Hydrophobic as Well as Charged Residues in Both MEK1 and ERK2 Are Important for Their Proper Docking

Abstract

Docking between MEK1 and ERK2 is required for their stable interaction and efficient signal transmission. The MEK1 N terminus contains the ERK docking or D domain that consists of conserved hydrophobic and basic residues. We mutated the hydrophobic and basic residues individually and found that loss of either type reduced MEK1 phosphorylation of ERK2 in vitroand its ability to bind to ERK2 in vivo. Moreover, ERK2 was localized in both the cytoplasm and the nucleus when co-expressed with MEK1 that had mutations in either the hydrophobic or the basic residues. We then identified two conserved hydrophobic residues on ERK2 that play roles in docking with MEK1. Mutating these residues to alanine reduced the interaction of ERK2 with MEK1 in cells. These mutations also reduced the phosphorylation of MEK1 by ERK2 but had little effect on phosphorylation of MBP by ERK2. Finally, we generated docking site mutants in ERK2-MEK1 fusion proteins. Although the mutation of the MEK1 D domain significantly reduced ERK2-MEK1 activity, mutations of the putatively complementary acidic residues and hydrophobic residues on ERK2 did not change its activity. However, both types of mutations decreased the phosphorylation of Elk-1 caused by ERK2-MEK1 fusion proteins. These findings suggest complex interactions of MEK1 D domains with ERK2 that influence its activation and its effects on substrates. Docking between MEK1 and ERK2 is required for their stable interaction and efficient signal transmission. The MEK1 N terminus contains the ERK docking or D domain that consists of conserved hydrophobic and basic residues. We mutated the hydrophobic and basic residues individually and found that loss of either type reduced MEK1 phosphorylation of ERK2 in vitroand its ability to bind to ERK2 in vivo. Moreover, ERK2 was localized in both the cytoplasm and the nucleus when co-expressed with MEK1 that had mutations in either the hydrophobic or the basic residues. We then identified two conserved hydrophobic residues on ERK2 that play roles in docking with MEK1. Mutating these residues to alanine reduced the interaction of ERK2 with MEK1 in cells. These mutations also reduced the phosphorylation of MEK1 by ERK2 but had little effect on phosphorylation of MBP by ERK2. Finally, we generated docking site mutants in ERK2-MEK1 fusion proteins. Although the mutation of the MEK1 D domain significantly reduced ERK2-MEK1 activity, mutations of the putatively complementary acidic residues and hydrophobic residues on ERK2 did not change its activity. However, both types of mutations decreased the phosphorylation of Elk-1 caused by ERK2-MEK1 fusion proteins. These findings suggest complex interactions of MEK1 D domains with ERK2 that influence its activation and its effects on substrates. mitogen-activated protein kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase MEK kinase extracellular signal-regulated kinase common docking myelin basic protein hemagglutinin epidermal growth factor glutathione S-transferase ribosomal S6 kinase MAP kinase-interacting kinase MAP1 kinase cascades are found in organisms ranging from yeast to human (1Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 2Pearson G. Gibson T.B. Xu B. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3543) Google Scholar, 3Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1404) Google Scholar). A typical MAP kinase pathway consists of at least three kinases: a MEKK that phosphorylates and activates the downstream kinase MEK; a MEK that phosphorylates and activates the MAP kinase; and the MAP kinase that has a variety of targets. The most studied mammalian MAP kinase pathway is the Raf/MEK/ERK pathway. Upon activation of the pathway, Raf-1 phosphorylates two serine residues in the activation loop of MEK1/2, and MEK1/2 in turn will phosphorylate a threonine and a tyrosine in the activation loop of ERK1/2. MAP kinase docking domains have been identified in ERK1/2 substrates, including transcription factors and protein kinases. The most thoroughly characterized MAP kinase docking domain is the D domain originally identified in the ternary complex factor Elk-1 (4Yang S.H. Yates P.R. Whitmarsh A.J. Davis R.J. Sharrocks A.D. Mol. Cell. Biol. 1998; 18: 710-720Crossref PubMed Scopus (233) Google Scholar). These docking domains have now been recognized not only in substrates but also in the upstream kinases, MEK1 and MEK2, and in dual specificity and phosphotyrosine phosphatases (5Sharrocks A.D. Yang S.H. Galanis A. Trends. Biochem. Sci. 2000; 25: 448-453Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 6Holland P.M. Cooper J.A. Curr. Biol. 1999; 9: R329-R331Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The typical D domain contains a cluster of basic residues followed by two conserved hydrophobic residues (e.g. KKKPTPIQL in MEK1). The MEK1 D domain is required for efficient phosphorylation and activation of ERK2 and for interaction with ERK2 in cells (7Fukuda M. Gotoh Y. Nishida E. EMBO J. 1997; 16: 1901-1908Crossref PubMed Scopus (331) Google Scholar, 8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 9Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (677) Google Scholar). Deletion of the MEK1 D domain decreases not only the feedback phosphorylation of MEK1 by ERK2 but also the phosphorylation of ERK2 by MEK1 in vitro (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). D domains are also necessary for inactivation of MAP kinases by some of the phosphatases. The protein kinase substrates of MAP kinases including RSKs and MNKs contain variants of the D domain motif, having a typical sequence LAQRR (5Sharrocks A.D. Yang S.H. Galanis A. Trends. Biochem. Sci. 2000; 25: 448-453Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar,10Smith J.A. Poteet-Smith C.E. Malarkey K. Sturgill T.W. J. Biol. Chem. 1999; 274: 2893-2898Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 11Smith J.A. Poteet-Smith C.E. Lannigan D.A. Freed T.A. Zoltoski A.J. Sturgill T.W. J. Biol. Chem. 2000; 275: 31588-31593Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 12Gavin A.C. Nebreda A.R. Curr. Biol. 1999; 9: 281-284Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). This domain on RSK interacts with ERK2 directly and is required for intracellular recognition by ERK2. Another ERK docking motif found in transcription factors such as Elk-1, SAP-1, and LIN-1 is the FXFP sequence (13Jacobs D. Glossip D. Xing H. Muslin A.J. Kornfeld K. Genes Dev. 1999; 13: 163-175Crossref PubMed Scopus (439) Google Scholar). It can work by itself or together with a D domain to mediate kinase-substrate interactions. Different experimental approaches have revealed multiple domains on ERKs that are involved in docking interactions with MEKs and substrates. Two groups independently identified a common docking (CD) motif, found in all known MAP kinase family members, that mediates interactions with MEKs, MAP kinase phosphatase 3, and the protein kinase substrate MNK1 (9Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (677) Google Scholar, 14Rubinfeld H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: 30349-30352Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The CD domain is located near the C termini of MAP kinases and contains two acidic residues that are thought to interact directly with the basic residues in D domains. Several studies with MAP kinase chimeras suggested that ERK1/2 residues in the N-terminal folding domain of the kinase are also important; these include the C helix and the ERK2 N terminus (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 15Brunet A. Pouysségur J. Science. 1996; 272: 1653-1655Crossref Scopus (119) Google Scholar, 16Wilsbacher J.L. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 1999; 274: 16988-16994Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Weber and co-workers (17Eblen S.T. Catling A.D. Assanah M.C. Weber M.J. Mol. Cell. Biol. 2001; 21: 249-259Crossref PubMed Scopus (52) Google Scholar) have also recently reported that a short sequence at the ERK2 N terminus is involved in MEK binding. In this study, we examined the importance of the conserved hydrophobic and basic residues in the MEK1 D domain to its ability to phosphorylate ERK2 in vitro and interact with ERK2 in vivo. In addition, two tyrosine residues near the CD domain on ERK2 were identified as being important for MEK1 interaction. We also tested the effects of the docking site mutations on the activation and activity of ERK2-MEK1 fusion proteins (18Robinson M.J. Stippec S.A. Goldsmith E. White M.A. Cobb M.H. Curr. Biol. 1998; 8: 1141-1150Abstract Full Text Full Text PDF PubMed Google Scholar). pCMV5-Myc-MEK1, pCEP-HA-ERK2, pCEP-HA-MEK1, and pCMV5-MEK1-R4F were as described (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 19Dang A. Frost J.A. Cobb M.H. J. Biol. Chem. 1998; 273: 19909-19913Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). pCMV5-Myc-ERK2-MEK1 and pCMV5-Myc-ERK2-MEK1(LA) were kindly provided by Megan Robinson (18Robinson M.J. Stippec S.A. Goldsmith E. White M.A. Cobb M.H. Curr. Biol. 1998; 8: 1141-1150Abstract Full Text Full Text PDF PubMed Google Scholar). pCMV5-Myc-ERK2 was provided by Jeff Frost (University of Texas, Houston, TX). pCMV5-Ras-V12, and pCMV5-His-Elk-1 were as described (18Robinson M.J. Stippec S.A. Goldsmith E. White M.A. Cobb M.H. Curr. Biol. 1998; 8: 1141-1150Abstract Full Text Full Text PDF PubMed Google Scholar). Site-directed mutagenesis was performed using the QuikChange kit (Stratagene) according to the manufacturer's instructions. The mutants used in this study are listed in TableI.Table IMutants used in this studyProteinNameMutationsMEK1ILAAI9A and L11AKKMMK3M and K4MAAMMI9A, L11A, K3M, and K4MERK2YYAAY314A and Y315ADDAAD316A and D319AAAAD316A, D319A, and E320A5AY314A, Y315A, D316A, D319A, and E320A Open table in a new tab GST-ERK2 (K52R) and His-MEK1 proteins were as described (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). His-MEK1 and were kindly provided by Ahn (University of basic protein was from was from the Cell was from the and were from complex kinase of proteins were from with the and protein were then three in and in followed by kinase MEK1 or ERK2 proteins were from of with the and of protein The were then three in and by was performed as described B. Wilsbacher J.L. Stippec Goldsmith E.J. Cobb M.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, Cobb M.H. Cell 1999; PubMed Scopus Google Scholar). cells were on with either or and used were was then used followed by to the were performed in of kinase at for the (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The were by of followed by for The were by and were as described using (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). cells were and as described (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). was in the for in in was to the The ERK2 was on the using the The MEK1 N terminus contains ERK docking or D this domain is also in phosphatases and substrates of ERK1/2 (5Sharrocks A.D. Yang S.H. Galanis A. Trends. Biochem. Sci. 2000; 25: 448-453Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). the MEK1 D a cluster of basic residues and two hydrophobic residues are conserved MEKs we that of the MEK1 N including the D reduced its ability to phosphorylate ERK2 in vitro (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In the study, we have examined the of these conserved residues in the MEK1 D domain by mutants cells were with type MEK1 or MEK1 The cells either were or were with to MEK1 was then using the and its kinase activity was using GST-ERK2 (K52R) as the substrate the of type MEK1 with the MEK1 mutants ERK2 in However, both the and the reduced activity either with or that both the conserved hydrophobic residues and the basic residues in the MEK1 D domain are required for efficient ERK2 These are to with MEK1 the D domain (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). We that a MEK1 N-terminal that contains the D domain was to the feedback phosphorylation of MEK1 by ERK2 (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). we examined this MEK1 phosphorylation of ERK2. MEK1 was used to phosphorylate GST-ERK2 (K52R) in the of of or the the D domain sequence of ERK2 phosphorylation by MEK1 was with the at was to was with the feedback phosphorylation (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). the the had little effect on MEK1 phosphorylation of ERK2 at the In had effect on the ability of not contain the D to phosphorylate GST-ERK2 (K52R) in vitro not These that a D domain MEK1 phosphorylation of ERK2. The N-terminal D domain of MEK1 is both required and for to ERK2 (7Fukuda M. Gotoh Y. Nishida E. EMBO J. 1997; 16: 1901-1908Crossref PubMed Scopus (331) Google Scholar, 9Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (677) Google Scholar). We that a MEK1 N-terminal to with ERK2 (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). we tested the ability of the MEK1 and mutants to with ERK2. cells were with and type or the MEK1 MEK1 proteins were with the and the of was examined by the with the Although type MEK1 with both the and the a decreased ability to with ERK2 These that both the hydrophobic and the basic residues in the MEK1 D domain are required for its with ERK2. ERK2 in the nucleus (7Fukuda M. Gotoh Y. Nishida E. EMBO J. 1997; 16: 1901-1908Crossref PubMed Scopus (331) Google Scholar, 14Rubinfeld H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: 30349-30352Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, S.T. Catling A.D. Assanah M.C. Weber M.J. Mol. Cell. Biol. 2001; 21: 249-259Crossref PubMed Scopus (52) Google Scholar). MEK1 can ERK2 to a in cells. the MEK1 D domain mutants were in with we tested to ERK2 to in the cells were with and either the or the type and The of was by using the In the of ERK2 was in both cytoplasm and nucleus as In the of type ERK2 was in the with the ability of MEK1 to ERK2 from the However, in the of either the MEK1 or the MEK1 ERK2 was the with a in the The is to that in cells in MEK1 was not These that both the hydrophobic and basic residues are necessary for MEK1 to ERK2 from the nucleus in cells. Several on ERK2 have been identified as being important for MEK1 interaction (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 9Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (677) Google Scholar, 14Rubinfeld H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: 30349-30352Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 15Brunet A. Pouysségur J. Science. 1996; 272: 1653-1655Crossref Scopus (119) Google Scholar, 16Wilsbacher J.L. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 1999; 274: 16988-16994Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, S.T. Catling A.D. Assanah M.C. Weber M.J. Mol. Cell. Biol. 2001; 21: 249-259Crossref PubMed Scopus (52) Google Scholar). the CD domain near the C terminus of ERK2 that contains two acidic residues and has been to interact directly with the basic residues in the MEK1 D domain (9Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (677) Google Scholar, 14Rubinfeld H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: 30349-30352Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). the two hydrophobic residues in the MEK1 D domain are important for we that hydrophobic residues near the CD domain on ERK2 that play roles in docking with MEK1. the sequence of ERK2 and MAP kinases, we that and in ERK2 are residues with or in all MAP kinase family The of ERK2 that the of both and are on the A. D. Cobb M.H. Goldsmith E.J. PubMed Scopus Google not the that involved in with proteins and were mutated to alanine to the ERK2 The CD domain residues and with also mutated to alanine to the ERK2 and were mutated to alanine to the ERK2 Finally, all residues were mutated to alanine to the ERK2 type or ERK2 mutants were cells with ERK2 proteins were with the and the of in the ERK2 was examined by with the type ERK2 a of MEK1 In all three ERK2 mutants tested in the MEK1 from the These findings suggest that both and are important for with MEK1 in cells. We examined the effects of these mutations on the kinase activity of ERK2. type and were cells with a of MEK1. ERK2 proteins were then with the and for their kinase activity using either MBP or His-MEK1 as substrates type ERK2 and the ERK2 mutants activity that all of the ERK2 mutants were and in cells by MEK1. His-MEK1 was used as the the a but decreased kinase activity with that by type ERK2. Although the a decreased the had activity MEK1. were when from or cells were used not These that not only the acidic residues of the CD domain but also the tyrosine residues near the CD domain are important for the interaction of ERK2 with MEK1. the docking interaction in the proteins are we mutations ERK2-MEK1 fusion proteins. A of ERK2 was by ERK2 to MEK1 (18Robinson M.J. Stippec S.A. Goldsmith E. White M.A. Cobb M.H. Curr. Biol. 1998; 8: 1141-1150Abstract Full Text Full Text PDF PubMed Google Scholar). A of the fusion protein ERK2-MEK1 residues in the MEK1 sequence with has activity the type fusion docking domain mutations the ERK2-MEK1 fusion we fusion protein mutants in both the MEK1 D domain and the ERK2 CD ERK2-MEK1 type and were from and for kinase activity using MBP as the that effects of ERK2 activation using a substrate that not contain a D domain MEK1 mutations reduced ERK2-MEK1 activity, the mutations reduced its activity These that the docking interaction is required for ERK2 activation when ERK2 and MEK1 are to the ERK2 the mutations had effect on ERK2 activity in the ERK2-MEK1 from these activity we also performed on the ERK2-MEK1 fusion proteins with the of protein were with from the kinase with MBP as were with both ERK2-MEK1 and ERK2-MEK1 fusion proteins and these docking site mutations in ERK2-MEK1 fusion proteins their ability to phosphorylate and the ternary complex transcription factor Elk-1, type and ERK2-MEK1 fusion were cells with that Elk-1 on was used to the of Elk-1 ERK2-MEK1 caused activation of Elk-1, the ERK2-MEK1 is from the had effect on Elk-1 (18Robinson M.J. Stippec S.A. Goldsmith E. White M.A. Cobb M.H. Curr. Biol. 1998; 8: 1141-1150Abstract Full Text Full Text PDF PubMed Google Scholar). In both the MEK1 D domain mutants and and the ERK2 mutants and decreased Elk-1 activation caused by ERK2-MEK1 the fusion proteins with D domain mutations contain ERK2 that is the type ERK2-MEK1 the reduced phosphorylation of Elk-1 a of reduced ERK2 activity in these fusion proteins. However, the of the fusion proteins the ERK2 mutations and are the as that of type ERK2-MEK1 their reduced Elk-1 suggest that these mutations the interaction of ERK2 with docking between MAP kinases and their substrates has as a to efficient and signal (5Sharrocks A.D. Yang S.H. Galanis A. Trends. Biochem. Sci. 2000; 25: 448-453Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 6Holland P.M. Cooper J.A. Curr. Biol. 1999; 9: R329-R331Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The recognition of the on substrates by MAP kinases also the specificity of pathway J.A. Poteet-Smith C.E. Lannigan D.A. Freed T.A. Zoltoski A.J. Sturgill T.W. J. Biol. Chem. 2000; 275: 31588-31593Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). It that multiple on MAP kinases are involved in substrate recognition and interactions (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (677) Google Scholar, 14Rubinfeld H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: 30349-30352Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 15Brunet A. Pouysségur J. Science. 1996; 272: 1653-1655Crossref Scopus (119) Google Scholar, 16Wilsbacher J.L. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 1999; 274: 16988-16994Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, S.T. Catling A.D. Assanah M.C. Weber M.J. Mol. Cell. Biol. 2001; 21: 249-259Crossref PubMed Scopus (52) Google Scholar, T. R. Adachi M. Nishida E. EMBO J. 2001; PubMed Scopus Google Scholar). we that both the hydrophobic and basic residues conserved in the MEK1 D domain are required for its efficient phosphorylation of ERK2 in A MEK1 N-terminal the D domain motif not only the feedback phosphorylation of MEK1 by also MEK1 phosphorylation of ERK2 in that either the hydrophobic or the basic residues are in to ERK2 in as by with type MEK1. Although type MEK1 is to ERK2 in the cytoplasm of these MEK1 mutants We also identified two conserved hydrophobic residues near the CD domain of ERK2 that to the interaction of ERK2 with MEK1. mutation of the identified acidic residues or these hydrophobic residues to MEK1 with that by type ERK2. both types of mutants phosphorylate MEK1 in The suggest that both hydrophobic and interactions are involved in The hydrophobic residues we identified in tyrosine and are in to the CD domain residues and in the and their are H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: 30349-30352Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) identified the CD domain on ERK2 as a a with the that has alanine for residues the that has alanine only for the three acidic and that are residues between and involved in MEK1 interaction. findings are with their and interact directly with the hydrophobic residues in the MEK1 D docking either by interaction with residues on MEK1 or by the on ERK2 that the docking ERK2 also with its substrates and phosphatases that the that MEK1. reduced phosphorylation of Elk-1 by the ERK2-MEK1 fusion protein in these tyrosine residues were mutated suggest that these hydrophobic residues are also important for substrate interactions. It will to the to and interact with D docking on proteins. RSK contains a docking domain from MEK1 or Elk-1, characterized as the LAQRR motif, and Elk-1 also contains the FXFP docking motif (5Sharrocks A.D. Yang S.H. Galanis A. Trends. Biochem. Sci. 2000; 25: 448-453Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). the of MAP kinases ranging from yeast to we found that residues to and in ERK2 are conserved in that all have at these It is to that the residues in MAP kinases also involved in docking with their The of that and are also near the two in the CD and their are R.J. J. Cobb M.H. Goldsmith E.J. Sci. A. 1997; PubMed Scopus Google and M. have to and on ERK2. but from studies using ERK2-MEK1 fusion proteins. in the MEK1 D domain in a of ERK2-MEK1 activity, mutations in the ERK2 CD domain had little the fusion ERK2 and MEK1 are their is not The that MEK1 D domain mutations decreased the fusion protein activity that the of docking is for the efficient phosphorylation and activation of ERK2. the mutation of and mutation of and of ERK2 in the fusion proteins their activity. These suggest that the D is the docking site on the CD domain and its on ERK2 are not for this of the of multiple docking domains on ERK2. with this and from groups that the ERK2 CD domain mutants are and in cells in and (9Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (677) Google Scholar, 14Rubinfeld H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: 30349-30352Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). from and Ahn Ahn N.G. 1998; PubMed Scopus Google Scholar) using that the MEK1 N terminus has the MEK1 D domain to the MEK1 N terminus that of the D domain have a effect on In the ERK2 is that of the CD domain not docking in the fusion protein and is are domains or residues on ERK2 necessary for docking with MEK1. Nishida and co-workers T. R. Adachi M. Nishida E. EMBO J. 2001; PubMed Scopus Google Scholar) recently reported the of the domain on and ERK2 near the substrate that mediates docking interactions with substrates with the CD However, did not the interaction with (17Eblen S.T. Catling A.D. Assanah M.C. Weber M.J. Mol. Cell. Biol. 2001; 21: 249-259Crossref PubMed Scopus (52) Google Scholar) recently that residues at the ERK2 N terminus are required for interaction with of chimeras also suggested in the N-terminal domain of MAP kinases that are necessary for interactions with MEKs (8Xu B. Wilsbacher J.L. Collisson T. Cobb M.H. J. Biol. Chem. 1999; 274: 34029-34035Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 15Brunet A. Pouysségur J. Science. 1996; 272: 1653-1655Crossref Scopus (119) Google Scholar, 16Wilsbacher J.L. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 1999; 274: 16988-16994Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). A of the interaction of ERK2 with MEK1 that of include from both of the domains of the kinase as as the MAP kinase in the domain J.L. Goldsmith E.J. Cobb M.H. J. Biol. Chem. 1999; 274: 16988-16994Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). the of ERK2 is for its recognition by MEK1 R. Ahn N.G. J. Cooper J.A. Cobb M.H. J. Biol. Chem. Full Text PDF PubMed Google mutations that the of ERK2 MEK recognition in a In of the of the proteins from mutagenesis We Gibson for and the and for Ahn for and for