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. G protein-coupled receptors (GPCRs) signal through Gq and G13-initiated MAPK cascades regulating c-Jun expression to induce cell transformation: Whereas the ability of GPCRs to stimulate normal and aberrant cell growth has been intensely investigated, the precise molecular mechanisms underlying their transforming potential are still not fully understood. Taking advantage of the potent mitogenic effect of thrombin and the focus-forming activity of one of its receptors, PAR-1, we have recently studied how this receptor, which is coupled to Galphai, Galphaq/11, and Galpha12/13, transduces signals from the membrane to the nucleus to initiate transcriptional events involved in cellular transformation. Using endogenous and transfected thrombin receptors, ectopic expression of GPCRs coupled to Galphaq and Galphai, as well as chimeric G proteins and murine fibroblasts knock out for Galphaq/11 and Galpha12/13, we found that although coupling to Galphai is sufficient to induce ERK activation, the ability to stimulate Galphaq and/or Galpha13 is necessary to induce c-jun expression and cellular transformation. Furthermore, we have observed that Galphaq and Galpha12/13 can initiate the activation of MAPK cascades, including JNK, p38, and ERK5, which in turn regulate transcription factors that control the expression from the c-jun promoter, and that c-Jun and the kinases regulating its expression are integral components of the transforming pathway initiated by PAR-1. These results suggest that the ability to stimulate c-jun expression can distinguish transforming from not transforming GPCRs. They also help explain how mitogens acting on GPCRs stimulate the expression of growth promoting genes. The small GTP-binding protein RhoA regulates c-Jun by a ROCK-JNK signaling axis. Rho GTPases, including RhoA, Rac1 and Cdc42, regulate gene expression in the nucleus in addition to their best known function in cytoskeletal control. In this regard, we have previously shown that RhoA stimulates the expression of c-jun, and that c-Jun is required for the RhoA-induced focus formation. In search for the mechanism by which RhoA stimulates c-jun, we found that RhoA signals to the nucleus by a biochemical route that bifurcates at the level of a kinase known as ROCK to initiate two independent pathways, one controlling the actin cytoskeleton and c-fos expression, and another leading to the activation of a novel MAPK cascade stimulating JNK that results in the activation of transcription factors pre-bound to the c-jun promoter and the expression of c-Jun. Ultimately, these divergent pathways acting downstream from ROCK converge in the nucleus to control the levels and transcriptional activity of AP1 complexes, thus coordinating RhoA-induced cytoskeletal changes with the transcriptional activation of genetic programs involved in key cellular processes, including normal and cancerous cell growth. Regulation of AP-1 by PDGF: ERK controls c-Fos expression and activity. GPCRs and polypeptide growth factors, such as PDGF, promote the re-initiation of DNA synthesis through multiple intracellular signaling pathways that converge in the nucleus to control the activity of the cell cycle machinery and transcription factors that regulate the expression of growth-promoting genes. Among the latter, the AP-1 family of transcription factors, including c-Fos and c-Jun family members, plays a key role. We took advantage of the observation that the stimulation of AP-1 by PDGF requires ERK activation to explore how this MAPK regulates AP-1. We fffound that PDGF stimulates the ERK-mediated phosphorylation of multiple residues in the carboxyl-terminal transactivation domain of c-Fos. By using an add-back mutational approach, we observed that this phosphorylation is required to stimulate c-Fos- and AP-1-dependent transcription. We also provided evidence that the ERK-dependent activation of c-Fos is an integral component of the mitogenic pathway utilized by PDGF. RGS-containing RhoGEFs: the link between transforming G proteins and Rho. Work done in our laboratory and in other institutions has identified a novel family of Rho (GEFs), including p115RhoGEF (p115), PDZ-RhoGEF (PRG), and LARG, that transduce signals from G??12/13 coupled receptors to RhoA. We have been making a concerted effort to better understand how these RhoGEFs contribute to signaling through G proteins. For example, we have recently found that RGS-RhoGEFs can form homo- and hetero-oligomers, through their unique C-terminal regions. Deletion of the C-terminal tail of these RhoGEFs resulted in a drastic increase in their ability to stimulate RhoA in vivo, and unleashed their full transforming potential, thus suggesting that RGS-RhoGEFs are negatively regulated by oligomerization and/or by inhibitory mechanisms acting via their C-terminal region. In search of the underlying molecular mechanism, we found that the serine-threonine kinase PAK4, an effector for Cdc42, binds to the inhibitory C-terminal region of PRG. This interaction results in the phosphorylation of PRG and diminishes its ability to mediate the accumulation of RhoA-GTP by G??13. Moreover, active PAK4 dramatically decreases RhoA-GTP loading in vivo and the formation of actin stress fibers in response to serum or lysophosphatidic acid (LPA). Thus, PAK4 can inhibit the activation of RhoA through a direct protein!Vprotein interaction with G protein-linked RhoGEFs. This provides a novel mechanism for cross talk among Rho GTPases. Based on the diversity of functions observed for these RhoGEFs in cell culture based experiments, we have recently engineered animals in which the gene for PRG or LARG has been disrupted (knock out), and are currently investigating their physiological consequences in vivo. Plexin B regulates RhoA through LARG and PRG: A potential link between axon guidance and tumor-induced angiogenesis. Using a yeast two-hybrid approach to screen for molecules interacting with the PDZ-domain of PRG, we identified the cytoplasmic tail of PlexinB2 as a candidate interacting molecule. Plexins constitute a novel family of transmembrane receptors that transduce attractive and repulsive signals mediated by the axon-guiding molecules semaphorins. By a number of complementary approaches, we showed that LARG and PRG provide 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. Interestingly, we have recently observed that Plexin B1 is highly expressed in endothelial cells, and that its ligand, Semaphorin 4D, is expressed in certain tumor cells, including squamous carcinomas. In a recent study, we have found that Semaphorin 4D is a potent angiogenic factor in vitro and in vivo, and that that Plexin B1 induces cell migration and tubulogenesis through PRG or LARG, acting on Rho-depedendent pathways. Whether Plexin B, LARG, and PRG play a role in developmental and tumor-induced angiogenesis is under current investigation.
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