IDENTIFICATION OF TUBERIN, THE TUBEROUS SCLEROSIS-2 PRODUCT - TUBERINPOSSESSES 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 r are malignancies. Two different genetic loci have been implicated in T SC; one of these loci, the tuberous sclerosis-2 gene (TSC2), encodes a n open reading frame with a putative protein product of 1784 amino aci ds. The putative TSC2 product (tuberin) contains a region of limited h omology 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 immunoprecipitati on and immunoblotting analyses. A wide variety of human cell lines exp ress the 180-kDa tuberin protein, and subcellular fractionation reveal ed that most tuberin is found in a membrane/particulate (100,000 x g) fraction. Immunoprecipitates of native tuberin contain an activity tha t specifically stimulates the intrinsic GTPase activity of Rap1a. Thes e results were confirmed in assays with a C-terminal fragment of tuber in, expressed in bacteria or Sf9 cells. Tuberin did not stimulate the GTPase activity of Rap2, Ha-Ras, Rac, or Rho. These results suggest th at the loss of tuberin leads to constitutive activation of Rap1 in tum ors of patients with tuberous sclerosis.