Constitutively activated mutants of the Ras small GTPase oncoprotein and the G-alpha12 heterotrimeric G protein subunit have been shown to cause growth transformation. Ras causes transformation by interaction with and activation of multiple downstream effectors, including Raf and phosphoinositide 3-kinase. G-alpha12 transformation has been attributed, in part, to activation of the RhoA small GTPase. Recent studies have implicated a novel phosphoinositide-specific phospholipase C, phospholipase C-epsilon (PLC-epsilon), as a downstream effector for Ras as well as G-alpha12. Therefore, PLC-epsilon serves as a point of convergence of diverse extracellular signaling mediated by receptor tyrosine kinase activation of Ras and G protein-coupled receptor activation of G-alpha12. PLC-epsilon activation generates two key second messengers, which in turn causes elevation of intracellular Ca2+ and activation of protein kinase C. Consequently, PLC-epsilon activation is likely to contribute significantly to the growth-promoting actions of Ras and G-alpha12. Our current knowledge on how PLC-epsilon is regulated is very limited and conflicting observations have been reported. PLC-epsilon also contains a second catalytic domain, a CDC25 homology domain, indicating that it functions as a guanine nucleotide exchange factor (GEF) and activator of Ras small GTPases. Whether Ras or G-alpha12 can regulate the GEF activity of PLC-epsilon has not been determined. We propose four specific aims to evaluate the mechanisms by which Ras and G-alpha12 regulate the distinct catalytic functions of PLC-epsilon and to assess the contribution of PLC-epsilon-mediated signaling in cellular transformation. We will determine (1) which Ras family GTPases regulate the PLC activity of PLC-epsilon, (2) if PLC-epsilon is an important effector of Ras transformation, (3) the GTPase specificity of the CDC25 homology domain of PLC-epsilon and whether activated Ras or G-alpha12 can regulate the GEF activity of PLC-epsilon, and finally (4) the structural basis for G-alpha12 interaction with PLC-epsilon and its role in regulating its PLC and GEF catalytic functions. Our studies will involve the expertise and experience of all components of this program project and will dissect the signaling and transforming (Der), catalytic (Harden), biochemical (Siderovski), and structural (Sondek) properties of PLC-epsilon.
Showing the most recent 10 out of 60 publications
