Rheb Inhibits C-Raf Activity and B-Raf/C-Raf Heterodimerization

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. 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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. 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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 TSC2 siRNA or Rheb siRNA or siRNA using The were and Cruz and and In were in kinase and or B-Raf or C-Raf kinase activity was using the B-Raf or C-Raf was with in the of or for at was using and was by For using phosphorylation was using the the cells were in The of C-Raf was performed using and B-Raf using for h at were with by and for For to in kinase cells were in kinase were with and two with kinase at independent were by to and as The was used to was at Rheb C-Raf at and C-Raf in a first whether Rheb influences the activity of C-Raf of C-Raf on is with C-Raf activity and is for C-Raf activation C.J. Cooper G. Marshall C.J. Marais R. J. PubMed Scopus Google Scholar, H. B. D. W. S. Marshall M.S. Nature. PubMed Scopus Google Scholar, R. Y. Paterson H.F. Marshall C.J. J. PubMed Scopus Google Scholar, J.R. Mol. Cell Biol. 13: PubMed Scopus Google Scholar). We Rheb in cells and C-Raf cells both and of of which was was strongly by the of phosphorylation of C-Raf was inhibited by Rheb whether Rheb inhibition of C-Raf phosphorylation is or we the cells with 20 nm rapamycin for 24 h. treatment not impact the of Rheb inhibition of C-Raf phosphorylation indicating that the inhibition is C-Raf phosphorylation at and kinase activity in a C-Raf was or Rheb decreased C-Raf phosphorylation at of on Rheb inhibition of C-Raf the activity of C-Raf was using as of Rheb inhibited C-Raf kinase activity and phosphorylation in the of by 24 h of rapamycin treatment not Rheb inhibition of C-Raf The results of independent were and by and mutant C-Raf were in The in kinase activity of was not inhibited by of tuberin down-regulation using siRNA decreased the activity of C-Raf in the of using as and using the or The results of independent were and Rheb down-regulation using siRNA the phosphorylation of C-Raf at Ser-338, in both and We of Rheb down-regulation at the with siRNA, as by not The results of independent were and a separate to Rheb inhibition of C-Raf we C-Raf with or Rheb, and the in kinase activity of C-Raf using as a Rheb decreased the activity and phosphorylation of C-Raf kinase in a The inhibition of C-Raf activity by Rheb in separate was We also a of S338D/Y341D/T491E/S494D H. J. Guan K.L. J. 2001; 20: PubMed Scopus Google Scholar). The mutant of Rheb, which more strongly mTOR K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1400) Google Scholar), to inhibit these Rheb we whether activation of Rheb inhibits C-Raf kinase Rheb, we used siRNA to tuberin GTPase-activating protein for in of tuberin decreased the in kinase activity of which was using as a to that C-Raf kinase is inhibited by Rheb, we Rheb using Rheb siRNA C-Raf phosphorylation by in and in Rheb of B-Raf and C-Raf in a the of C-Raf inhibition by Rheb, we whether Rheb interacts with C-Raf. We C-Raf and Rheb in cells and or both and We were not to between Rheb and C-Raf in to the between Rheb and B-Raf, which was by (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 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). data that Rheb inhibition of C-Raf and to whether inhibition of C-Raf B-Raf, as C-Raf and B-Raf Ras or (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, L. Rapp U.R. A. Dev. 2000; PubMed Scopus Google Scholar), which 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, L. Rapp U.R. A. Dev. 2000; PubMed Scopus Google Scholar, Garnett M.J. S. D. C.M. Marshall C.J. C.J. Barford D. Marais R. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). We that Rheb inhibition of C-Raf the of decreased B-Raf/C-Raf we B-Raf cells transfected with C-Raf. Rheb strongly inhibited the association of C-Raf with B-Raf, in both and cells Rheb inhibition of the B-Raf/C-Raf association was not by 24 h of rapamycin indicating that is mTOR-Raptor independent these we C-Raf cells transfected with B-Raf or with Rheb B-Raf association with and was with the of of of Rheb and that C-Raf inhibition by Rheb is a of decreased B-Raf activity and B-Raf/C-Raf thereby activation of C-Raf. The in B-Raf is in the of and in and cancers 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, M.S. P. M. M. I. R. E. M. J. Nicholson A. H. Cox C. G. Stephens P. Futreal P.A. Wooster R. Stratton M.R. Weber B.L. Cancer Res. 2002; Google Scholar). a activity than B-Raf 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). we that Rheb not inhibit 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 whether Rheb can inhibit the association of B-Raf with C-Raf. We B-Raf or B-Raf or with Rheb and C-Raf. Rheb strongly inhibited the of C-Raf with B-Raf in both and Rheb the of Ser-446 on B-Raf that of tuberin activation of is associated with decreased B-Raf we used siRNA to tuberin (TSC2) in tuberin down-regulation decreased the in kinase activity of B-Raf siRNA down-regulation of tuberin inhibited Ser-446 phosphorylation of B-Raf of Ser-446 is with B-Raf activity C.J. Cooper G. Marshall C.J. Marais R. J. PubMed Scopus Google Scholar, X. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), and as a kinase for X. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). whether Rheb inhibition of Ser-446 B-Raf phosphorylation is cells were and treated for with or or for with Rheb inhibition of Ser-446 phosphorylation was in Rheb also inhibited the Ser-446 phosphorylation of B-Raf However, the in kinase activity of B-Raf was not inhibited by Rheb that Ser-446 phosphorylation is not the which Rheb inhibits we generated a mutant of The in kinase activity of the B-Raf mutant was strongly inhibited by Rheb indicating that Rheb inhibition of B-Raf Ser-446 phosphorylation is independent of Rheb inhibition of B-Raf kinase Rheb also inhibited a mutant of B-Raf, not that inhibition of Ser-446 phosphorylation is not the which Rheb inhibits B-Raf, and are with a in which Rheb inhibits B-Raf to inhibition of phosphorylation Rheb not inhibit a of B-Raf, Guan K.L. J. 2000; PubMed Scopus Google the phosphorylation of B-Raf at the Ser-446 phosphorylation and in kinase activity of B-Raf were decreased siRNA down-regulation of tuberin in with siRNA, in in and in cells for with in the phosphorylation of protein was tuberin Rheb activation of B-Raf and Rheb were in Rheb inhibited B-Raf phosphorylation on Ser-446 in as as in cells for with or with The results of independent were and by B-Raf or mutant B-Raf was in cells with or Rheb and B-Raf was The Ser-446 phosphorylation of both and B-Raf was Rheb was with the of Rheb inhibited the activity of B-Raf, as using as Rheb not inhibit the activity of and B-Raf mutants were in The in kinase activity of and Ser-446 mutant of B-Raf not were inhibited by of Rheb, in both and The results of independent were and Our data are with a in which Rheb B-Raf resulting in of the association in a and inhibition of the heterodimerization of B-Raf with with inhibition of C-Raf Rheb Ras to to the between B-Raf and B-Raf in kinase activity is by the of Ras K. S. B. S. K. T. T. K. Matsumoto K. Y. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), and activated Ras 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 whether Rheb influences Ras to we that B-Raf is inhibited in Tsc2-/- in which Rheb is The in kinase activity of B-Raf kinase was inhibited in Tsc2-/- with Tsc2+/+ in both and We whether Rheb activation in the Tsc2-/- MEFs impacts the association of Ras with B-Raf by using siRNA to Rheb and Rheb down-regulation in Tsc2-/- MEFs the of and B-Raf we B-Raf and the Rheb in cells and H-Ras. Rheb strongly inhibited the association of association with B-Raf in both and Ras to B-Raf we found that Rheb inhibits both B-Raf Ser-446 phosphorylation A and and B-Raf association with and we whether B-Raf Ser-446 phosphorylation influences the We generated and forms of B-Raf and with B-Raf in The B-Raf than the or B-Raf that Ser-446 phosphorylation the between B-Raf and Rheb the between B-Raf and Rheb not the association between B-Raf and results that Ser-446 phosphorylation of B-Raf the association of B-Raf and to of B-Raf Taken together, data to a in which Rheb interacts with and inhibits B-Raf inhibits with decrease in C-Raf and Ser-446 B-Raf resulting in inhibition of the The signaling role in normal development and is activated in many forms of cancer. Rheb is known to inhibit the activity of B-Raf whether Rheb a impact on not previously Here, we that Rheb inhibits the activity of C-Raf the heterodimerization of B-Raf and and the of B-Raf with H-Ras. We found that Rheb C-Raf activity and C-Raf a that is strongly to C-Raf However, Rheb not that are also In to the that is at (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 not between C-Raf and Rheb in that Rheb inhibition of C-Raf is Rheb inhibition of B-Raf/C-Raf of B-Raf and C-Raf is as a 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). C-Raf in L. Rapp U.R. A. Dev. 2000; PubMed Scopus Google Scholar), and of B-Raf inhibits C-Raf activity S. K. H. Y. 2001; PubMed Scopus Google Scholar). It is that signaling in which B-Raf MEK activity regulation of signaling or 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 with a in which Rheb inhibition of C-Raf kinase activity is the of decreased B-Raf activity and decreased B-Raf/C-Raf Rheb is the first small shown to inhibit B-Raf/C-Raf We that Rheb to B-Raf a in B-Raf, which inhibits phosphorylation and Ras activation and inhibits B-Raf/C-Raf heterodimerization and C-Raf However, we the that Rheb inhibition of C-Raf is by a Raf Rheb also inhibits the of B-Raf and in a B-Raf data that one of Rheb to B-Raf is decreased Ser-446 resulting in B-Raf and inhibition of Ras The of Rheb inhibition of the in cells mutations in or TSC2 is not known. in TSC including renal and cell M. J. Henske E.P. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Henske E.P. J. 2004; PubMed Scopus Google Scholar), which by Raf kinases. in TSC and which is because have L. Rapp U.R. A. Dev. 2000; PubMed Scopus Google Scholar). B-Raf is for the of cells S. R. E.J. 2004; PubMed Scopus Google Scholar), is also that Rheb regulation of Raf kinases to the of which can and is used in for TSC Rheb inhibition of B-Raf and C-Raf is rapamycin-insensitive, of to the of TSC rapamycin is that the two independent of Rheb inhibition and mTOR the of Rheb to to the that the of in TSC are In we found that Rheb inhibits B-Raf/C-Raf and a of Raf signaling The activity of tuberin and Rheb are by and Therefore, data position Rheb as of to Raf of Rheb to the of in TSC including development and Raf kinase inhibition is of cancer with the Raf inhibitor approved for the treatment of renal cell in 2006 M. Patnaik A. Rowinsky E.K. J. Clin. Oncol. 2005; 23: 6771-6790Crossref PubMed Scopus (246) Google and other in the role of Rheb in the regulation of Raf kinases to for targeted Raf kinase inhibition. We are to and for critical of