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

A gene, PDE2, has been cloned from the yeast Saccharomyces cerevisiae that, when present in high copy, reverses the phenotypic effects of RAS2vad9, a mutant form of the RAS2 gene that renders yeast cells sensitive to heat shock and starvation. It has previously been shown that the RAS proteins are potent activators of yeast adenylate cyclase. We report here that PDE2 encodes a high-affinity cAMP phosphodiesterase that shares sequence homology with animal cell phosphodiesterases. These results therefore imply that the effects of RAS2vi19 are mediated through its changes in cAMP concentration.Our laboratory group has been studying the mechanism of growth control in the yeast Saccharomyces cerevisiae with particular concentration on the functions of the RAS] and RAS2 genes, which are structurally and functionally homologous to the ras oncogenes of mammalian cells (1-4). At least one RASI or RAS2 gene is required for the continued growth of yeast cells (5, 6) and it has been shown that RAS genes are essential controlling elements for adenylate cyclase in yeast (2,7,8). A mutant RAS2 gene has been constructed that encodes valine at the 19th codon position instead of glycine (5). This mutant (RAS2vall9) is analogous to the mutant and oncogenic human Ha-ras gene, which was first recognized in the T24/EJ bladder cell line (9-11). Yeast cells that express the mutant RAS2vall9 gene fail to synthesize glycogen, show an abnormal sensitivity to starvation (8), show a defective ability to arrest in the G1 phase of the cell cycle (8), and are sensitive to heat shock (unpublished results). To better understand the mechanism of these effects, we have searched for yeast genes that, when present in high copy, reverse these phenotypic effects. One such gene has been found, and it encodes the high-affinity cAMP phosphodiesterase (PDEase) of S. cerevisiae. We here present the nucleotide sequence of this gene and describe some of the phenotypic consequences of its perturbation.

Some of the characteristics of cancer cells are high rates of cell proliferation, cell survival, and the ability to invade surrounding tissue. The cytoskeleton has an essential role in these processes. Dynamic changes in the cytoskeleton are necessary for cell motility and cancer cells are dependent on motility for invasion and metastasis. The signaling pathways behind the reshaping and migrating properties of the cytoskeleton in cancer cells involve a group of Ras-related small GTPases and their effectors, including the p21-activated kinases (Paks). Paks are a family of serine/threonine protein kinases comprised of six isoforms (Pak 1–6), all of which are direct targets of the small GTPases Rac and Cdc42. Besides their role in cytoskeletal dynamics, Paks have recently been shown to regulate various other cellular activities, including cell survival, mitosis, and transcription. Paks are overexpressed and/or hyperactivated in several human tumors and their role in cell transformation makes them attractive therapeutic targets. Pak-targeted therapeutics may efficiently inhibit certain types of tumors and efforts to identify selective Pak-inhibitors are underway.

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The Ras oncogene regulates cellular proliferation, differentiation, transformation, and survival through multiple downstream signals. Ras signals through its effector phosphoinositide 3 (PI3) kinase to the Pak protein kinase (p65 pak ), but the steps from Ras to Pak remain to be elucidated. PI3 kinase can stimulate the small G protein, Rac, a direct activator of Pak, as well as the Akt proto-oncogene, a serine-threonine protein kinase. We found that activated Akt stimulated Pak, whereas a dominant negative Akt inhibited Ras activation of Pak in transfection assays. Akt stimulation of Pak was not inhibited by dominant negative mutants of either Rac or Cdc42 suggesting that Akt activated Pak through a GTPase-independent mechanism. We also developed a novel cell-free system to study Ras activation of Pak. In this system Ras activated Pak only in the presence of a crude cell extract but failed to activate Pak when Akt was immunodepleted from the extract. Akt protects cells from apoptosis through phosphorylation of downstream targets such as the Bcl-2 family member, Bad. We found that activated Pak decreased apoptosis and increased phosphorylation of Bad, whereas dominant negative Pak increased apoptosis and decreased phosphorylation of Bad. These studies define a new oncogene-mediated cell survival signal.