RhoA Binds to the Amino Terminus of MEKK1 and Regulates Its Kinase Activity

MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that can regulate the c-Jun amino-terminal kinase (JNK) MAP kinase cascade. MEKK1 is comprised of a kinase domain and a long amino-terminal regulatory domain. This amino-terminal domain has a scaffold function in that it can assemble modules of the JNK and ERK MAP kinase cascades. Recently, we have demonstrated that MEKK1 binds to p115 Rho GTPase-activating protein, which has GTPase-activating protein activity toward RhoA. Thus, we tested whether Rho GTPases interact with the regulatory domain of MEKK1. RhoA, but not Rac or Cdc42, binds to a site in the aminoterminal one-third of MEKK1, which includes its PHD domain. The interaction is prevented by mutation of the essential cysteine in the MEKK1 PHD domain. Rho-GTP stimulates the kinase activity of full-length MEKK1 as much as 10-fold toward MEK4 but does not appear to be ubiquitinated by MEKK1 under conditions that result in modification of ERK2. In summary, we have characterized a novel point at which Rho GTPases impinge upon the regulation and function of MEKK1. MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that can regulate the c-Jun amino-terminal kinase (JNK) MAP kinase cascade. MEKK1 is comprised of a kinase domain and a long amino-terminal regulatory domain. This amino-terminal domain has a scaffold function in that it can assemble modules of the JNK and ERK MAP kinase cascades. Recently, we have demonstrated that MEKK1 binds to p115 Rho GTPase-activating protein, which has GTPase-activating protein activity toward RhoA. Thus, we tested whether Rho GTPases interact with the regulatory domain of MEKK1. RhoA, but not Rac or Cdc42, binds to a site in the aminoterminal one-third of MEKK1, which includes its PHD domain. The interaction is prevented by mutation of the essential cysteine in the MEKK1 PHD domain. Rho-GTP stimulates the kinase activity of full-length MEKK1 as much as 10-fold toward MEK4 but does not appear to be ubiquitinated by MEKK1 under conditions that result in modification of ERK2. In summary, we have characterized a novel point at which Rho GTPases impinge upon the regulation and function of MEKK1. MAP/ERK 1The abbreviations used are: MAP, mitogen-activated protein; MAP3K, mitogen-activated protein kinase kinase kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun amino-terminal kinase; MEK, MAP kinase/ERK kinase; MEKK, MEK kinase; GAP, GTPase-activating protein; HEK, human embryonic kidney; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase. kinase kinase 1 (MEKK1), a MAP3 kinase, participates in a diverse array of responses to cellular stress (1.Karin M. Delhase M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9067-9069Crossref PubMed Scopus (206) Google Scholar, 2.Pearson G. Robinson F. Beers G.T. Xu B. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3619) Google Scholar). It is sensitive to numerous stimuli, including cold shock, UV light, lysophosphatidic acid, osmotic stress, and microtubule-disrupting drugs. In vitro MEKK1 activates the ERK and JNK MAP kinase pathways by phosphorylation of the relevant MAP2 kinases (3.Lin A. Minden A. Martinetto H. Claret F.-X. Lange-Carter C. Mercurio F. Johnson G. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (721) Google Scholar, 4.Xu S. Robbins D. Frost J. Dang A. Lange-Carter C. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6808-6812Crossref PubMed Scopus (151) Google Scholar). Overexpression of MEKK1 demonstrates that it may function not only in these MAP kinase pathways but also in regulation of NF-kB (5.Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar). MEKK1 plays a role in disease pathology. Notable is its binding to the human T cell lymphotrophic virus Tax protein, which enhances NF-kB activation (6.Yin M.J. Christerson L.B. Yamamoto Y. Kwak Y.T. Xu S. Mercurio F. Barbosa M. Cobb M.H. Gaynor R.B. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Findings in cells from animals in which the MEKK1 gene has been disrupted further support a role in regulating the JNK pathway in certain settings (7.Yujiri T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (284) Google Scholar, 8.Xia Y. Makris C. Su B. Li E. Yang J. Nemerow G.R. Karin M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5243-5248Crossref PubMed Scopus (237) Google Scholar). With nearly 1500 residues, MEKK1 is the longest of four related mammalian enzymes (9.Lange-Carter C.A. Pleiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (925) Google Scholar, 10.Xu S. Robbins D.J. Christerson L.B. English J.M. Vanderbilt C.A. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5291-5295Crossref PubMed Scopus (122) Google Scholar, 11.Blank J.L. Gerwins P. Elliott E.M. Sather S. Johnson G.L. J. Biol. Chem. 1996; 271: 5361-5368Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 12.Gerwins P. Blank J.L. Johnson G.L. J. Biol. Chem. 1997; 272: 8288-8295Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 13.Bonvin C. Guillon A. van Bemmelen M.X. Gerwins P. Johnson G.L. Widmann C. Cell Signal. 2002; 14: 123-131Crossref PubMed Scopus (59) Google Scholar). Binding partners identified with its amino-terminal domain include the MAP kinases JNK and ERK2, the MAP2 kinases MEK7 and MEK1, the MAP3 kinase Raf-1, and the MAP4 kinases Nck-interacting kinase and germinal center kinase (14.Xu S. Cobb M.H. J. Biol. Chem. 1997; 272: 32056-32060Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 15.Xia Y. Wu Z. Su B. Murray B. Karin M. Genes Dev. 1998; 12: 3369-3381Crossref PubMed Scopus (178) Google Scholar, 16.Karandikar M. Xu S. Cobb M.H. J. Biol. Chem. 2000; 275: 40120-40127Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 17.Su Y.-C. Han J. Xu S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (221) Google Scholar, 18.Chadee D.N. Yuasa T. Kyriakis J.M. Mol. Cell. Biol. 2002; 22: 737-749Crossref PubMed Scopus (75) Google Scholar). Thus, MEKK1 can assemble elements of signaling modules, in a manner reminiscent of the yeast scaffold Ste5p (19.Choi K.-Y. Satterberg B. Lyons D.M. Elion E.A. Cell. 1994; 78: 499-512Abstract Full Text PDF PubMed Scopus (167) Google Scholar, 20.Marcus S. Polverino A. Barr M. Wigler M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7762-7766Crossref PubMed Scopus (208) Google Scholar, 21.Printen J.A. Sprague G.F.J. Genetics. 1994; 138: 609-619Crossref PubMed Google Scholar). Through its PHD domain, MEKK1 can also ubiquitinate and promote the degradation of ERK2 (22.Lu Z. Xu S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Additional interactions with proteins such as α-actinin may impact MEKK1 localization, and with 14-3-3, reflect phosphorylation-dependent interactions (23.Christerson L.B. Vanderbilt C.A. Cobb M.H. Cell Motil. Cytoskeleton. 1999; 43: 186-198Crossref PubMed Scopus (89) Google Scholar, 24.Fanger G.R. Widmann C. Porter A.C. Sather S. Johnson G.L. Vaillancourt R.R. J. Biol. Chem. 1998; 273: 3476-3483Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The small G proteins Ras, Rac, and Cdc42 have also been shown to interact with the kinase domain of MEKK1 (25.Russell M. Lange-Carter C.A. Johnson G.L. J. Biol. Chem. 1995; 270: 11757-11760Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 26.Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (256) Google Scholar). Perhaps the clearest of functional changes as a result of these interactions is the activation of MEKK1 by the MAP4 kinase germinal center kinase elucidated by Kyriakis and co-workers (18.Chadee D.N. Yuasa T. Kyriakis J.M. Mol. Cell. Biol. 2002; 22: 737-749Crossref PubMed Scopus (75) Google Scholar). Rho family GTPases, including RhoA, B, C, Rac1, 2, and Cdc42, control cellular morphology and motility and MAP kinase signaling pathways and influence other processes, such as the cell cycle (27.Bar-Sagi D. Hall A. Cell. 2000; 103: 227-238Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar). Many of these effects derive directly or indirectly from their ability to regulate the cytoskeleton. The morphological phenotypes induced by active mutants of Rho GTPases have been well documented in different cell types. The most distinct effects of these GTPases were noted in Swiss 3T3 cells in which RhoA induces stress fiber formation and Rac1 induces membrane ruffling and lamellipodia, whereas Cdc42 induces spike-like filopodia (28.Ridley A.J. Paterson H.F. Johnson C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3155) Google Scholar). The connections between MEKK1 and the cytoskeleton are striking. Cells cultured from MEKK1–/–mice exhibit defects in cellular morphology and motility, suggesting that MEKK1 regulates cytoskeletal function (8.Xia Y. Makris C. Su B. Li E. Yang J. Nemerow G.R. Karin M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5243-5248Crossref PubMed Scopus (237) Google Scholar, 29.Yujiri T. Ware M. Widmann C. Oyer R. Russell D. Chan E. Zaitsu Y. Clarke P. Tyler K. Oka Y. Fanger G.R. Henson P. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7272-7277Crossref PubMed Scopus (209) Google Scholar). Drugs that target the cytoskeleton are among the most powerful activators of MEKK1 (16.Karandikar M. Xu S. Cobb M.H. J. Biol. Chem. 2000; 275: 40120-40127Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 30.Yujiri T. Fanger G.R. Garrington T.P. Schlesinger T.K. Gibson S. Johnson G.L. J. Biol. Chem. 1999; 274: 12605-12610Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). MEKK1 is localized not only to actin stress fibers and focal adhesions but also to microtubules, intermediate filaments, and around and in the nucleus (23.Christerson L.B. Vanderbilt C.A. Cobb M.H. Cell Motil. Cytoskeleton. 1999; 43: 186-198Crossref PubMed Scopus (89) Google Scholar, 26.Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (256) Google Scholar). As is the case for other signaling molecules, it is expected that its localization will have a major impact on its function (23.Christerson L.B. Vanderbilt C.A. Cobb M.H. Cell Motil. Cytoskeleton. 1999; 43: 186-198Crossref PubMed Scopus (89) Google Scholar, 31.Schlesinger, T. K., Bonvin, C., Jarpe, M. N., Fanger, G. R., Cardinaux, J. R., Johnson, G. L., and Widmann, C. (2002) J. Biol. Chem.Google Scholar). We recently found that MEKK1 recruits p115 RhoGAP to its amino terminus (32.Christerson L.B. Gallagher E. Vanderbilt C.A. Whitehurst A.W. Wells C. Kazempour R. Sternweis P.C. Cobb M.H. J. Cell. Physiol. 2002; 192: 200-208Crossref PubMed Scopus (26) Google Scholar). In view of the association of p115 RhoGAP with MEKK1, the importance of Rho to regulation of the cytoskeleton, and the linkage of MEKK1 to the cytoskeleton, we examined the possible interaction of Rho and other small GTPases with the regulatory amino-terminal portion of MEKK1. Cell Culture, Transfection, Plasmids, and Proteins—HEK 293 and Madin-Darby canine kidney cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. GST-MEKK1 221–559 (PHD domain) and GST-ERK2 (K52R) were produced in Escherichia coli. Full-length recombinant MEKK1, RhoA, and other small G proteins were produced in Sf9 cells (33.Gallagher E.D. Xu S. Moomaw C. Slaughter C.A. Cobb M.H. J. Biol. Chem. 2002; 277: 45785-45792Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 34.Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (743) Google Scholar). Transient transfection was as described (35.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) or with LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol. The following constructs were utilized in transfection experiments: pCMV5RhoA Myc, pCMV5RhoA L63Myc, and pCMV5RhoA N19Myc. Mutations were introduced into MEKK1 using the QuikChange kit (Stratagene). Antibodies and Immunoprecipitation—Antibodies were as follows: MEKK1 (Santa Cruz Biotechnology, 1–9C-2A and C-22), RhoA (Santa Cruz Biotechnology, 26C4,119-G and 119-R), Myc monoclonal 9E10 (National Cell Culture Center). HEK 293 cells were lysed, and immunoprecipitation was performed from soluble extracts using 20 μl of the C-22 rabbit polyclonal antibody, in the presence or absence of the C-22-P peptide (Santa Cruz Biotechnology), and 50 μl of protein A-Sepharose CL-4B (Amersham Biosciences) as described in detail previously (16.Karandikar M. Xu S. Cobb M.H. J. Biol. Chem. 2000; 275: 40120-40127Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Immunoprecipitates resolved by SDS-PAGE were immunoblotted as described (16.Karandikar M. Xu S. Cobb M.H. J. Biol. Chem. 2000; 275: 40120-40127Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In Vitro Ubiquitination Assays—Approximately 3 μg of the wild type MEKK1 PHD domain (GST-MEKK1 221–559 (22.Lu Z. Xu S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar)), His6-RhoA-GTP, and GST-ERK2 (K52R) were incubated with 50–500 nm His6-E1, 0.5–5 μm His6-E2, 10 μm ubiquitin, and 2 in and polyclonal and (Santa Cruz were used for His6-E1, His6-E2, and ubiquitin were from J. Chen of C. E. Yang A. J. Slaughter C. C. Chen Z.J. Cell. 2000; 103: Full Text Full Text PDF PubMed Scopus Google Scholar). protein kinase were performed in kinase 10 μm and 10 supplemented with of as described previously (16.Karandikar M. Xu S. Cobb M.H. J. Biol. Chem. 2000; 275: 40120-40127Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). were incubated at for the and resolved by were with with 10% and and by was performed as described previously (32.Christerson L.B. Gallagher E. Vanderbilt C.A. Whitehurst A.W. Wells C. Kazempour R. Sternweis P.C. Cobb M.H. J. Cell. Physiol. 2002; 192: 200-208Crossref PubMed Scopus (26) Google Scholar). The was with the following RhoA wild RhoA RhoA Rac1, Cdc42, Ras, and of p115 was on supplemented with RhoA with MEKK1 we found that p115 RhoGAP binds to a of MEKK1 amino-terminal to its kinase domain (32.Christerson L.B. Gallagher E. Vanderbilt C.A. Whitehurst A.W. Wells C. Kazempour R. Sternweis P.C. Cobb M.H. J. Cell. Physiol. 2002; 192: 200-208Crossref PubMed Scopus (26) Google Scholar). we whether Rho GTPases also interact with MEKK1 of its kinase domain. a yeast we found that a of MEKK1 with wild type RhoA, but not with Cdc42, Rac1, or of these GTPases to MEKK1 which with p115 As the were tested for interaction with the yeast protein and the protein kinase of MEKK1 with these molecules, with (23.Christerson L.B. Vanderbilt C.A. Cobb M.H. Cell Motil. Cytoskeleton. 1999; 43: 186-198Crossref PubMed Scopus (89) Google Scholar, B. English J.M. J.L. S. Cobb M.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). As α-actinin with and p115 RhoGAP with (23.Christerson L.B. Vanderbilt C.A. Cobb M.H. Cell Motil. Cytoskeleton. 1999; 43: 186-198Crossref PubMed Scopus (89) Google Scholar, L.B. Gallagher E. Vanderbilt C.A. Whitehurst A.W. Wells C. Kazempour R. Sternweis P.C. Cobb M.H. J. Cell. Physiol. 2002; 192: 200-208Crossref PubMed Scopus (26) Google of small G proteins with activation binding binding Rho in a We the that the binding of MEKK1 to RhoA was by the RhoA the we used of MEKK1 to a of the protein type RhoA to a of MEKK1, but not to the control In the RhoA which has a much for and to or was to of the MEKK1 The RhoA which is in its also but not of the other MEKK1 or the control are with the that MEKK1 most with Rho in its but not the that may also to MEKK1. only to a that we have shown a or domain (14.Xu S. Cobb M.H. J. Biol. Chem. 1997; 272: 32056-32060Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, E.D. Xu S. Moomaw C. Slaughter C.A. Cobb M.H. J. Biol. Chem. 2002; 277: 45785-45792Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). the of MEKK1 that with RhoA to we examined the amino of to possible RhoA binding of target proteins that interact with the GTPases Rac1 and Cdc42 are by the target is the found in a of of these small G proteins D. Hall A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). have been much to in RhoA by or the Rho is in the of the and K. A. T. Y. S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. T. G. K. P. S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar). A of MEKK1, to the Rho found in proteins and is among mammalian whether of the MEKK1 a functional Rho we introduced in that be in Rho binding 2, and C, and a interaction with RhoA. and to of cysteine was not to binding not suggesting of the cysteine with the of the Rho binding site with the PHD domain of MEKK1, to whether mutation of the cysteine for the ubiquitin activity of the MEKK1 PHD domain (22.Lu Z. Xu S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (276) Google influence Rho the also to RhoA and of RhoA with MEKK1 binding activation in a In between MEKK1 and RhoA in whether MEKK1 with RhoA in we examined MEKK1 for the presence of RhoA. We found that RhoA with MEKK1 The of the interaction was tested by using the peptide to MEKK1 immunoprecipitation from HEK 293 cell The peptide the immunoprecipitation of MEKK1 and the of RhoA. we found that MEKK1 with RhoA in extracts from Madin-Darby canine kidney cells A control to a interaction with MAP3K, has been demonstrated to to M.J. A. Science. 1993; 260: PubMed Scopus Google Scholar, J.A. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). MEKK1 RhoA in of MEKK1 to to RhoA that the binding of RhoA to the PHD domain of MEKK1 may its by MEKK1. whether can we used the PHD domain of MEKK1 in in vitro with RhoA. As a we used which was shown previously to be ubiquitinated by MEKK1 in vitro and in cells (22.Lu Z. Xu S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). A between the and protein which is the expected for ERK2, was upon the with the conditions with in of ERK2 as the modified of RhoA were by with that RhoA is not a of the MEKK1 PHD domain. the protein in the also to it is possible that other the to RhoA is a under other or it may the ability of MEKK1 to ubiquitinate other proteins in cells by binding to the MEKK1 PHD domain. is activation of Rho be expected to proteins that be ubiquitinated by MEKK1. In the we to RhoA the of MEKK1 in Vitro and in that MEKK1 to interact with we whether interaction the kinase activity of MEKK1. MEKK1 was from HEK 293 cells transfection with RhoA, RhoA and RhoA constructs of the Rho proteins were in the cells and of MEKK1 were of wild type RhoA or RhoA produced activation of MEKK1 as with In of RhoA in HEK 293 cells the activity of MEKK1 toward MEK4 nearly 10-fold whether the activation be in full-length recombinant MEKK1 in Sf9 cells was with to its activity (33.Gallagher E.D. Xu S. Moomaw C. Slaughter C.A. Cobb M.H. J. Biol. Chem. 2002; 277: 45785-45792Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). MEKK1 in Sf9 cells has in to The protein was in kinase with or RhoA, and activity toward MEK4 was was in the MEKK1 activity as much as with the in and that RhoA can directly the kinase activity of MEKK1. a in MEKK1 RhoA to with MEKK1 its ability to MEKK1 to further the that activity is by MEKK1 was incubated with Cdc42 or Rac1 with or Rac1 to or in activity as Cdc42 also MEKK1 activity to the as We have been to binding of Cdc42 to the aminoterminal of MEKK1. A used to that Rac1 and Cdc42 can to the MEKK1 kinase domain G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (256) Google to that the in activity we may be to interaction of Cdc42 with the kinase domain, a site distinct from the identified is it is that MEKK1 between Cdc42 and The of RhoA the activity of other protein including and Rho kinase K. A. T. Y. S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. T. G. K. P. S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar, T. M. Yamamoto T. K. M. M. T. K. A. K. EMBO J. 1996; PubMed Scopus Google Scholar, and Mol. Cell. Biol. Scholar). the of activation of MEKK1 induced by RhoA is that of kinases by Cdc42 or Rac1 E. T. H. 1994; PubMed Scopus Google Scholar, J.A. A. Cobb M.H. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google the activation of MEKK1 by RhoA is of a to that of these other kinases by RhoA. Thus, MEKK1 activity changes in with by other Rho We a nearly of the of MEKK1 and RhoA on activation of a suggesting a for Rho effects on MEKK1. kinases have previously been to activation of by Rho A. D. J. R. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). in the case of MEKK1, we have been to the between its kinase activity and activation of the a of MEKK1 the on activity in with Rho as does the wild type JNK activation by MEKK1 is not on MEKK1 kinase activity (14.Xu S. Cobb M.H. J. Biol. Chem. 1997; 272: 32056-32060Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Thus, the cellular of MEKK1 activity induced by Rho-GTP have not been The of MEKK1 to RhoA and a RhoGAP that Rho to MEKK1 will be by the This only activation of MEKK1 to of In the of Rho in the of MEKK1 may have a different active Rho in distinct to the of the MEKK1 of the JNK and ERK MAP kinase (14.Xu S. Cobb M.H. J. Biol. Chem. 1997; 272: 32056-32060Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 15.Xia Y. Wu Z. Su B. Murray B. Karin M. Genes Dev. 1998; 12: 3369-3381Crossref PubMed Scopus (178) Google Scholar, 16.Karandikar M. Xu S. Cobb M.H. J. Biol. Chem. 2000; 275: 40120-40127Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The that it also elements of Rho signaling We to the of these MEKK1 and to whether distinct In summary, we have a novel point at which Rho GTPases can impinge on the function of MEKK1. that MEKK1 to focal and stress is by that impact cytoskeletal and is for cell motility, MEKK1 a role in the regulation of the function of the cytoskeleton. It that interactions with Rho signaling pathways will to these of MEKK1.