The Rho GTP-binding proteins have been shown to regulate the assembly of actin stress fibers and focal adhesions, and Rho function has been implicated in regulation of cell motility. We have shown that Rho is able to regulate the cellular levels of PIP2 by modulating the activity of a novel PI-5 kinase. We have further established that Rho activity is necessary for normal cellular responsiveness to growth factors, and may be a critical component of anchorage-independent growth of transformed cells. Rho appears to be poised as a critical integrator of adhesion and growth signaling pathways. We propose in this study to determine the significance of PI-5 kinase regulation by Rho for basic cellular function. We will investigate the mechanisms involved in the control of Rho activation by upstream activators using transient and stable transfection to express Rho/Ras mutants and evaluate effects on PI-5 kinase, the cytoskeleton, and other signals. We will purify and characterize the PI-5 kinase that is regulated by Rho. This protein will serve as a useful means to validate hypotheses about the role of PIP2 in various cellular processes. The downstream consequences of the regulation of PIP2 levels by Rho will be explored. This will include examination of actin polymerization, growth factor responsiveness, and regulation of phospholipase D activity. In the long term, our goal is to understand how cellular function, particularly that of immune cells, is controlled by the GTP-binding proteins of the who family. The current studies should lead to new insights into the complex relationship between cell growth, transformation, and adhesion, with particular relevance to the development of tumors and the events involved in tumor metastasis.
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