Recently the tuberous sclerosis complex 2 (TSC2) tumor suppressor gene product has been identified as a negative regulator of protein synthesis upstream of the mTOR and ribosomal S6 kinases. Because of the homology of TSC2 with GTPase-activating proteins for Rap1, we examined whether a Ras/Rap-related GTPase might be involved in this process. TSC2 was found to bind to Rheb-GTP in vitro and to reduce Rheb GTP levels in vivo. Over-expression of Rheb but not Rap1 promoted the activation of S6 kinase in a rapamycin-dependent manner, suggesting that Rheb acts upstream of mTOR. The ability of Rheb to induce S6 phosphorylation was also inhibited by a farnesyl transferase inhibitor, suggesting that Rheb may be responsible for the Ras-independent anti-neoplastic properties of this drug.The Ras subfamily of small GTPases regulates a vast array of biological events that include cell growth, differentiation, and transformation (1). The prototypic Ras proteins, Ha-, K-, and N-Ras, are known to transduce mitogenic and differentiation signals from cell surface receptors to the nucleus (1). However, despite their conservation throughout eukaryotic evolution, the function of Ras-related GTPases such as Rap1, R-Ras, Ral, Rheb, and Rit remains, at best, poorly understood. Ras activity is regulated by a GDP/GTP cycle whereby guanine nucleotide exchange factors promote the release of GDP from inactive Ras, facilitating its loading with the more abundant GTP (2). Binding to GTP induces a conformational change enabling interaction with and activation of downstream effector proteins (1). The termination of this signal is regulated by GTPase-activating proteins (GAPs), 1 which greatly accelerate the intrinsic GTPase activity of Ras, returning it to its inactive state (2). Loss of Ras GAP, as occurs in the genetic disorder neurofibromatosis type 1, results in increased Ras GTP levels, which contribute to tumor development (3, 4).Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are additional diseases that could be linked to the loss of GTPase regulation. TSC is a genetic disorder that results in the formation of benign tumors known as hamartomas, most typically found in kidney, brain, heart, and lung (3,5). LAM is a devastating lung disease that affects mainly women and is characterized by proliferation of atypical smooth muscle cells within the lung parenchyma (6). The inactivation of two tumor suppressor genes has been associated with TSC and LAM: TSC1 encodes the protein hamartin (TSC1), and TSC2 that encodes tuberin (TSC2) (7-9). Although TSC1 contains coiled-coil domains that are important for the formation of a functional complex with TSC2, TSC2 shares sequence homology with a family of GAPs that regulate the Ras-related GTPase, Rap1 (5). Accordingly, TSC2 has been reported to increase the intrinsic GTPase activity of Rap1 and also Rab5 in vitro (10, 11). However, it is not known whether this activity occurs in vivo or if it contributes to the physiological function of the TSC1-TSC2 complex.Several recent studies...
Although the Ras subfamily of GTPases consists of ϳ20 members, only a limited number of guanine nucleotide exchange factors (GEFs) that couple extracellular stimuli to Ras protein activation have been identified. Furthermore, no novel downstream effectors have been identified for the M-Ras/R-Ras3 GTPase. Here we report the identification and characterization of three Ras family GEFs that are most abundantly expressed in brain. Two of these GEFs, MR-GEF (M-Ras-regulated GEF, KIAA0277) and PDZ-GEF (KIAA0313) bound specifically to nucleotide-free Rap1 and Rap1/Rap2, respectively. Both proteins functioned as Rap1 GEFs in vivo. A third GEF, GRP3 (KIAA0846), activated both Ras and Rap1 and shared significant sequence homology with the calcium-and diacylglycerol-activated GEFs, GRP1 and GRP2. Similarly to previously identified Rap GEFs, C3G and Smg GDS, each of the newly identified exchange factors promoted the activation of Elk-1 in the LNCaP prostate tumor cell line where B-Raf can couple Rap1 to the extracellular receptor-activated kinase cascade. MR-GEF and PDZ-GEF both contain a region immediately N-terminal to their catalytic domains that share sequence homology with Ras-associating or Ral-GDS/AF6 homology (RA) domains. By searching for in vitro interaction with Ras-GTP proteins, PDZ-GEF specifically bound to Rap1A-and Rap2B-GTP, whereas MR-GEF bound to M-Ras-GTP. C-terminally truncated MR-GEF, lacking the GEF catalytic domain, retained its ability to bind M-Ras-GTP, suggesting that the RA domain is important for this interaction. Co-immunoprecipitation studies confirmed the interaction of M-Ras-GTP with MR-GEF in vivo. In addition, a constitutively active M-Ras(71L) mutant inhibited the ability of MR-GEF to promote Rap1A activation in a dose-dependent manner. These data suggest that M-Ras may inhibit Rap1 in order to elicit its biological effects.
M-Ras is a Ras-related protein that shares ϳ55% identity with K-Ras and TC21. The M-Ras message was widely expressed but was most predominant in ovary and brain. Similarly to Ha-Ras, expression of mutationally activated M-Ras in NIH 3T3 mouse fibroblasts or C2 myoblasts resulted in cellular transformation or inhibition of differentiation, respectively. M-Ras only weakly activated extracellular signal-regulated kinase 2 (ERK2), but it cooperated with Raf, Rac, and Rho to induce transforming foci in NIH 3T3 cells, suggesting that M-Ras signaled via alternate pathways to these effectors. Although the mitogen-activated protein kinase/ ERK kinase inhibitor, PD98059, blocked M-Ras-induced transformation, M-Ras was more effective than an activated mitogen-activated protein kinase/ERK kinase mutant at inducing focus formation. These data indicate that multiple pathways must contribute to M-Ras-induced transformation. M-Ras interacted poorly in a yeast two-hybrid assay with multiple Ras effectors, including c-Raf-1, A-Raf, B-Raf, phosphoinositol-3 kinase ␦, RalGDS, and Rin1. Although M-Ras coimmunoprecipitated with AF6, a putative regulator of cell junction formation, overexpression of AF6 did not contribute to fibroblast transformation, suggesting the possibility of novel effector proteins. The M-Ras GTP/GDP cycle was sensitive to the Ras GEFs, Sos1, and GRF1 and to p120 Ras GAP. Together, these findings suggest that while M-Ras is regulated by similar upstream stimuli to Ha-Ras, novel targets may be responsible for its effects on cellular transformation and differentiation.The mammalian Ras superfamily is made up of over 60 GTPases that serve as molecular switches to regulate a diverse array of cellular functions. These include intracellular signal transduction for cell growth and differentiation (Ras subfamily), regulation of the actin cytoskeleton (Rho subfamily), membrane trafficking (Rab subfamily), and nuclear transport (Ran) (1-4). The Ras subfamily consists of Ha-, Ki-, and N-Ras; Krev-1/Rap1A and -1B; Rap2A and -2B; R-Ras; TC21(R-Ras2); Ral A and B; Rheb; Dex-Ras; Rin; and Rit that share several common features outside of the core GTP-binding domain (2).The classic/prototypic Ras proteins, Ha-, Ki-, and N-Ras, transduce signals for growth and differentiation from ligand-bound receptors to the nuclear transcriptional machinery and to the cytoskeleton (2, 3, 5, 6). These proteins can be constitutively activated by point mutation, contributing to the development of a broad spectrum of human malignancies (7). The introduction of equivalent activating mutations into the closely related TC21 and R-Ras proteins also results in transformation in tissue culture models (8, 9), and TC21 mutants have been identified in human tumor cell lines (10, 11). R-Ras has also been associated with apoptosis and integrin activation (12, 13). Overexpression of Rap1A/Krev-1 can induce transformation in some cells (14) but typically has been found to counter Rasinduced activities, due to competitive binding to Ras effectors (15,16). Rheb...
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