Abstract
Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome with manifestations that can include seizures, mental retardation, autism, and tumors in the brain, retina, kidney, heart, and skin. The products of the TSC1 and TSC2 genes, hamartin and tuberin, respectively, heterodimerize and inhibit the mammalian target of rapamycin (mTOR). We found that tuberin expression increases p42/44 MAPK phosphorylation and B-Raf kinase activity. Short interfering RNA down-regulation of tuberin decreased the p42/44 MAPK phosphorylation and B-Raf activity. Expression of Rheb, the target of the GTPase-activating domain of tuberin, inhibited wild-type B-Raf kinase but not activated forms of B-Raf. The interaction of endogenous Rheb with B-Raf was enhanced by serum and by Ras overexpression. A farnesylation-defective mutant of Rheb co-immunoprecipitated with and inhibited B-Raf but did not activate ribosomal protein S6 kinase, indicating that farnesylation is not required for B-Raf inhibition by Rheb and that B-Raf inhibition and S6 kinase activation are separable activities of Rheb. Consistent with this, inhibition of B-Raf and p42/44 MAPK by Rheb was resistant to rapamycin in contrast to Rheb activation of S6 kinase, which is rapamycin-sensitive. Taken together these data demonstrate that inhibition of B-Raf kinase via Rheb is an mTOR-independent function of tuberin.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Cell Differentiation
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Cell Division
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Cell Line
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Dimerization
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Down-Regulation
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Gene Expression Regulation, Enzymologic*
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Glutathione Transferase / metabolism
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Guanosine Diphosphate / metabolism
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Humans
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MAP Kinase Signaling System
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Mitogen-Activated Protein Kinase 1 / metabolism
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinases / metabolism
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Models, Biological
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Monomeric GTP-Binding Proteins / metabolism*
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Mutation
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Neuropeptides / metabolism*
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Phosphorylation
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Precipitin Tests
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Protein Binding
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Protein Kinases / metabolism*
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Protein Prenylation
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Proto-Oncogene Proteins B-raf
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Proto-Oncogene Proteins c-raf / metabolism*
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RNA / metabolism
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RNA, Small Interfering / metabolism
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Ras Homolog Enriched in Brain Protein
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Repressor Proteins / metabolism*
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Ribosomal Protein S6 Kinases / metabolism
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Sirolimus / pharmacology
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TOR Serine-Threonine Kinases
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Transfection
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Tuberous Sclerosis Complex 2 Protein
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Tumor Suppressor Proteins
Substances
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Neuropeptides
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RHEB protein, human
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RNA, Small Interfering
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Ras Homolog Enriched in Brain Protein
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Repressor Proteins
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TSC2 protein, human
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Tsc2 protein, rat
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Tuberous Sclerosis Complex 2 Protein
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Tumor Suppressor Proteins
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Guanosine Diphosphate
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RNA
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Glutathione Transferase
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Protein Kinases
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MTOR protein, human
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mTOR protein, rat
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Braf protein, rat
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Proto-Oncogene Proteins B-raf
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Proto-Oncogene Proteins c-raf
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Ribosomal Protein S6 Kinases
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TOR Serine-Threonine Kinases
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinases
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Monomeric GTP-Binding Proteins
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Sirolimus