No abstract

Mitogen stimulation of cytoskeletal changes and c-jun amino-terminal kinases is mediated by Rac small guanine nucleotide-binding proteins. Vav, a guanosine diphosphate (GDP)-guanosine triphosphate (GTP) exchange factor for Rac that stimulates the exchange of bound GDP for GTP, bound to and was directly controlled by substrates and products of phosphoinositide (PI) 3-kinase. The PI 3-kinase substrate phosphatidylinositol-4,5-bisphosphate inhibited activation of Vav by the tyrosine kinase Lck, whereas the product phosphatidylinositol-3,4,5-trisphosphate enhanced phosphorylation and activation of Vav by Lck. Control of Vav in response to mitogens by the products of PI 3-kinase suggests a mechanism for Ras-dependent activation of Rac.

We developed a method for immunoaffinity purification of Saccharomyces cerevisiae adenylyl cyclase based on creating a fusion with a small peptide epitope. Using oligonucleotide technology to encode the peptide epitope we constructed a plasmid that expressed the fusion protein from the S. cerevisiae alcohol dehydrogenase promoter ADHI. A monoclonal antibody previously raised against the peptide was used to purify adenylyl cyclase by affinity chromatography. The purified enzyme appeared to be a multisubunit complex consisting of the 200-kilodalton adenylyl cyclase fusion protein and an unidentified 70-kilodalton protein. The purified protein could be activated by RAS proteins. Activation had an absolute requirement for a guanine nucleoside triphosphate.We have been studying the two RAS genes, RASI and RAS2, of Saccsharony(ces cereivisiae as models for the mammalian ras oncogenes. The yeast RAS proteins are structurally, functionally, and biochemically similar to their mammalian counterparts and at least one of their effector systems is known (3,7,8,18,24,34,36,38). The yeast RAS genes were originally isolated by using mammalian ras genes to screen libraries from S. cerev,isiae (7,24). They encode proteins that are highly homologous to the mammalian ras proteins, particularly in their amino-terminal half. Both yeast and mammalian RAS proteins undergo similar processing events and localize to membrane fractions (6,11,25,30,42). All RAS proteins bind guanine nucleotides and possess an intrinsic GTPase activity. The GTPase activity is reduced in a number of oncogenic forms of mammalian ras proteins containing point mutations (12,21,29,33,34