Identification of an Extracellular Signal-regulated Kinase (ERK) Docking Site in Ribosomal S6 Kinase, a Sequence Critical for Activation by ERK in Vivo

Abstract

Glutathione S-transferase (GST)-fusion proteins containing the carboxyl-terminal tails of three p90 ribosomal S6 kinase (RSK) isozymes (RSK1, RSK2, and RSK3) interacted with extracellular signal-regulated kinase (ERK) but not c-Jun-NH2-kinase (JNK) or p38 mitogen-activated protein kinase (MAPK). Within the carboxyl-terminal residues of the RSK isozymes is a region of high conservation corresponding to residues722LAQRRVRKLPSTTL735 in RSK1. Truncation of the carboxyl-terminal 9 residues,727VRKLPSTTL735, completely eliminated the interaction of the GST-RSK1 fusion protein with purified recombinant ERK2, whereas the truncation of residues731PSTTL735 had no effect on the interaction with purified ERK2. ERK1 and ERK2 co-immunoprecipitated with hemagglutinin-tagged wild type RSK2 (HA-RSK2) in BHK cell cytosol. However, ERK did not co-immunoprecipitate with HA-RSK2(1–729), a mutant missing the carboxyl-terminal 11 amino acids, similar to the minimal truncation that eliminated in vitro interaction of ERK with the GST-RSK1 fusion protein. Kinase activity of HA-RSK2 increased 6-fold in response to insulin. HA-RSK2(1–729) had a similar basal kinase activity to that of HA-RSK2 but was not affected by insulin treatment. Immunoprecipitated HA-RSK2 and HA-RSK2(1–729) could be activated to the same extent in vitro by active ERK2, demonstrating that HA-RSK2(1–729) was properly folded. These data suggest that the conserved region of the RSK isozymes (722LAQRRVRKL730 of RSK1) provides for a specific ERK docking site approximately 150 amino acids carboxyl-terminal to the nearest identified ERK phosphorylation site (Thr573). Complex formation between RSK and ERK is essential for the activation of RSK by ERK in vivo. Comparison of the docking site of RSK with the carboxyl-terminal tails of other MAPK-activated kinases reveals putative docking sites within each of these MAPK-targeted kinases. The number and placement of lysine and arginine residues within the conserved region correlate with specificity for activation by ERK and p38 MAPKs in vivo. Glutathione S-transferase (GST)-fusion proteins containing the carboxyl-terminal tails of three p90 ribosomal S6 kinase (RSK) isozymes (RSK1, RSK2, and RSK3) interacted with extracellular signal-regulated kinase (ERK) but not c-Jun-NH2-kinase (JNK) or p38 mitogen-activated protein kinase (MAPK). Within the carboxyl-terminal residues of the RSK isozymes is a region of high conservation corresponding to residues722LAQRRVRKLPSTTL735 in RSK1. Truncation of the carboxyl-terminal 9 residues,727VRKLPSTTL735, completely eliminated the interaction of the GST-RSK1 fusion protein with purified recombinant ERK2, whereas the truncation of residues731PSTTL735 had no effect on the interaction with purified ERK2. ERK1 and ERK2 co-immunoprecipitated with hemagglutinin-tagged wild type RSK2 (HA-RSK2) in BHK cell cytosol. However, ERK did not co-immunoprecipitate with HA-RSK2(1–729), a mutant missing the carboxyl-terminal 11 amino acids, similar to the minimal truncation that eliminated in vitro interaction of ERK with the GST-RSK1 fusion protein. Kinase activity of HA-RSK2 increased 6-fold in response to insulin. HA-RSK2(1–729) had a similar basal kinase activity to that of HA-RSK2 but was not affected by insulin treatment. Immunoprecipitated HA-RSK2 and HA-RSK2(1–729) could be activated to the same extent in vitro by active ERK2, demonstrating that HA-RSK2(1–729) was properly folded. These data suggest that the conserved region of the RSK isozymes (722LAQRRVRKL730 of RSK1) provides for a specific ERK docking site approximately 150 amino acids carboxyl-terminal to the nearest identified ERK phosphorylation site (Thr573). Complex formation between RSK and ERK is essential for the activation of RSK by ERK in vivo. Comparison of the docking site of RSK with the carboxyl-terminal tails of other MAPK-activated kinases reveals putative docking sites within each of these MAPK-targeted kinases. The number and placement of lysine and arginine residues within the conserved region correlate with specificity for activation by ERK and p38 MAPKs in vivo. Mitogen-activated protein kinases (MAPKs) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; GST, glutathioneS-transferase; RSK, ribosomal S6 kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun-NH2-kinase; HA, hemagglutinin; HA-RSK2, HA-tagged wild type RSK2; MAPKAPK, MAPK-activated protein kinase; Mnk, MAPK-interacting kinase; PDB, phorbol-12,13-dibutyrate; PBS, phosphate-buffered saline. 1The abbreviations used are: MAPK, mitogen-activated protein kinase; GST, glutathioneS-transferase; RSK, ribosomal S6 kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun-NH2-kinase; HA, hemagglutinin; HA-RSK2, HA-tagged wild type RSK2; MAPKAPK, MAPK-activated protein kinase; Mnk, MAPK-interacting kinase; PDB, phorbol-12,13-dibutyrate; PBS, phosphate-buffered saline.transduce signals from the cell surface to the nucleus, altering the activity and subcellular localization of transcription factors. ERK, JNK, and p38 MAPKs lie in distinct signaling pathways that are activated by distinct stimuli. Whereas the minimal consensus phosphorylation sequence of these proline-directed kinases would suggest promiscuous phosphorylation of many proteins, the kinases play an integral role in the cellular growth machinery; therefore, substrate specificity must be tightly regulated. It is becoming clear that the substrate specificity of MAPKs with regard to transcription factors involves high affinity binding of MAPK to sequences within the substrate that are distinct from the consensus phosphorylation sequence (1Karin M. Curr. Opin. Cell Biol. 1994; 6: 415-424Crossref PubMed Scopus (359) Google Scholar, 2Davis R.J. Trends Biochem. Sci. 1994; 19: 470-473Abstract Full Text PDF PubMed Scopus (917) Google Scholar). Such an interaction has been described for JNK and the transcription factors c-Jun and activating transcription factor (ATF-2) (3Dai T. Rubie E. Franklin C.C. Kraft A. Gillespie D.A.F. Avruch J. Kyriakis J.M. Woodgett J.R. Oncogene. 1995; 10: 849-855PubMed Google Scholar, 4Kallunki T. Su B. Tsigelny I. Sluss H.K. Dérijard B. Moore G. Davis R.J. Karin M. Genes Dev. 1994; 8: 2996-3007Crossref PubMed Scopus (593) Google Scholar, 5Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (472) Google Scholar). Recently, a sequence within Elk-1 was shown to contain overlapping but distinct interaction sites for ERK and JNK (6Yang S.H. Whitmarsh A.J. Davis R.J. Sharrocks A.D. EMBO J. 1998; 17: 1740-1749Crossref PubMed Scopus (267) Google Scholar). Specific targeting interactions between MAPKs and substrates may not be limited to transcription factors. One possible ERK substrate for which targeting interactions might occur is p90 ribosomal S6 protein kinase (RSK). RSK is phosphorylated and activated by ERK in vitro (7Sturgill T.W. Ray L.B. Erikson E. Maller J.L. Nature. 1988; 334: 715-718Crossref PubMed Scopus (754) Google Scholar), and inhibition of the ERK pathway with the mitogen-activated protein/ERK kinase-specific inhibitor PD98059 prevents in vivo activation of RSK (8Lazar D.F. Wiese R.J. Brady M.J. Mastick C.C. Waters S.B. Yamauchi K. Pessin J.E. Cautrecasas P. Saltiel A.R. J. Biol. Chem. 1995; 270: 20801-20807Crossref PubMed Scopus (330) Google Scholar,9Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3248) Google Scholar). RSK is unusual in that it contains two distinct kinase catalytic domains within a single polypeptide chain (10Jones S.W. Erikson E. Blenis J. Maller J.L. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3377-3381Crossref PubMed Scopus (197) Google Scholar). In vivo ERK phosphorylation sites within RSK have been identified (11Sutherland C. Campbell D.G. Cohen P. Eur. J. Biochem. 1993; 212: 581-588Crossref PubMed Scopus (113) Google Scholar, 12Dalby K.N. Morrice N. Caudwell F.B. Avruch J. Cohen P. J. Biol. Chem. 1998; 273: 1496-1505Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar) (Fig.1 A). Two of these sites are essential for activation of RSK: 1) Ser363 in the linker between the two kinase domains, and 2) Thr573 in the activation loop of the carboxyl-terminal kinase domain (12Dalby K.N. Morrice N. Caudwell F.B. Avruch J. Cohen P. J. Biol. Chem. 1998; 273: 1496-1505Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). 2Dalby et al. (12Dalby K.N. Morrice N. Caudwell F.B. Avruch J. Cohen P. J. Biol. Chem. 1998; 273: 1496-1505Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar) report an extra codon for a glutamine at position 157 in the pMT2 construct encoding rat RSK1. Numbering for the phosphorylation sites of RSK1 in the present studies is in agreement with the published sequence of rat RSK1 (35Grove J.R. Price D.J. Banerjee P. Balasubramanyam A. Ahmed M.F. Avruch J. Biochemistry. 1993; 32: 7727-7738Crossref PubMed Scopus (55) Google Scholar). 2Dalby et al. (12Dalby K.N. Morrice N. Caudwell F.B. Avruch J. Cohen P. J. Biol. Chem. 1998; 273: 1496-1505Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar) report an extra codon for a glutamine at position 157 in the pMT2 construct encoding rat RSK1. Numbering for the phosphorylation sites of RSK1 in the present studies is in agreement with the published sequence of rat RSK1 (35Grove J.R. Price D.J. Banerjee P. Balasubramanyam A. Ahmed M.F. Avruch J. Biochemistry. 1993; 32: 7727-7738Crossref PubMed Scopus (55) Google Scholar).Physiological substrates for the three mammalian isozymes of RSK (RSK1, RSK2, and RSK3), which are encoded by separate genes (13Moller D.E. Xia C.H. Tang W. Zhu A.X. Jakubowski M. Am. J. Physiol. 1994; 266: C351-C359Crossref PubMed Google Scholar), are currently under investigation. RSK has been shown to phosphorylate the cAMP response element-binding protein (14Xing J. Ginty D.D. Greenberg M.E. Science. 1996; 273: 959-963Crossref PubMed Scopus (1082) Google Scholar, 15Pende M. Fisher T.L. Simpson P.B. Russell J.T. Blenis J. Gallo V. J. Neurosci. 1997; 17: 1291-1301Crossref PubMed Google Scholar), c-fos (16Chen R.H. Abate C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10952-10956Crossref PubMed Scopus (257) Google Scholar, 17Chen R.H. Juo P.C. Curran T. Blenis J. Oncogene. 1996; 12: 1493-1502PubMed Google Scholar), and the estrogen receptor (18Joel P.B. Smith J. Sturgill T.W. Fisher T.L. Blenis J. Lannigan D.A. Mol. Cell. Biol. 1998; 18: 1978-1984Crossref PubMed Scopus (310) Google Scholar), suggesting that RSK as well as ERK plays a role in transcriptional regulation. Whereas studies describing the existence of an interaction between ERK and RSK have been reported (19Scimeca J.C. Nguyen T.T. Filloux C. Van Obberghen E. J. Biol. Chem. 1992; 267: 17369-17374Abstract Full Text PDF PubMed Google Scholar, 20Hsiao K.M. Chou S.Y. Shih S.J. Ferrell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5480-5484Crossref PubMed Scopus (96) Google Scholar, 21Zhao Y. Bjorbæk C. Moller D.E. J. Biol. Chem. 1996; 271: 29773-29779Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), analyses of the site(s) of interaction and the in vivo significance of the interaction are essential. Examination of the RSK isozymes for the ability to interact with ERK revealed that ERK co-immunoprecipitated with RSK2 and RSK3, but not RSK1 (21Zhao Y. Bjorbæk C. Moller D.E. J. Biol. Chem. 1996; 271: 29773-29779Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The site of interaction between RSK and ERK was localized to the carboxyl-terminal 44 amino acids of RSK3 (21Zhao Y. Bjorbæk C. Moller D.E. J. Biol. Chem. 1996; 271: 29773-29779Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). MAPK-interacting kinase (Mnk) was recently identified as a serine/threonine kinase with sequence similarity to RSK that is phosphorylated and activated by MAPK (22Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. EMBO J. 1997; 16: 1909-1920Crossref PubMed Scopus (779) Google Scholar, 23Fukunaga R. Hunter T. EMBO J. 1997; 16: 1921-1933Crossref PubMed Scopus (552) Google Scholar). Mnk1 co-purified with (22Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. EMBO J. 1997; 16: 1909-1920Crossref PubMed Scopus (779) Google Scholar) and was phosphorylated by both ERK and p38 MAPK (22Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. EMBO J. 1997; 16: 1909-1920Crossref PubMed Scopus (779) Google Scholar, 23Fukunaga R. Hunter T. EMBO J. 1997; 16: 1921-1933Crossref PubMed Scopus (552) Google Scholar), whereas Mnk2 interacted specifically with ERK (22Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. EMBO J. 1997; 16: 1909-1920Crossref PubMed Scopus (779) Google Scholar). Truncation of the carboxyl-terminal 90 amino acids (residues 334–424) of Mnk1 eliminated phosphorylation and activation by ERK (23Fukunaga R. Hunter T. EMBO J. 1997; 16: 1921-1933Crossref PubMed Scopus (552) Google Scholar). Thus, the site of interaction between MAPK and the substrate kinases RSK and Mnk appears to lie within the carboxyl-terminal regions of the substrate. In the present studies, interaction between MAPK and three isozymes of RSK was examined. An ERK docking site critical to this interaction was identified, and the role of the ERK/RSK complex was assessed in vivo. Dulbecco's modified Eagle's medium (11965) and fetal calf serum were purchased from Life Technologies, Inc. Insulin (Humulin®) was obtained from Eli Lilly and Co. Phorbol-12,13-dibutyrate (PDB; P1269) was purchased from Sigma. ImmunoPure® protein A/G-agarose beads (20421) were from Pierce. Antibodies were obtained from the following sources: monoclonal anti-ERK antibody (M12320), Transduction Laboratories; polyclonal anti-ERK antibody (06-182), Upstate Biotechnology; anti-p38 MAPK antibody (SC-535, C-20) and anti-JNK antibody (SC-474, C-17), Santa Cruz Biotechnology, Inc.; polyclonal anti-HA antibody (PRB-101P), Berkley Antibody Co.; monoclonal anti-HA antibody (12CA5), the University of Virginia Lymphocyte Culture Center; and anti-mouse IgG:horseradish peroxidase-linked antibody (NA931) and anti-rabbit IgG:horseradish peroxidase-linked antibody (NA934), Amersham Life Science. COS-1 cells (African green monkey kidney cells, SV40 transformed; ATCC CRL-1650) and BHK-21 (C-13) cells (hamster kidney cells; ATCC CCL-1) were purchased from American Type Culture Collection. Glutathione-Sepharose 4B beads (17-0756) were from Pharmacia. Ribosomal S6 peptide (RRRLSSLRA, residues 231–239) was synthesized at the University of Virginia Biomolecular Research Facility. Expression vectors were generously provided by the following: pMT2.HA-RSK1 (rat), pMT2.HA-RSK2 (mouse), and pMT2.HA-RSK3 (human), Dr. Christian Bjørbæk (Harvard Medical School, Boston, MA); pEThis.MEK1/ERK2 (active ERK2) and pGexKG.SapKβ (K>A) (JNK3), Dr. Melanie H. Cobb (University of Texas Southwestern Medical Center, Dallas, TX); and pET14b.p38 WT (mouse), Dr. Research (K>A) was The encoding an was provided by Dr. (University of A. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). encoding the carboxyl-terminal residues of RSK2, and RSK3 were by chain of the of each was by chain of pMT2.HA-RSK2 and a site in which a RSK2 mutant in which the carboxyl-terminal 11 amino acids are was by a site and a to with The sequences were by used are on of proteins was to a for the BHK-21 (C-13) cells were in a containing in Dulbecco's modified Eagle's medium with fetal calf and The cells were on The cells were with of or as described for of in the the cells were for in by with insulin for at were with phosphate-buffered and the cells were 150 each of and and for complex kinase or 150 each of and and for The cells were by on for and the was by at for for complex kinase or at for COS-1 cells in fetal calf serum were in the or of for at The were with PBS, and cells were and were and an of 150 and inhibitor was to the cell The cell was and cells were by The was at for in a was in and at ERK2 ERK2) and active ERK2 were as described J. A.J. R. M.J. Sturgill T.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. S. J. P. E. Cobb J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Glutathione-Sepharose 4B beads were with The beads were with of or protein in and for at The beads were with COS-1 cell or ERK2 in for at with The beads were by and the was and The beads were three with of was to the beads and and the were for and for monoclonal antibody was used for BHK cell from cells HA-RSK2 or HA-RSK2(1–729) in the or of insulin for was by for with protein A/G-agarose beads were by and the was the was with of monoclonal anti-HA antibody at for by of the with anti-mouse IgG:horseradish peroxidase-linked for on protein A/G-agarose beads were with the for on a The beads were by and the was and The beads were with of and with of was to the beads and the and the were for and for with polyclonal anti-HA antibody and polyclonal anti-ERK the complex kinase of polyclonal anti-HA antibody was to of BHK cell or of polyclonal anti-HA antibody were to of BHK cell and for protein A/G-agarose beads were with the for The beads were by and the was and The beads were with of with of PBS, and with of kinase 150 protein kinase and S6 kinase 150 protein kinase 150 S6 was to the beads and at for of the were and with peptide substrate was by the in the of the was to the of the The were and for with polyclonal anti-HA The for protein sequences are as rat RSK2, RSK3, and proteins of the of three RSK isozymes were to the interaction between RSK and and the amino sequence from the of the carboxyl-terminal kinase domain catalytic to the carboxyl-terminal beads were with from COS-1 cells in the or of beads were for the of ERK, JNK, and p38 MAPKs revealed the of ERK from cells and active ERK from cells with each of the fusion JNK p38 MAPK was to be with the fusion proteins the with similar purified recombinant ERK2 to and wild type p38 MAPK ERK2 to the fusion proteins at that to not Thus, the carboxyl-terminal amino acids of the RSK isozymes specifically interact with et al. (21Zhao Y. Bjorbæk C. Moller D.E. J. Biol. Chem. 1996; 271: 29773-29779Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) reported that ERK not co-immunoprecipitate with However, activation of the three RSK isozymes is by the inhibition of mitogen-activated protein/ERK kinase (21Zhao Y. Bjorbæk C. Moller D.E. J. Biol. Chem. 1996; 271: 29773-29779Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Thus, the GST-RSK1 interaction with ERK is with the of an in the carboxyl-terminal of three The that ERK might to with affinity to HA-RSK2 or (21Zhao Y. Bjorbæk C. Moller D.E. J. Biol. Chem. 1996; 271: 29773-29779Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) is not the sequences for ERK of the identified region of RSK1 were An to containing the carboxyl-terminal amino acids, a region that is conserved between RSK and Mnk was for interaction with was to be for with ERK2 A). the residues within this sequence that were critical to ERK of the GST-RSK1 fusion protein were used of the GST-RSK1 fusion protein for two 1) a peptide amino acids to might ERK interaction to by of critical and 2) the carboxyl-terminal residues were for interaction with ERK, residues might be in of the carboxyl-terminal amino acids had no effect on the ability of the fusion protein to to ERK2 with that of of the carboxyl-terminal amino acids or of the carboxyl-terminal 9 amino acids which the conserved region RSK1) of the RSK completely eliminated the interaction of the fusion proteins with ERK2 of the conserved region of RSK was to the interaction of GST-RSK1 with ERK, it is that a critical of residues within this conserved region as the docking site for the sequence critical to in vitro interaction between and ERK was for interaction within the of the kinase in the interaction between ERK and HA-tagged wild type RSK (HA-RSK2) or mutant RSK2 in which the carboxyl-terminal 11 amino acids are was in BHK HA-RSK2(1–729) the minimal truncation of GST-RSK1 that in vitro interaction with in ERK1 and ERK2 co-immunoprecipitated with HA-RSK2 but did not co-immunoprecipitate with HA-RSK2(1–729) the in from cells with the Thus, the ERK docking site for in vitro interaction is essential for in vivo interaction of RSK and of the ERK docking site vivo activation of RSK, the kinase activity of HA-RSK2 or HA-RSK2(1–729) from BHK cells in the and of insulin was the ribosomal S6 peptide as substrate. The activity of HA-RSK2 was increased by insulin A). kinase activity of HA-RSK2(1–729) was similar to that of insulin had no effect on the kinase activity of A). These that identified ERK phosphorylation sites are in HA-RSK2(1–729), the under that Thus, the formation of a complex between ERK and the carboxyl-terminal of RSK is essential for ERK activation of RSK in the the ERK docking site has been from ERK have affinity for the consensus phosphorylation sequence to phosphorylate and RSK in vitro present at HA-RSK2 and HA-RSK2(1–729) were from BHK cells and in the or of purified recombinant active ERK2. ERK2 increased the kinase activity of HA-RSK2(1–729) which is to the of activation with HA-RSK2 that HA-RSK2(1–729) is a truncation of the ERK docking site did not the ability of ERK to a on as has been reported for wild type RSK 21Zhao Y. Bjorbæk C. Moller D.E. J. Biol. Chem. 1996; 271: 29773-29779Abstract Full Text Full Text PDF PubMed Scopus (143) Google MAPK-activated protein kinases have been identified for both ERK and and p38 MAPK and J. Cohen P. S. M. A. T. A.R. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, J. R. Science. 1994; PubMed Scopus Google Scholar, Jones E. S. J. J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, S. P.C. Van S. J.C. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, B. J. M. W. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, Y. M. K. Zhu W. Y. J. EMBO J. 1998; 17: PubMed Scopus Google Scholar). The recently described are M. A.D. J.M. D.R. EMBO J. 1998; 17: PubMed Scopus Google Scholar) and B. J. M. W. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google both have two kinase domains, and are of each are to be a activated by identified have sequence in catalytic domains to protein in in the carboxyl-terminal kinase of the ERK docking site of RSK1 with the carboxyl-terminal tails of and reveals a docking site within each of these MAPK-targeted kinases The of is with to the number of amino acids within the docking sites two for and for of for of the kinases and p38 as well as a that is by both ERK and p38 RSK2, RSK3, and Mnk2 are activated specifically by ERK The for this that the sequence amino may be for the specific binding and activation of these by and have been and have been to be activated specifically by p38 MAPKs in J. Cohen P. S. M. A. T. A.R. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, J. R. Science. 1994; PubMed Scopus Google Scholar, Jones E. S. J. J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, S. P.C. Van S. J.C. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). of is specific for p38 MAPK in vivo Y. M. K. Zhu W. Y. J. EMBO J. 1998; 17: PubMed Scopus Google Scholar). to was identified in a with MAPK and is activated by p38 MAPK and by ERK both in vitro and in cells B. J. M. W. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, of the conserved region of these the sequence amino amino with p38 MAPK consensus be for activated by both ERK and p38 MAPK and A.J. Flynn A. Proud C.G. Cooper J.A. EMBO J. 1997; 16: 1909-1920Crossref PubMed Scopus (779) Google and M. A.D. J.M. D.R. EMBO J. 1998; 17: PubMed Scopus Google Scholar), are Thus, of MAPK specificity with residues in the conserved regions correlate the number of amino acids with MAPK have two have three and and p38 have and is that the number and position of residues within the of the to be may be of analyses of these and sequences are to the effect on ERK and p38 MAPK The residues in the putative MAPK docking sites are and may localization sequences R. S. R. Curr. Biol. 1998; 8: Full Text Full Text PDF PubMed Scopus Google Scholar). Specific docking sites for MAPKs in the are to play at two in the targeting for ERK is for RSK2 activation in vivo. Thus, complex formation of MAPKs with the carboxyl-terminal tails of is to be a that is for the specific of these MAPK-targeted kinases by MAPKs in vivo. In docking sites may of a specific MAPK with a specific in to transcriptional and A. and G. for the of this and Dr. A. Lannigan for critical of the are to for and to E. and A. for