PHOSPHOLIPASE C AND KINETOCHORE FUNCTION

描述(申请人的描述) 我们实验室的长期目标是鉴定和鉴定真核生物。 磷脂酶C(PLC)调节细胞过程，这是一种发挥作用的酶 在信号转导通路中扮演重要角色。PLC水解物 磷脂酰肌醇4，5-二磷酸[PtdIns(4，5)P2]产生两个重要的 第二信使：三磷酸肌醇[INS(1，4，5)P3]，触发 从体内储存的钙释放，和二酰甘油(DG)，它 激活磷脂和钙离子依赖的蛋白激酶C。 我们最近的研究结果(Lin等人，2000)证明了酵母中的PLC 酿酒酵母(由plc1基因编码的plc1p蛋白)与 运动抑制并影响它们结合微管的能力。动粒是 一种特殊的细胞器，负责将染色体附着在纺锤体上 微管，因此对适当的染色体分离和细胞是必不可少的 循环递增。我们发现plc1基因缺失的细胞(Plc1Delta) 表现出更高的染色体丢失频率，诺可达唑敏感性，以及 有丝分裂延迟。此外，从p1c1Delta细胞中提取的染色质显示 微管与微染色体的结合减少。然而，它仍然是未知的 正常有丝分裂是否需要Plc1p的酶活性 功能，或者仅仅是这种蛋白质与动素中心的结合 足以满足正常行为，或者Plc1p的动粒结合和它的 两种酶的活性都是必需的。 我们的具体目标将解决这些替代可能性。我们会做好准备 两种类型的plc1p突变体：酶失活突变体 与动粒相互作用的能力，以及(Ii)保留酶的突变体 活动，但不能与动素相互作用。每个突变体Plc1p 将对蛋白质进行生化表征，酵母菌株将表达 这些突变的Plc1p蛋白将在保真度方面得到充分的表征 染色体传递，有丝分裂延迟，对诺可唑的敏感性，以及 微染色体结合微管的能力。结果将导致 更好地理解染色体调控的分子机制 有丝分裂、细胞增殖和肿瘤形成过程中的分离。

DESCRIPTION (applicant's description) The long-term goal of our laboratory is to identify and characterize eukaryotic cellular processes regulated by phospholipase C (PLC), an enzyme which plays vital roles in signal transduction pathways. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to produce two important second messengers: inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] which triggers release of calcium from internal stores, and diacylglycerol (DG) which activates the phospholipid- and Ca2+-dependent protein kinase C. Our recent results (Lin et al., 2000) demonstrate that PLC in Saccharomyces cerevisiae (Plc1p protein encoded by the PLC1 gene) associates with kinetochores and affects their ability to bind microtubules. The kinetochore is a specialized organelle that mediates chromosome attachment to spindle microtubules and hence is essential for proper chromosome segregation and cell cycle progression. We found that cells with deletion of PLC1 gene (plc1delta) display higher frequency of chromosome loss, nocodazole sensitivity, and mitotic delay. Furthermore, chromatin extracts from p1c1delta cells exhibit reduced microtubule binding to minichromosomes. However, it remains unknown whether the enzymatic activity of Plc1p is required for proper mitotic function, or whether the mere binding of this protein to kinetochores is sufficient for normal behavior, or whether Plc1p's kinetochore-binding and its enzymatic activity are both required. Our Specific Aims will resolve these alternative possibilities. We will prepare two types of Plc1p mutants: enzymatically inactive mutants with preserved ability to interact with kinetochores, and (ii) mutants which retain enzymatic activity but are unable to interact with kinetochores. Each mutant Plc1p protein will be characterized biochemically and the yeast strains expressing these mutant Plc1p proteins will be fully characterized in terms of fidelity of chromosome transmission, mitotic delay, sensitivity to nocodazole, and the ability of minichromosomes to bind microtubules. The results will lead to better understanding of the molecular mechanisms regulating chromosome segregation during mitosis, cell proliferation, and oncogenesis.