RAPAMYCIN BLOCKADE OF PROTEIN PHOSPHATASE SIGNALING

雷帕霉素是一种大环内酯类免疫抑制剂，可抑制T细胞 增殖和B细胞免疫球蛋白的产生。它能阻止蛋白质 在Gl期合成和阻止细胞，包括酵母的生长。 潜在的临床应用包括减弱移植物对宿主的作用 器官移植的反应与自身免疫性疾病的治疗 和某些癌症。这项研究的目的是阐明 雷帕霉素敏感的翻译激活途径 异常蛋白磷酸酶的作用。雷帕霉素与一种 称为FKBP的细胞内受体蛋白和药物-蛋白质复合体 抑制被称为雷帕霉素靶标(TOR)或FRAP的蛋白激酶。 酵母中的遗传分析发现了一种Tap42蛋白，它与 酵母蛋白磷酸酶Sit4和Pph21免疫沉淀 哺乳动物PP6和PP2A的酵母版本。雷帕霉素阻止结合 Tap42对磷酸酶的作用，但在突变的菌株中没有发生这种情况 在TOR中，表明Tap42在信令中位于TOR的下游 路径。令人惊讶的是，一种名为阿尔法-4的小鼠蛋白质与 Tap42的序列，作为一种磷酸蛋白被独立发现 与B细胞受体Ig-α蛋白相关。初步 研究表明，小鼠α-4与纯化的人PP2A结合，取代 其他调节亚基，并改变底物专一性。 标记α-4的表位在免疫共沉淀的COS细胞中的表达 PP2A并导致延伸因子EF2去磷酸化，而不是 对同样在下游运行的PHAs-1(eIF4E-BP1)或p70S6K的影响 是Tor的。该项目的具体目标是：1)定义 结合所需的α-4和PP2A/PP6的结构特征，使用 截短和突变重组融合蛋白及其表位标记 蛋白质在下拉和共沉淀分析中。产生突变形式 不能结合磷酸酶起显性负性作用的α-4。 2)测定α-4：PP2A的动力学和底物专一性 相对于生化分析中的AC二聚体，使用定义的底物 如EF2、EF2激酶、磷酸化酶、MBP和多肽等。3)快递 成纤维细胞及Jurkat T和Raji中α-4的显性阴性形式 B细胞，检测eEF2的磷酸化，启动因子， 进入S期，以及对雷帕霉素的敏感性。 4)发现与高度保守的N和C相关的蛋白质 α-4的末端结构域，在与其结合的区域之外 磷酸酶。这将揭示底物专一性和 靶向α-4：磷酸酶和可能的结合位点 α-4与Ig-α的相关性。这个项目将发现新的 蛋白质合成和细胞生长调控的分子机制 对雷帕霉素敏感的药物。

Rapamycin is a macrolide immunosuppressant that inhibits T cell proliferation and B cell immunoglobulin production. It blocks protein synthesis and arrests growth of cells, including yeast, in Gl phase. Potential clinical applications include attenuation of graft vs. host response in organ transplantation and treatment of autoimmune diseases and certain cancers. The goal of this research is to elucidate the rapamycin-sensitive pathway for activating translation through the action of unusual protein phosphatases. Rapamycin binds to an intracellular receptor protein called FKBP, and the drug-protein complex inhibits the protein kinase called target of rapamycin (TOR) or FRAP. Genetic analysis in yeast identified a Tap42 protein that co- immunoprecipitated with yeast protein phosphatases Sit4 and Pph21, the yeast versions of mammalian PP6 and PP2A. Rapamycin prevented binding of Tap42 to the phosphatases, but this did not occur in strains mutated in TOR, showing that Tap42 is downstream of TOR in the signaling pathway. Surprisingly, a mouse protein called alpha-4 is related in sequence to Tap42 and was discovered independently as a phosphoprotein associated with the B-cell receptor Ig-alpha protein. Preliminary studies show that murine alpha-4 binds purified human PP2A, displaces the other regulatory subunits, and changes substrate specificity. Epitope tagged alpha-4 expressed in COS cells co-immunoprecipitated with PP2A and caused dephosphorylation of the elongation factor EF2, without effects on PHAS-1 (eIF4E-BP1) or p70S6K, that also operate downstream of TOR. The specific aims of this project are to: 1) define the structural features of alpha-4 and PP2A/PP6 required for binding, using truncated and mutated recombinant fusion proteins and epitope-tagged proteins in pull-down and co-precipitation assays. Produce mutant forms of alpha-4 that will not bind phosphatases to act as dominant negatives. 2) determine the kinetics and substrate specificity of alpha-4: PP2A relative to the AC dimer in biochemical assays, using defined substrates such as EF2, EF2 kinase, phosphorylase, MBP and peptides. 3) express dominant-negative forms of alpha-4 in fibroblasts and Jurkat T and Raji B cells and measure the phosphorylation of eEF2, initiation factors, kinases, as well as entry into S phase and sensitivity to rapamycin. 4) discover proteins that associate with the highly conserved N and C terminal domains of alpha-4, outside of the regions that bind to phosphatases. This will reveal the basis for substrate specificity and targeting of the alpha-4: phosphatase and possibly a site for association of alpha-4 with Ig-alpha. This project will discover new molecular mechanisms for control of protein synthesis and cell growth that are sensitive to rapamycin.