Identification of Tuberin, the Tuberous Sclerosis-2 Product. TUBERIN POSSESSES SPECIFIC Rap1GAP ACTIVITY

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.

Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N)

“…Surprisingly, in spite of the 70% homology of Rap1 and Rap2 (Bokoch, 1993), C3G-CD does not react with Rap2A (Table 1) and only very weakly with Rap2B ( Figure 3; Table 1). A similar discrimination in favour of Rap1 over Rap2 has been reported for two di erent RapGAPs (Rubin®eld et al, 1991;Wienecke et al, 1995). These ®ndings suggest that Rap2 proteins most likely have their own regulatory proteins and their identification might help elucidating the biological function of Rap2.…”

A role of the tuberous sclerosis gene-2 product during neuronal differentiation

“…1993). It has been shown that tuberin, like Rap1GAP, possesses in vitro GAP activity for Rap1 (Wienecke et al, 1995). Rap1 is a member of the superfamily of Ras-related proteins, which are active when bound to GTP and inactive when bound to GDP (Lowy and Willumsen, 1993).…”

“…Mutations in the TSC2 gene have been described in patients with TSC (Kumar et al, 1995;Wilson et al, 1996) and loss of heterozygosity at the TSC2 locus has been demonstrated in TSC patient lesions, as well as in sporadic tumours of non-TSC patients (Green et al, 1994;Henske et al, 1995;Carbonara et al, 1996). The TSC2-encoded protein, designated tuberin, functions as a GTPase accelerating protein (GAP) for the small molecular weight GTPases Rap1a and Rab5 (Wienecke et al, 1995Xiao et al, 1997). Very recently, we have shown that the absence of tuberin can induce quiescent Go-arrested cells to re-enter the cell cycle.…”

The long and winding road to rational treatment of cancer associated with LKB1/AMPK/TSC/mTORC1 signaling

“…The two proteins interact through coiled-coil domains to form a stable, functional heterodimer (van Slegtenhorst et al, 1998). The C-terminal region of TSC2 shows homology to the Rap GTPase activating protein, and GTPase activating protein activity to various G-proteins (Wienecke et al, 1995;Xiao et al, 1997 NUAK1, NUAK2 SIK1, SIK2 QSK MARK1, MARK2, MARK3, MARK4 BRSK1, BRSK2 SNRK inactive active p la s m a m e m b r a n e Figure 1 Activation and translocation of LKB1. LKB1 is activated by its translocation from the nucleus to the cytoplasm.…”