BIOLOGICAL ROLES OF THE PROLYL ISOMERASE, PIN1

我们的总体目标是确定肽基脯氨酸异构酶Pin1作为有丝分裂负调节因子的方式，并评估该蛋白是否可能成为开发抗癌治疗药物的有吸引力的靶标。Pin1被发现是一种与NIMA相互作用的蛋白，NIMA是一种丝氨酸/苏氨酸激酶蛋白，在细粒曲霉从G2到有丝分裂的过程中是必不可少的。Pin1从酵母到人类都被保存下来，可能对有丝分裂过程至关重要。我们从曲霉和爪蟾中克隆了Pin1，并制造了该蛋白的抗体。我们发现，在爪蟾提取物中，Pin1将调节进入有丝分裂，并与p34cdc2调节途径的关键组分相互作用，如cdc25蛋白磷酸酶和plxl蛋白激酶的活性形式，以及MPM2抗体识别的许多其他有丝分裂磷酸化蛋白。我们建议使用非洲爪蟾卵/卵母细胞系统来解决有关Pin1的脯氨酸异构酶和蛋白质结合的关键问题，并阐明其作为细胞周期调节因子的关键细节。我们将评估Pin1在有丝分裂进入时间和控制G2/M转变的检查点操作中的作用，并检查Pin1是否在有丝分裂退出中发挥作用。同时，我们将确定Pin1是否对小鼠发育至关重要，并通过使用cre/lox系统在小鼠T淋巴细胞中整体或特异性地破坏Pin1基因，重点研究其在T淋巴细胞的发育、激活和/或增殖中的作用。我们将确定在任何脊椎动物系统中Pin1缺失的表型后果是否需要脯氨酸异构酶活性。这些研究的完成将明确Pin1在生长发育中的作用，确定其关键的细胞周期功能，并阐明Pin1脯氨酸异构酶是否是抗癌药物开发的良好候选物。

Our overall goal is to determine the manner by which the peptidyl-prolyl isomerase, Pin1, acts as a negative regulator of mitosis and evaluate whether this protein may be an attractive target for development of an anti-cancer therapeutic agent. Pin1 was discovered as a protein that interacts with NIMA, a protein Ser/Thr Kinase that is essential for progression from G2 to mitosis in Aspergillus nidulans. Pin1 has been conserved from yeast to man and may be critical for mitotic progression. We cloned Pin1 from Asperigillus and Xenopus, and made antibodies to the protein. We showed that Pin1 will regulate entry into mitosis in Xenopus extracts where it interacts with key components of the p34cdc2 regulatory pathway such as the active forms of the cdc25 protein phosphatase and the plxl protein kinase as well as a number of other mitotic phosphoproteins recognized by the MPM2 antibody. We propose to use the Xenopus Egg/oocyte system to address key issues regarding the prolyl isomerase and protein binding of Pin1 and elucidate critical details of its role(s) as a cell cycle regulator. We will evaluate the tole of Pin1 in the timing of mitotic entry and in the operation of checkpoints governing the G2/M transition as well as examine if Pin1 plays a role in exit from mitosis. In parallel, we will determine if Pin1 is essential for mouse development and focus on its role in development, activation and/or proliferation of T lymphocytes by using the cre/lox system to disrupt the Pin1 gene in mice either globally or specifically in T lymphocytes. We will determine if phenotypic consequences of Pin1 deletion in either vertebrate system requires the prolyl isomerase activity. Completion of these studies will define the role(s) of Pin1 in growth and development, determine its critical cell cycle functions and clarify whether the Pin1 prolyl isomerase is a good candidate for anti-cancer drug development.