Determination of Substrate Motifs for Human Chk1 and hCds1/Chk2 by the Oriented Peptide Library Approach
2002; Elsevier BV; Volume: 277; Issue: 18 Linguagem: Inglês
10.1074/jbc.m111705200
ISSN1083-351X
AutoresTed B. O'Neill, Lauren Giarratani, Ping Chen, Lakshmanan K. Iyer, Chang Hun Lee, Matthew Bobiak, Fumihiko Kanai, Bin‐Bing S. Zhou, Jay H. Chung, Gary Rathbun,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoMammalian Chk1 and Chk2 are two Ser/Thr effector kinases that play critical roles in DNA damage-activated cell cycle checkpoint signaling pathways downstream of ataxia telangiectasia-mutated and ataxia telangiectasia-related. Endogenous substrates have been identified for human hCds1/Chk2 and Chk1; however, the sequences surrounding the substrate residues appear unrelated, and consensus substrate motifs for the two Ser/Thr kinases remain unknown. We have utilized peptide library analyses to develop specific, highly preferred substrate motifs for hCds1/Chk2 and Chk1. The optimal motifs are similar for both kinases and most closely resemble the previously identified Chk1 and hCds1/Chk2 substrate target sequences in Cdc25C and Cdc25A, the regulation of which plays an important role in S and G2M arrest. Essential residues required for the definition of the optimal motifs were also identified. Utilization of the peptides to assay the substrate specificities and catalytic activities of Chk1 and hCds1/Chk2 revealed substantial differences between the two Ser/Thr kinases. Structural modeling analyses of the peptides into the Chk1 catalytic cleft were consistent with Chk1 kinase assays defining substrate suitability. The library-derived substrate preferences were applied in a genome-wide search program, revealing novel targets that might serve as substrates for hCds1/Chk2 or Chk1 kinase activity. Mammalian Chk1 and Chk2 are two Ser/Thr effector kinases that play critical roles in DNA damage-activated cell cycle checkpoint signaling pathways downstream of ataxia telangiectasia-mutated and ataxia telangiectasia-related. Endogenous substrates have been identified for human hCds1/Chk2 and Chk1; however, the sequences surrounding the substrate residues appear unrelated, and consensus substrate motifs for the two Ser/Thr kinases remain unknown. We have utilized peptide library analyses to develop specific, highly preferred substrate motifs for hCds1/Chk2 and Chk1. The optimal motifs are similar for both kinases and most closely resemble the previously identified Chk1 and hCds1/Chk2 substrate target sequences in Cdc25C and Cdc25A, the regulation of which plays an important role in S and G2M arrest. Essential residues required for the definition of the optimal motifs were also identified. Utilization of the peptides to assay the substrate specificities and catalytic activities of Chk1 and hCds1/Chk2 revealed substantial differences between the two Ser/Thr kinases. Structural modeling analyses of the peptides into the Chk1 catalytic cleft were consistent with Chk1 kinase assays defining substrate suitability. The library-derived substrate preferences were applied in a genome-wide search program, revealing novel targets that might serve as substrates for hCds1/Chk2 or Chk1 kinase activity. ataxia telangiectasia-mutated ataxia telangiectasia-related glutathione S-transferase dithiothreitol kinase-inactive protein kinase C embryonic stem adenosine 5′-(β,γ-imino)triphosphate In the presence of DNA damage or incomplete DNA replication, eukaryotic cells activate cell cycle checkpoints that temporarily halt the cell cycle to permit DNA repair or completion of DNA replication to take place. In the presence of extensive damage or absence of timely repair, these checkpoint signaling pathways may also trigger a pathway that effects programmed cell death or apoptosis (reviewed in Refs. 1Walworth N.C. Curr. Opin. Cell Biol. 2000; 12: 697-704Crossref PubMed Scopus (116) Google Scholarand 2O'Connell M.J. Walworth N.C. Carr A.M. 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ATM and ATR phosphorylation of certain residues within the TQ or SQ sequences modulates mammalian Chk2 and Chk1 kinase activity, respectively (23Rhind N. Russell P. J. Cell Sci. 2000; 113: 3889-3896Crossref PubMed Google Scholar). Analyses utilizing the yeast systems have been critical in understanding the roles of Chk1 and Chk2 in DNA damage responses. InS. pombe, Chk1 is the effector of the G2M DNA damage checkpoint pathway and is downstream of the Rad3 kinase (24Walworth N.C. Bernards R. Science. 1996; 271: 353-356Crossref PubMed Scopus (348) Google Scholar), whereas in S. cerevisiae, Chk1 is downstream of Mec1 (25Sanchez Y. Bachant J. Hu H. Wang F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Crossref PubMed Scopus (454) Google Scholar). 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Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2579-2584Crossref PubMed Scopus (59) Google Scholar). The Chk1 and Chk2 Ser/Thr kinases also play important roles in cell cycle checkpoint signaling pathways in higher organisms (20Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1082) Google Scholar, 35Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (193) Google Scholar). InXenopus and Drosophila, Chk1 functions not only in a checkpoint triggered by UV-damaged DNA but also in an S phase checkpoint triggered by a replication blockade (36Guo Z. Dunphy W.G. Mol. Biol. Cell. 2000; 11: 1535-1546Crossref PubMed Scopus (69) Google Scholar, 37Sibon O.C. Stevenson V.A. Theurkauf W.E. Nature. 1997; 388: 93-97Crossref PubMed Scopus (224) Google Scholar, 38Sibon O.C. Kelkar A. Lemstra W. Theurkauf W.E. Nat. 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Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (193) Google Scholar, 36Guo Z. Dunphy W.G. Mol. Biol. Cell. 2000; 11: 1535-1546Crossref PubMed Scopus (69) Google Scholar, 46Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (853) Google Scholar). Targeted mutation Chk2 and Chk1 in mice have demonstrated profound differences in cellular requirements for the two effector kinases. Both Chk1−/− and Chk2−/− ES cells exhibit defective G2/M DNA damage checkpoint function (35Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (193) Google Scholar,47Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. 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In this context, Chk2 functional loss is not ES cell or T cell lethal (47Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Crossref PubMed Scopus (1044) Google Scholar). Chk2−/− T cells fail to stabilize p53 after ionizing radiation and appear resistant to radiation-induced apoptosis (47Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Crossref PubMed Scopus (1044) Google Scholar). Loss of Chk1 function is ES cell lethal, and during development, Chk1 null cells appear to die of spontaneous apoptosis. Chk2 phosphorylates Ser988 of Brca-1 (49Lee J.S. Collins K.M. Brown A.L. Lee C.H. Chung J.H. Nature. 2000; 404: 201-204Crossref PubMed Scopus (461) Google Scholar) and Ser20 of p53 (50Chehab N.H. Malikzay A. Appel M. Halazonetis T.D. Genes Dev. 2000; 14: 278-288Crossref PubMed Google Scholar, 51Shieh S.Y. Ahn J. Tamai K. Taya Y. Prives C. Genes Dev. 2000; 14: 289-300Crossref PubMed Google Scholar) in response to double strand DNA breaks. Chk1 also phosphorylates Ser20 of p53 in vitro (47Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Crossref PubMed Scopus (1044) Google Scholar, 50Chehab N.H. Malikzay A. Appel M. Halazonetis T.D. Genes Dev. 2000; 14: 278-288Crossref PubMed Google Scholar, 51Shieh S.Y. Ahn J. Tamai K. Taya Y. Prives C. Genes Dev. 2000; 14: 289-300Crossref PubMed Google Scholar). Phosphorylation of Brca-1 by Chk2 in response to DNA damage is required for survival after DNA damage. Phosphorylation of p53 Ser20 blocks the ability of Mdm2 to complex with p53 and shunt the latter into a degradation pathway, allowing the G1 checkpoint to be activated by p53 (52Caspari T. Curr. Biol. 2000; 10: R315-R317Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar).In vitro, Chk1 and Chk2 both phosphorylate Ser216 of Cdc25C. Chk2 also phosphorylates Ser123 in Cdc25A after double-stranded DNA breaks in an ATM-dependent manner resulting in a replication stage S phase cell cycle checkpoint (53Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (868) Google Scholar). Chk1 has been shown to phosphorylate Cdc25A, Cdc25B, and Cdc25C in vitro (54Sanchez Y. Wong C. Thoma R.S. Wu R. Richman Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1121) Google Scholar). Whereas the substrate motifs in Cdc25C and Cdc25A are similar, the motifs of other endogenous substrates targeted by Chk2 and Chk1 appear unrelated (see Table I). Thus, the two Ser/Thr kinases are considered "versatile" protein kinases with a potentially wide range of acceptable substrate targets (51Shieh S.Y. Ahn J. Tamai K. Taya Y. Prives C. Genes Dev. 2000; 14: 289-300Crossref PubMed Google Scholar). We have utilized an oriented, degenerate peptide library approach to determine clear, unambiguous substrate specificities for human Chk2 and Chk1. Library-derived optimal peptides were compared with peptides representing the endogenous substrates in terms of Chk2 and Chk1 phosphorylation activities and utilized as probes to show both strong similarities as well as striking differences between the two Ser/Thr kinases. The substrate motifs established from peptide library analyses were also used in a genome-wide search program in an attempt to identify additional potential targets that might serve as novel substrates for Chk2 or Chk1.Table IEndogenous substrate targets of Chk2 and Chk1 lack a common motifCdc25CRSGLYRSPS216MPENLCdc25ACSPLKRSHS123DSLDHBrca-1 IPPLFPIKS988FVKTKp53 PPLSQETFS20DLWKLThe Cdc25C and Cdc25A isotypes contain motifs that are similar to each other but are dissimilar to Brca-1 and p53. Open table in a new tab The Cdc25C and Cdc25A isotypes contain motifs that are similar to each other but are dissimilar to Brca-1 and p53. Full-length Chk1 was subcloned into baculovirus expression vector pFASTBAC with glutathione S-transferase (GST) fused to N terminus of Chk1 via a linker containing a thrombin cleavage site. Spodoptera frugiperda (Sf9) cells expressing GST-Chk1 were scaled up, harvested, and then frozen until purification. To purify Chk1, a frozen cell pellet was resuspended on ice in lysis buffer containing 50 mm Tris-HCl, pH 7.5, 250 mm NaCl, 1 mm DTT, 0.1% Brij, a protease inhibitor mixture (2 μg/ml E64, 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, and 1 μg/ml pepstatin A), and 1 mm sodium orthovanadate, homogenized with a Tekmar tissuemizer, and disrupted through Microfluid fluidizer (M110-Y). The extracts were centrifuged at 100,000 × g for 30 min. The supernatant was added to glutathione-Sepharose 4B (Amersham Biosciences AB) beads equilibrated in wash buffer (20 mm Tris-HCl, pH 7.0, 10 mmMgCl2, 100 mm NaCl, 1 mm DTT, protease mixture) and rocked at 4 °C for 30 min. The suspension was transferred into a column and allowed to pack; the wash buffer was then applied extensively. The GST-Chk1 was eluted from the column with 10 mm glutathione in 50 mm Tris-HCl, pH 8.0, collected, and dialyzed into 25 mm HEPES, pH 7.5, 50 mm KCl, and 5 mm DTT. GST-Chk1 sample was further purified by Superdex 200 (equilibrated with the dialysis buffer), at 4 °C, and more than 90% purity was estimated based on SDS-PAGE and silver staining analysis (Silver Stain Plus; Bio-Rad). Recombinant human Chk2 was produced in baculovirus using a MaxBac2.0 transfection kit (Invitrogen) as described previously (18Brown A.L. Lee C.H. Schwarz J.K. Mitiku N. Piwnica-Worms H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3745-3750Crossref PubMed Scopus (237) Google Scholar). ST-Chk2 protein from the lysate was then purified using a StrepTactin-Sepharose column (Genosys Biotechnologies, Inc.). Briefly, the lysate (∼15 ml) was applied to the column (1 ml) in buffer containing 100 mm Tris-HCl, pH 8.0, 1 mm EDTA. After washing the column five times with the same buffer, Strep-tagged Cds1 was eluted with a buffer containing 2.5 mm desthiobiotin, 100 mm Tris-HCl, pH 8.0, 1 mm EDTA. Purity of the eluate was assessed by silver staining using Silver Stain Plus (Bio-Rad). Peptide libraries utilized in this analysis to derive consensus substrate motifs for Cds1 and Chk1 were designated as follows: RXXS, MAXXXXRXXSXXXXAKKK; SI, MAXXXXXSIAKKK; SF, MAXXXXXSFXXXXAKKK; SQ, MAXXXXXXSQXXXXAKKK; SP, MAXXXXSPXXXXAKKK; 4S4+ or 4S4−, MAXXXXSAKKK; 4T4+ or 4T4−, MAXXXXTXXXXAKKK; 4Y4+ or 4Y4−, MAXXXXYXXXXAKKK. Degenerate positions in the sequences are represented by X in which all amino acids were represented with the exception of Cys; + or − indicates the presence or absence of Tyr, Ser, and Thr at the degenerate positions. To assay peptide libraries with maximum degeneracy at non-fixed positions, and thus provide the truest preference values, we analyzed libraries that included Ser, Thr, and Tyr and compared the results to those with only the fixed target residue for phosphorylation by Chk1 and Chk2. We saw no striking differences between the two sets of libraries with fixed central Ser or Thr (see for example, Fig. 1B, and data not shown). Inclusion of Ser and Thr in the fixed Tyr library enhanced the phosphorylation of the 4Y4 library by Chk1 and Chk2 (data not shown) indicating preferences for aromatic hydrophobic residues N- or C-terminal to phosphorylated Ser or Thr. The reaction buffer for Chk2 consisted of a final concentration of 10 mm HEPES, pH 7.5, 75 mm KCl, 10 mm MgCl2 0.5 mm EDTA, 1.25 mm DTT, 100 μm cold ATP, 10 μCi of [γ-32P]ATP, 100 ng of recombinant human Chk2 in a 30-μl total reaction volume at 30 °C. Small scale peptide library analyses were conducted utilizing 100 μg of each library. To assay Chk1 phosphorylation activity, the reaction buffer utilized consisted of 20 mm HEPES, pH 7.5, 50 mm KCl,10 mm MgCl2, 0.1 mm EGTA, 100 μm cold ATP, 10 μCi of [γ-32P]ATP, 50–100 ng of Chk1 in a total volume of 30 μl at 30 °C. To monitor peptide phosphorylation by Chk2 or Chk1, 2 μl of the reaction mix was transferred to an Eppendorf tube containing an equal volume of 30% acetic acid to halt the reaction, and 2.5–3.0 μl were spotted onto to P-81 phosphocellulose paper squares and allowed to air dry. These samples were washed three times in 1% o-phosphoric acid (5 min per wash), placed in scintillation vials, and counted. Peptide phosphorylation experiments including kinetic assays and relative velocities of Chk2 and Chk1 were reproduced multiple times at several concentrations. Large scale analyses to determine preferred substrate motifs for Chk2 and Chk1 were performed using the RXXS, SF, SI, and SQ peptide libraries. Large scale peptide library assays were scaled up 10-fold and repeated at least twice for each library using the methods described previously (55Songyang 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 (531) Google Scholar, 56O'Neill T. Dwyer A.J. Ziv Y. Chan D.W. Lees-Miller S.P. Abraham R.H. Lai J.H. Hill D. Shiloh Y. Cantley L.C. Rathbun G.A. J. Biol. Chem. 2000; 275: 22719-22727Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Approximately 1% of each library was phosphorylated in the appropriate reaction conditions in the presence of 300 μmATP and 0.33 μCi of [γ-32P]ATP; the kinase reaction was terminated by the addition of acetic acid, and the reaction was desalted and partially purified using a 1 ml of DEAE-Sephacel (Sigma) column. Radiolabeled fractions were pooled, lyophilized, reconstituted in distilled H2O, and applied to a ferric chelation nitrilotriacetic acid-agarose column as described previously (56O'Neill T. Dwyer A.J. Ziv Y. Chan D.W. Lees-Miller S.P. Abraham R.H. Lai J.H. Hill D. Shiloh Y. Cantley L.C. Rathbun G.A. J. Biol. Chem. 2000; 275: 22719-22727Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 57Songyang Z. Cantley L.C. Methods Mol. Biol. 1998; 87: 87-98PubMed Google Scholar). Eluted phosphopeptides were pooled, lyophilized, reconstituted in distilled H2O, and sequenced. To determine amino acid preferences at each position in the sequenced peptides, each cycle was first internally normalized for all amino acids present at that cycle using a program designed to analyze relative amino acid abundance at each cycle (56O'Neill T. Dwyer A.J. Ziv Y. Chan D.W. Lees-Miller S.P. Abraham R.H. Lai J.H. Hill D. Shiloh Y. Cantley L.C. Rathbun G.A. J. Biol. Chem. 2000; 275: 22719-22727Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). To establish whether Chk1 and Chk2 exhibit preferences for distinct substrate motifs, we assayed phosphorylation of several different fixed, degenerate peptide libraries by the two Ser/Thr kinases. We included the most degenerate libraries fixed at only a central Ser, Thr, or Tyr (i.e.4S4, 4T4 and 4Y4, respectively) as part of an unbiased survey of Chk1 and Chk2 target phosphorylation preferences. Fig.1A shows that Chk2 prefers the RXXS peptide library that contains a fixed Arg at the −3-position (N-terminal) relative to the fixed Ser targeted by Chk2 kinase activity (Fig. 1A). Ser appears more highly selected than Thr as a target for phosphorylation by Chk2 in the context of the peptide libraries. A second tier of strongly selected libraries is that with hydrophobic residues (Phe and Ile) fixed at the +1-position (C-terminal) relative to Ser, as well as a degenerate library with a central SQ sequence (Fig. 1A). A library with Pro at +1 relative to Ser was not significantly phosphorylated by Chk2 (Fig.1A). Consistent with Chk2, Chk1 also selected the RXXS library but diverged from Chk2 in that the SI peptide library appeared equivalently phosphorylated compared with the RXXS library (Fig. 1B). The SF peptide library is also highly preferred; therefore, similar to Chk2, Chk1 also selects a hydrophobic residue at the +1-position. The SQ library (Fig. 1B) is modestly selected by Chk1; similar to Chk2, the SP library is less preferred and Chk1 also appeared to prefer Ser over Thr as the residue targeted for phosphorylation. Our results indicate that the −3- and +1-positions relative to a Ser targeted for phosphorylation appear to constitute a minimum selected motif for both Chk1 and Chk2. Fig. 2, A and B,shows that the kinase-inactive (KI) versions of Chk2 and Chk1 failed to phosphorylate the RXXS peptide library, indicating that phosphorylation was carried out by the catalytic site of Chk1 and Chk2 and not that of an associated enzyme. Chk2 KI continued to express an apparent low level autophosphorylation activity (data not shown). There was no detectable Ser/Thr autophosphorylation of Chk1 KI. The results shown in Fig. 1, A and B, indicated that we could utilize data from several unrelated peptide libraries in large scale analyses to develop optimal substrate motifs for Chk2 and Chk1. We assayed the most highly selected RXXS library to determine whether there was a strong selection for residues at positions in addition to the fixed Arg at position −3 as well as to confirm the predilection for hydrophobic residues at the C-terminal +1-position adjacent to Ser. To verify the strong preference of Chk1 and Chk2 for Arg or a basic residue at position −3, we employed peptide libraries fixed at the +1-position but degenerate at −3. For these latter analyses we assayed the SI, SF, and SQ libraries. An amino acid was considered strongly selected or preferred at a given cycle if its normalized value was greater than 1.00 (base line) (shown in Tables II andIII).Table IISelected amino acids at positions N- and C-terminal to the phosphorylated serine in degenerate peptide libraries that were quantitatively phosphorylated by Chk2Position −7Position −6Position −5Position −4Position −3Position −2Position −1Position +1Position +2Position +3Position +4RXXS+Arg, 1.15Met, 1.04Leu, 1.40XaaArgXaaGlu, 1.50Ser
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