RAPAMYCIN BLOCKADE OF PROTEIN PHOSPHATASE SIGNALING

雷帕霉素是一种大环内酯类免疫抑制剂， 增殖和B细胞免疫球蛋白产生。 它能阻止蛋白质 合成并阻止细胞（包括酵母）在G1期生长。 潜在的临床应用包括移植物与宿主的减毒 器官移植和自身免疫性疾病治疗中的反应 和某些癌症。 本研究的目的是阐明 雷帕霉素敏感性途径，通过 不寻常的蛋白磷酸酶的作用。 雷帕霉素结合于 细胞内受体蛋白FKBP和药物蛋白复合物 抑制称为雷帕霉素靶点（TOR）或FRAP的蛋白激酶。 酵母中的遗传分析鉴定了一种Tap 42蛋白， 用酵母蛋白磷酸酶Sit 4和Pph 21免疫沉淀， 哺乳动物PP 6和PP 2A的酵母版本。 雷帕霉素阻止结合 Tap 42的磷酸酶，但这并没有发生在菌株突变 在TOR中，显示Tap 42在信令中位于TOR的下游 通路 令人惊讶的是，一种名为alpha-4的小鼠蛋白质与 序列Tap 42和独立发现的磷蛋白 与B细胞受体Ig-α蛋白相关。 初步 研究表明，鼠α-4结合纯化的人PP 2A， 其他调节亚基，并改变底物特异性。 在COS细胞中表达的表位标记的α-4与 PP 2A并引起延伸因子EF 2的去磷酸化，而不 对PHAS-1（eIF 4 E-BP 1）或p70 S6 K的影响，也在下游发挥作用 的TOR。 该项目的具体目标是：1）确定 结合所需的α-4和PP 2A/PP 6的结构特征，使用 截短和突变的重组融合蛋白和表位标记的 在下拉和共沉淀测定中的蛋白质。 产生突变形式 不与磷酸酶结合的α-4蛋白作为显性负性蛋白。 2)确定α-4：PP 2A的动力学和底物特异性 相对于生化测定中的AC二聚体，使用规定的底物 如EF 2、EF 2激酶、磷酸化酶、MBP和肽。 3)表达 成纤维细胞和Jurkat T和Raji中α-4的显性阴性形式 B细胞，并测量eEF 2，起始因子， 激酶，以及进入S期和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，在结合 磷酸酶 这将揭示底物特异性的基础， α-4：磷酸酶的靶向和可能的位点， α-4与Ig-α的结合。 该项目将发现新的 控制蛋白质合成和细胞生长的分子机制 对雷帕霉素敏感

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.