MOLECULAR REGULATION OF INTERMEDIATE FILAMENT STRUCTURE

中间丝是主要的结构特征， 哺乳动物细胞的细胞骨架。 虽然这些细丝 动态结构，并在细胞分裂过程中重组， 我们知道调节纤维的分子机制 结构和组织。 提出的假设 实验表明，蛋白质磷酸化起着重要作用， 在调节中间纤维组织方面， 观察到的纤维磷酸化水平增加， 细胞分裂是这种调节的直接反映。 的 拟议的实验将使用中间丝蛋白 波形蛋白作为磷酸化调节丝的模型 organization. 我们将确定特定的波形蛋白丝氨酸位点 在非有丝分裂和有丝分裂培养小鼠中磷酸化 细胞以及在体外磷酸化后，用纯化的 蛋白激酶 我们将开发一个体外模型系统来研究 波形蛋白磷酸化对特异性相互作用的影响 与膜蛋白和核膜蛋白结合。 我们将评估 体外磷酸化对同型结合的影响 波形蛋白分子之间的相互作用导致了纤维的分解。 最后，我们将克隆波形蛋白cDNA，并产生cDNA， 在有丝分裂特异性丝氨酸磷酸化中特异性改变 位点通过寡核苷酸定向诱变。 这些重组 将cDNA插入表达载体中并用于 排除不表达波形蛋白的细胞。 稳定线 将分离转染的细胞并表征波形蛋白 丝蛋白含量和磷酸化。 细胞系， 然后将适当的表型用于细胞周期研究， 确定特定磷酸化位点的改变是否改变了 纤维组织或细胞重塑的能力 在细胞分裂期间的中间丝。 长期目标 是为了更好地理解蛋白质的作用 磷酸化在细胞质组织的控制和 这个过程与细胞增殖的关系。

Intermediate filaments are major structural features of the cytoskeleton of mammalian cells. Although the filaments are dynamic structures and are reorganized during cell division, little is known about the molecular mechanisms which regulate filament structure and organization. The hypothesis of the proposed experiments is that protein phosphorylation plays an important role in the regulation of intermediate filament organization, and that the increased level of filament phosphorylation observed in dividing cells is a direct reflection of this regulation. The proposed experiments will use the intermediate filament protein vimentin as a model for phosphorylation regulated filament organization. We will determine the specific vimentin serine sites that are phosphorylated in non-mitotic and mitotic cultured mouse cells as well as following in vitro phosphorylation with purified protein kinases. We will develop an in vitro model system to study the effect of vimentin phosphorylation on specific interactions with membrane and nuclear envelope proteins. We will evaluate the effect of in vitro phosphorylation on the homotypic associations between vimentin molecules that results in filament disassembly. Finally, we will clone vimentin cDNAs and produce cDNAs which are specifically altered at mitosis specific serine phosphorylation sites by oligonucleotide directed mutagenesis. These recombinant cDNAs will be inserted into an expression vector and used to transfect cells which do not express vimentin. Lines of stablely transfected cells will be isolated and characterized for vimentin filament protein content and phosphorylation. Cell lines with an appropriate phenotype will then be used in cell cycle studies to determine if alteration of specific phosphorylation sites alters filament organization or the ability of cells to remodel intermediate filaments during cell division. The long term goal of these studies is to better understand the role of protein phosphorylation in the control of cytoplasmic organization and the relation of this process to cellular proliferation.