MICROFILAMENT ORGANIZATION

这项建议的长期目标是了解分子机制 在有丝分裂过程中调节微丝重组，以及 在培养细胞的致癌转化过程中。呢我们一起来 有丝分裂特异性的生物学意义 钙调蛋白的磷酸化。我们第一次证明了， 非肌肉钙调素，一种相对分子质量Mr = 83000的蛋白质， 与肌动蛋白和钙调蛋白结合， 在有丝分裂期间，显然是磷酸化的结果。我们认为 这一过程可能会导致形状和结构的变化， 在经历有丝分裂的细胞中，因为钙调蛋白抑制肌动球蛋白ATP酶， 由于钙调蛋白和原肌球蛋白调节肌动蛋白， 凝溶胶蛋白的切割和封盖活性。我们将从生物化学的角度 描述有丝分裂特异性钙调蛋白磷酸化如何改变 微丝组装和肌动球蛋白的相互作用。体内 有丝分裂特异性钙调蛋白磷酸化的功能将被探索 通过显微注射磷酸化钙调蛋白， 磷酸化或去磷酸化，以及缺乏磷酸化或去磷酸化的突变型钙调蛋白。 磷酸化位点，进入培养的细胞。我们将观察 显微注射将干扰有丝分裂过程中微丝的组装 和其他细胞周期阶段。我们将研究磷酸化 细胞周期进程中钙调素的状态，详细了解是否 钙调素的磷酸化与微丝的变化相关 组装件.因为转化细胞的形态学改变 有丝分裂细胞中发现的那些，我们将使用大鼠-I细胞来检查， 钙调蛋白在细胞转化过程中是否被磷酸化。最后， 因为我们已经发现cdc 2激酶（有丝分裂促进因子）是一种 在有丝分裂期间磷酸化钙调蛋白的两种激酶活性中， 将描述CDC 2激酶和其他激酶活性 磷酸化钙调素。我们还将寻找其他 被这些激酶磷酸化的微生物相关蛋白 在有丝分裂期间。由于细胞周期调控与细胞周期调控密切相关， 转型，拟议的研究将帮助我们了解不仅如何 细胞分裂，以及为什么癌细胞失去对细胞分裂的控制。

The long term goal of this proposal is to understand molecular mechanisms that regulate microfilament reorganization during mitosis, as well as during oncogenic transformation of cultured cells. We will focus our studies on the biological significance of the mitosis-specific phosphorylation of caldesmon. We have demonstrated, for the first time, that nonmuscle caldesmon, a protein of relative molecular mass Mr = 83,000 which binds to actin and calmodulin, is disassociated from microfilaments during mitosis, apparently as a consequence of phosphorylation. We believe that this process may contribute to the changes of shape and structure seen in cells undergoing mitosis because caldesmon inhibits actomyosin ATPase, and because caldesmon, together with tropomyosin, regulates the actin severing and capping activities of gelsolin. We will biochemically characterize how the mitosis-specific phosphorylation of caldesmon changes microfilament assembly and actomyosin interactions in vitro. In vivo functions of mitosis-specific phosphorylation of caldesmon will be explored through microinjection of phosphorylated caldesmon, antibodies that block phosphorylation or dephosphorylation, and mutant caldesmons lacking the phosphorylation sites, into cultured cells. We will observe whether the microinjection will perturb the assembly of microfilaments during mitosis and other cell cycle stages. We will examine changes in the phosphorylation state of caldesmon during cell cycle progression in detail to see whether the phosphorylation of caldesmon correlates with changes in microfilament assembly. Because transformed cells show morphological alterations similar to those found in mitotic cells, we will examine, using ts Rat-I cells, whether caldesmon is phosphorylated during cell transformation. Finally, because we have found that cdc2 kinase (mitosis promotion factor) is one of two kinase activities that phosphorylate caldesmon during mitosis, we will characterize how cdc2 kinase and the other kinase activity phosphorylate caldesmon. We will also search for other microfilament-associated proteins that are phosphorylated by these kinases during mitosis. Because cell cycle control is intimately related to cell transformation, the proposed studies will help us understand not only how cells divide, but also why cancer cells lose control of cell division.