MEKK1 Binds Raf-1 and the ERK2 Cascade Components

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 (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. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (282) Google Scholar, 6Robinson M.J. Cheng M. Khokhlatchev A. Ebert D. Ahn N. Guan K. Stein B. Goldsmith E. Cobb M.H. J. Biol. Chem. 1996; 271: 29734-29739Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Protein scaffolds offer a mechanism to organize cascade components to aid in signaling specificity and targeting. Many proteins that localize kinases and phosphatases to sites of action, such as the glycogen-binding G protein and A kinase anchoring proteins, have been identified in PKA and protein kinase C systems (7Rubin C.S. Biochim. Biophys. Acta. 1994; 1224: 467-479PubMed Google Scholar, 8Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1900) Google Scholar, 9Mochly-Rosen D. Kauvar L.M. Semin. Immunol. 2000; 12: 55-61Crossref PubMed Scopus (34) Google Scholar). 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). Kinase suppressor of Ras also binds multiple kinases of the ERK1/2 MAP kinase module; these interactions are essential for its function in Drosophila andCaenorhabditis elegans (16Therrien M. Chang H.C. Solomon N.M. Karim F.D. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 879-888Abstract Full Text PDF PubMed Scopus (340) Google Scholar, 17Therrien M. Michaud N.R. Rubin G.M. Morrison D.K. Genes Dev. 1996; 10: 2684-2695Crossref PubMed Scopus (210) Google Scholar, 18Kornfeld K. Hom D.B. Horvitz H.R. Cell. 1995; 83: 903-913Abstract Full Text PDF PubMed Scopus (250) Google Scholar). Specificity may be aided by the association of pairs of enzymes in MAP kinase cascades through stable binding sites (19Zanke B.W. Rubie E.A. Winnett E. Chan J. Randall S. Parsons M. Boudreau K. McInnis M. Yan M. Templeton D.J. Woodgett J.R. J. Biol. Chem. 1996; 271: 29876-29881Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 20Fukuda M. Gotoh Y. Nishida E. EMBO J. 1997; 16: 1901-1908Crossref PubMed Scopus (331) Google Scholar). MAP kinases recognize targeting domains such as the D domain (21Yang 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, 22Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (681) Google Scholar, 23Jacobs D. Glossip D. Xing H. Muslin A.J. Kornfeld K. Genes Dev. 1999; 13: 163-175Crossref PubMed Scopus (441) Google Scholar). This type of mechanism apparently facilitates the association of ERK2 not only with its substrates but also with its activator MEK1 and inactivators such as the phosphatase PTP-SL (22Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (681) Google Scholar, 24Pulido R. Zuniga A. Ullrich A. EMBO J. 1998; 17: 7337-7350Crossref PubMed Scopus (272) Google Scholar). 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. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (875) Google Scholar). The full-length cDNA revealed that MEKK1 is a 195-kDa protein with a large noncatalytic N terminus (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). The N terminus mediates protein-protein interactions affecting its behavior and function. Several MEKK1-interacting proteins, such as JNK/SAPK, 14-3-3, the Nck-interacting kinase NIK, and α-actinin, have been shown to bind residues in the N terminus, although the physiological and regulatory impact of these interactions is uncertain (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, 33Fanger 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, 34Su Y.-C. Han J. Xu S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (218) Google Scholar, 35Christerson L.B. Vanderbilt C.A. Cobb M.H. Cell Motil. Cytoskelet. 1999; 43: 186-198Crossref PubMed Scopus (89) Google Scholar). MEKK1 has been implicated in the activation of the ERK1/2 and the JNK/SAPK pathways based on its ability to activate these kinases when overexpressed (31Lange-Carter C.A. Pleiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (875) Google Scholar, 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, 36Yan M. Dal T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (658) Google Scholar, 37Minden A. Lin A. McMahon M. Lange-Carter C. Dérijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1011) Google Scholar). Most attention has focused on JNK/SAPK, in part because MEKK1 can induce apoptosis under certain circumstances and JNK/SAPK has been implicated in this process, and because Raf isoforms appear to be the major, if not only, MEKKs for the ERK1/2 pathway (38Johnson N.L. Gardner A.M. Diener K.M. Lange-Carter C.A. Gleavy J. Jarpe M.B. Minden A. Karin M. Zon L.I. Johnson G.L. J. Biol. Chem. 1996; 271: 3227-3229Google Scholar, 39Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5036) Google Scholar, 40Dent P. Haser W. Haystead T.A.J. Vincent L.A. Roberts T.M. Sturgill T.W. Science. 1992; 257: 1404-1407Crossref PubMed Scopus (499) Google Scholar, 41Kyriakis J.M. App H. Zhang X.-F. Banerjee P. Brautigan D.L. Rapp U.R. Avruch J. Nature. 1992; 358: 417-421Crossref PubMed Scopus (977) Google Scholar). The most compelling evidence to implicate MEKK1 in the ERK1/2 pathway comes from experiments using embryonic stem cells lacking MEKK1 expression. In the MEKK1-deficient cells, the activation of JNK/SAPK and ERK1/2 by sorbitol, serum, and lysophosphatidic acid was significantly diminished, suggesting that these stimuli activate JNK/SAPK and ERK1/2 at least in part through MEKK1 (5Yujiri T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (282) Google Scholar). In vitro the catalytic domain of MEKK1 activates MEK1 by phosphorylating it on the same sites that Raf-1 phosphorylates, consistent with the idea that MEKK1 activates ERK1/2 directly through MEK1 (42Gardner A.M. Vaillancourt R.R. Lange-Carter C.A. Johnson G.L. Mol. Biol. Cell. 1994; 5: 193-201Crossref PubMed Scopus (80) Google Scholar, 43Yan M. Templeton D.J. J. Biol. 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 to Raf-1 protein was 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 MEKK1 fragments were on 30 μl of in the of 10 bovine and then incubated with or in ml of lysis buffer with 1% Triton for 2 h at 4 °C. were washed three times with 1 ml of 50 mm Tris-Cl, pH 8.0, 150 mm NaCl, 0.1% 1% and 0.5% for a total of h. Immunoprecipitates were washed as described in and incubated with 7 of purified with of in kinase buffer mm HEPES, pH 10 10 mm MgCl2, and 10 for 30 min at 30 °C. of was by and of We and previously showed that MEKK1 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 of the heterotrimeric G protein that activates this pathway Wu C. T. K. A. Thomas D.Y. E. Science. 1995; 269: PubMed Scopus Google Scholar, Genes Dev. 1998; 12: PubMed Scopus Google Scholar, R. Johnson J. Genetics. 1996; PubMed Google Scholar, Y. E. Elion E.A. Curr. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar). the also bind the kinase upstream of the MAP kinase to the module T. Wu C. J.D. M. Thomas D.Y. E. Nature. 1998; PubMed Scopus Google Scholar). then the MEKK to activation of the kinase cascade C. M. Thomas D.Y. E. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). To the that MEKK1 may be in a we examined its ability to bind the protein kinase, of the mammalian of E. T. H. Nature. 1994; PubMed Scopus Google Scholar, A. Frost J. Yang P. Hutchison M. A.M. Cobb M.H. S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). with this has been shown to activate the JNK/SAPK pathway and to activation of the ERK1/2 pathway through 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, A. Frost J. Yang P. Hutchison M. A.M. Cobb M.H. S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, S. Dérijard B. Davis R.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, A.J. H. B. D. W. S. Nature. 1998; PubMed Scopus Google Scholar). we another of that is a MEKK Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google and Raf-1 the MEKK in the ERK1/2 shown in with to Raf-1 was as a protein. We that of the overexpressed MEKK1 was to The that and the control to MEKK1 the idea that Raf-1 binding is not to a We the of MEKK1 with Raf-1 is in a the activation of type MEKK1 that is into cells is active in the of a (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); we the of MEKK1 activity by the of Raf-1 with wild type and MEKK1 2 that Raf-1 with both wild type and In to MEKK1, wild type Raf-1 has activity when it is expressed in to the of Raf catalytic activity on the we the of MEKK1 with the activated Raf-1 Raf-1 BXB and Raf-1 and the wild type protein. All three of Raf-1 were in MEKK1 indicating that MEKK1 interacts with Raf-1 in a of the activation of These that Raf and MEKK1 may be in cells the enzymes are activated or The also the that at least a portion of Raf-1 in a cell is with In other we that the of MEKK1 is to or than that of J. R. and M. H. for To the that the has in cells, we the association of the endogenous proteins. Raf-1 was in of MEKK1 to Jurkat or 293 cells antibodies MEKK1 Raf-1, indicating that the is not the result of protein or of Raf-1 in the MEKK1 were washed with salt and with to the of the 1 m NaCl 0.1% were to the These demonstrate that endogenous MEKK1 and Raf-1 with proteins. We also examined the possible of the by the activation of the endogenous of the kinases were in complexes using and MEK1 as substrates for MEKK1 and Raf-1, MEKK1 is inactive in Jurkat T cells and is activated by and (5Yujiri T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (282) Google 3 Raf-1 is in MEKK1 of the activation of In experiments was or no in the of Raf-1 in the MEKK1 although a small was with To the of Raf-1 activity on its association with MEKK1, we used 293 cells, because Jurkat T cells not in Raf-1 is active in Jurkat T cells and in 293 its activity is by not A of endogenous Raf-1 interacts with endogenous MEKK1 in or 293 cells 3 that their association is of the activation of We the domains by encoding kinase with fragments of the The binding site on MEKK1 was first to a fragment including residues the N-terminal and fragments not 4 A fragment containing residues still binds to Raf-1, but residues not indicating that residues the Raf-1 binding This portion of the regulatory N terminus of MEKK1 a but the of residues for Raf-1 was not experiments with the and domains of Raf-1 that the which the catalytic domain of Raf-1, residues is to bind to MEKK1 4 Raf-1 and MEKK1 bind a of proteins within cells. it is possible that the these kinases is mediated by or proteins. To this fragments and Raf-1 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 protein in bacteria using this fusion A.J. H. B. D. W. S. Nature. 1998; PubMed Scopus Google Scholar). In with the from 293 cells, MEKK1 fragment 221–559 to purified Raf-1 This result that MEKK1 binds Raf-1 because no other proteins were in these binding was also using full-length Raf-1 expressed in Sf9 cells that Raf-1 and MEKK1 are to a under all we to evidence the of their MEKK1 can activate the ERK1/2 pathway, we the that MEKK1 also binds to MEK1 and 293 cells were with MEK1 or with encoding fragments or full-length The MEKK1 proteins were using the and were for MEK1 with MEKK1, both full-length and an N-terminal residues For we examined the ability of MEKK1 to with of the JNK/SAPK binds to a fragment containing residues We showed previously that directly with MEKK1 but p38 not (28Xu S. Cobb M.H. J. Biol. Chem. 1997; 272: 32056-32060Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). MEKK1 N-terminal fragments and ERK2 both expressed in bacteria bind in vitro not To the ability of MEKK1 to bind to ERK2 in cells, 293 cells were