RAPAMYCIN BLOCKADE OF PROTEIN PHOSPHATASE SIGNALING

描述（由申请方提供）：雷帕霉素是一种具有多种临床应用的大环内酯类天然产物。雷帕霉素作为器官移植后的免疫抑制剂，作为肿瘤的化学疗法和作为动脉再狭窄的抑制剂。然而，雷帕霉素作为抗增殖剂的作用机制仍不完全清楚，对其作用的抗性破坏了临床应用。雷帕霉素与一种称为FKBP 12的普遍存在的细胞内受体蛋白结合，并抑制雷帕霉素靶蛋白激酶（TOR），细胞周期检查点激酶家族的成员。TOR在所有真核生物中是保守的，并且在控制细胞生长和细胞增殖中起作用。酵母遗传学已经揭示，TOR下游的信号传导需要必需蛋白质Ser/Thr磷酸酶SIT 4（哺乳动物PP 6），其控制G1细胞周期蛋白的诱导和细胞周期进程。此外，必需的酵母蛋白TAP 42和最近发现的TIP 41调节SIT 4磷酸酶，并且还可能调节酵母中的PP 2A磷酸酶。该修订后的继续申请提出了四个特定目的：1）阐明雷帕霉素和TOR调节蛋白磷酸酶PP 6和PP 2A的机制，涉及磷酸化和/或亚基交换。2)使用截短和突变蛋白质的瞬时表达和共沉淀，定义人α-4（TAP 42）及其结合伴侣AlP（TIP 41）之间相互作用所需的结构决定因素。3)确定PP 6（SIT 4）独特N末端的功能，该功能赋予该磷酸酶通过产生融合蛋白和嵌合磷酸酶促进G1期至S期进展的能力，以检测特异性定位、显性负干扰或催化特性变化。4)通过使用转染和siRNA敲低增加和消耗细胞中AlP和α-4水平，确定细胞对雷帕霉素的抗性取决于α-4与PP 6（SIT 4）磷酸酶结合的可用性。拟议的研究将为研究不足的磷酸酶信号通路提供新的见解，并提供有关雷帕霉素作用和耐药性的分子基础的信息。

DESCRIPTION (provided by applicant): Rapamycin is a macrolide natural product with multiple clinical applications. Rapamycin serves as an immunosuppressant following organ transplantation, as chemotherapy for tumors and as an inhibitor of restenosis of arteries. Yet, the mechanism of action of rapamycin as an antiproliferative agent remains incompletely understood and resistance to its effects undermines the clinical applications. Rapamycin binds to a ubiquitous intracellular receptor protein called FKBP12 and inhibits the protein kinase Target Of Rapamycin (TOR), a member of the family of cell cycle checkpoint kinases. TOR is conserved among all eucaryotes and functions in control cell growth and cell proliferation. Yeast genetics has revealed that signaling downstream of TOR requires the essential protein Ser/Thr phosphatase SIT4 (mammalian PP6), which controls induction of G1 cyclins and cell cycle progression. In addition, the essential yeast protein TAP42 and recently found TIP41 regulate the SIT4 phosphatase and possibly also the PP2A phosphatase in yeast. This revised continuation application proposes four Specific Aims: 1) Elucidate the mechanism for rapamycin and TOR regulation of protein phosphatases PP6 and PP2A involving phosphorylation and/or subunit interchange. 2) Define the structural determinants required for interaction between human alpha-4 (TAP42) and its binding partner AlP (TIP41) using transient expression of truncated and mutated proteins and co-precipitation. 3) Determine the function of the unique N terminus of PP6 (SIT4) that confers the ability of this phosphatase to promote G1 to S phase progression by producing fusion proteins and chimeric phosphatases to test for specific localization, dominant negative interference or change in catalytic properties. 4) Establish that cellular resistance to rapamycin depends on availability of alpha-4 to associate with PP6 (SIT4) phosphatase by increasing and depleting cellular levels of AlP and alpha-4 using transfection and siRNA knock-down. The proposed studies will give new insights into an under-studied phosphatase-signaling pathway and provide information on the molecular basis for action of and resistance to rapamycin.