Members of the family of serine/threonine-specific protein kinase known collectively as protein kinase C (PKC) are thought to play a pivotal role in the regulation of metazoan cell proliferation through their activation by growth factors and other agonists. The long-range goal of this project is to use a combined molecular genetic and biochemical approach to understand the role of PKC in the growth control of the budding yeast, S. cerevisiae. The PKC1 gene of S. cerevisiae encoded a homolog of mammalian isozyme of PKC.Loss of PKC1 function results in a cell cycle-specific osmotic stability defect. Specifically, mutants in PKC1 undergo cell lysis shortly after bud emergence. Thin section electron microscopy of pkc1-arrested cells reveals small holes in the cell walls located at the bud tips - the site of new growth in budding yeast. This suggests a defect in the ability of pkc1 mutants to remodel their cell walls to accommodate new growth. Electron micrographs also reveal a thinning of the beta-glucan layer of the cell wall in the region surrounding the bud tip. The level of beta- glucan synthase and beta-glucanase activities in yeast cell extracts lacking PKC1 function will be examined to determine if either activity is regulated by PKC1. A class of dominant extragenic suppressors of a pkc1 deletion results from activation of novel protein kinase that may act downstream of Pkc1p. Deletion of the suppressor gene, designated BCK1, results in a cell lysis defect similar to that of pkc1 mutants. The sequence changes identified in suppressor alleles of BCK1 suggest that, under normal conditions, Bck1 may be activated in response to phosphorylation by Pkc1p. To test this hypothesis, protein kinase assays for Pkc1 and for Bck1p will be developed initially. Then, the ability of Pkc1p to phosphorylate Bck1 will be tested, and the effect of such phosphorylation on the protein kinase activity associated with Bck1p will be examined. Finally, if these initial experiments indicate that Bck1p is regulated by Pkc1p, dominant suppressor forms of Bck1p will be tested for Pkc1p-independent activity. A distinguishing feature of PKC isozymes is their requirement of several and 3) diacylgylcerol (DAG). PKC isozymes possess a catalytic domain, which is responsible for their protein kinase activity and a regulatory domain to which activating cofactors bind. Exogenous phorbolester tumor promoter can substitute for DAG in the activation of PKC. it is this feature of phorbol esters that is believed to be responsible for their tumorigenic properties. Mutant alleles of the PKC1 gene will be generated by oligonucleotide-directed mutagenesis for use in structure/function analysis of the regulatory domain of Pkc1p. The biological effects of these mutations (as measured in yeast cells bearing chromosomal deletion of PKC1) should provide insight into the specific amino acid residues involved in cofactor activation of Pkc1p. Enzymological studies of mutant Pkc1 forms will allow direct examination of the cofactor requirements of these enzymes. Existing pkc1 mutants will be exploited for the purpose of isolating additional pathway components. Expression of presumptive cofactor-independent forms of Pkc1p results in growth inhibition of yeast cells. Genes that are involved in the regulation of Pkc1p, or that encode proteins that modulate the effects of pkc1 activity, such as specific protein phosphatases, will be isolated through their ability to suppress this growth defect. Other known cell lysis mutants will also be examined for genetic interactions with PKC1 and BCK1.
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