Screening And Analysis Of Genes Influencing Expression Of Protein Kinase Rck2 In Saccharomyces Cerevisiae

RCK2 encode protein kinase Rck2. And it is the substrate of protein kinase Hog1 in the high osmolarity glycerol pathway(HOG) in Saccharomyces cerevisiae. It is activated through phosphorylation thereby in response to changes in the extracellular environment. In order to explore genes involved in the regulation of Rck2 expression, we introduced the plasmid pHAC111-RCK2-HA, expression a RCK2-HA fusion protein, into deletion mutants for all nonessential genes in S.cerevisiae. Our preliminary study has identified 13 genes that are required for Rck2 expression by Western Blot analysis. Functions of these 13 genes are related to cell wall integrity, mitochondrial function and cell division. These genes are involved in regulating the transcriptional expression of RCK2 to influence Rck2 levels. Except SAP190 and KRE2, deletion of other genes reduces the Hog1 phosphorylation levels, indicating that they are involved in the HOG pathway. In addition, we found deletion of RTF1, KRE6, EMC5 or KRE28 causes cells sensitive to cell wall interfering agents by phenotypic test. Lack of MTC3, RTF1, KRE6, EMC5, KRE2 or MRLP39 reduces Slt2 phosphorylation levels under cadmium stress, demonstrating that these genes are involved in cadmium-induced activation of the Cell wall integrity(CWI) pathway. Among the four protein phosphatases genes PTP2, PTP3, SDP1 and MSG5 that negatively regulating Slt2, deletion of PTP2 or MSG5 also causes cells sensitive to cell wall interfering agents. And in cadmium induced gene deletion MSG5 make Slt2 phosphorylation level increases, and the cells have a certain tolerance to cadmium. Msg5 is the major protein phosphatase regulating the cell wall integrity pathway MAP kinase Slt2 in response to cadmium stress.