Members of the family of protein kinases known collectively as protein kinase C (PKC) are thought to play a pivotal role in the regulation of a host of cellular functions in metazoans. The PKC1 gene of S. cerevisiae encodes a homolog of mammalian isozymes of PKC that is essential for cell growth. Loss of PKC1 function results in a cell lysis defect that is due to a deficiency in cell wall construction. PKC1 regulates a phosphorylation cascade that culminates in the stimulation of the MPK1 MAP kinase. This phosphorylation cascade is known as the """"""""cell integrity pathway"""""""", because mutants that are deficient in protein kinase signaling display cell lysis defects that are exacerbated by growth at high temperature and by pheromone-induced morphogenesis. The long-range goal of this project is to use a combined molecular/genetic and biochemical approach to delineating the entire cell integrity pathway from the plasma membrane-associated receptor molecules to the ultimate nuclear and cytoplasmic targets of signaling. It is anticipated that this signaling pathway will serve as a paradigm for understanding the role of PKC signaling in growth control and stress responses in animal cells. It is also expected that identification of the components of the cell integrity pathway that are involved in cell wall construction will provide potential targets for the development of antifungal drugs that are specific to fungal species.
The specific aims of this project are to: 1) identify additional genes whose expression is controlled by the cell integrity pathway. Genes that have been implicated in cell wall construction will be examined for induced expression in response to cell integrity pathway signaling. Additionally, a """"""""promoter trap"""""""" screen has been devised to identify novel genes whose regulation is controlled by this pathway. From these studies, it is anticipated that PKC1/MPK1-dependent upstream activating sequences (UASs) will be identified in the promoter regions of regulated genes. 2) To identify and characterize PKC1/MPK1-regulated transcription factors. We will screen genomic and cDNA libraries for sequences that, when overexpressed, increase the basal level of MPK-1 dependent transcription. Potential MPK-1 regulated transcription factors will be characterized biochemically with respect to their ability to interact with UASs identified in Aim #1. 3) To determine the role of RHO1 in PKC1 signaling. The small G-protein encoded by RHO1 is required for MPK1 activation, probably by interacting with and stimulating PKC1. This notion will be tested in vitro using purified PKC1 and RHO1. The GTPase-activating protein anticipated to interact with RHO1 will also be identified using glucan synthase activity as an assay for RHO1 function. 4) To identify additional upstream components of the cell integrity pathway. Novel signaling components will be identified through synthetic lethal and suppressor screens using a conditional allele of the SIT4 gene, which encodes a Pl-4 kinase that has been implicated in PKC1 signaling.
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