof.
- hYAKl activity or hYAKl polypeptide activity or “biological activity of the hYAKl or hYAKl polypeptide” refers to the metabolic or physiologic function of said hYAKl including similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are antigenic and lmmunogenic activities of said hYAKl.
- hYAKl gene refers to a polynucleotide having the nucleotide sequence set forth m SEQ
- hYAK3a refers, among others, generally to a polypeptide having the amino acid sequence set forth m SEQ ID NO: 6 or an allehc variant thereof.
- hYAK3a activity or hYAK3a polypeptide activity or “biological activity of the hYAK3a or hYAK3a polypeptide” refers to the metabolic or physiologic function of said hYAK3a including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and lmmunogenic activities of said hYAK3a.
- hYAK3a gene refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 5 or allehc va ⁇ ants thereof and/or their complements.
- hYAK3b refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 8 or an allehc va ⁇ ant thereof.
- hYAK3b activity or hYAK3b polypeptide activity or “biological activity of the hYAK3b or hYAK3b polypeptide” refers to the metabolic or physiologic function of said hYAK3b including similar activities or improved activities or these activities with decreased undesirable side-effects. Also mcluded are antigenic and lmmunogenic activities of said hYAK3b
- hYAK3b gene refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:7 or allehc va ⁇ ants thereof and/or their complements.
- Yeast YAKl refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 or an allehc variant thereof.
- yeast YAKl activity or yYAKl polypeptide activity or “biological activity of the yYAKl or yYAKl polypeptide” refers to the metabolic or physiologic function of said yYAKl including similar activities or improved activities or these activities with decreased undesirable side -effects Also included are antigenic and lmmunogenic activities of said yYAKl.
- Yeast YAKl gene refers to a polynucleotide having the nucleotide sequence set forth m SEQ ID NO: 1 or allehc va ⁇ ants thereof and/or their complements.
- Antibodies as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobulm expression library.
- Isolated means altered “by the hand of man” from the natural state. If an "isolated” composition or substance occurs m nature, it has been changed or removed from its o ⁇ gmal environment, or both. For example, a polynucleotide or a polypeptide naturally present m a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated", as the term is employed herein.
- Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeoxy ⁇ bonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
- Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comp ⁇ smg DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
- polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
- the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases include, for example, t ⁇ tylated bases and unusual bases such as inosine. A va ⁇ ety of modifications has been made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide” also embraces relatively short polynucleotides, often referred to as ohgonucleotides.
- Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
- Polypeptide refers to both short chains, commonly referred to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
- Polypeptides include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art.
- Modifications can occur anywhere m a polypeptide, including the peptide backbone, the ammo acid side-chains and the ammo or carboxyl termini. It will be appreciated that the same type of modification may be present m the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branching.
- Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods Modifications include acetylation, acylation, ADP- ⁇ bosylation, amidation, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a hpid or hpid derivative, covalent attachment of phosphotidylmositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-lmks, formation of cystme, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lod ation, methylation, my ⁇ stoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulf
- Variant is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
- a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result m ammo acid substitutions, additions, deletions, fusions and truncations m the polypeptide encoded by the reference sequence, as discussed below.
- a typical variant of a polypeptide differs in ammo acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the va ⁇ ant are closely similar overall and, in many regions, identical.
- a va ⁇ ant and reference polypeptide may differ m ammo acid sequence by one or more substitutions, additions, or deletions in any combination.
- a substituted or inserted ammo acid residue may or may not be one encoded by the genetic code.
- a va ⁇ ant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc va ⁇ ant, or it may be a variant that is not known to occur naturally.
- Non-naturally occur ⁇ ng va ⁇ ants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
- Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences
- identity refers to an exact nucleotide to nucleotide or ammo acid to ammo acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared
- a "% identity" may be determined In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences.
- a % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length
- Similarity is a further, more sophisticated measure of the relationship between two polypeptide sequences
- similar ⁇ ty means a comparison between the ammo acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact correspondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other. This likelihood has an associated 'score' from which the "% simila ⁇ ty" of the two sequences can then be determined.
- BESTFIT is more suited to compa ⁇ ng two polynucleotide or two polypeptide sequences that are dissimilar m length, the program assuming that the shorter sequence represents a portion of the longer In compa ⁇ son, GAP aligns two sequences, finding a "maximum similarity", according to the algo ⁇ thm of Neddleman and Wunsch (J Mo I Biol , 48, 443-453, 1970) GAP is more suited to compa ⁇ ng sequences that are approximately the same length and an alignment is expected over the entire length.
- the parameters "Gap Weight” and “Length Weight” used m each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively.
- % identities and simila ⁇ ties are determined when the two sequences being compared are optimally aligned.
- Proc Nat Acad ci USA, 89: 10915-10919 (1992)) is used in polypeptide sequence compa ⁇ sons including where nucleotide sequences are first translated into ammo acid sequences before compa ⁇ son.
- the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a polynucleotide or a polypeptide sequence of the present invention, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore desc ⁇ bed.
- a polynucleotide sequence having, for example, at least 95% identity to a reference polynucleotide sequence is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference sequence.
- Such point mutations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion.
- point mutations may occur at the 5 ' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides m the reference sequence or in one or more contiguous groups withm the reference sequence.
- a polynucleotide sequence having at least 95% identity to a reference polynucleotide sequence up to 5% of the nucleotides of the m the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described The same applies mutatis mutandis for other % identities such as 96%, 97%, 98%, 99% and 100%
- a polypeptide sequence having, for example, at least 95% identity to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include up to five point mutations per each 100 ammo acids of the reference sequence.
- Such point mutations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion. These point mutations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups withm the reference sequence.
- a sequence polypeptide sequence having at least 95% identity to a reference polypeptide sequence up to 5% of the ammo acids of the m the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described The same applies mutatis mutandis for other % identities such as 96%, 97%, 98%, 99%, and 100%.
- Polynucleotide embodiments further include an isolated polynucleotide comp ⁇ smg a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO.1 , (or SEQ ID NOS: 3, 5, or 7) wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides m the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides m SEQ
- n n is the number of nucleotide alterations
- x n is the total number of nucleotides m SEQ ID NO: 1
- any non-mteger product of x n and y is rounded down to the nearest integer p ⁇ or to subtracting it from x n .
- Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations m this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
- Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, (or SEQ ID NOS: 4, 6, or 8-18)wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO:2 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids m the reference sequence or in one or more contiguous groups withm the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO:2 by the integer defining the percent identity divided by 100 and then subtract
- n a is the number of ammo acid alterations
- x a is the total number of ammo acids in SEQ ID NO: 1
- y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-mteger product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
- the present invention relates to the Serl64 peptides.
- the Serl64 peptides include the peptide of the sequence m Figure 3; as well as peptide analogs which were made to study the structure activity relationship of Ser 164 phosphorylation by these PSTK's. These analogs include but are not limited to peptides 1 to 8 below (modified residues are bolded):
- Serl64 as the site on MBP phosphorylated by the YAKs PSTKs:
- hYAKl By screening several proteins as substrates, we determined that hYAKl, hYAK3a and 3b and yeast YAKl preferred MBP as a substrate over casein, enolase, poly-Gly/Tyr, and several forms of histone.
- bovme MBP was incubated for 30 minutes at 30 °C with 100 ⁇ M ATP, 10 mM MgC12, and purified hYAKl, pu ⁇ fied hYAK3b, or yeast YAKl immunoprecipitated (using anti-HA Ab) from crude extracts of Saccharomyces cerevisiae transfected with HA-tagged yeast YAKl .
- Such an in vitro phosphorylated MBP was digested with endoprotemase Lys-C for 2 hours at 38 °C in 50 mM T ⁇ s pH 8.8 An aliquot of the unfractionated protein digest was analyzed by MALDI TOF for the presence of phosphorylated peptides.
- MALDI mass spectra (Annan, and Carr, (1996) Anal Chem. 68, 3413-3421) were recorded on a Micromass TofSpec SE single-stage, g ⁇ dless reflectron time-of-flight mass spectrometer equipped with a time-lag-focusmg source Samples were prepared by mixing the digest with an equal volume of mat ⁇ x solution which contained two internal mass standards at a concentration of 200 fmol/ ⁇ l.
- Peptides are eluted using acetomt ⁇ le:water:0.1% trifluoroacetic acid gradients at 50 ⁇ l/rmn.
- the column eluent was split post detector such that 5 ⁇ l/mm was sent to a Micromass QTOF quadrupole time-of-flight mass spectrometer, and 45 ⁇ l/min went to a fraction collector taking one minute fractions.
- Phosphopeptides were sequenced by tandem MS (Verma, et al. (1997) Science, 278, 455-560.) using the QTOF mass spectrometer equipped with a nanoelectrospray source (Wilm, and Mann, (1994) Analytical Chem 66, 4390-4399 1994).
- the Lys-C digests were fractionated by reverse phase HPLC using MS as an on-line readout of the fractions. Fractions containing the two putative phosphopeptides were analyzed by nanoES, and the appropriate precursor ions sequenced by colhsion-mduced dissociation tandem mass spectrometry. The spectrum obtained for the 1570 9 peptide (Fig. 1 (labelled B)) is consistent with two phosphorylated forms of the sequence NIVTPRTPPPSQGK (SEQ ID NO: 18), which corresponds to residues 91-104 of bovme MBP. The major phosphorylation site was found to be Thr97 The 181.04 Da mass difference between the bg and > ⁇ ions indicates the presence of phosphothreonme at this position.
- the tandem mass spectrum of the 1681 peptide (Fig. 1 (labelled C)) is consistent with a monophosphorylated form of LGGRDSRSGSPMARR (SEQ ID NO:9), corresponding to the C- terminal residues 155-169 of bov e MBP.
- the tandem MS data clearly mdicate that Serl60 is not phosphorylated, it was unable to distinguish between Serl62 or Ser 164 as the sole site of phosphorylation in this peptide.
- Assignment of the phosphorylation site as Serl64 is based on the presence of an S/TP sequence at this site, which is analogous to the motif found at Thr94 and Thr97.
- the source of Ser 164 peptide It was custom-ordered from California Peptide Research Inc. (Napa, CA), and its pu ⁇ ty was determined by HPLC. The peptide contained 15 ammo acids, and its calculated molecular mass was 1601.82 daltons. Solid sample was dissolved at 5 mM in ice-cold kmase assay buffer (see later), aliquoted, and stored at -20 °C until use.
- Figure 1 shows a determination of YAKl -induced phosphorylation sites on myehn basic protein.
- A Partial MALDI mass spectrum of an unfractionated Lys-C digest of in vitro phosphorylated MBP. Peptides modified by a single mole of phosphate show an increase in mass of 80 Da.
- B ES-CID tandem mass spectrum of the M ⁇ + ion (m/z 786.4) from the 1570 Da phosphorylated peptide shown above. Ions marked b n ⁇ have the structure b n -H3P04.
- C ES-CID tandem mass spectrum of the M ⁇ + ion (m/z
- Figure 2 shows the activity of hYAKl, hYAK3, and yeast YAKl against MBP and the Serl64 peptide.
- 5 ng pu ⁇ fied hYAKl and 100 ng purified hYAK3 were used per assay.
- Anti-HA mAb immune complex kmase assay was performed on 100 ⁇ g protein from crude extracts of yeast cells expressing either FL or ⁇ N yeast YAKl. Concentration of ATP was 100 ⁇ M, Serl64 was used at 0.5 mM, and MBP was at 10 ⁇ g/reaction (18.5 uM)
- Figure 3 shows double reciprocal plots of enzyme velocity vs. ATP or Ser 164 concentration.
- hYAKl DETl/DET2-tagged full length hYAKl was expressed in Drosophila sf9 cells and pu ⁇ fied to >95% pu ⁇ ty using Ni column chromatography. The pu ⁇ fied protein migrated on SDS gels as a single band with an apparent molecular mass of 62 kDa. Samples were stored at -80 °C until use.
- hYAK3b Glutathione-S-Transferase (GST)/Factor Xa-tagged hYAK3b was expressed in baculovirus cells and pu ⁇ fied to about 50% pu ⁇ ty using Glutathione Sepharose 4B column chromatography, followed by Ni-NTA column chromatography. Samples were stored at -80 °C until use.
- Yeast YAKl Full length and an ammo-terminally truncated (ammo acids 148-807, termed ⁇ N) hemagglutmm (HA)-tagged yeast YAKl was each expressed m a strain of S cerevisiae lacking the endogenous YAKl gene and all three PKA genes.
- cell pellets were thawed and resuspended at 1 ml/100 ml of original culture m lysis buffer (LB) containing 50 mM T ⁇ s pH 7.5, 150 mM NaCl, 10 ⁇ g/ml each aprotin , leupeptin and TLCK, 0.1 mM PMSF, 50 mM NaF, 1 mM NaVanadate, 10 mM ⁇ -glycerophosphate. Following the addition of 0.5 ml ste ⁇ le acid-washed glass beads, cells were disrupted via ten, 30 second intervals of vortexmg.
- LB original culture m lysis buffer
- NP40 was added to a 2% final concentration followed by rocking at 4 °C for 30-50 minutes. Lysates were cla ⁇ fied by high-speed centrifugation, and the supernatants were stored at -80 °C until use. Each form of yeast YAKl was immunoprecipitated from the detergent extracts using anti-HA mAb.
- Yeast cellular extracts were immunoprecipitated by rocking overnight at 4°C with 4 ⁇ g of the anti-HA tag antibody and 100 ⁇ l of 20% suspension of protein A agarose (GIBCO-BRL) in LB that contained 1% NP-40. Samples were then washed twice with LB and once with basic kmase assay buffer (25 mM Hepes, pH 7.5; 1 mM DTT; 10 mM ⁇ -glycerol phosphate; 0.2 mM NaV).
- basic kmase assay buffer 25 mM Hepes, pH 7.5; 1 mM DTT; 10 mM ⁇ -glycerol phosphate; 0.2 mM NaV.
- Washed immune complexes were suspended m 20 ⁇ l of basic kmase assay buffer that contained 0.1 mM ATP, 3 ⁇ Ci of [ ⁇ -32p]ATP, 10 mM MgCl2, plus either bovme MBP or the Ser 164 peptide. After incubation for 15 minutes at
- the reactions were stopped by adding 20 ⁇ l of 0.15 M phosphoric acid.
- Phosphorylated substrates were isolated by spotting 20 ⁇ l of each sample on phosphocellulose (p81) filters. Filters were washed 3 times with 75 mM phosphoric acid followed by 3 times with H2O, and counted for 2p incorporation using a ⁇ -scmtillation counter.
- Kinase assay of pu ⁇ fied hYAKl and hYAK3 The assay was performed in 96 well Mmisorp plates (Costar, Catalog No 3356). Reaction (in 30 ⁇ l volume) mixtures contained in final concentrations, 25 mM Hepes buffer, pH 7.5; 0.2 mM sodium vandate; 10 mM MgCl2; 1 mM DTT;
- This same assay can be performed on a FlashPlate format m which the plate is coated with MBP or with the SI 64 peptide by incubation overnight at 4 °C in 100 ⁇ l of either substrate dissolved in Sodium Carbonate buffer, pH 8.8.
- MBP a solution of 100 ⁇ g/ml MBP was used to coat wells with 100 ⁇ l (10 ⁇ g) MBP per well.
- Serl64 a solution of 0.4 mg/ml (0.25 mM) was used to coat wells with 100 ⁇ l (40 ⁇ g) Serl64 per well.
- An example of a FlashPlate assay protocol and typical results are given below:
- FlashPlate (FP) Protocol Coat Maxisorp plates with MBP or Serl 64 as above.
- FlashPlate Typical results of FlashPlate are shown below FlashPlate Typical results
- SKF-108752 IC50 0.19 ⁇ M (0.1 ⁇ g hYAKl)
- the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides of the present invention may be employed in a screening process for compounds, which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides of the present invention.
- polypeptides of the invention may also be used to assess or identify agonists or antagonists from, for example, cells, cell-free preparations, chemical hbra ⁇ es, and natural product mixtures.
- agonists or antagonists may be natural substrates, hgands, receptors, etc., as the case may be, of the polypeptides of the present invention; or may be structural or functional mimetics of the polypeptide of the present invention. See Coligan et al , Current Protocols in Immunology 1(2): Chapter 5 (1991). hYAKl, hYAK3a and 3b , and yeast YAKl polypeptides are ubiquitous in the mammalian host and are responsible for many biological functions, including many pathologies.
- agonists are employed for therapeutic and prophylactic purposes for such conditions as bone loss including osteoporosis; inflammatory diseases such as Adult Respiratory Disease Syndrome (ARDS), Rheumatoid arth ⁇ tis, Osteoarth ⁇ tis, Inflammatory Bowel Disease (IBD), pso ⁇ asis, dermatitis, asthma, allergies; infections such as bacte ⁇ al, fungal, protozoan and viral infections, particularly infections caused by HTV-1 or HIV-2; HlV-associated cachexia and other immunodeficiency disorders; septic shock; pam; injury; cancers; anorexia; bulimia; Parkinson's disease; cardiovascular disease including restenosis, atherosclerosis, acute heart failure, myocardial infarction; hypotension; hypertension; u ⁇ nary retention; angina pecto ⁇ s; ulcers; benign prostatic hypertrophy; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, deh ⁇ um, dementia, severe mental retardation and dyskmesi
- Antagonists may be employed for a va ⁇ ety of therapeutic and prophylactic purposes for such conditions as bone loss including osteoporosis; inflammatory diseases such as Adult Respiratory Disease Syndrome (ARDS), Rheumatoid arth ⁇ tis, Osteoarth ⁇ tis, Inflammatory Bowel Disease (IBD), pso ⁇ asis, dermatitis, asthma, allergies; infections such as bacte ⁇ al, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; HlV-associated cachexia and other immunodeficiency disorders; septic shock; pam; injury; cancers; anorexia, bulimia; Parkinson's disease; cardiovascular disease including restenosis, atherosclerosis, acute heart failure, myocardial infarction; hypotension; hypertension; u ⁇ nary retention; angina pecto ⁇ s; ulcers; benign prostatic hypertrophy; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, deh ⁇ um, dementia, severe mental retard
- screenmg procedures may involve using approp ⁇ ate cells which express the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides or respond to hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide of the present invention.
- Such cells include cells from mammals, yeast, Drosoph a or E coh Cells which express the hYAKl , hYAK3a and 3b, and yeast YAKl polypeptides (or cell membrane containing the expressed polypeptides) or respond to hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
- the ability of the cells, which were contacted with the candidate compounds is compared with the same cells which were not contacted for hYAKl, hYAK3a and 3b, and yeast YAKl activity.
- hYAKl, hYAK3a and 3b, and yeast YAKl encodes a protein kmase
- Separation methods include lmmunoprecipitation, conjugation of substrate to a bead allowing separation by cent ⁇ fugation or determination of incorporation by scintillation proximity assay, SDS-PAGE followed by autoradiography or biosensor analysis.
- candidates include hYAKl , hYAK3a and 3b, and yeast YAKl themselves (autophosphorylation), myehn basic protein, casem, histone and HSP27.
- Other substances might be discovered by incubating hYAKl, hYAK3a and 3b, and yeast YAKl with random peptides conjugated to solid supports or displayed on the surface of phage or by incubation of hYAKl, hYAK3a and 3b, and yeast YAKl with mammalian cell lysates and ⁇ -32p. ATP, followed by separation of the labelled target proteins, and sequencmg.
- the protein kmase activity of hYAKl , hYAK3a and 3b, and yeast YAKl may require incubation with a specific upstream effector. This may be achieved by preincubatmg hYAKl, hYAK3a and 3b, and yeast YAKl with lysates from a va ⁇ ety of stimulated eukaryotic cells and ATP.
- These assays permit the discovery and modification of compounds which inhibit hYAKl, hYAK3a and 3b, and yeast YAKl kmase activity in vitro and would be expected to have effects on proliferation of osteoblasts, chondrocytes, cardiac myocytes or skeletal myoblasts. Any inhibitors so identified would be expected to have up-regulatory effects on proliferation and be useful as a therapeutic for the treatment and prevention of diseases such as osteoporosis, osteoarth ⁇ tis, cardiomyopathy and cachexia.
- This invention contemplates the treatment and/or amelioration of such diseases by adrmmste ⁇ ng a hYAKl, hYAK3a and 3b, or yeast YAKl inhibiting amount of a compound.
- adrmmste ⁇ ng a hYAKl, hYAK3a and 3b, or yeast YAKl inhibiting amount of a compound without wishing to be bound by any particular theory of the functioning of the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides of this invention, it is believed that among the useful inhibitors of hYAKl, hYAK3a and 3b, and yeast YAKl function are those compounds which inhibit the kmase activity of the hYAKl, hYAK3a and 3b, and yeast YAKl .
- the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide, using detection systems appropriate to the cells bearing the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide.
- Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Standard methods for conducting such screening assays are well understood in the art.
- Examples of potential hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide antagonists include antibodies or, in some cases, ohgonucleotides or proteins which are closely related to the hgands, substrates, receptors, etc., as the case may be, of the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide, e.g., a fragment of the hgands, substrates, receptors, or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
- This invention provides methods of treating an abnormal condition related to both an excess of and insufficient amounts of hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide activity.
- hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides are m excess.
- One approach comp ⁇ ses administe ⁇ ng to a subject an inhibitor compound (antagonist) as hereinabove desc ⁇ bed along with a pharmaceutically acceptable earner in an amount effective to inhibit activation by blocking binding of hgands to the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
- soluble forms of hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides still capable of binding the hgand m competition with endogenous hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides may be administered.
- Typical embodiments of such competitors comp ⁇ se fragments of the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides.
- expression of the gene encoding endogenous hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide can be inhibited using expression blocking techniques.
- Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988).
- ohgonucleotides which form triple helices with the gene can be supplied. See.
- hYAKl For treating abnormal conditions related to an under-expression of hYAKl, hYAK3a and 3b, and yeast YAKl and its activity, several approaches are also available.
- One approach comp ⁇ ses administering to a subject a therapeutically effective amount of a compound which activates hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide, i.e., an agonist as desc ⁇ bed above, m combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition.
- gene therapy may be employed to effect the endogenous production of hYAKl, hYAK3a and 3b, and yeast YAKl by the relevant cells m the subject.
- a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above.
- the retroviral expression construct may then be isolated and introduced into a packagmg cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
- These producer cells may be administered to a subject for engmeenng cells in vivo and expression of the polypeptide in vivo.
- gene therapy see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therem) m Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996).
- Peptides such as the soluble form of hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides, and agonists and antagonist peptides or small molecules, may be formulated m combination with a suitable pharmaceutical earner.
- a suitable pharmaceutical earner Such formulations compnse a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient.
- earners include but are not limited to, salme, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
- the formulation should suit the mode of administration, and is well withm the skill of the art.
- the invention further relates to pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
- Polypeptides and other compounds of the present invention may be employed alone or m conjunction with other compounds, such as therapeutic compounds.
- systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection.
- Other injection routes such as subcutaneous, intramuscular, or intrape ⁇ toneal, can be used.
- Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
- penetrants such as bile salts or fusidic acids or other detergents.
- oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels and the like.
- the dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 ⁇ g/kg of subject. Wide variations in the needed dosage, however, are to be expected m view of the vanety of compounds available and the differing efficiencies of vanous routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Vanations m these dosage levels can be adjusted using standard empi ⁇ cal routines for optimization, as is well understood m the art.
- Polypeptides used m treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above.
- cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
- Example 1 is earned out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise desc ⁇ bed in detail. The examples illustrate, but do not limit the invention. Example 1
- a partial clone (ATG-355, HGS EST # 454640) was initially identified through random searches of the Human Genome Sciences database. This partial clone ( ⁇ lkb) showed significant homology to YAKl from S cerevisiae.
- To get the full length cDNA A total of 1M plaques were screened from a Human Osteoblast cDNA library (Stratagene, LaJolla CA) using the insert of the above partial clone as a probe. Library screening procedure is described by (Elgin, et al. Strategies 4: 8-9, 1991). The probes were ⁇ -32P labeled, using Random Pnmed Labeling Kit (Bohe ⁇ nger Manheim, Germany, Cat. # 1585584 ) and punfied by running over Sephadex G-50 columns
- Human Leukocyte "Marathon Ready" cDNA (Clontech, Palo Alto, CA) was used as a template to amplify a fragment corresponding to the 5' region of hYAKl using a 5' anchor pnmer-1 (Clontech) and a reverse gene specific p ⁇ mer. This fragment was T/A cloned into pCR2.1 (Invitrogen), and multiple isolates were sequenced. An m-frame stop codon was identified upstream of the predicted initiation codon confirming that the full-length cDNA had been obtained.
- Northern analysis was earned out to determine the distnbution of hYAKl mRNA in human tissues.
- a fragment contammg the 3' untranslated region of hYAKl was isolated from SEQ ID NO: 1 using standard techniques. The isolated fragment was radiolabelled with ⁇ -32p-dATP using a randomly pnmed labelling kit.
- Membranes contammg mRNA from multiple human tissues (Clontech #7760-1) were hyb ⁇ dized with the probe and washed under high st ⁇ ngency conditions as directed. Hyb ⁇ dized mRNA was visualized by exposing the membranes for 4 days to X-ray film.
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Abstract
Description
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PCT/US2000/030542 WO2001032918A1 (en) | 1999-11-05 | 2000-11-06 | Specific peptide substrate for human yak1, yak3a, and yak3b and yeast yak1 protein kinases |
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