Substrate Discrimination among Mitogen-activated Protein Kinases through Distinct Docking Sequence Motifs

Abstract

Mitogen-activated protein kinases (MAPKs) mediate cellular responses to a wide variety of extracellular stimuli. MAPK signal transduction cascades are tightly regulated, and individual MAPKs display exquisite specificity in recognition of their target substrates. All MAPK family members share a common phosphorylation site motif, raising questions as to how substrate specificity is achieved. Here we describe a peptide library screen to identify sequence requirements of the DEF site (docking site for ERK FXF), a docking motif separate from the phosphorylation site. We show that MAPK isoforms recognize DEF sites with unique sequences and identify two key residues on the MAPK that largely dictate sequence specificity. Based on these observations and computational docking studies, we propose a revised model for MAPK interaction with substrates containing DEF sites. Variations in DEF site sequence requirements provide one possible mechanism for encoding complex target specificity among MAPK isoforms. Mitogen-activated protein kinases (MAPKs) mediate cellular responses to a wide variety of extracellular stimuli. MAPK signal transduction cascades are tightly regulated, and individual MAPKs display exquisite specificity in recognition of their target substrates. All MAPK family members share a common phosphorylation site motif, raising questions as to how substrate specificity is achieved. Here we describe a peptide library screen to identify sequence requirements of the DEF site (docking site for ERK FXF), a docking motif separate from the phosphorylation site. We show that MAPK isoforms recognize DEF sites with unique sequences and identify two key residues on the MAPK that largely dictate sequence specificity. Based on these observations and computational docking studies, we propose a revised model for MAPK interaction with substrates containing DEF sites. Variations in DEF site sequence requirements provide one possible mechanism for encoding complex target specificity among MAPK isoforms. Mitogen-activated protein kinases (MAPKs) 4The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; WT, wild type; GST, glutathione S-transferase; r.m.s.d., root mean square deviation; MKK, MAPK kinase. lie at the bottom of conserved three-component phosphorylation cascades that integrate cellular responses to a wide variety of extracellular stimuli, including growth factors, cytokines, UV irradiation, and oxidative stress (1Raman M. Chen W. Cobb M.H. Oncogene. 2007; 26: 3100-3112Crossref PubMed Scopus (1173) Google Scholar). Canonical MAPKs are classified into three major subfamilies, extracellular signal-regulated kinases (ERK), p38 MAPK (p38), and c-Jun N-terminal kinases (JNK), based on sequence homology, shared upstream kinases, and activating stimuli. In addition, the different MAPK subfamilies phosphorylate a distinct set of protein substrates. These substrates act as the critical effectors that enable cells to mount the appropriate responses to varied stimuli. Analysis of reported MAPK substrate phosphorylation sites suggests a nearly absolute requirement for a Pro residue immediately downstream of the Ser or Thr phosphoacceptor. Extensive peptide library studies on ERK1, ERK2, and p38α have defined a common phosphorylation site motif for these MAPKs. In addition to the requirement for a Pro residue at the +1 position relative to the phosphorylation site, this motif includes a weak preference for Pro and other aliphatic residues at the -2 position (2Songyang Z. Lu K.P. Kwon Y.T. Tsai L.H. Filhol O. Cochet C. Brickey D.A. Soderling T.R. Bartleson C. Graves D.J. DeMaggio A.J. Hoekstra M.F. Blenis J. Hunter T. Cantley L.C. Mol. Cell. Biol. 1996; 16: 6486-6493Crossref PubMed Scopus (493) Google Scholar, 3Hutti J.E. Jarrell E.T. Chang J.D. Abbott D.W. Storz P. Toker A. Cantley L.C. Turk B.E. Nat. Meth. 2004; 1: 27-29Crossref PubMed Scopus (291) Google Scholar, 4Manke I.A. Nguyen A. Lim D. Stewart M.Q. Elia A.E. Yaffe M.B. Mol. Cell. 2005; 17: 37-48Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Mutagenesis experiments with the JNK substrate JunB have also suggested additional specificity for JNK family MAPKs at positions downstream of the phosphorylation site (5Kallunki T. Deng T. Hibi M. Karin M. Cell. 1996; 87: 929-939Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). MAPKs have affinities for short peptide substrates that are at least an order of magnitude lower than for full-length protein substrates (4Manke I.A. Nguyen A. Lim D. Stewart M.Q. Elia A.E. Yaffe M.B. Mol. Cell. 2005; 17: 37-48Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 6Hawkins J. Zheng S. Frantz B. LoGrasso P. Arch. Biochem. Biophys. 2000; 382: 310-313Crossref PubMed Scopus (29) Google Scholar, 7Fantz D.A. Jacobs D. Glossip D. Kornfeld K. J. Biol. Chem. 2001; 276: 27256-27265Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 8Rainey M.A. Callaway K. Barnes R. Wilson B. Dalby K.N. J. Am. Chem. Soc. 2005; 127: 10494-10495Crossref PubMed Scopus (40) Google Scholar, 9Callaway K.A. Rainey M.A. Riggs A.F. Abramczyk O. Dalby K.N. Biochemistry. 2006; 45: 13719-13733Crossref PubMed Scopus (41) Google Scholar, 10Fernandes N. Bailey D.E. Vanvranken D.L. Allbritton N.L. ACS Chem. Biol. 2007; 2: 665-673Crossref PubMed Scopus (24) Google Scholar). Thus although phosphorylation site motifs likely serve to direct the MAPK to phosphorylate a specific serine or threonine residue within a protein, they are insufficient to fully account for protein substrate recognition. Although mechanisms such as subcellular localization and the use of scaffolding proteins can contribute to kinase substrate targeting in vivo, MAPKs also exhibit a high degree of target specificity in vitro (11Knebel A. Morrice N. Cohen P. EMBO J. 2001; 20: 4360-4369Crossref PubMed Scopus (212) Google Scholar, 12Yang S.H. Galanis A. Sharrocks A.D. Mol. Cell. Biol. 1999; 19: 4028-4038Crossref PubMed Scopus (227) Google Scholar, 13Zhao M. New L. Kravchenko V.V. Kato Y. Gram H. di Padova F. Olson E.N. Ulevitch R.J. Han J. Mol. Cell. Biol. 1999; 19: 21-30Crossref PubMed Scopus (384) Google Scholar). For MAPKs, substrate specificity is ensured the use of docking sequence motifs in the substrate from the phosphorylation site and of the kinase of the site (1Raman M. Chen W. Cobb M.H. Oncogene. 2007; 26: 3100-3112Crossref PubMed Scopus (1173) Google Scholar, Biochem. J. PubMed Scopus Google Scholar, R. T. Chem. 2007; PubMed Scopus Google Scholar). common of docking have MAPKs and their substrates that serve to and specificity. interaction of short sequence motifs within substrates with a or on the kinase. docking site MAPKs is the to as the or of two or residues a short and a of the MAPK and substrate have and T. M. T. Nat. Biol. 2000; 2: PubMed Scopus Google Scholar, B.E. R. Cobb M.H. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Y. J. Chen L. K.A. Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, T. L. J. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. S. A. 2006; Scholar). a on the of the MAPK relative to the site that includes and to the are in ERK, JNK, and p38 substrates and have in specific targeting the different In addition, that MAPKs, including their activating MAPK kinases and MAPK also that are critical for specific with these kinases T. M. T. Nat. Biol. 2000; 2: PubMed Scopus Google Scholar). major MAPK docking site, as the DEF site (docking site for ERK also the in a of ERK substrates. DEF sites are as an and to the phosphorylation site. experiments with in complex with DEF site have the DEF interaction site on ERK to to a to the site T. Y. J. Chen L. K.A. Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). of this to docking sites is to of the kinase. MAPKs are phosphorylation of threonine and residues within a conserved as the R. T. Chem. 2007; PubMed Scopus Google Scholar, B. S. Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). a of the residues for the site, and the DEF site interaction A. Cobb M.H. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). reported substrates DEF sites are factors, including and D. Glossip D. H. A.J. Kornfeld K. 1999; PubMed Scopus Google Scholar, S. Chen Blenis J. Nat. Biol. PubMed Scopus Google Scholar, Blenis J. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar, M. A. A. C. A. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). DEF sites have also in proteins with such as the ERK scaffolding protein D. Glossip D. H. A.J. Kornfeld K. 1999; PubMed Scopus Google the J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google the family protein R. K. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus (41) Google the protein J. S. N. Mol. Cell. Biol. 2005; PubMed Scopus Google and the protein M. A. T. K. N. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In addition to a in substrate DEF sites can also mediate with of ERK, including the and that of the kinase T. Y. J. Chen L. K.A. Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, Y. K. Cobb M.H. S. A. PubMed Scopus Google Scholar). Although as a docking site for ERK MAPKs, the DEF site within the also in to phosphorylation p38α A. S.H. Sharrocks A.D. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). In to phosphorylation site motif, peptide of DEF site for ERK are based on a of sites that have in protein substrates. In the of a the sequence requirements that an DEF site for ERK are is MAPKs other than ERK can with DEF sites in or they recognize distinct DEF site sequence these we peptide library to sequences for kinase docking sites. We in the DEF site docking motifs MAPK family library with a of to identify critical of specificity within the of the kinase. distinct sequence motifs different MAPKs contribute to targeting these kinases to their unique substrates. for library on a and used All to peptide and from the peptide in to a of based on at and at the into into in with and at on a and high at the in and at to peptide and at ERK2, and MAPK sequences from into for as sequences as with the immediately downstream of the site and the DEF site and specificity in these sequences for the S. K. PubMed Scopus Google and J. A.J. T. B. R.J. Mol. Cell. Biol. 1996; 16: PubMed Scopus Google into with their immediately downstream of the site. All sequence of the MAPK and of MAPKs as proteins in with activating kinases to A. S. J. P. Cobb M.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google with the with the and with from and in with and and as A. S. J. P. Cobb M.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Full for protein and are in the site specificity a peptide library as B.E. J.E. Cantley L.C. Nat. 2006; 1: PubMed Scopus Google Scholar). For the DEF site in in with peptide in of and of kinase for at of used to from immediately the in in for to in in and in in the to a screen for and on a phosphorylation for of the as the of the individual signal to the signal for at that DEF site position experiments with DEF site in a with in phosphorylation of MAPKs for individual as Cohen P. Nat. 2006; 1: PubMed Scopus Google is in the docking individual MAPK we the of phosphorylation of for kinase. All p38 and DEF site interaction to of and docking site in vitro a containing the MAPK phosphorylation sites in as a protein D.A. Jacobs D. Glossip D. Kornfeld K. J. Biol. Chem. 2001; 276: 27256-27265Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). and the as the peptide phosphorylation and to with and from or and to protein For as a protein in the sequence encoding the of into the cells as the cells with into containing and for with or for to with containing or as cells with and into in and at and to with the DEF the docking R. Olson A.J. J. Chem. 19: Scopus Google Scholar). the to used to set the docking and the of the docking a and of the of used as the and a used as the of with a of on the on the T. Y. J. Chen L. K.A. Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). We used the the of the with a of set to docking set to the in as the of in set to the of and the of complex into based on their from one the MAPKs a MAPKs are as kinases, in that they have a major preference for Pro at the +1 position at their phosphorylation sites. members of the family have fully with to and in at other positions the phosphorylation site MAPKs from one We targeting of distinct protein substrates different MAPKs to in their phosphorylation site We a peptide library that of the residues and at positions a J.E. Jarrell E.T. Chang J.D. Abbott D.W. Storz P. Toker A. Cantley L.C. Turk B.E. Nat. Meth. 2004; 1: 27-29Crossref PubMed Scopus (291) Google Scholar). this we among of MAPKs and in their phosphorylation site sequences the phosphorylation site motif reported for and with a Ser or Thr an Pro at the +1 position and a preference for Pro at -2 J.E. Jarrell E.T. Chang J.D. Abbott D.W. Storz P. Toker A. Cantley L.C. Turk B.E. Nat. Meth. 2004; 1: 27-29Crossref PubMed Scopus (291) Google Scholar, 4Manke I.A. Nguyen A. Lim D. Stewart M.Q. Elia A.E. Yaffe M.B. Mol. Cell. 2005; 17: 37-48Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). other residues the are with the of Pro at the position and residues at the -2 motif is common in and with phosphorylation site motifs of kinases Z. S. N. Hoekstra M.F. H. Cantley L.C. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). that the phosphorylation site motif to the unique target of the MAPK isoforms. of MAPK to of the kinase site, and targeting specific MAPKs recognition of distinct docking site of the DEF site to the site on that a MAPK phosphorylation site and a DEF site docking sequence can in a peptide D.A. Jacobs D. Glossip D. Kornfeld K. J. Biol. Chem. 2001; 276: 27256-27265Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, D. Glossip D. H. A.J. Kornfeld K. 1999; PubMed Scopus Google Scholar). We a substrate for DEF phosphorylation based on of a ERK phosphorylation site and a DEF site D. Glossip D. H. A.J. Kornfeld K. 1999; PubMed Scopus Google Scholar). In these we varied the sequence the and the the phosphorylation site and DEF docking site. In we the phosphorylation containing an DEF site and with a within the site that ERK phosphorylation to target site other and to of phosphorylation of these at of the DEF site to ERK phosphorylation of the peptide to the sequence of the Pro residue at the -2 position with the phosphorylation than the phosphorylation of and of the DEF site in the of this the phosphorylation an additional residue the Ser and the DEF site the peptide phosphorylation and the DEF site and These suggested that the phosphorylation a DEF site is in peptide substrates a phosphorylation site sequence or a DEF site that residues downstream of the Ser of these into a peptide library to sequences at the DEF site DEF peptide library of in of the positions within the sequence is one at a with the possible in a and to kinase with a the of the a and to the of phosphorylation of peptide We the DEF peptide library with ERK2, the DEF motif peptide library a sequence for the DEF site a preference for residues at positions and and and motif DEF sites in ERK protein and of the of the residues within the DEF site D.A. Jacobs D. Glossip D. Kornfeld K. J. Biol. Chem. 2001; 276: 27256-27265Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, D. Glossip D. H. A.J. Kornfeld K. 1999; PubMed Scopus Google Scholar). Although Pro to the residue at the of other residues at this position a on the of is with the of other residues at this position in DEF sites in and J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, R. K. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Although the with with protein we in DEF site sequences other MAPKs. We the DEF peptide library with a of MAPKs, including p38 isoforms and We that p38α a DEF site motif to ERK2, for residues at the and with a preference for at sites. In aliphatic residues and residues at the a preference at We to such a members of a of MAPKs. the p38 can based on sequence into two of and we that DEF site specificity specificity although these other p38 isoforms are of aliphatic and residues at the and of the in the library with that use this docking site. residues at positions are in of the library are in site motifs for MAPKs at position within the DEF site library are for in as in the to with than are and positions residues to such an are with an with than are in in a DEF for peptide substrates for MAPKs to have high and different members of the and specific MAPK we individual for p38α and to as and These substrates share a common N-terminal sequence with an MAPK phosphorylation site on the from a DEF site sequence to for kinase. of these kinase an order of magnitude than a peptide a DEF site or a peptide with at and of the DEF site p38α and their to of substrate and we to and for We to these for p38α phosphorylation of of and suggests that the of phosphorylation p38α an DEF site is of in and of phosphorylation of and for p38 isoforms at a For the relative of phosphorylation of the two their in the DEF peptide library DEF for of also the requirement for specific DEF site motifs for phosphorylation of the of the DEF site motif residues in from to phosphorylation at a on the of phosphorylation D.A. Jacobs D. Glossip D. Kornfeld K. J. Biol. Chem. 2001; 276: 27256-27265Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, T. Y. J. Chen L. K.A. Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). We that with the sequence also phosphorylation at in in the DEF site sequence for phosphorylation of at as the the at that site. These that specific DEF site sequences are for phosphorylation of MAPK in vitro and in of MAPK DEF of and the p38 isoforms show a high degree of of residues within the DEF defined T. Y. J. Chen L. K.A. Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google of residues the phosphorylation of substrates with DEF sites in vitro and in T. Y. J. Chen L. K.A. Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, W. Blenis J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). We of a of these residues in p38α and and their on phosphorylation of DEF site containing at or in p38α phosphorylation of relative to For of or a although the a phosphorylation to the kinase. We the of these the DEF peptide library are in of in p38α to a of at the position at a for at to the kinase at in at the position for aliphatic at in p38α and at and an of the the residues to and of are conserved in p38α and are with and in and We that these residues for the in and this we and in p38α to and and the and in Analysis with the DEF peptide library that p38α at with a preference for aliphatic including residues a a preference for and a of aliphatic at specificity of the two phosphorylation of the and substrates Although p38α with a preference for the is specific for the the to phosphorylate these experiments a of in the residue residues to and of ERK2, the residue with of p38α and have we docking of a DEF site the DEF interaction site of the of A. Cobb M.H. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). docking with of the on the with root mean of than relative to In to the docking model T. Y. J. Chen L. K.A. Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google to DEF site residues into the this revised model the and residues within the the Pro is to of interaction is with peptide for the and residues with a preference for the Pro at the Based on the we can the distinct DEF site for the different p38 isoforms. the and p38α two and of ERK2, are at the of the and to and of residue into the p38α to have DEF site specificity to with of ERK2, the of in is likely to the of the in the that with the the of the to interaction with aliphatic kinase specificity is in recognition of sequences immediately the site of phosphorylation the kinase site. these phosphorylation site sequence motifs peptide library J.E. Jarrell E.T. Chang J.D. Abbott D.W. Storz P. Toker A. Cantley L.C. Turk B.E. Nat. Meth. 2004; 1: 27-29Crossref PubMed Scopus (291) Google Scholar, Z. S. N. Hoekstra M.F. H. Cantley L.C. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. L. J. Chem. 2004; PubMed Scopus Google Scholar, K. Y. J. Chen L. J. S. S. A. 2004; PubMed Scopus Google Scholar, Chem. 2007; PubMed Scopus Google Scholar, D. L. M. ACS Chem. Biol. 2007; 2: PubMed Scopus Google Scholar). short sequence motifs can at high in as we have for MAPKs, kinases site specificity is insufficient to direct a kinase to a specific protein kinase substrate and specificity is that of the site. protein kinases high protein interaction such as and and among T. Nat. Mol. Biol. 2006; PubMed Scopus Google Scholar). These can a in subcellular localization of the kinase and substrate direct affinities of these protein interaction for peptide to peptide library of direct Z. M. T. F. T. S. R.J. J.E. H. B. Cantley L.C. Cell. Full Text PDF PubMed Scopus Google Scholar, H. Chen S. Cell. Full Text PDF PubMed Scopus Google Scholar, A.E. P. K. D. Cantley L.C. Yaffe M.B. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, N. Elia A.E. T. Cantley L.C. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). weak affinities of docking within the kinase the use of such an for docking specificity. that the phosphorylation site specificity of protein kinases of peptide substrates. peptide substrates with a and a docking site, we have the of peptide to weak docking within the kinase peptide substrates for MAPKs have also N. Bailey D.E. Vanvranken D.L. Allbritton N.L. ACS Chem. Biol. 2007; 2: 665-673Crossref PubMed Scopus (24) Google that this for docking sequence motifs these We have that MAPK family members can with DEF sites and that they exhibit distinct sequence for DEF docking sites. DEF sites have in ERK with a that p38α also can use DEF sites. that DEF site are to these two MAPKs likely from the that the other p38 in and are specific for These isoforms a separate within the p38 with distinct unique protein and A. S. Biophys. 2007; PubMed Scopus Google Scholar). is likely that DEF sites within specific substrates for these isoforms that contribute to their In to the p38 family we an of specificity for within the DEF mean that use this docking site, and on other docking to substrate specificity. is possible that a DEF site sequence specificity that is from the sequence in that with of the within the library that we the residue to in the DEF of is conserved p38 isoforms is a residue in JNK isoforms of this residue the in JNK, or the of the that can the residues in the DEF library is also possible that this of to docking sites on substrates an For the of kinase a with that are in a than B. C. A. R. C. A.D. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, R. M. L.H. EMBO J. PubMed Scopus Google Scholar). of docking sites is to phosphorylation substrate and the of MAPKs for their substrates to from docking than phosphorylation site M.A. Callaway K. Barnes R. Wilson B. Dalby K.N. J. Am. Chem. Soc. 2005; 127: 10494-10495Crossref PubMed Scopus (40) Google Scholar, 9Callaway K.A. Rainey M.A. Riggs A.F. Abramczyk O. Dalby K.N. Biochemistry. 2006; 45: 13719-13733Crossref PubMed Scopus (41) Google Scholar). DEF site have reported to lower the for phosphorylation of ERK and in D.A. Jacobs D. Glossip D. Kornfeld K. J. Biol. Chem. 2001; 276: 27256-27265Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, D. Glossip D. H. A.J. Kornfeld K. 1999; PubMed Scopus Google Scholar). We show for p38α that the of a DEF site the and mean that of the DEF site different for ERK and p38α that on on the substrate that with DEF sites is the kinase and is in the in the of docking to the to the in DEF site to the substrate such that phosphorylation site is in a that docking have also in of MAPK distinct to ERK, JNK, and p38α their to and MAPK B.E. R. Cobb M.H. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Y. J. Chen L. K.A. Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, T. L. J. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. S. A. 2006; Scholar, S. EMBO J. 2004; PubMed Scopus Google Scholar). of the to the MAPK two of the including the and and of the kinase A. Lim 2006; PubMed Scopus Google Scholar). mechanisms substrate is a for MAPK docking and to a of with kinase of docking sites is to the specificity of and sequence requirements for interaction with docking sites can to identify kinase substrates. of proteins in the have at least one of the MAPK phosphorylation site motif and the phosphorylation site motif is insufficient to direct specific protein for MAPK sequences to substrates or for substrates (4Manke I.A. Nguyen A. Lim D. Stewart M.Q. Elia A.E. Yaffe M.B. Mol. Cell. 2005; 17: 37-48Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). of docking sites can substrate targeting specific kinases, and the of docking site motifs to identify substrates computational for the kinase a for the phosphorylation site and docking site sequences to the of substrates M. Cohen C. J. Yaffe M.B. Chem. Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). a of proteins have an DEF sequence within residues downstream of the MAPK phosphorylation site, this is to for MAPK substrates as targeting of MAPKs is of of the ERK, and JNK have for including and A. S. Biophys. 2007; PubMed Scopus Google Scholar, J. 2007; PubMed Scopus Google Scholar, 2005; PubMed Scopus Google Scholar). peptide substrates such as are to high and are for of these into also provide a for M. C. J. ACS Chem. Biol. 2006; 1: PubMed Scopus Google Scholar, J. Chem. Biol. 2005; PubMed Scopus (24) Google Scholar, M. Y. Y. Chem. 2007; PubMed Scopus Google Scholar). In addition, docking an to the site as a for targeting with A. A.D. P. J. Chem. 2005; PubMed Scopus Google Scholar). of in docking specificity the MAPKs mechanisms of substrate also suggests an for of We and for on the We from We for a of protein and for of the site peptide model and the and with