RAPAMYCIN BLOCKADE OF PROTEIN PHOSPHATASE SIGNALING

说明书(申请人提供)：雷帕霉素是一种具有多种临床应用的大环内酯类天然产品。雷帕霉素可作为器官移植后的免疫抑制剂、肿瘤的化疗药物和动脉再狭窄的抑制剂。然而，雷帕霉素作为一种抗增殖剂的作用机制仍不完全清楚，对其作用的耐药性破坏了其临床应用。雷帕霉素与一种名为FKBP12的普遍存在的细胞内受体蛋白结合，并抑制雷帕霉素的蛋白激酶靶标(TOR)，TOR是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，在控制细胞生长和细胞增殖方面起着重要作用。酵母遗传学研究表明，TOR下游的信号传递需要必需的蛋白质Ser/Thr磷酸酶SIT4(哺乳动物PP6)，它控制G1周期蛋白的诱导和细胞周期的进展。此外，酵母必需蛋白TAP42和新近发现的TIP41还调节酵母中的SIT4磷酸酶和PP2A磷酸酶。这一修订的继续申请提出了四个具体目的：1)阐明雷帕霉素和TOR调节涉及磷酸化和/或亚单位交换的蛋白磷酸酶PP6和PP2A的机制。2)利用截短和突变蛋白的瞬时表达和共沉淀，确定人α-4(TAP42)与其结合伙伴ALP(TIP41)相互作用所需的结构决定因素。3)确定PP6的唯一N末端(SIT4)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来促进G1期向S期进展的能力，以测试其特异性定位、显性负干扰或催化性质的变化。4)证实细胞对雷帕霉素的耐药依赖于α-4与PP6(SIT4)磷酸酶结合的可用性，方法是通过转染和siRNA敲除来增加和耗尽细胞中ALP和α-4的水平。建议的研究将为研究不足的磷酸酶信号通路提供新的见解，并提供关于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.