CALCIUM, PROTEIN PHOSPHORYLATION, AND CELL CYCLE CONTROL

本申请中概述的实验的目的是提供， 从细胞生物学的角度来看，关于这些因素的新信息 在细胞周期中控制细胞的进程。众多人 各种癌症的共同特征之一是不受控制 癌细胞分裂。因此细胞反复分裂，在 不需要的数量和地点，造成很大损害的。这个 这里提出的实验将继续生物化学和分子 一种与微管稳定有关的蛋白质的鉴定 在有丝分裂期间，因此可能是细胞分裂的关键调节因子。 具体地说，提出的实验旨在定义 一种新近发现的蛋白质(MREL=62kD)的特性 后期开始钙依赖的磷酸化底物。 在活细胞中，一个钙脉冲在中期和后期被释放。 这种转变被认为会触发细胞内持续的转运 周而复始。为了更全面地了解这一触发因素的性质，62 kD 将对蛋白质进行表征。要做到这一点，丰度和分布 对这些实验至关重要的蛋白质抗体中的 纯化和特性将在体外和体内进行测定。这个 以前的一系列实验将确定蛋白质的能力 在体外组装系统中与微管相互作用，并测试 蛋白质参与了微管的解体。对于活体内 实验中，纯化的蛋白质将被微感染到活细胞中 在细胞周期的不同时间测试蛋白质对 正常的细胞周期进程。最后，编码62kD蛋白的克隆 将从海胆表达文库中分离出来开始分子 对该蛋白质进行分析，以将其与其他已知的参与 细胞周期控制。此应用程序中建议的实验包括 很重要，因为它们将提供新的和明确的信息 关于细胞分裂的控制，通过开始鉴定 一系列事件中涉及的因素标志着 有丝分裂的后期，最终完成细胞分裂。

The purpose of the experiments outlined in this application is to provide, from a cell biology point of view, new information concerning the factors controlling the progression of cells through the cell cycle. The many forms of cancer have as one of their common characteristics uncontrolled division of the cancerous cells. Thus the cells divide repeatedly, in amounts and place where they are not required, causing much damage. The experiment proposed here will continue a biochemical and molecular characterization of one protein that is involved in microtubule stability during mitosis and thus may be a key regulator of cell division. Specifically, the experiments proposed are designed to define the characteristics of a recently identified protein (Mrel = 62 kD) that is the substrate for calcium dependent phosphorylation at the start of anaphase. In living cells, a pulse of calcium is released at the metaphase-anaphase transition which is thought to trigger continued transit through the cell cycle. To understand the nature of this trigger more completely, the 62 kD protein will be characterized. To do this, the abundance and distribution of the protein antibodies essential for these experiments have been purified and characteristics will be determined in vitro and in vivo. The former series of experiments will determine the ability of the protein to interact with microtubules in an in vitro assembly system, and test how the protein is involved in microtubule disassembly. For the in vivo experiments, the purified protein will be microinfected into living cells at various times during the cell cycle to test the affect of the protein on normal cell cycle progression. Finally, clones encoding the 62 kD protein will be isolated from a sea urchin expression library to begin molecular analysis of the protein to compare it to other known proteins involved in cell cycle control. The experiments proposed in this application are important because they will provide new and definitive information concerning the control of cell division by beginning an identification of the factors involved in the cascade of events that signal the start of anaphase of mitosis, and, ultimately, the completion of cell division.