P190 RhoGAP (p190) is a multi-domain, multi-functional protein that regulates cell growth and survival as well as neural development and homeostasis. Structurally, it consists of an N-terminal domain with homology to small GTPases, a Middle Domain with multiple protein-protein interactions motifs, and a C terminal GAP (GTPase activating protein) domain for Rho family members. Most of the actions of p190 have been linked to its ability to negatively regulate Rho, enhancing the hydrolysis of active RhoGTP to inactive RhoGDP and thereby promoting actin stress fiber disassembly. This activity of p190 affects actin cytoskeletal rearrangements in response to growth factor stimulation and integrin engagement, developmental morphogenesis of neural tissue, and axon branching. Multiple lines of evidence also implicate p190 in apoptosis and suppression of tumor growth. To pursue the mechanism by which p190 RhoGAP regulates cell growth, we conditionally overexpressed p190 in human breast cancer cells and observed the formation of multinucleated cells, suggesting that p190 may be involved in cytokinesis. It is clear from the literature that Rho plays a critical role during cytokinesis and that regulation of its activity is key to the successful completion of the process. It has been shown that Rho is positively regulated by guanine nucleotide exchange factors (GEFs), and evidence is beginning to emerge that RhoGAPs, such as p190, can provide the negative arm of control. But little is understood about how the regulators of Rho are themselves regulated during this stage of the cell cycle. All GEFs and GAPs of Rho known to date contain structural domains that are not catalytic, suggesting that these domains may play a role in regulating their own localization, activity, or abundance, and thereby regulating the activity of Rho. In this grant we focus on p190 RhoGAP and on specific aspects of its regulation. We outline Aims that are designed to reveal the localization of endogenous vs. overexpressed p190 throughout the process of cytokinesis, what domains regulate that localization, what proteins bind p190 or are regulated by it, and what regulates the striking reduction in p190 protein levels we observe in late mitosis. Multiple strategies, including conventional and real-time confocal microscopy, are proposed to determine at which stage of cytokinesis both endogenous and overexpressed p190 function. The mechanism by which p190 mediates its effects will be explored by analyzing the effect of over- or under-expression of p190 on the activity or localization of known participants in cytokinesis. Efforts will also be taken to identify by mass spectrometry novel participants in this process that co-associate with p190 at different stages of mitosis. Finally, the role of ubiquitination in mediating the degradation of endogenous p190 will be explored. The goal of these studies is to gain insights into the mechanism of action and regulation of a potentially novel type of tumor suppressor, whose function can eventually be exploited for the treatment of cell cycle-based diseases, such as cancer.
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