MEKK1 Binds Raf-1 and the ERK2 Cascade Components
2000; Elsevier BV; Volume: 275; Issue: 51 Linguagem: Inglês
10.1074/jbc.m005926200
ISSN1083-351X
AutoresMahesh Karandikar, Shuichan Xu, Melanie H. Cobb,
Tópico(s)Protein Tyrosine Phosphatases
ResumoMitogen-activated protein (MAP) kinase cascades are involved in transmitting signals that are generated at the cell surface into the cytosol and nucleus and consist of three sequentially acting enzymes: a MAP kinase, an upstream MAP/extracellular signal-regulated protein kinase (ERK) kinase (MEK), and a MEK kinase (MEKK). Protein-protein interactions within these cascades provide a mechanism to control the localization and function of the proteins. MEKK1 is implicated in activation of the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and ERK1/2 MAP kinase pathways. We showed previously that MEKK1 binds directly to JNK/SAPK. In this study we demonstrate that endogenous MEKK1 binds to endogenous ERK2, MEK1, and another MEKK level kinase, Raf-1, suggesting that it can assemble all three proteins of the ERK2 MAP kinase module. Mitogen-activated protein (MAP) kinase cascades are involved in transmitting signals that are generated at the cell surface into the cytosol and nucleus and consist of three sequentially acting enzymes: a MAP kinase, an upstream MAP/extracellular signal-regulated protein kinase (ERK) kinase (MEK), and a MEK kinase (MEKK). Protein-protein interactions within these cascades provide a mechanism to control the localization and function of the proteins. MEKK1 is implicated in activation of the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and ERK1/2 MAP kinase pathways. We showed previously that MEKK1 binds directly to JNK/SAPK. In this study we demonstrate that endogenous MEKK1 binds to endogenous ERK2, MEK1, and another MEKK level kinase, Raf-1, suggesting that it can assemble all three proteins of the ERK2 MAP kinase module. mitogen-activated protein extracellular signal-regulated protein kinase MAP kinase/ERK kinase (also called MAP kinase kinase or MKK) MEK kinase 1 c-Jun N-terminal kinase stress-activated protein kinase glutathioneS-transferase CAL, calmodulin-binding protein cAMP-dependent protein kinase Mitogen-activated protein (MAP)1 kinases mediate responses to a wide array of cellular stimuli (1Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 2English J. Pearson G. Wilsbacher J. Swantek J. Karandikar M. Xu S. Cobb M.H. Exp. Cell Res. 1999; 253: 255-270Crossref PubMed Scopus (377) Google Scholar). MAP kinases lie in a three-kinase module consisting of a MAP kinase or extracellular signal-regulated kinase (ERK) activated by a MAP/ERK kinase (MEK) activated by a MEK kinase (MEKK). These modules are controlled by upstream protein kinases, small and heterotrimeric G proteins, and other regulatory mechanisms. Nearly 20 mammalian MAP kinases have been identified that compose at least six modules. Among these the stress response pathways mediated by the MAP kinases c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK) and p38 MAP kinases, and the ERK1/2 pathway often coupled to proliferation and differentiation of cells have been most thoroughly studied. The specificities of protein kinases in vitro are often broader than their functions in cells. For example, phosphoinositide-dependent protein kinase 1 will phosphorylate several protein kinases including cAMP-dependent protein kinase (PKA) on their activation loops to stabilize or activate them; however, PKA is still phosphorylated on this site in phosphoinositide-dependent protein kinase 1-deficient cells, indicating that it is not the major PKA kinase (3Cheng X. Ma Y. Moore M. Hemmings B.A. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9849-9854Crossref PubMed Scopus (191) Google Scholar, 4Williams M.R. Arthur J.S. Balendran A. van der Kaay J. Poli V. Cohen P. Alessi D.R. Curr. Biol. 2000; 10: 439-448Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Among many examples in MAP kinase cascades, p38 MAP kinase appears to be activated normally in MEKK1-deficient cells, despite the fact that the p38 activators MEK3 and MEK6 are MEKK1 substrates in vitro (5Yujiri T. Sather S. Fanger G.R. Johnson G.L. 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One result is the likely restriction of possible substrates to those that bind to or are concentrated in the immediate vicinity of the complex. Cascade complexes may be assembled by binding to separate scaffold proteins or by binding to sites contained within the enzymes of the cascade. The first scaffold identified for MAP kinases was yeast Ste5p (10Choi K.-Y. Satterberg B. Lyons D.M. Elion E.A. Cell. 1994; 78: 499-512Abstract Full Text PDF PubMed Scopus (165) Google Scholar, 11Marcus S. Polverino A. Barr M. Wigler M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7762-7766Crossref PubMed Scopus (202) Google Scholar, 12Printen J.A. Sprague G.F.J. Genetics. 1994; 138: 609-619Crossref PubMed Google Scholar). Ste5p binds all three kinases in the MAP kinase module of the pheromone response pathway but has no known enzymatic activity itself. A mammalian scaffold protein, JIP-1, may be functionally similar to Ste5p in that it binds the MEKK, mixed lineage kinase 3, MEK7, and JNK1, forming a JNK/SAPK module (13Whitmarsh A.J. Davis R.J. Trends Biochem. Sci. 1998; 23: 481-485Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). In the ERK1/2 pathway, MP-1 links ERK1 to MEK1, although it is probably too small to assemble the complete cascade (14Schaeffer H.J. Catling A.D. Eblen S.T. Collier L.S. Krauss A. Weber M.J. Science. 1998; 281: 1668-1671Crossref PubMed Scopus (384) Google Scholar). The adapter Grb10, which binds both Raf-1 and MEK, may have a related role (15Nantel A. Mohammad-Ali K. Sherk J. Posner B.I. Thomas D.Y. J. Biol. Chem. 1998; 273: 10475-10484Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). 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The yeast MEK, Pbs2p, and the mammalian MEKKs, TAO1/2 and MEKK1/2, also contain binding sites for kinases in their respective MAP kinase modules (25Posas F. Saito H. Science. 1997; 276: 1702-1705Crossref PubMed Scopus (467) Google Scholar, 26Hutchison M. Berman K. Cobb M.H. J. Biol. Chem. 1998; 273: 28625-28632Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 27Chen Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 28Xu S. Cobb M.H. J. Biol. Chem. 1997; 272: 32056-32060Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 29Xia Y. Wu Z. Su B. Murray B. Karin M. Genes Dev. 1998; 12: 3369-3381Crossref PubMed Scopus (176) Google Scholar, 30Cheng J. Yang J. Xia Y. Karin M. Su B. Mol. Cell. Biol. 2000; 20: 2334-2342Crossref PubMed Scopus (67) Google Scholar). MEKK1 was identified based on its sequence similarity to the yeast MEKK, Ste11p (31Lange-Carter C.A. Pleiman C.M. 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Chem. 1994; 269: 19067-19073Abstract Full Text PDF PubMed Google Scholar). In the following study we have examined the interactions of MEKK1 with components of the ERK1/2, JNK/SAPK, and p38 MAP kinase pathways. The results show that endogenous MEKK1 interacts with both the JNK/SAPK and the ERK1/2 modules, suggesting that MEKK1 may be a scaffold for two separate MAP kinase cascades. MEKK1 fragments 30–220 and 221–559 were prepared from bacteria as described (28Xu S. Cobb M.H. J. Biol. Chem. 1997; 272: 32056-32060Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Other MEKK1 constructs were generated using polymerase chain reaction with cDNA encoding the full-length wild type protein in pCMV5-Myc. A point mutation in MEKK1, D1369A, renders the kinase inactive by disrupting the conserved aspartic acid residue responsible for binding Mg2+ and was generated as described (32Xu 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). Mutation of serine 259 to aspartate in Raf-1 is believed to result in an active kinase by eliminating an inhibitory site for 14-3-3 binding, and Raf-1 BXB lacks a portion of its regulatory N terminus and is as described (44Frost J.A. Steen H. Shapiro P.S. Lewis R. Ahn J. Shaw P.E. Cobb M.H. EMBO J. 1997; 16: 6426-6438Crossref PubMed Scopus (362) Google Scholar). All other Raf-1 constructs were generated using polymerase chain reaction with pCMV5-Raf-1 as template. To express Raf-1 in bacteria, its cDNA was inserted at the EcoRI-XhoI sites into pCAL-n (Stratagene), which will incorporate a calmodulin-binding protein fragment at the N terminus of the expressed protein. cDNAs encoding MEK1 and ERK2 were cloned into pCEP4/HA as described (45Xu 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 (148) Google Scholar). 293 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Transfected cells were harvested after 48 h and lysed in 50 mm Tris-Cl, pH 8.0, 150 mm NaCl, 1% Triton X-100, 1 mm sodium orthovanadate, 80 mm β-glycerophosphate, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 7 μg/ml aprotinin. The cells had been starved for 26 h in serum-free medium prior to harvest. Jurkat T cells were grown in RPMI (HYClone) containing 10% fetal bovine serum, harvested at a density of 106 cells/ml, and lysed in hypotonic buffer containing 20 mm HEPES, pH 7.6, 10 mm NaCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 80 mm β-glycerophosphate, and 1 mm sodium orthovanadate. Nuclear fractions were sedimented at 200 × g. The supernatants were adjusted to a final salt concentration of 1 m NaCl and homogenized using a Dounce homogenizer. After 30 min on ice, the supernatants used for immunoprecipitation were collected by ultracentrifugation for 30 min at 100,000 × g. 293 cell lysates (0.5 ml at 3–4 mg/ml) were incubated with appropriate antibodies and 30 μl of protein A-Sepharose at 4 °C for 2 h with constant rotation. The beads were washed three times with 1 ml of 293 lysis buffer for a total of 3 h. Immunoprecipitates from Jurkat cells were washed for 3 h as above and also with 20 mm HEPES, pH 7.6, 0.3m NaCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 80 mmβ-glycerophosphate, and 0.5% Triton X-100. Immunoprecipitates were then blotted for the indicated proteins. Antibodies recognizing the indicated molecules were as follows: MEKK1, C22 (Santa Cruz); Raf-1, SC-133 (Santa Cruz); MEK1, A2227 (45Xu 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 (148) Google Scholar); ERK2, A249/p42 (46Boulton T.G. Cobb M.H. Cell Regul. 1991; 2: 357-371Crossref PubMed Scopus (282) Google Scholar); ERK2, pTEpY (NEB); p38, Sc-535 (Santa Cruz); ERK3, A654 (47Cheng M. Boulton T.G. Cobb M.H. J. Biol. Chem. 1996; 271: 8951-8958Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar); HA, 12 CA-5 (Babco); and Myc (Cell Culture Center). MEKK1 fragments 30–220 and 221–559 were purified as glutathione S-transferase (GST) fusion proteins. Bacteria were induced using 100 μmisopropyl-1-thio-β-d-galactopyranoside for 8 h at 30 °C. Cells were lysed using chicken lysozyme, and DNase was added to degrade DNA. The recombinant baculovirus expressing Raf-1 containing a FLAG epitope was kindly provided by D. Morrison (NIH, Frederick). Purification of Raf-1 from Sf9 cells was performed as described (48Morrison D.K. Heidecker G. Rapp U.R. Copeland T.D. J. Biol. Chem. 1993; 268: 17309-17316Abstract Full Text PDF PubMed Google Scholar). Raf-1 was purified from Escherichia coli strain BL21DE3pLys. The cells were induced for 4 h at room temperature, harvested, and lysed as above in five volumes of 50 mmTris-Cl, pH 8, 0.15 m NaCl, 1 mm magnesium acetate, 10 mm β-mercaptoethanol, 0.5 mmimidazole, 2 mm CaCl2, 30% glycerol, and 0.1% Triton X-100. The lysate was clarified by sedimentation, and the supernatant was applied to calmodulin affinity resin. Raf-1 protein was eluted with 50 mm Tris-Cl, pH 8, 0.15 m NaCl, 10 mm β-mercaptoethanol, 30% glycerol, 0.1% Triton X-100, and 2 mm EGTA. MEKK1 fragments (0.39 mg) were immobilized on 30 μl of glutathione-agarose in the presence of 10 mg/ml bovine serum albumin and then incubated with 0.34 mg FLAG-Raf-1 or CAL-Raf-1 in 0.2 ml of lysis buffer with 1% Triton X-100 for 2 h at 4 °C. Beads were washed three times with 1 ml of 50 mm Tris-Cl, pH 8.0, 150 mm NaCl, 0.1% SDS, 1% Nonidet P-40, and 0.5% deoxycholate for a total of 6 h. Immunoprecipitates were washed as described above, resuspended in water, and incubated with 7 μg of purified MEK4 K131M along with 5 μCi of [γ-32P]ATP in kinase buffer (20 mm HEPES, pH 7.8, 10 μm ATP, 10 mm MgCl2, and 10 mmβ-glycerophosphate) for 30 min at 30 °C. Incorporation of radioactive phosphate was determined by autoradiography and liquid scintillation counting of excised bands. We and others previously showed that MEKK1 acts as a scaffold for the JNK/SAPK pathway by binding directly to JNK/SAPK isoforms (28Xu S. Cobb M.H. J. Biol. Chem. 1997; 272: 32056-32060Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 29Xia Y. Wu Z. Su B. Murray B. Karin M. Genes Dev. 1998; 12: 3369-3381Crossref PubMed Scopus (176) Google Scholar). Ste5p binds the kinases of the yeast MAP kinase module in the pheromone response pathway (10Choi K.-Y. Satterberg B. Lyons D.M. Elion E.A. Cell. 1994; 78: 499-512Abstract Full Text PDF PubMed Scopus (165) Google Scholar, 11Marcus S. Polverino A. Barr M. Wigler M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7762-7766Crossref PubMed Scopus (202) Google Scholar, 12Printen J.A. Sprague G.F.J. Genetics. 1994; 138: 609-619Crossref PubMed Google Scholar). Ste5p also binds to subunits of the heterotrimeric G protein that activates this pathway (49Whiteway M.S. Wu C. Leeuw T. Clark K. Fourest-Lieuvin A. Thomas D.Y. Leberer E. Science. 1995; 269: 1572-1575Crossref PubMed Scopus (153) Google Scholar, 50Pryciak P.M. Huntress F.A. Genes Dev. 1998; 12: 2684-2697Crossref PubMed Scopus (197) Google Scholar, 51Akada R. Kallal L. Johnson D.I. Kurjan J. Genetics. 1996; 143: 103-117Crossref PubMed Google Scholar, 52Feng Y. Song L.Y. Kincaid E. Mahanty S.K. Elion E.A. Curr. Biol. 1998; 8: 267-278Abstract Full Text Full Text PDF PubMed Google Scholar). Because the subunits also bind Ste20p, the kinase upstream of the MAP kinase module, they link Ste20p to the scaffolded module (53Leeuw T. Wu C. Schrag J.D. Whiteway M. Thomas D.Y. Leberer E. Nature. 1998; 391: 191-195Crossref PubMed Scopus (183) Google Scholar). Ste20p then phosphorylates the MEKK Ste11p, thus leading to activation of the kinase cascade (54Wu C. Whiteway M. Thomas D.Y. Leberer E. J. Biol. Chem. 1995; 270: 15984-15992Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). To test the possibility that MEKK1 may be regulated in a comparable manner, we examined its ability to bind the p21-activated protein kinase, PAK1, one of the mammalian orthologs of Ste20p (55Manser E. Leung T. Salihuddin H. Zhao Z.-S. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1300) Google Scholar, 56Polverino A. Frost J. Yang P. Hutchison M. Neiman A.M. Cobb M.H. Marcus S. J. Biol. Chem. 1995; 270: 26067-26070Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Consistent with this idea, PAK has been shown to activate the JNK/SAPK pathway and to enhance activation of the ERK1/2 pathway through effects on Raf-1 and MEK1 (44Frost J.A. Steen H. Shapiro P.S. Lewis R. Ahn J. Shaw P.E. Cobb M.H. EMBO J. 1997; 16: 6426-6438Crossref PubMed Scopus (362) Google Scholar, 56Polverino A. Frost J. Yang P. Hutchison M. Neiman A.M. Cobb M.H. Marcus S. J. Biol. Chem. 1995; 270: 26067-26070Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 57Bagrodia S. Dérijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 58King A.J. Sun H. Diaz B. Barnard D. Miao W. Bagrodia S. Marshall M.S. Nature. 1998; 396: 180-183Crossref PubMed Scopus (385) Google Scholar). As controls we employed TAO2, another relative of Ste20p, that is itself a MEKK (27Chen Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), and Raf-1 the MEKK in the ERK1/2 pathway. As shown in Fig. 1, neither PAK1 nor TAO2 coimmunoprecipitated with cotransfected MEKK1. However, to our surprise, Raf-1 was readily detected as a MEKK1-associated protein. We estimate that 50% of the overexpressed MEKK1 was bound to Raf-1. The observation that PAK1, TAO2, and the GST control failed to coimmunoprecipitate MEKK1 supports the idea that Raf-1 binding is not due to a nonspecific association. We next ascertained whether the interaction of MEKK1 with Raf-1 is regulated in a manner dependent upon the activation state of either enzyme. Wild type MEKK1 that is transfected into cells is active even in the absence of a stimulus (32Xu 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); thus we tested the effect of MEKK1 activity by comparing the amount of Raf-1 coprecipitating with wild type and kinase-dead MEKK1 (D1369A). Fig. 2 shows that Raf-1 coimmunoprecipitates equally with both wild type and kinase-dead MEKK1. In contrast to MEKK1, wild type Raf-1 has low activity when it is expressed in serum-starved cells; thus, to test the effect of Raf catalytic activity on the Raf-MEKK1 association, we compared the amount of MEKK1 coprecipitating with the activated Raf-1 mutants, Raf-1 BXB and Raf-1 S259D, and the inactive, wild type protein. All three forms of Raf-1 were present in MEKK1 immunoprecipitates (Fig. 2), indicating that MEKK1 interacts with Raf-1 in a manner independent of the activation state of Raf-1. These findings suggest that Raf and MEKK1 may be associated in cells whether the enzymes are activated or inactive. The data also support the notion that at least a portion of Raf-1 in a cell is constitutively associated with MEKK1. In other experiments, we estimated that the abundance of MEKK1 is close to or less than that of Raf-1. 2M. Karandikar, J. R. Woodgett, and M. H. Cobb, submitted for publication. To support the conclusion that the MEKK1-Raf-1 interaction has relevance in cells, we probed the potential association of the endogenous proteins. Raf-1 was found in immunoprecipitates of MEKK1 native to Jurkat or 293 cells (Fig. 3). Control antibodies neither immunoprecipitated MEKK1 nor coimmunoprecipitated Raf-1, indicating that the interaction is not the result of protein aggregation or nonspecific trapping of Raf-1 in the precipitates (Fig. 3). MEKK1 immunoprecipitates were washed more stringently with high salt and with detergent to assess qualitatively the strength of the interaction. Neither 1 m NaCl nor 0.1% SDS were sufficient to disrupt the interaction (not shown). These data demonstrate that endogenous MEKK1 and Raf-1 indeed interact strongly, confirming findings with transfected proteins. We also examined the possible regulation of the MEKK1-Raf-1 interaction by modulating the activation states of the endogenous enzymes. Activities of the kinases were measured in immune complexes using kinase-inactive MEK4 (K131M) and MEK1 (K97M) as substrates for MEKK1 and Raf-1, respectively. Endogenous MEKK1 is inactive in unstimulated Jurkat T cells and is activated by nocodazole, colchicine, and sorbitol (5Yujiri T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (282) Google Scholar) (Fig. 3 A). Raf-1 is present in MEKK1 immunoprecipitates regardless of the activation state of MEKK1. In numerous experiments there was little or no change in the amount of Raf-1 in the MEKK1 precipitates, although a small decrease was occasionally observed with sorbitol. To test the effect of Raf-1 activity on its association with MEKK1, we used serum-starved 293 cells, because Jurkat T cells did not withstand serum starvation in our hands. Raf-1 is active in cycling Jurkat T cells and in unstarved 293 cells; its activity is reduced by serum deprivation (data not shown). A population of endogenous Raf-1 interacts with endogenous MEKK1 in serum-starved or stimulated 293 cells (Fig. 3 B), demonstrating that their association is independent of the activation state of either enzyme. We next mapped the interacting domains by cotransfecting plasmids encoding each kinase with fragments of the other. The binding site on MEKK1 was first narrowed to a fragment including residues 221–559; the N-terminal region () and more C-terminal fragments do not interact (Fig. 4 A). A fragment containing residues 221–370 still binds to Raf-1, but residues 371–559 do not bind, indicating that residues 221–370 comprise the Raf-1 binding domain. This portion of the regulatory N terminus of MEKK1 includes a proline-rich region, but the requirement of individual residues for Raf-1 interaction was not explored. Similar experiments with the N- and C-terminal domains of Raf-1 indicate that the C-terminal half, which contains the catalytic domain of Raf-1, residues 303–648, is sufficient to bind to MEKK1 (Fig. 4 B). Both Raf-1 and MEKK1 bind a variety of proteins within cells. Thus, it is possible that the interaction between these kinases is mediated by one or more accessory proteins. To test this possibility, GST-MEKK1 fragments and Raf-1 linked to a fragment of a calmodulin-binding protein were expressed and purified from bacteria as fusion proteins. The catalytic domain of Raf-1 was previously expressed as a soluble protein in bacteria using this fusion system (58King A.J. Sun H. Diaz B. Barnard D. Miao W. Bagrodia S. Marshall M.S. Nature. 1998; 396: 180-183Crossref PubMed Scopus (385) Google Scholar). In accord with the data from transfected 293 cells, MEKK1 fragment 221–559 bound specifically to purified Raf-1 (Fig. 5). This result demonstrates that MEKK1 binds Raf-1 directly, because no other eukaryotic proteins were present in these preparations. Direct binding was also found using full-length Raf-1 expressed in Sf9 cells (not shown). Having established that Raf-1 and MEKK1 are associated to a significant extent under all conditions examined, we wished to gain additional evidence supporting the significance of their association. Because MEKK1 can activate the ERK1/2 pathway, we tested the possibility that MEKK1 also binds to MEK1 and ERK2. 293 cells were transfected with HA-tagged MEK1 alone or together with plasmids encoding Myc-tagged fragments or full-length MEKK1. The MEKK1 proteins were immunoprecipitated using the anti-Myc antibody, and precipitates were probed for MEK1. MEK1 does indeed associate with MEKK1, both full-length and an N-terminal fragment, residues 1–482 (not shown). For comparison we examined the ability of MEKK1 to interact with MEK7 of the JNK/SAPK pathway. MEK7 binds to a fragment containing residues 30–400 (not shown). We showed previously that JNK associates directly with MEKK1 but p38 does not (28Xu S. Cobb M.H. J. Biol. Chem. 1997; 272: 32056-32060Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Purified MEKK1 N-terminal fragments and ERK2 both expressed in bacteria bind in vitro (data not shown). To test the ability of MEKK1 to bind to ERK2 in intact cells, 293 cells were tr
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