Certain alterations of proteins involved in mitogenic signaling are known to exert profound effects on cellular behavior, including malignant transformation. Our overall objective is to explore the molecular bases of cancer, approaching this problem through the study of normal and aberrant functioning of molecules that participate in the transduction of proliferative signals. Potent activation of RhoA by Galphaq and Gq-coupled Receptors: Heterotrimeric G proteins of the Gi, Gs, and Gq family control a wide array of physiological functions, primarily by regulating the activity of key intracellular second messenger-generating systems. Alpha subunits of the G12 family, Galpha12 and Galpha13, however, can promote cellular responses, including cell transformation, that are independent of conventional second messengers, but that result from the activation of small GTP-binding proteins of the Rho family and their downstream targets. These findings led to the identification of a novel family of guanine-nucleotide exchange factors (GEFs) that provides a direct link between Galpha12/13 and Rho stimulation. Recent observations suggest that many cellular responses elicited by Gq and its coupled receptors also require the functional activity of Rho. However, available evidence suggests that Galphaq may act on pathways downstream from Rho rather than by promoting Rho activation. These seemingly conflicting observations and the recent development of sensitive assays to assess the in vivo levels of active Rho prompted us to ask whether Galphaq and its coupled receptors can stimulate endogenous Rho. Indeed, we observed that the expression of activated forms of G?q and the stimulation of Gq-coupled receptors or chimeric Galphaq molecules that respond to Gi-linked receptors can promote a robust activation of endogenous Rho. Interestingly, this response was not prevented by molecules interfering with the ability of Galpha13 to stimulate its linked Rho-GEFs, together suggesting the existence of a novel molecular mechanism by which Galpha?q and the large family of Gq-coupled receptors can regulate the activity of Rho and its downstream signaling pathways. Regulation of G protein-linked guanine nucleotide exchange factors for Rho, PDZ-RhoGEF and LARG, by tyrosine phosphorylation: We have recently identified a family of guanine nucleotide exchange factors for Rho that includes PDZ-RhoGEF, LARG, and p115-RhoGEF, which exhibits a unique structural feature consisting in the presence of area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a structural motif by which heterotrimeric G proteins alpha subunits of the Galpha12 family can bind and regulate the activity of RhoGEFs. Hence, these newly discovered RGL-domain containing Rho GEFs provide a direct link from Galpha12 and Galpha13 to Rho. Recently available data suggest, however, that tyrosine kinases can regulate the ability of GPCRs to stimulate Rho, although the underlying molecular mechanisms are still unknown. In a recent study, we found that the activation of thrombin receptors endogenously expressed in HEK-293T cells leads to a remarkable increase in the levels of GTP-bound Rho within one min (11 fold), and a more limited but sustained activation (4 fold) thereafter, which lasts even several hours. Interestingly, tyrosine kinase inhibitors did not affect the early phase of Rho activation, immediately after thrombin addition, but diminished the levels of GTP-bound Rho during the delayed phase. We obtained evidence that the tyrosine kinase FAK can be activated by thrombin, Galpha12, Galpha13, and Galphaq through both Rho-dependent and Rho-independent mechanisms, and that PDZ-RhoGEF and LARG can in turn be tyrosine phosphorylated through FAK in response to thrombin, thereby enhancing their ability to stimulate Rho. These data indicate that FAK may act as a component of a positive feedback loop that results in the sustained activation of Rho by GPCRs, thus providing evidence of the existence of a novel biochemical route by which tyrosine kinases may regulate the activity of Rho through the tyrosine phosphorylation of RGL-containing RhoGEFs. Plexin B regulates Rho through the guanine nucleotide exchange factors LARG and PDZ-RhoGEF: Plexins represent a novel family of transmembrane receptors that transduce attractive and repulsive signals mediated by the axon-guiding molecules semaphorins. Emerging evidence implicates Rho GTPases in these biological events. However, Plexins lack any known catalytic activity in their conserved cytoplasmic tails, and how they transduce signals from semaphorins to Rho is still unknown. Using a yeast two-hybrid approach to screen for molecules interacting with the PDZ-domain of PDZ-RhoGEF, we identified the cytoplasmic tail of PlexinB2 as a candidate interacting molecule. Indeed, we found that Plexin B2 can associate directly with both PDZ-RhoGEF and LARG. This physical interaction was mediated by their PDZ domains and a PDZ-binding motif found only in Plexins of the B family. In addition, we observed that ligand-induced dimerization of Plexin B is sufficient to stimulate endogenous RhoA potently, and to induce the reorganization of the cytoskeleton. Moreover, overexpression of the PDZ domain of PDZ-RhoGEF but not its RGS domain prevented the cell rounding and neurite retraction of differentiated PC12 cells induced by activation of endogenous Plexin B1 by semaphorin 4D. The association of Plexins with LARG and PDZ-RhoGEF thus provides a direct molecular mechanism by which semaphorins acting on Plexin B can control Rho, thereby regulating the actin-cytoskeleton during axonal guidance and cell migration. Identification of H-Ras, RhoA, Rac1 and Cdc42 Responsive Genes: The superfamily of small GTP-binding proteins has expanded dramatically in recent years. The Ras family has long been associated with signaling pathways contributing to normal and aberrant cell growth, while Rho related protein function is to integrate extracellular signals with specific targets regulating cell morphology, cell aggregation, tissue polarity, cell motility, and cytokinesis. Recent findings suggest that certain Rho proteins, including RhoA, Rac1 and Cdc42, can also play a role in signal transduction to the nucleus and cell growth control. However, the nature of the genes regulated by Ras and Rho GTPases, as well as their contribution to their numerous biological effects is still largely unknown. To approach these questions, we investigated the global gene expression pattern induced by activated forms of H-Ras, RhoA, Rac1 and Cdc42 using cDNA microarrays comprising 19,117 unique elements. Using this approach, we identified 1,184 genes that were up- or down- regulated by at least two fold. Hierarchical cluster analysis revealed the existence of patterns of gene regulation both unique and common to H-Ras V12, RhoA QL, Rac1 QL and Cdc42 QL activation. For example, H-Ras V12 up-regulated osteopontin and Akt 1 while Rac1 QL and Cdc42 QL up-regulated extracellular matrix and cell adhesion proteins such as alpha-actinin 4, pro-collagen type I and V, and neuropilin. Furthermore, H-Ras V12 down-regulated by > 8-fold 52 genes compared to only 3 genes by RhoA QL, Rac1 QL and Cdc42 QL. These results provide key information to begin unraveling the complexity of the molecular mechanisms underlying the transforming potential, as well as the morphological and cell cycle effects induced by these small GTPases.
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