K. Ferguson

A family of human phosphodiesterases homologous to the dunce learning and memory gene product of Drosophila melanogaster are potential targets for antidepressant drugs.

We have isolated cDNAs for four human genes (DPDE1 through DPDE4) closely related to the dnc learning and memory locus of Drosophila melanogaster. The deduced amino acid sequences of the Drosophila and human proteins have considerable homology, extending beyond the putative catalytic region to include two novel, highly conserved, upstream conserved regions (UCR1 and UCR2). The upstream conserved regions are located in the amino-terminal regions of the proteins and appear to be unique to these genes. Polymerase chain reaction analysis suggested that these genes encoded the only homologs of dnc in the human genome. Three of the four genes were expressed in Saccharomyces cerevisiae and shown to encode cyclic AMP-specific phosphodiesterases. The products of the expressed genes displayed the pattern of sensitivity to inhibitors expected for members of the type IV, cyclic AMP-specific class of phosphodiesterases. Each of the four genes demonstrated a distinctive pattern of expression in RNA from human cell lines.

Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein

Adenylyl cyclase from S. cerevisiae contains at least two subunits, a 200 kd catalytic subunit and a subunit with an apparent molecular size of 70 kd, which we now call CAP (cyclase-associated protein). We cloned a cDNA encoding CAP by screening a yeast cDNA expression library in E. coli with antisera raised against the purified protein. The cDNA contained an open reading frame capable of encoding a 526 amino acid protein that is not homologous to any sequences in the current data bases. Adenylyl cyclase activity in membranes from cells that lacked CAP was not stimulated by RAS2 proteins in vitro. These results suggest that CAP is required for at least some aspects of the RAS-responsive signaling system. Mutants lacking CAP had four additional phenotypes that appear to be unrelated to effects of the RAS/adenylyl cyclase pathway: the inability to grow on rich medium (YPD), temperature sensitivity on minimal medium, sensitivity to nitrogen starvation, and a swollen cell morphology.

CAP is a bifunctional component of the Saccharomyces cerevisiae adenylyl cyclase complex.

CAP, a protein from Saccharomyces cerevisiae that copurifies with adenylyl cyclase, appears to be required for yeast cells to be fully responsive to RAS proteins. CAP also appears to be required for normal cell morphology and responsiveness to nutrient deprivation and excess. We describe here a molecular and phenotypic analysis of the CAP protein. The N-terminal domain is necessary and sufficient for cellular response to activated RAS protein, while the C-terminal domain is necessary and sufficient for normal cellular morphology and responses to nutrient extremes. Thus, CAP is a novel example of a bifunctional component involved in the regulation of diverse signal transduction pathways.

Genetic analysis of mammalian GAP expressed in yeast

We have designed a vector to express the mammalian GAP protein in the yeast S. cerevisiae. When expressed in yeast, GAP inhibits the function of the human H-rasgly12 protein, but not that of the H-rasval12 protein, and complements the loss of IRA1. IRA1 is a yeast gene that encodes a protein with homology to GAP and acts upstream of RAS. Mammalian GAP can therefore function in yeast and interact with yeast RAS. Because expression of GAP complements ira1-mutants, we propose that GAP shares some biochemical functions with IRA1. Other studies indicate that IRA1 controls the level of RAS activity, presumably by regulating GTP hydrolysis. By analogy, we propose that GAP may play a similar role.

Studies of RAS Function in the Yeast Saccharomyces cerevisiae

The three mammalian RAS genes, Ha-ras, Ki-ras, and N-ras, are capable of the malignant transformation of cultured animal cells (Barbacid 1987). Mutations in these genes have been linked to a large number of human cancers (Barbacid 1987). These genes encode closely related proteins that bind guanine nucleotides (Scolnick et al. 1979; Shih et al. 1980; Ellis et al. 1981) and are localized to the inner surface of the plasma membrane (Willingham et al. 1980; Papageorge et al, 1982). Normal RAS proteins also slowly hydrolyze GTP (Gibbs et al. 1984; McGrath et al. 1984; Sweet et al. 1984). These properties are similar to those of the G proteins, which has led to the widespread expectation that RAS proteins, like G proteins, are involved in the transduction of membrane signals that are linked to cellular proliferation or differentiation.