C3G (Crk SH3-domain-binding guanine-nucleotide-releasing factor) is a ubiquitously expressed member of a class of molecules called GEFs (guanine-nucleotide-exchange factor) that activate small GTPases and is involved in pathways triggered by a variety of signals. It is essential for mammalian embryonic development and many cellular functions in adult tissues. C3G participates in regulating functions that require cytoskeletal remodelling such as adhesion, migration, maintenance of cell junctions, neurite growth and vesicle traffic. C3G is spatially and temporally regulated to act on Ras family GTPases Rap1, Rap2, R-Ras, TC21 and Rho family member TC10. Increased C3G protein levels are associated with differentiation of various cell types, indicating an important role for C3G in cellular differentiation. In signalling pathways, C3G serves functions dependent on catalytic activity as well as protein interaction and can therefore integrate signals necessary for the execution of more than one cellular function. This review summarizes our current knowledge of the biology of C3G with emphasis on its role as a transducer of signals to the actin cytoskeleton. Deregulated C3G may also contribute to pathogenesis of human disorders and therefore could be a potential therapeutic target.

Endothelial senescence in conjunction with mitochondrial dysfunction orchestrates age-associated cardiovascular disorders. In this study we investigated the causal link between these two processes and studied the molecular mechanisms by which metformin acts to coordinate the delay of endothelial senescence via enhancing mitochondrial biogenesis/function. AMPK activators metformin and AICAR delayed endothelial senescence via SIRT1-mediated upregulation of DOT1L, leading to increased trimethylation of H3K79 (H3K79me3). Treatment of cells with either siAMPK or siSIRT1 repressed DOT1L-mediated enhancement of H3K79me3. Moreover, the increase in SIRT3 expression and mitochondrial biogenesis/function by AMPK activators was H3K79me-dependent as H3K79N mutant or siDOT1L abrogated these effects. This was confirmed by the enrichment of H3K79me3 in the SIRT3 promoter with AMPK activation. Intriguingly, enhanced PGC-1α expression by SIRT3 via AMPK activation was responsible for increased hTERT expression and delayed endothelial senescence. In contrast, SIRT3 knockdown caused increased oxidative stress and premature senescence, possibly by depleting hTERT expression. Furthermore, a chronic low dose administration of metformin significantly attenuated vascular aging and inhibited age-associated atherosclerotic plaque formation in ApoE mice. Overall, the results of this study show a novel regulation of mitochondrial biogenesis/function, and cellular senescence by H3K79me acting through SIRT3, thus providing a molecular basis for metformin-mediated age-delaying effects.

The guanine nucleotide exchange factor C3G (RAPGEF1) regulates proliferation, migration, and differentiation of cells and is essential for mammalian embryonic development. The molecular effectors of C3G dependent functions are poorly understood. Here we report that C3G functions as a negative regulator of β-catenin, a major player in pathways known to be deregulated in human cancers. In mammalian cells, C3G is present in a complex with cellular β-catenin. The proline rich Crk binding region of C3G and residues 90-525 of β-catenin are sufficient for the interaction. Knockdown of cellular C3G stimulated, and its overexpression repressed, β-catenin/TCF transcription activity. C3G acts by destabilizing β-catenin protein and inhibiting its nuclear accumulation. Nuclear extracts of C3G overexpressing cells showed reduced binding to TCF consensus oligos. C3G exerts its effects independent of its function as an exchange factor. It also inhibits stability and activity of an N-terminal deletion construct of β-catenin that is not subject to GSK3β dependent phosphorylation, suggesting that C3G exerts its effect independent of GSK3β. β-catenin repression by C3G was not significantly altered in the presence of proteasome inhibitors, MG132 or lactacystin, suggesting that alternate mechanisms are engaged by C3G to cause β-catenin turnover. C3G expression represses β-catenin target gene expression, and stable clones of MCF-7 breast cancer cells expressing C3G showed reduced migration. Activation of cellular β-catenin or expression of constitutively active β-catenin resulted in reduced C3G expression, indicating that C3G gene expression is negatively regulated by β-catenin. Our results identify a novel property of C3G in functioning as a negative regulator of β-catenin signaling by promoting its degradation. In addition, we show that β-catenin inhibits C3G expression, forming a feedback loop. KeywordsC3G, β-catenin, guanine nucleotide exchange factor, signaling, GSK3βOriginal Article C3G interacts with and inhibits β-catenin function / Dayma et al.

The chimeric oncoprotein BCR-Abl exhibits deregulated protein tyrosine kinase activity and is responsible for the pathogenesis of certain human leukemias, such as chronic myelogenous leukemia. The activities of cellular Abl (c-Abl) and BCR-Abl are stringently regulated and the cellular mechanisms involved in their inactivation are poorly understood. Protein tyrosine phosphatases can negatively regulate Abl mediated signaling by dephosphorylating the kinase and/or its substrates. This study investigated the ability of the intracellular T cell protein tyrosine phosphatase (TCPTP/PTPN2) to dephosphorylate and regulate the functions of BCR-Abl and c-Abl. TCPTP is expressed as two alternately spliced isoforms - TC48 and TC45, which differ in their C-termini and localize to the cytoplasm and nucleus respectively. We show that TC48 dephosphorylates BCR-Abl but not c-Abl and inhibits its activity towards its substrate, CrkII. Y1127 and Y1294 residues whose phosphorylation corresponds with BCR-Abl activation status were the primary sites targeted by TC48. Co-localization and immunoprecipitation experiments showed that TC48 interacted with BCR-Abl but not with c-Abl, and BCR domain was sufficient for interaction. TC48 expression resulted in the stabilization of Bcr-Abl protein dependent on its phosphatase activity. Inactivation of cellular TC48 in K562 cells by stable expression of a dominant negative catalytically inactive mutant TC48, enhanced proliferation. TC48 expressing K562 clones showed reduced proliferation and enhanced sensitivity to STI571 compared to control clones suggesting that TC48 can repress the growth of CML cells. This study identifies a novel cellular regulator that specifically inhibits the activity of oncogenic BCR-Abl but not that of the cellular Abl kinase.