INHIBITION OF CYTOKINE SIGNALING BY PIS PROTEINS

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The long term goal of this proposal is to understand the regulation of gene expression by cytokines. The applicant has recently cloned cDNA for two members of a novel family of proteins, term originally PIS (Protein Interacting with STAT) and now changed to PIAS (Protein Interacting with Activated STAT). Preliminary data are presented to suggest that individual PIAS proteins display specificity for particular STATs in that PIAS1 and PIAS3 interact with STAT1 and STAT3 respectively. In addition, PIAS proteins appear to inhibit STAT-DNA interactions and STAT-induced gene expression. The applicant proposes to characterize further the PIAS proteins. Three specific aims are proposed. Firstly the molecular basis of the PIAS-STAT interaction will be defined, to examine which segments of the PIAS proteins are necessary and sufficient for the interaction with STATs. Secondly, the mechanisms by which PIAS proteins inhibit STAT function will be addressed. The applicant will use cross linking to determine if PIAS proteins block dimerization of STATs and will express PIAS proteins in cells to examine effects on the nuclear translocation of STATs. An examination of the effects of cytokines on PIAS function and a search for additional PIAS proteins is also proposed. Finally, the effects of PIAS3 on IL6 function in M1 cells and PIAS1 on IFNg function in U3A-STAT1 cells is proposed, which will involve creation of stable cell lines overexpressing the PIAS proteins or alternatively the application of anti-sense strategies.