MOLECULAR REGULATION OF INTERMEDIATE FILAMENT STRUCTURE

中间丝是纤维的主要结构特征。 哺乳动物细胞的细胞骨架。尽管细丝是 动态结构，并在细胞分裂期间进行重组，很少 已知调节细丝的分子机制 结构和组织。提出的假设 实验表明，蛋白质的磷酸化起着重要的作用 在调节中间丝组织方面，以及 观察到细丝磷酸化水平的增加 细胞分裂就是这一规律的直接反映。这个 拟议的实验将使用中间丝蛋白 波形蛋白作为磷酸化调控纤维模型的研究进展 组织。我们将确定特定的波形蛋白丝氨酸位置 在非有丝分裂和有丝分裂培养的小鼠中被磷酸化 细胞以及以下纯化的体外磷酸化 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 有膜蛋白和核膜蛋白。我们将评估 体外磷酸化对同型关联的影响 导致细丝分解的波形蛋白分子之间的相互作用。 最后，我们将克隆Vimentin cDNA并生产出cDNA 有丝分裂特异性丝氨酸磷酸化的特异性改变 由寡核苷酸引导的突变位点。这些重组人 CDNA将被插入到表达载体中并用于 将其导入不表达波形蛋白的细胞。行稳致远 将分离转基因细胞并对其进行波形蛋白鉴定 细丝蛋白质含量和磷酸化。细胞系有一种 然后在细胞周期研究中使用适当的表型来 确定特定磷酸化位点的改变是否会改变 细丝组织或细胞重塑的能力 细胞分裂过程中的中间丝。长期目标 这些研究的目的是更好地理解蛋白质的作用 磷酸化在细胞质组织调控中的作用 这一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.