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• W2004259745 abstract "Growth factors activate Raf-1 by engaging a complex program, which requires Ras binding, membrane recruitment, and phosphorylation of Raf-1. The present study employs the microtubule-depolymerizing drug nocodazole as an alternative approach to explore the mechanisms of Raf activation. Incubation of cells with nocodazole leads to activation of Pak1/2, kinases downstream of small GTPases Rac/Cdc42, which have been previously indicated to phosphorylate Raf-1 Ser338. Nocodazole-induced stimulation of Raf-1 is augmented by co-expression of small GTPases Rac/Cdc42 and Pak1/2. Dominant negative mutants of these proteins block activation of Raf-1 by nocodazole, but not by epidermal growth factor (EGF). Thus, our studies define Rac/Cdc42/Pak as a module upstream of Raf-1 during its activation by microtubule disruption. Although it is Ras-independent, nocodazole-induced activation of Raf-1 appears to involve the amino-terminal regulatory region in which the integrity of the Ras binding domain is required. Surprisingly, the Raf zinc finger mutation (C165S/C168S) causes a robust activation of Raf-1 by nocodazole, whereas it diminishes Ras-dependent activation of Raf-1. We also show that mutation of residues Ser338 to Ala or Tyr340-Tyr341 to Phe-Phe immediately amino-terminal to the catalytic domain abrogates activation of both the wild type and zinc finger mutant Raf by both EGF/4β-12-O-tetradecanoylphorbol-13-acetate and nocodazole. Finally, an in vitro kinase assay demonstrates that the zinc finger mutant serves as a better substrate of Pak1 than the wild type Raf-1. Collectively, our results indicate that 1) the zinc finger exerts an inhibitory effect on Raf-1 activation, probably by preventing phosphorylation of 338SSYY341; 2) such inhibition is first overcome by an unknown factor binding in place of Ras-GTP to the amino-terminal regulatory region in response to nocodazole; and 3) EGF and nocodazole utilize different kinases to phosphorylate Ser338, an event crucial for Raf activation. Growth factors activate Raf-1 by engaging a complex program, which requires Ras binding, membrane recruitment, and phosphorylation of Raf-1. The present study employs the microtubule-depolymerizing drug nocodazole as an alternative approach to explore the mechanisms of Raf activation. Incubation of cells with nocodazole leads to activation of Pak1/2, kinases downstream of small GTPases Rac/Cdc42, which have been previously indicated to phosphorylate Raf-1 Ser338. Nocodazole-induced stimulation of Raf-1 is augmented by co-expression of small GTPases Rac/Cdc42 and Pak1/2. Dominant negative mutants of these proteins block activation of Raf-1 by nocodazole, but not by epidermal growth factor (EGF). Thus, our studies define Rac/Cdc42/Pak as a module upstream of Raf-1 during its activation by microtubule disruption. Although it is Ras-independent, nocodazole-induced activation of Raf-1 appears to involve the amino-terminal regulatory region in which the integrity of the Ras binding domain is required. Surprisingly, the Raf zinc finger mutation (C165S/C168S) causes a robust activation of Raf-1 by nocodazole, whereas it diminishes Ras-dependent activation of Raf-1. We also show that mutation of residues Ser338 to Ala or Tyr340-Tyr341 to Phe-Phe immediately amino-terminal to the catalytic domain abrogates activation of both the wild type and zinc finger mutant Raf by both EGF/4β-12-O-tetradecanoylphorbol-13-acetate and nocodazole. Finally, an in vitro kinase assay demonstrates that the zinc finger mutant serves as a better substrate of Pak1 than the wild type Raf-1. Collectively, our results indicate that 1) the zinc finger exerts an inhibitory effect on Raf-1 activation, probably by preventing phosphorylation of 338SSYY341; 2) such inhibition is first overcome by an unknown factor binding in place of Ras-GTP to the amino-terminal regulatory region in response to nocodazole; and 3) EGF and nocodazole utilize different kinases to phosphorylate Ser338, an event crucial for Raf activation. Ras binding domain cysteine-rich zinc finger domain mitogen-activated protein kinase extracellular regulated kinase mitogen-activated protein kinase andextracellular regulated kinase kinase glutathione S-transferase polyacrylamide gel electrophoresis epidermal growth factor 4β-12-O-tetradecanoylphorbol-13-acetate p21-activated kinase myelin basic protein hemagglutinin amino acid(s) The proto-oncogene raf-1, first identified as a cellular counterpart of the oncogene v-raf, encodes a serine/threonine protein kinase. Raf-1 is ubiquitously expressed and plays an important role in cell proliferation and differentiation (1Rapp U.R. Cleveland J.L. Bonner T.I. Storm S.M. Reddy E.P. Skalka A.M. Curran T. The Oncogene Handbook. Elsevier Science Publishers B.V., Amsterdam1988: 213-253Google Scholar). The mechanism by which Raf-1 is activated by growth factors is still incompletely understood, although it is known to be preassembled as a complex with 14-3-3 and heat shock proteins 90/50 (2Avruch J. Zhang X.F. Kyriakis J.M. Trends Biochem. Sci. 1994; 19: 279-283Abstract Full Text PDF PubMed Scopus (541) Google Scholar, 3Morrison D.K. Cutler Jr., R.E. Curr. Opin. Cell Biol. 1997; 9: 174-179Crossref PubMed Scopus (537) Google Scholar, 4Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 536-539Crossref Scopus (388) Google Scholar, 5Thorson J.A., Yu, L.W.K. Hsu A.L. Shih N.-Y. Graves P.R. Tanner J.W. Allen P.M. Piwnica-Worms H. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar, 6Schulte T.W. Blagosklonny M.V. Romanova L. Mushinski J.F. Monia B.P. Johnston J.F. Nguyen P. Trepel J. Neckers L.M. Mol. Cell. Biol. 1996; 16: 5839-5845Crossref PubMed Scopus (256) Google Scholar). It involves multiple steps including Ras-GTP binding, membrane recruitment, and phosphorylation. Raf-1 consists of an amino-terminal regulatory domain and a carboxyl-terminal kinase domain. The amino-terminal moiety of Raf-1 exerts an inhibitory effect on the catalytic activity, since amino-terminal truncations lead to progressive increases in its transforming ability (7Stanton Jr., V.P. Nichols D.W. Laudano A.P. Cooper G.M. Mol. Cell. Biol. 1989; 9: 639-647Crossref PubMed Scopus (189) Google Scholar, 8Heidecker G. Huleihel M. Cleveland J.L. Kolch W. Beck T.W. Lloyd P. Pawson T. Rapp U.R. Mol. Cell. Biol. 1990; 10: 2503-2512Crossref PubMed Scopus (206) Google Scholar). The amino-terminal regulatory region of Raf-1 contains a Ras binding domain (RBD)1 and a cysteine-rich zinc finger domain (CRD), both of which participate in binding to Ras. The first interaction engages Raf RBD ranging from aa 50 to 150 and the effector loop of Ras-GTP, which is essential for activation of Raf-1 (9Zhang X.F. Settleman J. Kyriakis J.M. Takeuchi-Suzuki E. Elledge S.J. Marshall M.S. Bruder J.T. Rapp U.R. Avruch J. Nature. 1993; 364: 308-313Crossref PubMed Scopus (687) Google Scholar, 10Chuang E. Barnard D. Hettich L. Zhang X.F. Avruch J. Marshall M.S. Mol. Cell. Biol. 1994; 14: 25-5318Crossref Scopus (158) Google Scholar, 11Scheffler J.E. Waugh D.S. Bekesi E. Kiefer S.E. LoSardo J.E. Neri A. Prinzo K.M. Tsao K.L. Wegrzynski B. Emerson S.D. J. Biol. Chem. 1994; 269: 22340-22346Abstract Full Text PDF PubMed Google Scholar). Raf CRD, located between aa 139 and 184, binds to an epitope involving Ras residues Asn26 and Val45 outside the effector loop in prenylated Ras in a GTP-independent manner (12Hu C.D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30274.7Crossref PubMed Scopus (127) Google Scholar, 13Williams J.G. Drugan J.K. Yi G.S. Clark G.J. Der C.J. Campbell S.L. J. Biol. Chem. 2000; 275: 22172-22179Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 14Marshall M.S. Trends Biochem. Sci. 1993; 18: 250-254Abstract Full Text PDF PubMed Scopus (194) Google Scholar, 15Luo Z. Diaz B. Marshall M.S. Avruch J. Mol. Cell. Biol. 1997; 17: 46-53Crossref PubMed Scopus (106) Google Scholar). The strength of this second interaction is much lower than the first one (12Hu C.D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30274.7Crossref PubMed Scopus (127) Google Scholar, 13Williams J.G. Drugan J.K. Yi G.S. Clark G.J. Der C.J. Campbell S.L. J. Biol. Chem. 2000; 275: 22172-22179Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Nevertheless, it is crucial for the formation of a productive complex, as the mutation of residues necessary for this interaction on either proteins abolishes their functions, such as the ability to transform cells (14Marshall M.S. Trends Biochem. Sci. 1993; 18: 250-254Abstract Full Text PDF PubMed Scopus (194) Google Scholar) and to activate MAPK kinase (15Luo Z. Diaz B. Marshall M.S. Avruch J. Mol. Cell. Biol. 1997; 17: 46-53Crossref PubMed Scopus (106) Google Scholar). Thus, these studies suggest that growth factor-stimulated GTP charging of Ras initiates the association of the Raf RBD with the effector loop of Ras, which ensures the second productive interaction between the CRD and Ras. Considerable evidence indicates that phosphorylation plays an important role in Raf activation. Incubation of Raf-1 activated in vivo with serine/threonine or tyrosine protein phosphatases leads to inactivation of Raf-1 (16Kovacina K.S. Yonezawa K. Brautigan D.L. Tonks N.K. Rapp U.R. Roth R.A. J. Biol. Chem. 1990; 265: 12115-12118Abstract Full Text PDF PubMed Google Scholar, 17Turner B.C. Tonks N.K. Rapp U.R. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5544-5548Crossref PubMed Scopus (38) Google Scholar, 18Dent P. Jelinek T. Morrison D.K. Weber M. Sturgill T.W. Science. 1995; 269: 1902-1906Crossref Scopus (173) Google Scholar, 19Jelinek T. Dent P. Sturgill T.W. Weber M.J. Mol. Cell. Biol. 1996; 16: 1027-1034Crossref PubMed Scopus (123) Google Scholar). Indeed, a number of serine/threonine and tyrosine protein kinases have been implicated in the activation of Raf-1 (3Morrison D.K. Cutler Jr., R.E. Curr. Opin. Cell Biol. 1997; 9: 174-179Crossref PubMed Scopus (537) Google Scholar). Raf-1 Ser259 and Ser621 are the major phosphorylation sites which are critical determinants for binding to 14-3-3 (20Morrison D.K. Heidecker G. Rapp U.R. Copeland T.D. J. Biol. Chem. 1993; 268: 17309-17316Abstract Full Text PDF PubMed Google Scholar). It appears that binding of 14-3-3 to the amino-terminal site (Ser(P)259) plays an inhibitory role, whereas the binding to the carboxyl-terminal site (Ser(P)621) is indispensable for the Raf kinase activity (21Michaud N.R. Fabian J.R. Mathes K.D. Morrison D.K. Mol. Cell. Biol. 1995; 15: 3390-3397Crossref PubMed Scopus (190) Google Scholar, 22Rommel C. Radziwill G. Lovric J. Noeldeke J. Heinicke T. Jones D. Aitken A. Moelling K. Oncogene. 1996; 12: 609-619PubMed Google Scholar, 23Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1188) Google Scholar). Other crucial residues include338SSYY341, whose mutation to alanine or phenylalanine severely inhibits Raf-1 activation and to aspartic acid or glutamic acid results in an increase in the basal Raf-1 activity (24Fabian J.R. Daar I.O. Morrison D.K. Mol. Cell. Biol. 1993; 13: 7170-7179Crossref PubMed Scopus (304) Google Scholar, 25Marais R. Light Y. Paterson H.P. Marshall C.J. EMBO J. 1995; 14: 3136-3145Crossref PubMed Scopus (527) Google Scholar, 26Diaz B. Barnard D. Filson A. MacDonald S. King A. Marshall M.S. Mol. Cell. Biol. 1997; 17: 4509-4516Crossref PubMed Scopus (163) Google Scholar, 27Mason C.S. Springer C.J. Cooper R.G. Superti-Furga G. Mashall C.J. Marais R. EMBO J. 1999; 18: 2137-2148Crossref PubMed Scopus (366) Google Scholar). In B-Raf, the two residues corresponding to Raf-1 Tyr340-Tyr341 are replaced by aspartic acids, which may account for the increased kinase activity (27Mason C.S. Springer C.J. Cooper R.G. Superti-Furga G. Mashall C.J. Marais R. EMBO J. 1999; 18: 2137-2148Crossref PubMed Scopus (366) Google Scholar). p21-activated kinases (Paks), mammalian homologs of Ste20-like Ser/Thr protein kinases, are activated by signals that increase the level of GTP-bound form of Rac and Cdc42 GTPases, although the GTPase-independent pathway also exists (28Knaus U.G. Bokoch G.M. Int. J. Biochem. Cell Biol. 1998; 30: 857-862Crossref PubMed Scopus (159) Google Scholar, 29Bagrodia S. Cerione R.A. Trends Cell Biol. 1999; 9: 350-355Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). The Pak family, consisting of Pak1 (Pakα), Pak2 (Pakγ), Pak3 (Pakβ), and Pak4, has been implicated in a variety of cellular functions, including regulation of cell proliferation, apoptosis, the cell cycle, stress response, oxidant generation, cell adhesion and motility, and cytoskeletal dynamics. Pak1 participates in V12Ras-induced transformation (30Tang Y. Chen Z. Ambrose D. Liu J. Gibbs J.B. Chernoff J. Field J. Mol. Cell. Biol. 1997; 17: 4454-4464Crossref PubMed Scopus (187) Google Scholar, 31Tang Y. Marwaha S. Rutkowski J.L. Tennekoon G.I. Phillips P.C. Field J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5139-5144Crossref PubMed Scopus (91) Google Scholar) and cooperates with Raf-1, leading to a maximal activation of MEK1 by phosphorylating the latter (32Frost J.A. Steen H. Shapiro P. Lewis T. Ahn N. Shaw P.E. Cobb M.H. EMBO J. 1997; 16: 6426-6438Crossref PubMed Scopus (362) Google Scholar). Recent data reveal that Pak2 can phosphorylate Ser338 (33King A.J. Sun H. Diaz B. Barnard D. Miao W. Bagrodia S. Marshall M.S. Nature. 1998; 396: 180-184Crossref PubMed Scopus (385) Google Scholar) and contribute to Raf-1 activation by V12Ras or a constitutively active mutant of phosphatidylinositol 3-kinase (34Sun H. King A.J. Diaz B. Marshall M.S. Curr. Biol. 2000; 10: 281-284Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In addition, integrin-induced activation of Raf-1 has been shown to be mediated by Pak1 (35Chaudhary A. King W.G. Mattalino M.D. Frost J.A. Diaz B. Morrison D.K. Cobb M.H. Marshall M.S. Brugge J.S. Curr. Biol. 2000; 10: 551-554Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). The requirement of multiple factors for receptor tyrosine kinase-stimulated activation of Raf-1 has greatly challenged us in precise elucidation of its regulation. Recent studies on Raf-1 activation by disrupting microtubule integrity that can elude the Ras binding and membrane recruitment may shed a light on uncovering this mystery (36Ziogas A. Lorenz I.C. Moelling K. Radziwill G. J. Biol. Chem. 1998; 273: 24108-24114Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 37Laird A.D. Morrison D.K. Shalloway D. J. Biol. Chem. 1999; 274: 4430-4439Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). We have shown that nocodazole, a microtubule-depolymerizing drug, activates Raf-1 and increases its binding to 14-3-3, while inducing mitosis and hyperphosphorylation of Raf-1 (38Hayne C. Tzivion T. Luo Z. J. Biol. Chem. 2000; 275: 31878-31882Abstract Full Text Full Text PDF Scopus (94) Google Scholar). Furthermore, the activation of Raf-1 is necessary for entry of the cell cycle into mitosis (38Hayne C. Tzivion T. Luo Z. J. Biol. Chem. 2000; 275: 31878-31882Abstract Full Text Full Text PDF Scopus (94) Google Scholar). In the present study, we have characterized this Ras-independent, nocodazole-induced activation of Raf-1. Here we first show that nocodazole utilizes Rac/Cdc42/Pak to activate Raf-1, in which a crucial step is the phosphorylation of Ser338 by Pak, while EGF activates Raf-1 by a different Ser338 kinase. We also find that, although Raf-1 activation by nocodazole is Ras-independent, the integrity of the Raf RBD, but not the CRD, is still required. Moreover, mutation of Cys165-Cys168 to Ser-Ser within the CRD causes a robust activation of Raf-1 by nocodazole and an increased ability of Raf to be phosphorylated in vitro by Pak, suggesting that the zinc finger structure plays an inhibitory role in Raf activation. Nocodazole and 4β-12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma. Human recombinant epidermal growth factor (EGF) was from Calbiochem (San Diego, CA). Glutathione (GSH)-Sepharose 4B was purchased from Amersham Pharmacia Biotech. Monoclonal antibody against Raf-1 (E10), monoclonal antibody against GST (B14), and horseradish peroxidase-conjugated second antibodies and protein A/G-agarose were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody against Raf-1 phospho-Ser338 were from Upstate Biotechnology (Lake Placid, NY). Antibodies against phospho-14-3-3 pan binding sites, phospho-MAPK (Erk1/2) and MAPK, and Akt phospho-Thr473 and Akt were from New England Biolabs (Beverly, MA). cDNAs encoding Raf-1 variants, wild type, Ras binding site mutant (84–87AAAA), zinc finger mutant (165S/168S) and kinase-dead mutant (K375M) were constructed into pMT2Myc as described previously (15Luo Z. Diaz B. Marshall M.S. Avruch J. Mol. Cell. Biol. 1997; 17: 46-53Crossref PubMed Scopus (106) Google Scholar). cDNAs for wild type Raf, amino-terminally truncated kinase domain (BXBRaf, aa 1–25/303–648) (39Bruder J.T. Hedcker G. Rapp U.R. Genes Dev. 1992; 6: 545-556Crossref PubMed Scopus (396) Google Scholar), amino-terminal regulatory region (C4, aa 1–259) (40Luo Z.J. Zhang X.F. Rapp U. Avruch J. J. Biol. Chem. 1995; 270: 23681-23687Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and Pak1 variants were inserted into pEBG (40Luo Z.J. Zhang X.F. Rapp U. Avruch J. J. Biol. Chem. 1995; 270: 23681-23687Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). cDNA for Raf-1 84–87AAAA was also inserted into pBJMFPK3E in which Src myristoylation sequence was tagged to the amino terminus of three copies of FKBP followed by the cDNA encoding a hemagglutinin epitope and the Raf mutant (41Luo Z. Tzivion G. Belshaw P.J. Vavvas D. Marshall M. Avruch J. Nature. 1996; 383: 181-185Crossref PubMed Scopus (204) Google Scholar). Double site mutants, 165S/168S-380A/339A and 165S/168S-340F/341F, were made by replacing the wild typeSalI fragment in pMTMyc-Raf 165S/168S with the one containing the mutation S338A/S339A or Y340F/Y341F. N17HaRas, V12Rac and V12Cdc42 were constructed in pCMV5flag plasmid (15Luo Z. Diaz B. Marshall M.S. Avruch J. Mol. Cell. Biol. 1997; 17: 46-53Crossref PubMed Scopus (106) Google Scholar). N17Rac and N17Cdc42 in pRK5Myc were obtained from W. Xiong, and Pak2 constructs (in pRK5Myc) were from G. M. Bokoch. Raf 338A/339A and 340F/341F constructs were from M. S. Marshall. Human embryonic kidney 293 cells (HEK293T) and COS7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transfection of plasmid DNA into HEK293T cells was carried out using the calcium phosphate precipitation method. LipofectAMINE reagents (Life Technologies, Inc.) were used for transfection of plasmids into COS7 cells according to manufacturer's protocol. Forty-eight hours after transfection, cells were serum-starved in Dulbecco's modified Eagle's medium containing 0.1% fetal bovine serum for 16–20 h and were treated with nocodazole, EGF, or TPA as indicated in figure legends. Cells were then lysed in a lysis buffer (20 mm Tris-HCl, pH 7.8, 1 mm EDTA, 1 mm EGTA, 1 mm Na3VO4, 25 mm β-glycerol phosphate, 1 mmdithiothreitol, 1% Nonidet P-40, and protease inhibitors) (40Luo Z.J. Zhang X.F. Rapp U. Avruch J. J. Biol. Chem. 1995; 270: 23681-23687Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Cell debris were removed by centrifugation at 14,000 × gfor 15 min at 4 °C, and protein concentrations in cell lysates were measured using a Bio-Rad protein assay kit. For immunoprecipitation, cell lysates were incubated with specific antibodies and protein A/G-agarose at 4 °C overnight. The precipitates were washed once with the lysis buffer, twice with the buffer containing 0.5 m NaCl, and twice with a kinase buffer (25 mm Tris-HCl, pH 7.5, 50 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol). For purification of recombinant GST-Raf, the lysates were incubated with GSH beads and washed as immunoprecipitation. For Western blotting, samples were separated by 8% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore) in a transfer buffer consisting of 154 mmglycine, 20 mm Tris, and 20% methanol. The membranes were blocked and incubated with specific antibodies and then with a horseradish peroxidase-conjugated second antibody. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection system. Raf-1 kinase activity was measured by a coupled enzyme assay in which bacterially expressed recombinant GST-MEK1 and kinase-dead mutant of ERK2 were sequentially added to the Raf preparation in the presence of [γ-32P]ATP (100 μm, 2000 cpm/pmol), as described previously (40Luo Z.J. Zhang X.F. Rapp U. Avruch J. J. Biol. Chem. 1995; 270: 23681-23687Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The reaction was stopped by the addition of a SDS-PAGE sample buffer, and the labeled mixture was resolved by 8% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and visualized by autoradiography. The radiolabeled Erk2 bands were excised and quantified by liquid scintillation counting. Recombinant GST-Pak1 and Myc-tagged Pak2 were transiently expressed in HEK293T cells. After stimulation with 300 nm nocodazole for 1 h, cell lysates were prepared as described above. GST-Pak1 was purified by GSH beads and Myc-Pak2 immunoprecipitated with anti-Myc antibody (9E10). Pak activity was analyzed at 30 °C for 30 min using 1 μg of myelin basic protein (MBP) as a direct substrate. For in vitro phosphorylation of Raf-1, recombinant GST-Pak1 was purified by GSH beads and eluted with 5 mm GSH. The immunoprecipitated Myc-Raf-1 was incubated with Pak1 and ATP. The phosphorylation was visualized by anti-Ser(P)338 blotting (27Mason C.S. Springer C.J. Cooper R.G. Superti-Furga G. Mashall C.J. Marais R. EMBO J. 1999; 18: 2137-2148Crossref PubMed Scopus (366) Google Scholar). Recently, we have shown that nocodazole activates Raf-1 in a time-dependent manner and such activation is necessary for transition of the cell cycle from G2 to M phase (38Hayne C. Tzivion T. Luo Z. J. Biol. Chem. 2000; 275: 31878-31882Abstract Full Text Full Text PDF Scopus (94) Google Scholar). To further study the mechanism of nocodazole-induced Raf activation, we first determined the dose effect of nocodazole on activation of Raf-1. In doing so, cDNAs encoding the wild type and the kinase-defective mutant (K375M) of Raf-1 were transiently expressed in HEK293T cells, and Raf-1 kinase activity was assayed after cells were treated with nocodazole in different doses as indicated in Fig.1A. Raf-1 was progressively activated by increasing doses of nocodazole with a maximal effect at 300 nm under the condition of equal expression of the polypeptides. The inability of the kinase-defective mutant Raf to be activated indicated that the nocodazole-increased kinase activity was not due to kinases copurifying with Raf-1. The dose-dependent activation of Raf-1 is consistent with the ability of nocodazole to disrupt the microtubule structure (data not shown). To differentiate nocodazole-induced activation of Raf-1 from the Ras-dependent one by EGF, Myc-tagged Raf-1 was co-expressed with RafC4 (aa 1–259), the amino-terminal regulatory region containing RBD and CRD, or N17HaRas, a dominant negative mutant of Ras. This experiment was done in COS7 cells since this cell line exhibits better response to EGF than HEK293T cells. As shown in Fig.1B, co-expression of C4 or N17Ras reproducibly inhibited EGF activation of Raf-1 by about 40%, which is similar to our previous findings (41Luo Z. Tzivion G. Belshaw P.J. Vavvas D. Marshall M. Avruch J. Nature. 1996; 383: 181-185Crossref PubMed Scopus (204) Google Scholar). In contrast, these mutants did not have significant effect on Raf-1 activation by nocodazole, despite the fact that the immunoblot showed equal expression of C4 or N17Ras under these two treatments (data not shown). Interestingly, we observed a 40% increase in Raf activation by nocodazole in the presence of N17 Ras, as compared with that in its absence (Fig. 1B, lanes 1and 3). Overall, our results indicate that nocodazole up-regulates Raf kinase via a Ras-independent mechanism, which is consistent with previous publications (36Ziogas A. Lorenz I.C. Moelling K. Radziwill G. J. Biol. Chem. 1998; 273: 24108-24114Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 37Laird A.D. Morrison D.K. Shalloway D. J. Biol. Chem. 1999; 274: 4430-4439Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Since Ser338 has been previously shown to be phosphorylated by Pak (33King A.J. Sun H. Diaz B. Barnard D. Miao W. Bagrodia S. Marshall M.S. Nature. 1998; 396: 180-184Crossref PubMed Scopus (385) Google Scholar, 34Sun H. King A.J. Diaz B. Marshall M.S. Curr. Biol. 2000; 10: 281-284Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 35Chaudhary A. King W.G. Mattalino M.D. Frost J.A. Diaz B. Morrison D.K. Cobb M.H. Marshall M.S. Brugge J.S. Curr. Biol. 2000; 10: 551-554Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), we attempted to evaluate the role of Rac/Cdc42 and Pak in nocodazole-induced Raf-1 activation. In the first assay, we introduced GST-Pak1 and Myc-Pak2 into HEK293T cells and examined activation of these kinases by nocodazole (Fig.2A). The recombinant Pak1 was activated by 3-fold, whereas Pak2 activity was increased by about 50%. The lesser activation of Pak2 might be due to its high basal activity or partial activation caused by the basic substrate MBP (49Clark G.J. Drugan J.K. Rossman K.L. Carpenter J.W. Rogers-Graham K. Fu H. Der C.J. Campbell S.L. J. Biol. Chem. 1997; 272: 20990-20993Crossref PubMed Scopus (104) Google Scholar). In the next experiment, a constitutively active mutant of Rac, V12Rac was co-expressed with the wild type Raf and its activation was examined after treatment of cells with nocodazole. Coexpression of V12Rac led to about 2.5-fold increase in basal kinase activity of Raf (Fig. 2B, lanes 1 and 4) and more potent activation by nocodazole so that the Raf activity was increased by an additional 7-fold (Fig. 2B, lanes 1, 4,and5). Similar activation profiles were obtained by expression of a constitutively active mutant of Cdc42, Pak1, and Pak2 (Fig. 2, C and D). To further establish the role of Rac/Cdc42/Pak pathway in activation of Raf-1 by disrupting the microtubule integrity, we next co-transfected Raf with a dominant negative mutant of Rac, N17Rac, or a kinase-defective mutant of Pak2. Fig. 3shows that, whereas the mutants had no effect on EGF-induced activation of Raf-1, they strongly inhibited Raf-1 activation by nocodazole. The same inhibitory effect was also achieved by co-expression of dominant negative mutants of Cdc42 and Pak1 with Raf-1 (data not shown). These results clearly place the Rac/Cdc42/Pak lineage as an upstream module in the activation of Raf-1 by microtubule depolymerization and suggest that growth factor-dependent activation of Raf-1 occurs through different regulators. To evaluate the role of Ser338-Ser339 and Tyr340-Tyr341 in nocodazole-induced activation of Raf-1, we transfected Raf mutants 338A/339A and 340F/341F into COS7 cells and examined their kinase activities, as compared with the wild type Raf-1 (Fig. 4A). Both mutations greatly inhibited the activation of Raf-1 by EGF and nocodazole. Additionally, co-expression of Pak was without effect on Raf-1 activity if Ser338 was mutated to Ala (Fig.4B). Thus, these results suggest that phosphorylation of this region is critical to both Ras-dependent and independent activation of Raf-1. To verify whether Raf residue Ser338 is phosphorylated during Raf activation, endogenous Raf-1 was immunoprecipitated and blotted with phospho-Ser338 antibody. Fig.5A shows that the phosphorylation of Ser338 was enhanced by both TPA and nocodazole. When Raf-1 was co-expressed with the active mutant of Pak2, Raf Ser338 was highly phosphorylated (Fig. 5B), which held with its stimulatory role in Raf-1 activation by nocodazole (Figs. 2 and 3). The phosphorylation of Raf-1 by Pak seemed to be site-specific, as the phosphorylation of the 14-3-3 binding sites was not altered under the same conditions (Fig. 5B). To ascertain whether the phosphorylation and activation of Raf-1 by nocodazole is a specific cellular event, we examined the phosphorylation of Akt Ser473, an indicator for its activation. Fig. 5C shows that phosphorylation of Akt Ser473 was not changed in HEK 293T cells treated with nocodazole and TPA, while MAPK/Erk was significantly activated. Another piece of experimental evidence was the failure of nocodazole to activate cAMP-dependent protein kinase (data not shown). To assess whether the amino-terminal moiety is necessary for nocodazole-induced Raf-1 activation, we compared the activation of full-length Raf-1 and BXB-Raf-1 containing the entire carboxyl-terminal kinase domain. Fig.6A shows that the full-length Raf-1was activated well by both EGF and nocodazole, whereas the activity of BXB-Raf-1 was barely affected by these agents. The same results were obtained by using both HEK293T and COS7 cells. Thus, our findings demonstrate that the amino-terminal regulatory domain encompassing the Ras binding site is also necessary for Ras-independent activation of Raf-1. We next tested whether RBD is required for the action of nocodazole. To this regard, Raf-1 with the 84KLAK87 to AAAA mutation in the RBD was assayed for its kinase activity. Although the wild type Raf-1 was activated normally by both TPA and nocodazole, the mutation blunted its activation (Fig. 6B), even in the presence of co-expressed Pak1 (Fig. 6C). To ascertain whether the inability of thi" @default.
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• W2004259745 title "Microtubule Integrity Regulates Pak Leading to Ras-independent Activation of Raf-1" @default.
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