ONCOGENESIS & CONTROL OF PHOSPHOINOSITIDE CYCLE/KINASE C

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The goals of this proposal are to understand the regulation of the p2l Ras protoncogene protein by Ras-GRF (Guanine Nucleotide Release Factor) and pl20 Ras-GAP (GTPase Activating Protein), and to elucidate the function of the GAP SR3 domain. There are three major aims: (l) mapping the sequences in Ras that specify sensitivity to GRF; (2) the ramifications of a nev model for the interaction of Ras with GAP; and (3) the mechanism of interaction of the GAP SH3 domain with signal transduction via muscarinic receptors, using a novel biological assay for this interaction. The first aim will complete ongoing efforts to map region(s) of the Ras protein that confer sensitivity to the Ras-specific GRF, by alanine- scanning mutagenesis. Mutants that abolish sensitivity to GRF will be tested biologically by introduction into dominant-negative form of Ras (S17N). The purpose of the second aim is to understand the mechanism of interaction of Ras with GAP. A model will be tested which predicts that when p2l Ras associates with GAP, it induces a conformational change that exposes the SH2/3 domains of GAP, enabling them to engage targets such as specific Tyrosine-phosphorylated proteins. The GAP-target complex is proposed to possess an downstream effector function. The affinity of GAP for Tyr-phosphorylated proteins is expected to be lower in the absence than in the presence of p2l Ras:GTP. This prediction will be tested both in intact cells, using the dominant negative N17Ras to suppress Ras:GTP formation; and in vitro, using GST-fusions of GAP fragments, phospho- peptides and p2l Ha-c-Ras. The interaction of GAP with specific lipids will also be investigated to determine whether lipids interfere with the putative conformational change that allows access to the GAP SH2/3 domain. The third aim utilizes a new, focus suppression assay for GAP function to study the role of the GAP SH3 domain. Expression of isolated SH3 domain inhibits muscarinic receptor-dependent transformation of NIH 3T3 cells. Specificity will be determined using SH3 domains from other genes and site-directed mutagenesis. The inhibitory mechanism will be investigated. Proteins that interact specifically with the GAP SH3 domains will be identified using recombinant SH3 as a probe, and cloned using the yeast two-hybrid system. These studies will provide important new information on SH3 domain function, and on the mechanism of signal transduction by muscarinic receptors.