INHIBITION OF CYTOKINE SIGNALING BY PIS PROTEINS

本提案的长期目标是了解 通过细胞因子的基因表达。 申请人最近克隆了cDNA 一个新的蛋白质家族的两个成员， （与STAT相互作用的蛋白质），现在变更为皮亚斯（蛋白质 与激活的STAT交互）。初步数据已提交给 表明单个皮亚斯蛋白对特定 PIAS 1和PIAS 3与STAT 1和STAT 3相互作用 分别 此外，皮亚斯蛋白似乎抑制STAT-DNA 相互作用和STAT诱导的基因表达。 申请人建议 以进一步表征皮亚斯蛋白。 三个具体目标是 提出了 首先，PIAS-STAT相互作用的分子基础将 以检查皮亚斯蛋白的哪些片段是必需的 并足以与STAT相互作用。 第二，机制 通过该方法将解决皮亚斯蛋白抑制STAT功能的问题。 的 申请人将使用交联来确定皮亚斯蛋白是否阻断 STAT的二聚化，并将在细胞中表达皮亚斯蛋白，以检查 对STAT核转位的影响。 的检查 细胞因子对皮亚斯功能的影响及对其他皮亚斯的研究 还提出了蛋白质。 最后，研究PIAS 3对IL 6的影响。 PIAS 1对M1细胞中IFNg功能的影响以及PIAS 1对U3 A-STAT 1细胞中IFNg功能的影响。 这将涉及建立稳定的细胞系， 过表达所述皮亚斯蛋白，或者 反义策略。

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.