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Identification of tuberin, the tuberous sclerosis-2 product. Tuberin possesses specific Rap1GAP activity
R Wienecke1, A König, J E DeClue
1Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland 20892-4040, USA.
Abstract:
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.
Insights
Tuberous sclerosis (TSC) is a genetic disorder. The tuberin protein (TSC2) regulates Rap1a GTPase activity, and its loss may cause Rap1 activation in TSC tumors.
Area of Science:
- Genetics
- Molecular Biology
- Biochemistry
Background:
- Tuberous sclerosis (TSC) is a human genetic syndrome.
- TSC is linked to two genetic loci, including the TSC2 gene.
- The TSC2 gene encodes the tuberin protein, which has homology to Rap1GAP.
Purpose of the Study:
- To characterize the TSC2 protein, tuberin.
- To investigate tuberin's function in GTPase activity.
- To understand the molecular mechanisms underlying TSC.
Main Methods:
- Generated antisera against tuberin N- and C-terminal portions.
- Performed immunoprecipitation and immunoblotting to detect tuberin.
- Subcellular fractionation to localize tuberin.
- Assayed GTPase activity of Rap1a with tuberin.
Main Results:
- Successfully generated antisera recognizing a 180-kDa tuberin protein.
- Tuberin is primarily found in the membrane/particulate fraction.
- Tuberin specifically stimulates the intrinsic GTPase activity of Rap1a, but not Rap2, Ha-Ras, Rac, or Rho.
- These effects were confirmed using bacterially and Sf9-expressed tuberin fragments.
Conclusions:
- Tuberin directly regulates Rap1a GTPase activity.
- Loss of tuberin function may lead to constitutive Rap1 activation in TSC-associated tumors.
- This provides insight into the pathogenesis of tuberous sclerosis.