Communications
The Tuberous Sclerosis 2 Gene Product, Tuberin, Functions as a Rab5 GTPase Activating Protein (GAP) in Modulating Endocytosis*

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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.

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*

This work was supported by National Institutes of Health Grant CA61889, American Cancer Society Grant CN-161, National Tuberous Sclerosis Association Grant 95-3, and an appropriation from the Commonwealth of Pennsylvania. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement− in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

1

The abbreviations used are:

    TSC

    tuberous sclerosis complex

    CHAPS

    3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

    PAGE

    polyacrylamide gel electrophoresis

    GST

    glutathione S-transferase

    GAP

    GTPase activating protein

    HRP

    horseradish peroxidase

    DTT

    dithiothreitol.