40% Effort. Molecular dissection of the pathway linking growth factor receptors to the nucleus: their role in normal cell growth and cancer. Mitogen-activated protein kinase (MAPK) signaling pathways play an essential role in the transduction of environmental stimuli to the nucleus, therefore regulating a variety of cellular processes, including cell proliferation, differentiation, programmed cell death, and neoplastic transformation. The aberrant activation of the MAPK extracellular activated protein kinase (ERK) is a frequent event among highly prevalent human cancers. Thus, the components of the ERK kinase cascade represent an attractive target for cancer therapy. The MAPK mechanism of catalysis is still poorly understood due to the lack of complex structures of MAPKs with their bound targets in the active site. Recently, we performed the first computational study of the phosphoryl transfer reaction catalyzed by ERK2 using a Quantum Mechanics/Molecular Mechanics (QM/MM) approach. We modeled the interaction of ERK2 with a target peptide and analyzed the specificity towards S/T-P motifs. Our results provided a detailed description of the molecular events involved in the phosphorylation catalyzed by ERK2. This dynamic model of how MAPKs function may contribute to the understanding of how these kinases phosphorylate their growth promoting downstream targets. Key molecules linking GPCRs to the activation of AP1: GPCRs stimulate cell proliferation by regulating the activity of transcription factors, including AP1, whose aberrant activation has been implicated in tumor initiation and progression. To investigate how GPCRs regulate AP1-dependent gene transcription, we performed a genome-wide high-throughput RNAi screen in Drosophila S2 cells expressing muscarinic 1 receptors, a typical GPCR, and an AP-1 reporter system. Both Jun and Fos, members of the AP1 dimer, were hits on our screen. Interestingly, we found that members of the Rho family of GTPases, specifically Rho and Rac, and their downstream effectors such as Pak and multiple regulators of Jun N-terminal kinase (JNK) were integral to AP1 activation, while a JNK-specific phosphatase puckered, was a negative regulator. While molecules linking GPCRs to the hydrolysis of phosphatidylinositol and PKC activation were dispensable, we found that Trio, a Rho guanine nucleotide exchange factor (GEF) that binds directly to Gq, is essential for AP-1 activation by M1. We have now extended these studies in mammalian cells, and obtained evidence that Gq stimulates the activity of multiple MAPK cascades, including JNK, p38, and ERK5 through Rho GTPases and their downstream targets. Collectively, the emerging information indicates that GPCRs are linked to the activation of AP-1 through a Rho-GTPase network that is governed by highly specific protein-protein interactions and phosphorylation events rather than by diffusible second messengers. 40% Effort. Molecular basis of developmental and tumor-induced angiogenesis. PRK and ROCK are required for thrombin-induced endothelial cell permeability downstream from G12/13 and G11/q and RhoA: Increase in vascular permeability occurs under many physiological conditions such as wound repair, inflammation, and thrombotic reactions, and is central in diverse human pathologies, including tumor-induced angiogenesis. Thrombin is a pro-coagulant serine protease, which causes the local loss of endothelial barrier integrity thereby enabling the rapid extravasation of plasma proteins and the local formation of fibrin-containing clots. We used pharmacological inhibitors, knockdown approaches, and the emerging knowledge on how permeability factors affect endothelial junctions to investigate in detail the mechanism underlying thrombin-induced endothelial permeability. We showed that thrombin signals through PAR-1 and its coupled G proteins G12/13 and G11/q to induce RhoA activation and intracellular calcium elevation, and that these events are interrelated. In turn, this leads to the stimulation of ROCK and PRK, two Rho-dependent serine/threonine kinases that are required for endothelial permeability and the remodeling of cell-extracellular matrix and cell-cell adhesions. The signal initiated by thrombin bifurcates at the level of RhoA to promote changes in the cytoskeletal architecture through ROCK, and the remodeling of focal adhesion components through PRK. Ultimately, both pathways converge to cause cell-cell junction disruption and provoke vascular leakage. A role for a CXCR2/PI3Kgamma signaling axis in acute and chronic vascular permeability: Aberrant angiogenesis and vessel leakiness occur in numerous pathological conditions, such as in acute and chronic inflammation, tumor-induced angiogenesis and metastasis, and is central in the progression of many ocular diseases. Of interest, IL-8 (CXCL-8) exerts multiple functions in angiogenesis by acting directly on endothelial cell growth, permeability and migration and by serving as a potent chemo-attractant factor for macrophages and neutrophils. In search for the underlying mechanism by which IL-8 promotes vascular leakage, we found that IL-8 stimulates the redistribution of VE-cadherin, a key endothelial cell-cell adhesion molecule, by a signaling pathway involving CXCR2, the small GTPase Rac1, and its downstream target, PAK, resulting in the phosphorylation-dependent internalization of VE-cadherin. This process required the activation of a particular PI(3)K catalytic isoform, PI3Kgamma. Indeed, we obtained evidence that IL-8 induces a rapid and severe vascular leakage that can be efficiently prevented by the pharmacological inhibition of CXCR2 and PI3Kgamma in vivo. These results prompted us to investigate the potential role of IL-8 signaling in a mouse model for retinal vascular hyper-permeability. We showed that interfering with the CXCR2/PI3Kgamma signaling axis may represent a novel strategy to prevent and treat retinal hyper-permeability, found in numerous ocular diseases and inflammation conditions. These findings may also raise the possibility of targeting IL-8 signaling in the tumor microenvironment, as many angiogenic mechanisms are similarly deregulated in ocular diseases and tumor vascularization. 20% Effort. AIDS-associated Kaposi s sarcoma: molecular mechanisms. Kaposis sarcoma (KS) is the most common cancer arising in HIV-infected patients. HHV-8 or KS associated herpesvirus (KSHV) is the infectious cause of KS. In prior studies, we have shown that a KSHV-encoded constitutively active GPCR, vGPCR, can initiate the formation of KS lesions in mice, and that vGPCR promotes the expression of VEGF-A under the normoxic conditions that characterize KS. We took advantage of the observation that the chemokine IL-8, which acts on CXCR2 that is the closest human homolog of vGPCR, can rescue the proangiogenic phenotype in HIF-1 deficient cancer cells, probably by promoting VEGF expression and secretion by a HIF-1-independent mechanism, to investigate how GPCRs regulate VEGF-A expression in endothelial cells. Indeed, we showed here that IL-8 induces the expression of VEGF message and protein, thereby promoting the autocrine activation of its receptor, VEGFR2, in endothelial cells. Surprisingly, this effect was found to be independent on HIF-1, but instead involves the activation of NFkappaB by the CXCR2 chemokine receptor, which utilizes the CBM (Carma3/Bcl10/Malt1) complex to stimulate IKKalpha/beta, thus causing the nuclear activation of NFkappaB. These findings have important implications regarding tumor and inflammation-induced angiogenesis, and may explain how vGPCR stimulates the release of VEGF-A in KS.
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