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The tuberous sclerosis 2 gene product, tuberin, functions as a Rab5 GTPase activating protein (GAP) in modulating
G H Xiao1, F Shoarinejad, F Jin
1Divisions of Medical, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Abstract:
The tuberous sclerosis complex 2 (TSC2) is a tumor suppressor gene that plays a causative role in the autosomal dominant syndrome of tuberous sclerosis. The latter is characterized by the development of hamartomas and occasional malignancies. Expression of the wild-type gene in TSC2 mutant tumor cells inhibits proliferation and tumorigenicity. This "suppressor" activity is encoded by functional domain(s) in the C terminus that contains homology to Rap1GAP. Using a yeast two-hybrid assay to identify proteins that interact with the C-terminal domain of tuberin, the product of TSC2, a cytosolic factor, rabaptin-5, was found to associate with a distinct domain lying adjacent to the TSC2 GAP homology region. Rabaptin-5 also binds the active form of GTPase Rab5. Immune complexes of native tuberin, as well as recombinant protein, possessed activity to stimulate GTP hydrolysis of Rab5. Tuberin GAP activity was specific for Rab5 and showed no cross-reactivity with Rab3a or Rab6. Cells lacking tuberin possessed minimal Rab5GAP activity and were associated with an increased uptake of horseradish peroxidase. Re-expression of tuberin in TSC2 mutant cells reduced the rate of fluid-phase endocytosis. These findings suggest that tuberin functions as a Rab5GAP in vivo to negatively regulate Rab5-GTP activity in endocytosis.
Insights
Tuberous sclerosis complex 2 (TSC2) acts as a tumor suppressor by inhibiting cell growth. This study reveals TSC2 functions as a Rab5 GTPase-activating protein (Rab5GAP), regulating endocytosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Tuberous sclerosis complex 2 (TSC2) is a tumor suppressor gene linked to tuberous sclerosis, characterized by hamartomas and malignancies.
- The C-terminal domain of TSC2, containing homology to Rap1GAP, encodes its tumor suppressor activity.
Purpose of the Study:
- To identify proteins interacting with the TSC2 C-terminal domain.
- To elucidate the molecular mechanism underlying TSC2's tumor suppressor function, specifically its role in regulating GTPase activity and endocytosis.
Main Methods:
- Yeast two-hybrid assay to identify interacting proteins.
- In vitro assays to assess GTPase-activating protein (GAP) activity.
- Cellular assays measuring endocytosis rates.
Main Results:
- Tuberin (TSC2 protein) interacts with rabaptin-5 and exhibits GTPase-activating protein (GAP) activity specific for Rab5.
- Cells lacking tuberin show reduced Rab5GAP activity and increased fluid-phase endocytosis.
- Re-expression of tuberin in TSC2-deficient cells normalizes endocytosis rates.
Conclusions:
- Tuberin functions as a Rab5GAP in vivo, negatively regulating Rab5-GTP activity.
- This regulation is crucial for controlling endocytosis, suggesting a mechanism for TSC2's tumor suppressor role.