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W1978168320 abstract "The Ras-Raf-MEK signaling cascade is critical for normal development and is activated in many forms of cancer. We have recently shown that B-Raf kinase interacts with and is inhibited by Rheb, the target of the GTPase-activating domain of the tuberous sclerosis complex 2 gene product tuberin. Here, we demonstrate for the first time that activation of Rheb is associated with decreased B-Raf and C-Raf phosphorylation at residues Ser-446 and Ser-338, respectively, concomitant with a decrease in the activities of both kinases and decreased heterodimerization of B-Raf and C-Raf. Importantly, the impact of Rheb on B-Raf/C-Raf heterodimerization and kinase activity are rapamycin-insensitive, indicating that they are independent of Rheb activation of the mammalian target of rapamycin-Raptor complex. In addition, we found that Rheb inhibits the association of B-Raf with H-Ras. Taken together, these results support a central role of Rheb in the regulation of the Ras/B-Raf/C-Raf/MEK signaling network. The Ras-Raf-MEK signaling cascade is critical for normal development and is activated in many forms of cancer. We have recently shown that B-Raf kinase interacts with and is inhibited by Rheb, the target of the GTPase-activating domain of the tuberous sclerosis complex 2 gene product tuberin. Here, we demonstrate for the first time that activation of Rheb is associated with decreased B-Raf and C-Raf phosphorylation at residues Ser-446 and Ser-338, respectively, concomitant with a decrease in the activities of both kinases and decreased heterodimerization of B-Raf and C-Raf. Importantly, the impact of Rheb on B-Raf/C-Raf heterodimerization and kinase activity are rapamycin-insensitive, indicating that they are independent of Rheb activation of the mammalian target of rapamycin-Raptor complex. In addition, we found that Rheb inhibits the association of B-Raf with H-Ras. Taken together, these results support a central role of Rheb in the regulation of the Ras/B-Raf/C-Raf/MEK signaling network. B-Raf, A-Raf, and C-Raf (also called Raf-1) are members of the Raf kinase family, which regulate the activation of the MEK 2The abbreviations used are: MEK, MAPK/ERK-activating kinase; MAPK, mitogen-activating protein kinase; ERK, extracellular signal-regulated kinase; TSC2, tuberous sclerosis complex 2; mTOR, mammalian target of rapamycin; siRNA, small interference RNA; HA, hemagglutinin; MEF, mouse embryonic fibroblast.2The abbreviations used are: MEK, MAPK/ERK-activating kinase; MAPK, mitogen-activating protein kinase; ERK, extracellular signal-regulated kinase; TSC2, tuberous sclerosis complex 2; mTOR, mammalian target of rapamycin; siRNA, small interference RNA; HA, hemagglutinin; MEF, mouse embryonic fibroblast. (MAPK/ERK-activating kinase)/MAPK signaling cascade and share Ras as a common activator. B-Raf and C-Raf heterodimerize, thereby increasing the activity of both kinases (1Weber C.K. Slupsky J.R. Kalmes H.A. Rapp U.R. Cancer Res. 2001; 61: 3595-3598PubMed Google Scholar, 2Garnett M.J. Rana S. Paterson H. Barford D. Marais R. Mol. Cell. 2005; 20: 963-969Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Both B-Raf and C-Raf mutations are associated with human cancers (3Wellbrock C. Karasarides M. Marais R. Nat. Rev. Mol. Cell Biol. 2004; 5: 875-885Crossref PubMed Scopus (919) Google Scholar, 4Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix-Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8215) Google Scholar, 5Zebisch A. Staber P.B. Delavar A. Bodner C. Hiden K. Fischereder K. Janakiraman M. Linkesch W. Auner H.W. Emberger W. Wind-passinger C. Schimek M.G. Hoefler G. Troppmair J. Sill H. Cancer Res. 2006; 66: 3401-3408Crossref PubMed Scopus (83) Google Scholar). B-Raf mutations, which occur in melanoma, thyroid, and other cancers and in developmental disorders, including cardio-facio-cutaneous syndrome (6Rodriguez-Viciana P. Tetsu O. Tidyman W.E. Estep A.L. Conger B.A. Cruz M.S. McCormick F. Rauen K.A. Science. 2006; 311: 1287-1290Crossref PubMed Scopus (460) Google Scholar, 7Schubbert S. Zenker M. Rowe S.L. Boll S. Klein C. Bollag G. van der Burgt I. Musante L. Kalscheuer V. Wehner L.E. Nguyen H. West B. Zhang K.Y. Sistermans E. Rauch A. Niemeyer C.M. Shannon K. Kratz C.P. Nat. Genet. 2006; 38: 331-336Crossref PubMed Scopus (589) Google Scholar, 8Niihori T. Aoki Y. Narumi Y. Neri G. Cave H. Verloes A. Okamoto N. Hennekam R.C. Gillessen-Kaesbach G. Wieczorek D. Kavamura M.I. Kurosawa K. Ohashi H. Wilson L. Heron D. Bonneau D. Corona G. Kaname T. Naritomi K. Baumann C. Matsumoto N. Kato K. Kure S. Matsubara Y. Nat. Genet. 2006; 38: 294-296Crossref PubMed Scopus (454) Google Scholar), are more common than C-Raf mutations. However, because of the heterodimerization of C-Raf and B-Raf, which impacts the activity of both kinases (1Weber C.K. Slupsky J.R. Kalmes H.A. Rapp U.R. Cancer Res. 2001; 61: 3595-3598PubMed Google Scholar, 2Garnett M.J. Rana S. Paterson H. Barford D. Marais R. Mol. Cell. 2005; 20: 963-969Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar), mutations in one kinase can influence the activity of the other. A small molecule inhibitor of Raf was approved in 2006 for the treatment of renal cancer (9Staehler M. Haseke N. Schoppler G. Stadler T. Adam C. Stief C.G. Urologe. A. 2006; 45 (quiz 111-112): 99-110Crossref PubMed Scopus (8) Google Scholar, 10Beeram M. Patnaik A. Rowinsky E.K. J. Clin. Oncol. 2005; 23: 6771-6790Crossref PubMed Scopus (246) Google Scholar). We and others have previously shown that Rheb (Ras homolog enriched in brain) inhibits B-Raf kinase activity (11Im E. von Lintig F.C. Chen J. Zhuang S. Qui W. Chowdhury S. Worley P.F. Boss G.R. Pilz R.B. Oncogene. 2002; 21: 6356-6365Crossref PubMed Scopus (116) Google Scholar, 12Karbowniczek M. Cash T. Cheung M. Robertson G.P. Astrinidis A. Henske E.P. J. Biol. Chem. 2004; 279: 29930-29937Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Rheb, a member of the Ras/Rap/Ral subfamily of Ras proteins (13Yamagata K. Sanders L.K. Kaufmann W.E. Yee W. Barnes C.A. Nathans D. Worley P.F. J. Biol. Chem. 1994; 269: 16333-16339Abstract Full Text PDF PubMed Google Scholar), is inhibited by the GTPase-activating domain of the tuberous sclerosis complex 2 (TSC2) gene product, tuberin (14Stocker H. Radimerski T. Schindelholz B. Wittwer F. Belawat P. Daram P. Breuer S. Thomas G. Hafen E. Nat. Cell Biol. 2003; 5: 559-565Crossref PubMed Scopus (429) Google Scholar, 15Saucedo L.J. Gao X. Chiarelli D.A. Li L. Pan D. Edgar B.A. Nat. Cell Biol. 2003; 5: 566-571Crossref PubMed Scopus (530) Google Scholar, 16Zhang Y. Gao X. Saucedo L.J. Ru B. Edgar B.A. Pan D. Nat. Cell Biol. 2003; 5: 578-581Crossref PubMed Scopus (712) Google Scholar, 17Tee A.R. Manning B.D. Roux P.P. Cantley L.C. Blenis J. Curr. Biol. 2003; 13: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (940) Google Scholar, 18Inoki K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1400) Google Scholar, 19Garami A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. Mol. Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar, 20Castro A.F. Rebhun J.F. Clark G.J. Quilliam L.A. J. Biol. Chem. 2003; 278: 32493-32496Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Activation of Rheb also results in activation of the mammalian target of rapamycin (mTOR) (14Stocker H. Radimerski T. Schindelholz B. Wittwer F. Belawat P. Daram P. Breuer S. Thomas G. Hafen E. Nat. Cell Biol. 2003; 5: 559-565Crossref PubMed Scopus (429) Google Scholar, 15Saucedo L.J. Gao X. Chiarelli D.A. Li L. Pan D. Edgar B.A. Nat. Cell Biol. 2003; 5: 566-571Crossref PubMed Scopus (530) Google Scholar, 16Zhang Y. Gao X. Saucedo L.J. Ru B. Edgar B.A. Pan D. Nat. Cell Biol. 2003; 5: 578-581Crossref PubMed Scopus (712) Google Scholar, 17Tee A.R. Manning B.D. Roux P.P. Cantley L.C. Blenis J. Curr. Biol. 2003; 13: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (940) Google Scholar, 18Inoki K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1400) Google Scholar, 19Garami A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. Mol. Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar, 20Castro A.F. Rebhun J.F. Clark G.J. Quilliam L.A. J. Biol. Chem. 2003; 278: 32493-32496Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar), resulting in translational initiation and cell growth. Rheb inhibition of B-Raf kinase is insensitive to rapamycin (12Karbowniczek M. Cash T. Cheung M. Robertson G.P. Astrinidis A. Henske E.P. J. Biol. Chem. 2004; 279: 29930-29937Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), which is a specific inhibitor of the mTOR-Raptor complex (21Sabatini D.M. Erdjument-Bromage H. Lui M. Tempst P. Snyder S.H. Cell. 1994; 78: 35-43Abstract Full Text PDF PubMed Scopus (1211) Google Scholar, 22Brown E.J. Albers M.W. Shin T.B. Ichikawa K. Keith C.T. Lane W.S. Schreiber S.L. Nature. 1994; 369: 756-758Crossref PubMed Scopus (1647) Google Scholar, 23Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1717) Google Scholar). Therefore, Rheb appears to have two separate functions: mTOR activation and B-Raf inhibition. However, whether Rheb influences the activity of other members of the Raf kinase family is not known. It is also not known whether Rheb inhibition of B-Raf impacts the association of B-Raf with Ras and C-Raf. We demonstrate here that Rheb activation inhibits the kinase activity of C-Raf and strongly inhibits the heterodimerization of B-Raf and C-Raf. We also found that Rheb inhibits the association between B-Raf and H-Ras. Our data position Rheb as a key regulator of the Ras/B-Raf/C-Raf/MEK signaling cascade, with implications for the pathogenesis of TSC and for the design of targeted strategies to inhibit Raf kinases. Cells, Constructs, siRNA, and Antibodies—HEK293 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen). The Tsc2-/- p53-/- and Tsc2+/+ p53-/- MEFs were the gifts of Dr. David Kwiatkowski (Dept. of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA). When indicated, cells were serum-deprived for 18 h. Transfections were performed using Lipofectamine 2000 (Invitrogen). Where indicated, cells were treated for 24 h with 20 nm rapamycin (Biomol Research Laboratories, Plymouth Meeting, PA). The B-Raf kinase mutants were generated by site-directed mutagenesis using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA) and fully sequence-confirmed. The C-Raf cDNA and the C-Raf mutant S338D/Y341D/T491E/S494D (DEDD) were the gifts of Dr. Kun-Liang Guan (Life Sciences Institute, Dept. of Biological Chemistry, Institute of Gerontology, University of Michigan, Ann Arbor, MI). For siRNA down-regulation of TSC2 or Rheb, cells were transfected with 100 nm of either TSC2 SMARTpool siRNA or Rheb SMARTpool siRNA or nonspecific control siRNA (both from Dharmacon, Lafayette, CO) using Trans-IT TKO transfection reagent (Mirus, Madison WI). The following antibodies were used: anti-tuberin C20, anti-B-Raf (F-7), anti-C-Raf (E10), and anti-H-Ras (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-Ser-235/236 S6 ribosomal protein, anti-Myc, anti-HA, and anti-phospho-Ser-217/222 MEK1, anti-phospho-Thr-202/Tyr-204-p42/44 MAPK (Cell Signaling, Beverly, MA); anti phospho-Raf-1 (Ser-338) (Upstate Biotechnology, Lake Placid, NY); and anti-HA-agarose, anti-FLAG EZview (Sigma). In Vitro Kinase Assays—Cells were lysed in ice-cold kinase assay lysis buffer (25 mm HEPES, pH 7.5, 5 mm EDTA, 50 mm NaCl, 50 mm NaF, 30 mm Na4P2O7, 10% glycerol, 1% Nonidet P-40, protease and phosphatase inhibitors) after serum deprivation, growth factor, or serum stimulation. B-Raf or C-Raf kinase activity was measured using the following direct method. Immunoprecipitated B-Raf or C-Raf was incubated with recombinant MEK1 (Upstate) in the presence of either [32P]ATP or cold ATP for 30 min at 30 °C. MEK1 was then resolved using SDS-PAGE, and 32P incorporation was visualized by autoradiography. For experiments using cold ATP, MEK1 phosphorylation was detected using the phospho-MEK1 (Ser-217/222) antibody. Immunoprecipitation—For the co-immunoprecipitation analyses, cells were lysed in cold buffer containing 50 mm HEPES, pH 7.5, 50 mm NaCl, 5 mm EDTA, 1% Triton, 50 mm NaF, 10 mm Na4P2O7. The immunoprecipitation of endogenous C-Raf was performed using anti-C-Raf (E10) and endogenous B-Raf using anti-B-Raf (F7) for 1.5 h at 4 °C. Immune complexes were washed three times with lysis buffer, separated by SDS-PAGE, and analyzed for co-immunoprecipitated proteins. For immunoprecipitations prior to in vitro kinase assays, cells were lysed in kinase assay lysis buffer. Immune complexes were washed four times with lysis buffer and two times with kinase buffer (50 mm Tris-HCl, pH 7.5, 0.1 mm EGTA, 15 mm dithiothreitol). Statistics—Data from at least three independent experiments were quantified by densitometry, normalized to circumvent inter-experimental differences, and expressed as means ± S.E. The paired Student t test was used to detect statistically significant differences. Significance was achieved at p < 0.05. Rheb Decreases C-Raf Phosphorylation at Ser-338 and C-Raf Kinase Activity in a Rapamycin-insensitive Manner—We first asked whether Rheb influences the activity of C-Raf kinase. Phosphorylation of C-Raf on Ser-338 is correlated with C-Raf activity and is required for serum-induced C-Raf activation (24Mason C.S. Springer C.J. Cooper R.G. Superti-Furga G. Marshall C.J. Marais R. EMBO J. 1999; 18: 2137-2148Crossref PubMed Scopus (364) Google Scholar, 25King A.J. Sun H. Diaz B. Barnard D. Miao W. Bagrodia S. Marshall M.S. Nature. 1998; 396: 180-183Crossref PubMed Scopus (382) Google Scholar, 26Marais R. Light Y. Paterson H.F. Marshall C.J. EMBO J. 1995; 14: 3136-3145Crossref PubMed Scopus (520) Google Scholar, 27Fabian J.R. Daar I.O. Morrison D.K. Mol. Cell Biol. 1993; 13: 7170-7179Crossref PubMed Scopus (302) Google Scholar). We expressed Rheb in HEK293 cells and immunoprecipitated endogenous C-Raf from cells under both serum starvation and serum stimulation conditions. Phosphorylation of Ser-338 of endogenous C-Raf, which was low under serum-deprivation conditions, was strongly stimulated by the addition of fetal bovine serum. This serum-induced phosphorylation of endogenous C-Raf was inhibited ∼3.5-fold by Rheb expression (Fig. 1A). To determine whether Rheb inhibition of C-Raf Ser-338 phosphorylation is mTOR-Raptor-dependent or independent, we preincubated the cells with 20 nm rapamycin for 24 h. Rapamycin treatment did not impact the magnitude of Rheb inhibition of C-Raf Ser-338 phosphorylation (Fig. 1A), indicating that the inhibition is mTOR-Raptor-independent.FIGURE 1Rheb decreases C-Raf phosphorylation at Ser-338 and kinase activity in a rapamycin-resistant manner. A, endogenous C-Raf was immunoprecipitated from serum-starved or serum-stimulated HEK293 cells. Rheb expression decreased C-Raf phosphorylation at Ser-338 ∼3-fold after 10 min of serum stimulation. Rapamycin had no effect on Rheb inhibition of C-Raf phosphorylation. B, the activity of immunoprecipitated C-Raf was measured using MEK1 as substrate. Co-expression of Rheb inhibited C-Raf kinase activity and Ser-338 phosphorylation in the presence of serum by ∼2-fold. 24 h of rapamycin treatment did not block Rheb inhibition of C-Raf activity. The results of three independent experiments were normalized and compared (*, p < 0.05 by Student's paired t test). C, wild-type and DDED mutant C-Raf were expressed in HEK 293 cells. The in vitro kinase activity of C-Raf-DDED was not inhibited by co-expression of Q64L Rheb. D, tuberin down-regulation using siRNA decreased the activity of C-Raf in the presence of serum, using MEK1 as substrate and detected using either the phospho-MEK1 antibody or 32P-labeled MEK1 (upper panel). The results of three independent experiments were normalized and compared. E, Rheb down-regulation using siRNA increased the phosphorylation of endogenous C-Raf at Ser-338, in both serum-starved and serum-stimulated conditions (upper panel). We achieved ∼70% of Rheb down-regulation at the mRNA level compared with control siRNA, as determined by real-time PCR (data not shown). The results of three independent experiments were normalized and compared.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As a separate means to confirm Rheb inhibition of C-Raf activation, we expressed C-Raf with or without Rheb, and measured the in vitro kinase activity of C-Raf using recombinant MEK1 as a substrate. Rheb expression decreased the activity and Ser-338 phosphorylation of C-Raf kinase in a rapamycin-resistant manner (Fig. 1B). The average inhibition of C-Raf activity by Rheb in five separate experiments was 2.6-fold (p < 0.05). We also tested a constitutively active form of C-Raf, S338D/Y341D/T491E/S494D (C-Raf-DDED) (28Chong H. Lee J. Guan K.L. EMBO J. 2001; 20: 3716-3727Crossref PubMed Scopus (194) Google Scholar). The Q64L mutant form of Rheb, which more strongly activates mTOR (18Inoki K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1400) Google Scholar), failed to inhibit C-Raf-DDED (Fig. 1C). Because these experiments involve Rheb overexpression, we next asked whether activation of endogenous Rheb inhibits C-Raf kinase activity. To activate endogenous Rheb, we used siRNA to down-regulate tuberin (the GTPase-activating protein for Rheb) in HEK293 cells. Down-regulation of tuberin decreased the in vitro kinase activity of endogenous C-Raf, which was measured using recombinant MEK1 as a substrate (Fig. 1D). Finally, to confirm that C-Raf kinase is inhibited by endogenous Rheb, we down-regulated Rheb using siRNA. Rheb siRNA increased C-Raf phosphorylation by ∼3.1-fold in serum deprivation and 3.3-fold in serum stimulation conditions (Fig. 1E). Rheb Expression Inhibits Heterodimerization of B-Raf and C-Raf in a Rapamycin-insensitive Manner—To investigate the mechanism of C-Raf inhibition by Rheb, we asked whether Rheb physically interacts with C-Raf. We expressed tagged C-Raf and Rheb in HEK293 cells and immunoprecipitated either Myc-Rheb or FLAG-C-Raf, under both serum starvation and serum stimulation conditions. We were not able to detect an interaction between Rheb and C-Raf (Fig. 2A), in contrast to the interaction between Rheb and B-Raf, which was demonstrated by our group (12Karbowniczek M. Cash T. Cheung M. Robertson G.P. Astrinidis A. Henske E.P. J. Biol. Chem. 2004; 279: 29930-29937Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and others (11Im E. von Lintig F.C. Chen J. Zhuang S. Qui W. Chowdhury S. Worley P.F. Boss G.R. Pilz R.B. Oncogene. 2002; 21: 6356-6365Crossref PubMed Scopus (116) Google Scholar). These data suggest that Rheb inhibition of C-Raf involves an indirect mechanism and led us to ask whether Rheb-dependent inhibition of C-Raf could be mediated via B-Raf, as C-Raf and B-Raf form functional heterodimers upon Ras or serum stimulation (1Weber C.K. Slupsky J.R. Kalmes H.A. Rapp U.R. Cancer Res. 2001; 61: 3595-3598PubMed Google Scholar, 2Garnett M.J. Rana S. Paterson H. Barford D. Marais R. Mol. Cell. 2005; 20: 963-969Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 29Wojnowski L. Stancato L.F. Larner A.C. Rapp U.R. Zimmer A. Mech. Dev. 2000; 91: 97-104Crossref PubMed Scopus (96) Google Scholar), which synergistically enhances the activity of both kinases (1Weber C.K. Slupsky J.R. Kalmes H.A. Rapp U.R. Cancer Res. 2001; 61: 3595-3598PubMed Google Scholar, 29Wojnowski L. Stancato L.F. Larner A.C. Rapp U.R. Zimmer A. Mech. Dev. 2000; 91: 97-104Crossref PubMed Scopus (96) Google Scholar, 30Wan P.T. Garnett M.J. Roe S.M. Lee S. Niculescu-Duvaz D. Good V.M. Jones C.M. Marshall C.J. Springer C.J. Barford D. Marais R. Cell. 2004; 116: 855-867Abstract Full Text Full Text PDF PubMed Scopus (2211) Google Scholar). We hypothesized, therefore, that Rheb inhibition of C-Raf might be the consequence of decreased B-Raf/C-Raf heterodimerization. To test this, we immunoprecipitated endogenous B-Raf from cells transfected with C-Raf. Rheb expression strongly inhibited the association of C-Raf with endogenous B-Raf, in both serum-starved and serum-stimulated cells (Fig. 2B). Rheb inhibition of the B-Raf/C-Raf association was not blocked by 24 h of rapamycin treatment, indicating that it is mTOR-Raptor independent (Fig. 2B). To confirm these results, we immunoprecipitated endogenous C-Raf from cells transfected with B-Raf alone or with Rheb. Rheb expression almost completely blocked B-Raf association with endogenous C-Raf, and again this was rapamycin-insensitive (Fig. 2C). These results, together with the lack of evidence of co-immunoprecipitation of Rheb and C-Raf, suggest that C-Raf inhibition by Rheb is a consequence of decreased B-Raf activity and B-Raf/C-Raf heterodimerization, thereby preventing full activation of C-Raf. The V600E mutation in B-Raf is present in the majority of malignant melanomas and in some ovarian, colorectal, and thyroid cancers (4Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix-Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8215) Google Scholar, 31Brose M.S. Volpe P. Feldman M. Kumar M. Rishi I. Gerrero R. Einhorn E. Herlyn M. Minna J. Nicholson A. Roth J.A. Albelda S.M. Davies H. Cox C. Brignell G. Stephens P. Futreal P.A. Wooster R. Stratton M.R. Weber B.L. Cancer Res. 2002; 62: 6997-7000PubMed Google Scholar). V600E has a basal activity ∼10-fold higher than wild-type B-Raf (4Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix-Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8215) Google Scholar). Previously, we demonstrated that Rheb does not inhibit V600E B-Raf kinase activity (12Karbowniczek M. Cash T. Cheung M. Robertson G.P. Astrinidis A. Henske E.P. J. Biol. Chem. 2004; 279: 29930-29937Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Here, we asked whether Rheb can inhibit the association of V600E B-Raf with C-Raf. We expressed either wild-type B-Raf or V600E B-Raf alone or with Rheb and immunoprecipitated endogenous C-Raf. Rheb expression strongly inhibited the interaction of endogenous C-Raf with V600E B-Raf in both serum deprivation and serum stimulation conditions (Fig. 2D). Rheb Negatively Regulates the Phosphorylation of Ser-446 on B-Raf Kinase—To verify that loss of endogenous tuberin (and consequent activation of endogenous Rheb) is associated with decreased B-Raf activity, we used siRNA to down-regulate endogenous tuberin (TSC2) in HEK293 cells. As expected, tuberin down-regulation decreased the in vitro kinase activity of endogenous B-Raf (Fig. 3A). Interestingly, siRNA down-regulation of tuberin inhibited Ser-446 phosphorylation of endogenous B-Raf (Fig. 3A). Phosphorylation of Ser-446 is correlated with B-Raf activity (24Mason C.S. Springer C.J. Cooper R.G. Superti-Furga G. Marshall C.J. Marais R. EMBO J. 1999; 18: 2137-2148Crossref PubMed Scopus (364) Google Scholar, 32Tran N.H. Wu X. Frost J.A. J. Biol. Chem. 2005; 280: 16244-16253Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), and Pak 1 has been identified as a kinase for this site (32Tran N.H. Wu X. Frost J.A. J. Biol. Chem. 2005; 280: 16244-16253Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). To determine whether Rheb inhibition of Ser-446 B-Raf phosphorylation is growth factor-specific, HEK293 cells were serum-starved overnight and then treated for 15 min with platelet-derived growth factor or hepatocyte growth factor or for 30 min with epidermal growth factor. Rheb inhibition of Ser-446 phosphorylation was evident in all three conditions (Fig. 3B). Rheb also inhibited the Ser-446 phosphorylation of V600E B-Raf (Fig. 3C). However, the in vitro kinase activity of V600E B-Raf was not significantly inhibited by Rheb expression (Fig. 3D), suggesting that Ser-446 phosphorylation is not the mechanism through which Rheb inhibits B-Raf. To directly test this, we generated a S446D (phospho-mimetic) mutant form of B-Raf. The in vitro kinase activity of the S446D B-Raf mutant was strongly inhibited by Rheb (Fig. 3E), indicating that Rheb inhibition of B-Raf Ser-446 phosphorylation is independent of Rheb inhibition of B-Raf kinase activity. Rheb also inhibited a double phospho-mimetic mutant form of B-Raf, S446D/S447D (data not shown). These findings indicate that inhibition of Ser-446 phosphorylation is not the mechanism through which Rheb inhibits B-Raf, and are instead consistent with a model in which Rheb directly inhibits B-Raf kinase, leading to inhibition of phosphorylation (see Fig. 5). Rheb did not inhibit a constitutively active form of B-Raf, T599E/S602D (33Zhang B.H. Guan K.L. EMBO J. 2000; 19: 5429-5439Crossref PubMed Scopus (206) Google Scholar) (Fig. 3E).FIGURE 3Rheb negatively regulates the phosphorylation of B-Raf at S446. A, the Ser-446 phosphorylation and in vitro kinase activity of immunoprecipitated endogenous B-Raf were decreased after siRNA down-regulation of tuberin in HEK293 cells, compared with control siRNA, in serum starvation, continuous growth in serum, and in cells stimulated for 15 min with hepatocyte growth factor. An increase in the phosphorylation of ribosomal protein S6 was seen after tuberin down-regulation, reflecting Rheb activation of mTOR. B, B-Raf and Rheb were expressed in HEK293 cells. Rheb inhibited B-Raf phosphorylation on Ser-446 in serum-starved cells, as well as in serum-starved cells stimulated for 15 min with platelet-derived growth factor (25 ng/ml), hepatocyte growth factor (50 ng/ml), or 30 min with epidermal growth factor (25 ng/ml). The results of four independent experiments were normalized and compared (*, p < 0.05 by Student's paired t test). C, wild-type B-Raf or V600E mutant B-Raf was expressed in HEK293 cells with or without Rheb and B-Raf was immunoprecipitated. The Ser-446 phosphorylation of both wild-type and V600E B-Raf was markedly reduced when Rheb was expressed compared with the vector control. D, expression of Rheb inhibited the activity of wild-type B-Raf, as measured using MEK1 as substrate. Rheb did not inhibit the activity of V600E B-Raf. E, wild-type, S446D and T599E/S602D B-Raf mutants were expressed in HEK 293 cells. The in vitro kinase activity of wild-type and Ser-446 mutant form of B-Raf (S446D), but not T599E/S602D, were inhibited by co-expression of Rheb, in both serum starvation (left panel) and serum stimulation (right panel) conditions. The results of three independent experiments were normalized and compared.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Working model. Our data are consistent with a model in which Rheb directly binds B-Raf resulting in disruption of the B-Raf/H-Ras association in a S445-dependent manner, and inhibition of the heterodimerization of B-Raf with C-Raf, with subsequent inhibition of C-Raf activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Rheb Inhibits Ras Binding to B-Raf—We next turned our attention to the interaction between B-Raf and Ras. B-Raf in vitro kinase activity is enhanced by the addition of Ras (34Shimizu K. Kuroda S. Yamamori B. Matsuda S. Kaibuchi K. Yamauchi T. Isobe T. Irie K. Matsumoto K. Takai Y. J. Biol. Chem. 1994; 269: 22917-22920Abstract Full Text PDF PubMed Google Scholar), and activated Ras stimulates the heterodimerization of B-Raf and C-Raf (1Weber C.K. Slupsky J.R. Kalmes H.A. Rapp U.R. Cancer Res. 2001; 61: 3595-3598PubMed Google Scholar). Therefore, we asked whether Rheb influences Ras binding to B-Raf. First, we confirmed that B-Raf is inhibited in Tsc2-/- MEFs, in which endogenous Rheb is activated. The in vitro kinase activity of endogenous B-Raf kinase was inhibited in Tsc2-/- MEFs, compared with Tsc2+/+ MEFs, in both serum starvation and serum stimulation conditions (Fig. 4A). We then asked whether Rheb activation in the Tsc2-/- MEFs impacts the association of Ras with B-Raf by using siRNA to down-regulate Rheb and immunoprecipitating endogenous B-Raf. Rheb down-regulation in Tsc2-/- MEFs significantly enhanced the binding of endogenous H-Ras and B-Raf (Fig. 4B). To confirm this finding, we expressed B-Raf and the hyperactive Q64L Rheb in HEK293 cells and immunoprecipitated endogenous H-Ras. Rheb very strongly inhibited the association of endogenous H-Ras association with B-Raf in both serum starvation and serum stimulation conditions (Fig. 4C). Ras Binding to B-Raf Is S446-dependent—Because we had found that Rheb inhibits both B-Raf Ser-446 phosphorylation (Fig. 3, A and B) and B-Raf association with H-Ras (Fig. 4, B and C), we next asked whether B-Raf Ser-446 phosphorylation influences the B-Raf/H-Ras interaction. We generated S446A (non-phosphorylatable) and S446D (phospho-mimetic) forms of B-Raf and expressed them with B-Raf in HEK293 cells. The S446A B-Raf bound substantially less H-Ras than the S446D or wild-type B-Raf (Fig. 4D), suggesting that Ser-446 phosphorylation regulates the interaction between B-Raf and Ras. Furthermore, whereas Rheb expression disrupted the interaction between wild-type B-Raf and H-Ras (Fig. 4C), Rheb did not disrupt the association between S446D B-Raf and H-Ras (Fig. 4E). These results suggest that Ser-446 phosphorylation of B-Raf promotes the association of B-Raf and Ras, possibly due to conformational changes and/or release of B-Raf autoinhibition. Taken together, our data lead to a model (Fig. 5) in which Rheb directly interacts with and inhibits B-Raf kinase. This direct interaction inhibits C-Raf/B-Raf heterodimerization, with consequent decrease in C-Raf activity, and decreases Ser-446 B-Raf phosphorylation, resulting in inhibition of the B-Raf-H-Ras interaction. The Ras/B-Raf/C-Raf signaling network plays an essential role in normal development and is activated in many forms of cancer. Rheb is known to inhibit the activity of B-Raf kinase, but whether Rheb has a broader impact on this network has not been previously addressed. Here, we report that Rheb inhibits the activity of C-Raf kinase, the heterodimerization of B-Raf and C-Raf, and the interaction of B-Raf with H-Ras. We found that Rheb decreases C-Raf activity and C-Raf Ser-338 phosphorylation, a site that is strongly linked to C-Raf activity. However, Rheb did not completely block Ser-338 phosphorylation, suggesting that Rheb-independent factors are also involved. In contrast to the B-Raf-Rheb interaction that is evident at endogenous expression levels (12Karbowniczek M. Cash T. Cheung M. Robertson G.P. Astrinidis A. Henske E.P. J. Biol. Chem. 2004; 279: 29930-29937Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), we did not detect an interaction between C-Raf and Rheb in vivo. This suggests that Rheb inhibition of C-Raf is indirect, potentially mediated via Rheb inhibition of B-Raf/C-Raf heterodimerization. Heterodimerization of B-Raf and C-Raf is recognized as a significant mechanism of Raf kinase regulation (1Weber C.K. Slupsky J.R. Kalmes H.A. Rapp U.R. Cancer Res. 2001; 61: 3595-3598PubMed Google Scholar, 2Garnett M.J. Rana S. Paterson H. Barford D. Marais R. Mol. Cell. 2005; 20: 963-969Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Epidermal growth factor does stimulate C-Raf in B-Raf-deficient fibroblasts (29Wojnowski L. Stancato L.F. Larner A.C. Rapp U.R. Zimmer A. Mech. Dev. 2000; 91: 97-104Crossref PubMed Scopus (96) Google Scholar), and depletion of B-Raf inhibits C-Raf activity (35Mizutani S. Inouye K. Koide H. Kaziro Y. FEBS Lett. 2001; 507: 295-298Crossref PubMed Scopus (20) Google Scholar). It is hypothesized that this intricate signaling network, in which B-Raf modifies MEK activity through C-Raf, provides subtle regulation of signaling intensity or duration (2Garnett M.J. Rana S. Paterson H. Barford D. Marais R. Mol. Cell. 2005; 20: 963-969Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Our data are consistent with a model in which Rheb inhibition of C-Raf kinase activity is the result of decreased B-Raf activity and subsequent decreased B-Raf/C-Raf heterodimerization. To our knowledge, Rheb is the first small GTPase shown to inhibit B-Raf/C-Raf heterodimerization. We hypothesize that Rheb binding to B-Raf induces a conformational change in B-Raf, which inhibits S446-B-Raf phosphorylation and subsequent Ras activation and inhibits B-Raf/C-Raf heterodimerization and subsequent C-Raf activity. However, we cannot exclude the possibility that Rheb inhibition of C-Raf is mediated by a Raf phosphatase. Rheb also inhibits the interaction of B-Raf and H-Ras in a B-Raf S446-dependent manner. These data suggest that one consequence of Rheb binding to B-Raf is decreased Ser-446 phosphorylation, resulting in enhanced B-Raf autoinhibition and inhibition of Ras binding. The clinical consequences of Rheb inhibition of the Ras/B-Raf/C-Raf network in cells carrying mutations in TSC1 or TSC2 is not yet known. Tumors in TSC patients, including renal angiomyolipomas and subependymal giant cell astrocytomas exhibit aberrant differentiation patterns (36Karbowniczek M. Yu J. Henske E.P. Am. J. Pathol. 2003; 162: 491-500Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 37Astrinidis A. Henske E.P. J. Child Neurol. 2004; 19: 710-715Crossref PubMed Scopus (17) Google Scholar), which may be mediated by Raf kinases. Abnormal angiogenesis occurs in TSC angiomyolipomas and angiofibromas, which is intriguing because B-Raf-deficient mice have vascular defects (29Wojnowski L. Stancato L.F. Larner A.C. Rapp U.R. Zimmer A. Mech. Dev. 2000; 91: 97-104Crossref PubMed Scopus (96) Google Scholar). B-Raf is required for the neuronal differentiation of PC12 cells (38Yoon S. Seger R. Choi E.J. Yoo Y.S. Biochemistry (Mosc.). 2004; 69: 799-805Crossref PubMed Scopus (4) Google Scholar), so it is also possible that Rheb regulation of Raf kinases contributes to the neurological manifestations of TSC, which can include mental retardation and autism. Rapamycin is currently being used in clinical trials for TSC patients. Because Rheb inhibition of B-Raf and C-Raf is rapamycin-insensitive, it will be of particular interest to examine the phenotypes of TSC tumors after rapamycin therapy. Finally, it is possible that the two independent growth-related functions of Rheb (B-Raf/C-Raf inhibition and mTOR activation) may naturally limit the ability of Rheb to induce tumor growth, contributing to the fact that the vast majority of tumors in TSC patients are benign. In conclusion, we found that Rheb inhibits C-Raf, B-Raf/C-Raf heterodimerization, and B-Raf/H-Ras association, revealing a novel mechanism of Raf signaling regulation. The activity of tuberin and Rheb are regulated by growth factor signals, nutrient availability, and energy status. Therefore, our data position Rheb as an integrator of upstream cues to modulate Raf activity. This function of Rheb may be related to the unusual aspects of tumorigenesis in TSC patients, including aberrant vascular development and abnormal cellular differentiation patterns. Raf kinase inhibition is an active area of cancer research, with the Raf inhibitor Sorafenib approved for the treatment of renal cell carcinoma in 2006 (10Beeram M. Patnaik A. Rowinsky E.K. J. Clin. Oncol. 2005; 23: 6771-6790Crossref PubMed Scopus (246) Google Scholar) and other pharmacological inhibitors in development. Defining the precise role of Rheb in the regulation of Raf kinases could lead to novel approaches for targeted Raf kinase inhibition. We are grateful to Drs. Jon Chernoff, Erica Golemis, and Victoria Robb for their critical reviews of this manuscript." @default.
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W1978168320 cites W1666849257 @default.
W1978168320 cites W191846108 @default.
W1978168320 cites W1977747566 @default.
W1978168320 cites W1983247384 @default.
W1978168320 cites W1985046929 @default.
W1978168320 cites W198944900 @default.
W1978168320 cites W2010890298 @default.
W1978168320 cites W2011063821 @default.
W1978168320 cites W2023819606 @default.
W1978168320 cites W2037048868 @default.
W1978168320 cites W2044247583 @default.
W1978168320 cites W2044407816 @default.
W1978168320 cites W2046902755 @default.
W1978168320 cites W2047885346 @default.
W1978168320 cites W2056137479 @default.
W1978168320 cites W2057106513 @default.
W1978168320 cites W2064850685 @default.
W1978168320 cites W2070204715 @default.
W1978168320 cites W2074089133 @default.
W1978168320 cites W2081526354 @default.
W1978168320 cites W2082853003 @default.
W1978168320 cites W2087128490 @default.
W1978168320 cites W2102646127 @default.
W1978168320 cites W2103508362 @default.
W1978168320 cites W2109263914 @default.
W1978168320 cites W2114859236 @default.
W1978168320 cites W2122228538 @default.
W1978168320 cites W2124662474 @default.
W1978168320 cites W2140998787 @default.
W1978168320 cites W2163188200 @default.
W1978168320 cites W2166692731 @default.
W1978168320 cites W2171529739 @default.
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