A Predictive Scale for Evaluating Cyclin-dependent Kinase Substrates

Protein phosphorylation by members of the Cdk (cyclin-dependent kinase) family of protein kinases is necessary for progression through the cell cycle. However, the primary sequence determinants of Cdk substrate specificity have yet to be examined quantitatively. We have used a panel of glutathione S-transferase peptide fusions to investigate the fine-structure specificity of p33cdk2 and p34cdc2. Our data indicate that the generally held consensus sequences for p34cdc2 represent a significant oversimplification of its true specificity and that this specificity is conserved between species. p33cdk2 and p34cdc2 have similar but distinct substrate specificities that are affected modestly by the associated cyclin subunit. We derive specific values of phosphorylation efficiencies by these enzymes that can be used to estimate the phosphorylation potential of proposed Cdk substrates. Protein phosphorylation by members of the Cdk (cyclin-dependent kinase) family of protein kinases is necessary for progression through the cell cycle. However, the primary sequence determinants of Cdk substrate specificity have yet to be examined quantitatively. We have used a panel of glutathione S-transferase peptide fusions to investigate the fine-structure specificity of p33cdk2 and p34cdc2. Our data indicate that the generally held consensus sequences for p34cdc2 represent a significant oversimplification of its true specificity and that this specificity is conserved between species. p33cdk2 and p34cdc2 have similar but distinct substrate specificities that are affected modestly by the associated cyclin subunit. We derive specific values of phosphorylation efficiencies by these enzymes that can be used to estimate the phosphorylation potential of proposed Cdk substrates. INTRODUCTIONThe cell cycle consists of a series of strictly ordered steps, requiring the completion of one event before the next can occur. The protein kinases that control entry into and progression through various stages of the cell cycle are members of the Cdk 1The abbreviations used are: Cdkcyclin-dependent kinaseGSTglutathione S-transferase. (cyclin-dependent kinase) subfamily of protein kinases. Cdk activities fluctuate as a result of post-translational modifications and protein-protein interactions. An active Cdk is formed after binding to a cyclin partner and phosphorylation on a key threonine (Thr-161 in human p34cdc2). In vertebrates, Cdk4-cyclin D is necessary for passage through G1, p33cdk2-cyclin E is necessary for the transition from G1 to S phase, p33cdk2-cyclin A is necessary for progression through S, and p34cdc2-cyclin B is necessary for the transition from G2 to M phase (1Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (492) Google Scholar).Crucial to our understanding of the cell cycle is the ability to identify for the various Cdk-cyclin complexes the key substrates whose phosphorylation leads to the progression through a particular cellular event. Many of these downstream effects could be caused directly by the Cdk; for example, p34cdc2-cyclin B can phosphorylate lamins thus leading to their disassembly (2Peter M. Nakagawa J. Dorée M. Labbé J.C. Nigg E.A. Cell. 1990; 61: 591-602Abstract Full Text PDF PubMed Scopus (548) Google Scholar, 3Dessev G. Iovcheva-Dessev C. Bischoff J.R. Beach D. Goldman R. J. Cell Biol. 1991; 112: 523-533Crossref PubMed Scopus (102) Google Scholar, 4Peter M. Heitlinger E. Haner M. Aebi U. Nigg E.A. EMBO J. 1991; 10: 1535-1544Crossref PubMed Scopus (140) Google Scholar), an important event in the initiation of mitosis. Other effects could be indirect, the result of a cascade of events initiated by the Cdk; for example, Cdk4-cyclin D phosphorylates Rb, thus releasing E2F to promote the transcription of many genes important for DNA replication (5Kato J.-y. Matsushime H. Hiebert S.W. Ewen M.E. Sherr C.J. Genes Dev. 1993; 7: 331-342Crossref PubMed Scopus (1086) Google Scholar).An understanding of the basis of substrate specificities of different Cdk-cyclin complexes is of central importance as specificity can be influenced by many factors. Obviously the choice of a phosphorylation target site will be influenced strongly by inherent differences in the substrate binding region of a particular Cdk (6Knighton D.R. Zheng J. Eyck L.F.T. Ashford V.A. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1439) Google Scholar, 7Knighton D.R. Zheng J. Eyck L.F.T. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-420Crossref PubMed Scopus (804) Google Scholar, 8DeBondt H.L. Rosenblatt J. Jancarik J. Jones H.D. Morgan D.O. Kim S.-H. Nature. 1993; 363: 595-602Crossref PubMed Scopus (828) Google Scholar, 9Songyang Z. Blechner S. Hoagland N. Hoekstra M.F. Piwnica-Worms H. Cantley L.C. Curr. Biol. 1994; 4: 973-982Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar). In addition, the cyclin subunit could influence substrate specificity in any of the following ways: by binding a potential substrate and bringing it into contact with the Cdk; by targeting the Cdk to a particular subcellular location where it has access to only a limited number of potential substrates (10Pines J. Hunter T. J. Cell Biol. 1991; 115: 1-17Crossref PubMed Scopus (673) Google Scholar, 11Pines J. Hunter T. EMBO J. 1994; 13: 3772-3781Crossref PubMed Scopus (224) Google Scholar, 12Jackman M. Firth M. Pines J. EMBO J. 1995; 14: 1646-1654Crossref PubMed Scopus (218) Google Scholar); or by restricting Cdk activities to a narrow window within the cell cycle so that the kinase can only affect those substrates present and able to be activated during that stage (1Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (492) Google Scholar). Most likely, the substrate is recognized by a combination of the Cdk substrate binding pocket and long range interactions with surface residues of the cyclin subunit (13Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1209) Google Scholar). The majority of substrates would be recognized by the Cdk in association with any cyclin, but certain subsets might be recognized or preferred by a specific Cdk-cyclin pair (14Minshull J. Golsteyn R. Hill C.S. Hunt T. EMBO J. 1990; 9: 2865-2875Crossref PubMed Scopus (255) Google Scholar). Several recent studies have indeed demonstrated that the identity of the cyclin partner can influence substrate specificity significantly (14Minshull J. Golsteyn R. Hill C.S. Hunt T. EMBO J. 1990; 9: 2865-2875Crossref PubMed Scopus (255) Google Scholar, 15Thomas L. Clarke P.R. Pagano M. Gruenberg J. J. Biol. Chem. 1992; 267: 6183-6187Abstract Full Text PDF PubMed Google Scholar, 16Hoffmann I. Clarke P.R. Marcote M.J. Karsenti E. Draetta G. EMBO J. 1993; 12: 53-63Crossref PubMed Scopus (562) Google Scholar, 17Peeper D.S. Parker L.L. Ewen M.E. Toebes M. Hall F.L. Xu M. Zantema A. van der Eb A.J. Piwnica-Worms H. EMBO J. 1993; 12: 1947-1954Crossref PubMed Scopus (148) Google Scholar).Several loose consensus substrate sequences have been reported for p34cdc2 based on a limited number of known in vivo and in vitro p34cdc2 substrates (for review, see 18Nigg E.A. Semin. Cell Biol. 1991; 2: 261-270PubMed Google Scholar). These include (K/R)(S/T)PX(K/R), where X is any amino acid (18Nigg E.A. Semin. Cell Biol. 1991; 2: 261-270PubMed Google Scholar) or a polar amino acid (19Moreno S. Nurse P. Cell. 1990; 61: 549-551Abstract Full Text PDF PubMed Scopus (359) Google Scholar), and (S/T)PX(K/R), where X is any amino acid (20Nigg E.A. Trends Cell Biol. 1993; 3: 296-301Abstract Full Text PDF PubMed Scopus (206) Google Scholar). It has generally been assumed that p33cdk2 has a similar specificity. The few studies investigating the substrate specificity of the Cdks have been performed primarily on p34cdc2 (14Minshull J. Golsteyn R. Hill C.S. Hunt T. EMBO J. 1990; 9: 2865-2875Crossref PubMed Scopus (255) Google Scholar, 18Nigg E.A. Semin. Cell Biol. 1991; 2: 261-270PubMed Google Scholar, 20Nigg E.A. Trends Cell Biol. 1993; 3: 296-301Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 21Erikson E. Maller J.L. J. Biol. Chem. 1989; 264: 19577-19582Abstract Full Text PDF PubMed Google Scholar, 22Marin O. Meggio F. Draetta G. Pinna L.A. FEBS Lett. 1992; 301: 111-114Crossref PubMed Scopus (64) Google Scholar, 23Yamakita Y. Yamashiro S. Matsumura F. J. Cell Biol. 1994; 124: 129-137Crossref PubMed Scopus (133) Google Scholar) and have examined only a small number of peptides or sites in diverse proteins. A systematic study of protein kinase substrate specificity was carried out recently by Songyang et al. (9Songyang Z. Blechner S. Hoagland N. Hoekstra M.F. Piwnica-Worms H. Cantley L.C. Curr. Biol. 1994; 4: 973-982Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar) using a peptide library containing approximately 2.5 billion unique peptides, with a fixed serine as the phosphate acceptor, as substrates for various kinases including p33cdk2-cyclin A and p34cdc2-cyclin B. This method identified a sequence similar to one of the consensus sites as the optimal substrate for p34cdc2-cyclin B, (K/R)SP(R/P)(R/K/H).We have investigated the substrate specificity of p33cdk2 bound to cyclin A or E and of p34cdc2 bound to cyclin A or B using a systematic series of specifically defined peptide substrates appended to the COOH terminus of glutathione S-transferase, constructed by polymerase chain reaction using degenerate oligonucleotides. These substrates allowed us to determine quantitatively the role of the primary sequence of a target site in substrate utilization. Our panel of altered target sites has allowed us to compare the inherent differences in substrate recognition between p33cdk2 and p34cdc2 as well as to examine the effects of the cyclin regulatory subunits on specificity. In addition, we have found that the data generated from these experiments can be used to predict the potential utilization of novel phosphorylation sites.DISCUSSIONWe have used a panel of GST fusion proteins containing systematic alterations of a canonical p34cdc2 phosphorylation site to determine the fine specificity of p34cdc2 and p33cdk2 bound to various cyclins. Understanding the similarities and differences in the specificities of these enzymes is an essential first step toward evaluating potential substrates that could play important roles in cell cycle progression. Previous studies of p34cdc2 phosphorylation sites have involved compilations of sites found in diverse proteins and examination of modest numbers of synthetic peptide substrate variants. Recently a peptide selection approach has been used to define a p34cdc2 consensus site as (K/R)SP(R/P)(R/K/H). Although this method is extremely useful for rapidly determining optimal phosphorylation sites, it is not as well suited for determining which amino acids are poorly tolerated or excluded, it does not analyze all 20 amino acids, and it systematically overestimates the phosphorylation of suboptimal substrates (9Songyang Z. Blechner S. Hoagland N. Hoekstra M.F. Piwnica-Worms H. Cantley L.C. Curr. Biol. 1994; 4: 973-982Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar). Our approach benefits from using a comprehensive collection of variant substrates within the same protein context. The fusion proteins are inexpensive and readily purified, and additional mutant phosphorylation sites can be engineered quite easily. We also expect that our panel of substrates will prove useful for determining the substrate specificity of other Cdks, including those involved in cell cycle control as well as those involved in other processes.Overall, we found that Xenopus p34cdc2 was least sensitive to substitutions at the -1 position of the wild type sequence KSPRK (with respect to the phosphorylated Ser), fairly sensitive to substitutions at the +2 position, and most sensitive to substitutions at the +3 position. Although this general pattern is consistent with widely held consensus sites for phosphorylation by p34cdc2, our data significantly alter our view of what sequences can constitute good, fair, or poor phosphorylation targets. Our finding that the -1 position can accommodate any amino acid, but that there is about a 2-fold variation in phosphorylation efficiency, is closer to the consensus that posits no specificity than to those that place a basic residue at this position. At the +2 position, we found that neither consensus view, either specifying a polar residue or tolerating all amino acids, adequately fit the data. There was a strong degree of specificity at this position since some substitution mutants were phosphorylated almost 20-fold more efficiently than others. However, we have been unable to discern any simple pattern to explain this specificity. Clearly, though, both polar and nonpolar amino acid side chains could form excellent substrates, and some polar amino acids yielded quite poor substrates. At the +3 position our data are in agreement with the consensus view that basic residues are best. However, by focusing on the best sites, the consensus view fails to distinguish among the poorer sites. We would divide the substitutions at the +3 position into four classes: basic residues, which form excellent sites (about 100% of wild type phosphorylation efficiency); His and Pro, which can form surprisingly strong sites (about 20% of wild type); most other amino acids, which form weak but significant sites (about 5% of wild type); and acidic groups, which form virtually unphosphorylatable sites. The approximately 20-fold reduction in binding affinity on substitution of Ala and most other amino acids at the +3 position corresponds to a weakening of the interaction by about 1.8 kcal/mol, which is consistent with the loss of a single ionic interaction linking the +3 basic residue to p34cdc2.We observed two classes of modest effects of the cyclin partner on phosphorylation of the substrates. First, we noted that the cyclin A-containing complex of p33cdk2 had a consistently ~2-fold higher Km for our substrates than the cyclin E-containing complex (Table I). The Km was not measured for all substrates, but if phosphorylation efficiencies reflect changes in Km, and not in Vmax, then this difference in Km probably applies to nearly all of the substrates and not just to those shown in Table I. This result may indicate that cyclin E is a “better” cyclin in that it may be able to induce a better geometry of the binding pocket in p33cdk2 for substrates. We saw no comparable effect of cyclin A versus cyclin B in the p34cdc2-containing complexes. We also noted a more sporadic effect of cyclin partner on phosphorylation efficiencies that we are inclined to attribute to weak longer range interactions between the cyclin and individual target sequences.Although the specificities of p34cdc2 and p33cdk2 were generally similar, we were surprised to find a number of instances where p33cdk2 was far more selective. p33cdk2 was much less of or at the +2 position than was p34cdc2. This effect was approximately or on the cyclin The differences were at the +3 position, where p33cdk2 not phosphorylate than of wild type any substrate not containing or at this which a p34cdc2 only poor substrates (about 5% wild type for The of any amino acid other than or at the +3 position of a Cdk phosphorylation site can be as a strong that p34cdc2, than phosphorylates that The of or at the +2 position, or of at the +3 position, would also to toward p34cdc2 as the protein data are to be useful for the of potential substrates of p34cdc2 and of We were able to predict the phosphorylation efficiencies of site substitutions fairly since the effects of the single amino acid substitution mutants on phosphorylation were (Table We that a of potential phosphorylation sites on novel proteins will to experiments toward the most sites. example, our data predict that an poor target would be phosphorylated almost as efficiently by Xenopus p34cdc2-cyclin B as an excellent versus of wild type We not that our will be for all sites in all protein Clearly, many to determine the phosphorylation of a A excellent site could be within a protein or in an an weak site could be and in a interactions between either subunit of the Cdk and a substrate could influence specificity. these and in particular their are to we that our an for examination of Cdk substrates. the phosphorylation efficiencies in are readily from the INTRODUCTIONThe cell cycle consists of a series of strictly ordered steps, requiring the completion of one event before the next can occur. The protein kinases that control entry into and progression through various stages of the cell cycle are members of the Cdk 1The abbreviations used are: Cdkcyclin-dependent kinaseGSTglutathione S-transferase. (cyclin-dependent kinase) subfamily of protein kinases. Cdk activities fluctuate as a result of post-translational modifications and protein-protein interactions. An active Cdk is formed after binding to a cyclin partner and phosphorylation on a key threonine (Thr-161 in human p34cdc2). In vertebrates, Cdk4-cyclin D is necessary for passage through G1, p33cdk2-cyclin E is necessary for the transition from G1 to S phase, p33cdk2-cyclin A is necessary for progression through S, and p34cdc2-cyclin B is necessary for the transition from G2 to M phase (1Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (492) Google Scholar).Crucial to our understanding of the cell cycle is the ability to identify for the various Cdk-cyclin complexes the key substrates whose phosphorylation leads to the progression through a particular cellular event. Many of these downstream effects could be caused directly by the Cdk; for example, p34cdc2-cyclin B can phosphorylate lamins thus leading to their disassembly (2Peter M. Nakagawa J. Dorée M. Labbé J.C. Nigg E.A. Cell. 1990; 61: 591-602Abstract Full Text PDF PubMed Scopus (548) Google Scholar, 3Dessev G. Iovcheva-Dessev C. Bischoff J.R. Beach D. Goldman R. J. Cell Biol. 1991; 112: 523-533Crossref PubMed Scopus (102) Google Scholar, 4Peter M. Heitlinger E. Haner M. Aebi U. Nigg E.A. EMBO J. 1991; 10: 1535-1544Crossref PubMed Scopus (140) Google Scholar), an important event in the initiation of mitosis. Other effects could be indirect, the result of a cascade of events initiated by the Cdk; for example, Cdk4-cyclin D phosphorylates Rb, thus releasing E2F to promote the transcription of many genes important for DNA replication (5Kato J.-y. Matsushime H. Hiebert S.W. Ewen M.E. Sherr C.J. Genes Dev. 1993; 7: 331-342Crossref PubMed Scopus (1086) Google Scholar).An understanding of the basis of substrate specificities of different Cdk-cyclin complexes is of central importance as specificity can be influenced by many factors. Obviously the choice of a phosphorylation target site will be influenced strongly by inherent differences in the substrate binding region of a particular Cdk (6Knighton D.R. Zheng J. Eyck L.F.T. Ashford V.A. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1439) Google Scholar, 7Knighton D.R. Zheng J. Eyck L.F.T. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-420Crossref PubMed Scopus (804) Google Scholar, 8DeBondt H.L. Rosenblatt J. Jancarik J. Jones H.D. Morgan D.O. Kim S.-H. Nature. 1993; 363: 595-602Crossref PubMed Scopus (828) Google Scholar, 9Songyang Z. Blechner S. Hoagland N. Hoekstra M.F. Piwnica-Worms H. Cantley L.C. Curr. Biol. 1994; 4: 973-982Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar). In addition, the cyclin subunit could influence substrate specificity in any of the following ways: by binding a potential substrate and bringing it into contact with the Cdk; by targeting the Cdk to a particular subcellular location where it has access to only a limited number of potential substrates (10Pines J. Hunter T. J. Cell Biol. 1991; 115: 1-17Crossref PubMed Scopus (673) Google Scholar, 11Pines J. Hunter T. EMBO J. 1994; 13: 3772-3781Crossref PubMed Scopus (224) Google Scholar, 12Jackman M. Firth M. Pines J. EMBO J. 1995; 14: 1646-1654Crossref PubMed Scopus (218) Google Scholar); or by restricting Cdk activities to a narrow window within the cell cycle so that the kinase can only affect those substrates present and able to be activated during that stage (1Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (492) Google Scholar). Most likely, the substrate is recognized by a combination of the Cdk substrate binding pocket and long range interactions with surface residues of the cyclin subunit (13Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1209) Google Scholar). The majority of substrates would be recognized by the Cdk in association with any cyclin, but certain subsets might be recognized or preferred by a specific Cdk-cyclin pair (14Minshull J. Golsteyn R. Hill C.S. Hunt T. EMBO J. 1990; 9: 2865-2875Crossref PubMed Scopus (255) Google Scholar). Several recent studies have indeed demonstrated that the identity of the cyclin partner can influence substrate specificity significantly (14Minshull J. Golsteyn R. Hill C.S. Hunt T. EMBO J. 1990; 9: 2865-2875Crossref PubMed Scopus (255) Google Scholar, 15Thomas L. Clarke P.R. Pagano M. Gruenberg J. J. Biol. Chem. 1992; 267: 6183-6187Abstract Full Text PDF PubMed Google Scholar, 16Hoffmann I. Clarke P.R. Marcote M.J. Karsenti E. Draetta G. EMBO J. 1993; 12: 53-63Crossref PubMed Scopus (562) Google Scholar, 17Peeper D.S. Parker L.L. Ewen M.E. Toebes M. Hall F.L. Xu M. Zantema A. van der Eb A.J. Piwnica-Worms H. EMBO J. 1993; 12: 1947-1954Crossref PubMed Scopus (148) Google Scholar).Several loose consensus substrate sequences have been reported for p34cdc2 based on a limited number of known in vivo and in vitro p34cdc2 substrates (for review, see 18Nigg E.A. Semin. Cell Biol. 1991; 2: 261-270PubMed Google Scholar). These include (K/R)(S/T)PX(K/R), where X is any amino acid (18Nigg E.A. Semin. Cell Biol. 1991; 2: 261-270PubMed Google Scholar) or a polar amino acid (19Moreno S. Nurse P. Cell. 1990; 61: 549-551Abstract Full Text PDF PubMed Scopus (359) Google Scholar), and (S/T)PX(K/R), where X is any amino acid (20Nigg E.A. Trends Cell Biol. 1993; 3: 296-301Abstract Full Text PDF PubMed Scopus (206) Google Scholar). It has generally been assumed that p33cdk2 has a similar specificity. The few studies investigating the substrate specificity of the Cdks have been performed primarily on p34cdc2 (14Minshull J. Golsteyn R. Hill C.S. Hunt T. EMBO J. 1990; 9: 2865-2875Crossref PubMed Scopus (255) Google Scholar, 18Nigg E.A. Semin. Cell Biol. 1991; 2: 261-270PubMed Google Scholar, 20Nigg E.A. Trends Cell Biol. 1993; 3: 296-301Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 21Erikson E. Maller J.L. J. Biol. Chem. 1989; 264: 19577-19582Abstract Full Text PDF PubMed Google Scholar, 22Marin O. Meggio F. Draetta G. Pinna L.A. FEBS Lett. 1992; 301: 111-114Crossref PubMed Scopus (64) Google Scholar, 23Yamakita Y. Yamashiro S. Matsumura F. J. Cell Biol. 1994; 124: 129-137Crossref PubMed Scopus (133) Google Scholar) and have examined only a small number of peptides or sites in diverse proteins. A systematic study of protein kinase substrate specificity was carried out recently by Songyang et al. (9Songyang Z. Blechner S. Hoagland N. Hoekstra M.F. Piwnica-Worms H. Cantley L.C. Curr. Biol. 1994; 4: 973-982Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar) using a peptide library containing approximately 2.5 billion unique peptides, with a fixed serine as the phosphate acceptor, as substrates for various kinases including p33cdk2-cyclin A and p34cdc2-cyclin B. This method identified a sequence similar to one of the consensus sites as the optimal substrate for p34cdc2-cyclin B, (K/R)SP(R/P)(R/K/H).We have investigated the substrate specificity of p33cdk2 bound to cyclin A or E and of p34cdc2 bound to cyclin A or B using a systematic series of specifically defined peptide substrates appended to the COOH terminus of glutathione S-transferase, constructed by polymerase chain reaction using degenerate oligonucleotides. These substrates allowed us to determine quantitatively the role of the primary sequence of a target site in substrate utilization. Our panel of altered target sites has allowed us to compare the inherent differences in substrate recognition between p33cdk2 and p34cdc2 as well as to examine the effects of the cyclin regulatory subunits on specificity. In addition, we have found that the data generated from these experiments can be used to predict the potential utilization of novel phosphorylation sites.