A potential p120 GTPase-activating protein (RasGAP) effector, G3BP (RasGAP Src homology 3 [SH3] binding protein), was previously identified based on its ability to bind the SH3 domain of RasGAP. Here we show that G3BP colocalizes and physically interacts with RasGAP at the plasma membrane of serumstimulated but not quiescent Chinese hamster lung fibroblasts. In quiescent cells, G3BP was hyperphosphorylated on serine residues, and this modification was essential for its activity. Indeed, G3BP harbors a phosphorylation-dependent RNase activity which specifically cleaves the 3-untranslated region of human c-myc mRNA. The endoribonuclease activity of G3BP can initiate mRNA degradation and therefore represents a link between a RasGAP-mediated signaling pathway and RNA turnover.The Ras protein belongs to a family of low-molecular-weight GTPases which are essential components of multiple receptormediated signal transduction pathways controlling cell proliferation, differentiation, and cytoskeletal organization (23). Activated Ras is bound to GTP, while the GDP-bound form of Ras is inactive (27). Extracellular stimuli induce the exchange of GDP for GTP on Ras through a series of protein-protein interactions involving activated receptors, adaptor proteins (such as Grb2 or Shc), and Ras guanine nucleotide exchange factors (5,9,33,38). Mutations in the Ras gene which lock Ras in the GTP-bound form lead to cell growth in the absence of mitogenic signals and are associated with an oncogenic phenotype (17). Physiological inactivation of Ras involves interaction with GTPase-activating proteins (GAPs) (40), such as p120 (RasGAP) (41,43) or the product of the NF1 gene (neurofibromin) (26,44), which accelerate the hydrolysis of Ras-associated GTP, thereby converting Ras from an active to an inactive form. Disruption of either the RasGAP or the NF1 gene in mice results in an embryonic lethal phenotype (3, 14), indicating that Ras inactivation is a key process in normal cell signaling and development.In addition to being a negative regulator of Ras, RasGAP may also represent a downstream target of Ras (35). RasGAP is a widely expressed modular protein which comprises several structural features that likely enable it to function in the transduction cascade (29). While the carboxyl-terminal domain of RasGAP constitutes a catalytic domain (25), the N-terminal region is believed to mediate interactions with other signaling proteins (20). The N-terminal region is characterized by a Src homology 3 (SH3) domain flanked by two SH2 domains, as well as pleckstrin homology (PH) and calcium-dependent lipid binding domains (4, 34). Upon activation of many growth factor receptors, RasGAP becomes phosphorylated and associates with cytosolic proteins as well as with the autophosphorylated tyrosine kinase receptors (19). RasGAP has been shown to form a complex with G3BP (RasGAP SH3 binding protein) in a Ras-GTP-dependent manner (32). G3BP is composed of 466 amino acid and has a predicted molecular mass of 52 kDa; the carboxyl-terminal region contai...

IB␣ inhibits the transcriptional activity of NF-B both in the cytoplasm by preventing the nuclear translocation of NF-B and in the nucleus where it dissociates NF-B from DNA and transports it back to the cytoplasm. Cytoplasmic localization of inactive NF-B/IB␣ complexes is controlled by mutual masking of nuclear import sequences of NF-B p65 and IB␣ and active CRM1-mediated nuclear export. Here, we describe an additional mechanism accounting for the cytoplasmic anchoring of IB␣ or NF-B/IB␣ complexes. The N-terminal domain of IB␣ contains a sequence responsible for the cytoplasmic retention of IB␣ that is specifically recognized by G3BP2, a cytoplasmic protein that interacts with both IB␣ and IB␣/NF-B complexes. G3BP2 is composed of an N-terminal domain homologous to the NTF2 protein, followed by an acidic domain sufficient for the interaction with the IB␣ cytoplasmic retention sequence, a region containing five PXXP motifs and a C-terminal domain containing RNA-binding motifs. Overexpression of G3BP2 directly promotes retention of IB␣ in the cytoplasm, indicating that subcellular distribution of IB␣ and NF-B/IB␣ complexes likely results from a equilibrium between nuclear import, nuclear export, and cytoplasmic retention. The molecular organization of G3BP2 suggests that this putative scaffold protein might connect the NF-B signal transduction cascade with cellular functions such as nuclear transport or RNA metabolism.Rel/NF-B transcription factors play a major role in inducible expression of a number of cellular genes involved in immune, inflammatory, and anti-apoptotic responses (1-3). Human NF-B is composed of a homo-or heterodimer of proteins that belong to the multigene family of transcription factors comprising p50, p52, p65/RelA, c-Rel, and RelB (4 -12). The prototypical NF-B is a heterodimeric p50/p65 molecule. Each member of NF-B/Rel family of proteins contains a Rel homology domain that is responsible for nuclear translocation, dimerization, and sequence-specific DNA binding. In most unstimulated cells, NF-B is retained in an inactive form in the cytoplasm through its association with the IB inhibitor proteins (13-16). IBs also belong to a multigene family of proteins including IB␣, IB␤, IB⑀, Bcl-3, and also the C-terminal domains of p50 and p52 precursors (p105 and p100, respectively) that in isolation are known as IB␥ and IB␦, respectively (17-25). Members of the IB family contain multiple conserved ankyrin repeat domains that interact with NF-B factors such that their nuclear localization sequences (NLS) 1 are masked, leading to cytoplasmic retention of the complex. IB proteins are also characterized by their ability to inhibit NF-B DNA binding activity.IB␣ is composed of a surface-exposed N-terminal domain, a central region containing six ankyrin repeat domains, and a highly acidic C-terminal domain. Upon stimulation of cells with appropriate signals such as tumor necrosis factor or interleukin 1, a signaling cascade is initiated leading to activation of two IB␣ kinases, IKK-1 and IKK-2, which phosph...

Sam68 is the main tyrosine-phosphorylated and Srcassociated protein in mitotic cells. Sam68 exhibits a conserved functional KH (hnRNPK homology) RNA binding domain and binds single strand nucleic acids. Tyrosine phosphorylation mediates the interaction of Sam68 with many SH3-and SH2-containing proteins and negatively regulates its nucleic acid binding properties. But the function and the impact of Sam68 on cell signaling and cell proliferation remains elusive. We report here the identification of a natural isoform of Sam68 with a deletion within the KH domain. This isoform, called Sam68⌬KH, is specifically expressed at growth arrest upon confluency in normal cells. In cells that do not enter quiescence at confluency such as Src-transformed cells, no recruitment of Sam68⌬KH is observed. Transfected Sam68⌬KH inhibits serum-induced DNA synthesis and cyclin D1 expression. Sam68 overcomes these effects, suggesting that isoforms of Sam68 are involved, through KH domain signaling, in cell proliferation, and more precisely in G 1 /S transition.

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Controversy exists as to whether the interaction of a guanosine triphosphatase activating protein (GAP) with Ras proteins functions both to initiate and to terminate Ras-dependent signaling events or only to terminate them. GAP-C, a carboxyl-terminal fragment of GAP that is sufficient to stimulate GTPase activity, inhibited the stimulation of transcription produced by some oncoproteins (v-Src, polyoma middle T, wild-type Ras, and oncogenic Ras) but not that produced by v-Mos. Wild-type GAP did not affect transcription induced by oncogenic Ras but reversed the inhibitory effect of GAP-C on transcription induced by oncogenic Ras. These results indicate that GAP is a negative regulator of wild-type Ras and elicits a downstream signal by interacting with Ras-GTP (guanosine triphosphate).