In the yeast S. cerevisiae, G1 progression depends on nutrient availability and the function of the cAMP-dependent protein kinase (PKA) pathway. Although the molecular details of this pathway have been described, the growth-and cell-cycle-specific effectors and processes regulated by PKA are unknown. By identifying the gene products that mediate PKA control, we hope to understand how yeast coordinate G1 progression with growth. Because the primary step of cell cycle control in most eukaryotes is in G1, with exceptions including tumor cell lines, information gained will likely be of both general and specific interest. Several genes specifying potential PKA effectors have recently been defined by suppressor analysis. One of the genes, SOK2, bears some of the hallmarks of a repressor of transcription, suggesting a role for PKA- regulated gene expression in growth control and proliferation. Experiments described here will examine the localization and potential phosphorylation of the SOK2 gene product. Genes known to be regulated by SOK2 will be used to define the DNA-responsive element of the Sok2 protein, which in turn will be used to establish an in vitro DNA binding assay for Sok2. Since the known Sok2 regulated genes are not essential for growth, we outline an enhancer-trap approach aimed at identifying new Sok2- (and PKA-) regulated genes. Finally, a genetic screen is described that will allow us to identify functional homologs of Sok2. Suppressor analysis has also identified two genes, YAK1 and SOK1, that may define a linear pathway that is partially redundant with that of PKA. Localization of Sok1 to the nucleus suggests Sok1 activation might alter the expression of several PKA- (and Sok2-?) regulated genes. This possibility will be addressed by molecular analysis of genes known to be regulated by PKA and Sok2. We will also determine if Sok1 is phosphorylated, and hence regulated, by Yak1 (or PKA). Finally, to extend our analysis of the downstream effectors of the PKA pathway, we will isolate and characterize dominant suppressors of the PKA growth defect. PKA and Sok2 have also been implicated in the dimorphic switch to pseudohyphal growth. Several genetic and physiological tests are proposed to examine the role of PKA in this switch. In addition, tests of epistasis will determine the level at which PKA, Sok2, and several members of the MAP kinase pathway regulate the switch to pseudohyphal growth.
