Identification of tuberin, the tuberous sclerosis-2 product. Tuberin possesses specific Rap1GAP activity.
Tuberous sclerosis (TSC) is a human genetic syndrome characterized by the development of benign tumors in a variety of tissues, as well as rare malignancies. Two different genetic loci have been implicated in TSC; one of these loci, the tuberous sclerosis-2 gene (TSC2), encodes an open reading frame with a putative protein product of 1784 amino acids. The putative TSC2 product (tuberin) contains a region of limited homology to the catalytic domain of Rap1GAP. We have generated antisera against the N-terminal and C-terminal portions of tuberin, and these antisera specifically recognize a 180-kDa protein in immunoprecipitation and immunoblotting analyses. A wide variety of human cell lines express the 180-kDa tuberin protein, and subcellular fractionation revealed that most tuberin is found in a membrane/particulate (100,000 x g) fraction. Immunoprecipitates of native tuberin contain an activity that specifically stimulates the intrinsic GTPase activity of Rap1a. These results were confirmed in assays with a C-terminal fragment of tuberin, expressed in bacteria or Sf9 cells. Tuberin did not stimulate the GTPase activity of Rap2, Ha-Ras, Rac, or Rho. These results suggest that the loss of tuberin leads to constitutive activation of Rap1 in tumors of patients with tuberous sclerosis.
Wienecke
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Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland 20892-4040, USA.
König
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A
DeClue
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JE
D002451:Cell Compartmentation; D002458:Cell Fractionation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D016147:Genes, Tumor Suppressor / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D011233:Precipitin Tests; D011506:Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012097:Repressor Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D014402:Tuberous Sclerosis / Q000235:genetics* / Q000378:metabolism; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D020703:ras GTPase-Activating Proteins; D019204:GTP-Binding Proteins