Assembly of a Ras-responsive adenylyl cyclase complex

The RAS oncogene is mutated in approximately 20-30% of human tumors making it one of the most frequently mutated oncogenes. The Ras protein has been shown to be an important regulator of fundamental biological processes such as cell growth and differentiation. Ras is the prototypic member of the small GTPase family. Ras functions as a molecular switch turning on and off signal transduction pathways.; We have chosen to study RAS in S. cerevisiae in order to gain a deeper understanding of how RAS is regulated. Our studies have focused on determining the essential components necessary to activate the conversion of ATP to cAMP by adenylyl cyclase. These studies have led us to characterize adenylyl cyclase, Cap/Srv2 (Cyclase Associated protein) and Cdc25p (Ras guanine nucleotide exchange factor).; We demonstrate that Cap/Srv2 is not necessary for Ras to activate adenylyl cyclase in vitro. In addition, we map the adenylyl cyclase binding site to the amino one third of Cap/Srv2p (amino acids 1-168) and the Cap/Srv2p binding site on adenylyl cyclase to the carboxyl terminus of adenylyl cyclase (amino acids 1768-2026). We also find that the 66 C-terminal amino acids of adenylyl cyclase are necessary for Ras activation in vitro.; In chapter 4 we identify a critical residue, glycine 116, in the SH3 domain of the RAS guanine nucleotide exchange protein, Cdc25p that is necessary for proper Ras/cAMP signaling. We go on to investigate the molecular targets of Cdc25p's SH3 domain. We present evidence for the direct binding of Cdc25p's SH3 domain to the 417 C-terminal amino acids of adenylyl cyclase. In support of our in vivo studies, this interaction is abolished by the same point mutation that fails to full respond to glucose. Interestingly this region of adenylyl cyclase does not contain the PXXP motif commonly found in SH3 targets. Using our assay system we detected a synergistic effect of SH3 domains and Ras on adenylyl cyclase activity in vitro. Our work allows us to suggest a model for how Ras, Cap/Srv2p, adenylyl cyclase and Cdc25p assemble into a cAMP signaling complex.