PHOSPHORYLATION OF CYTOSKELETAL PROTEINS IN LENS

我建议对晶状体中的蛋白激酶系统进行表征 使细胞骨架蛋白磷酸化。细胞骨架的磷酸化 蛋白质与维持细胞形状和细胞形状有关 改变，从而可能参与了晶状体纤维的分化和 保持镜头的清晰度。 添加~(32)P-正磷酸盐的全晶状体器官培养体系 用于检测细胞骨架磷酸化的各个方面。这个 晶状体细胞骨架磷蛋白的发展和老化方面将是 在胚胎和孵化后的雏鸡以及上皮细胞中进行检查 诱导伸长的细胞内磷酸化效应物(即cAMP 和cGMP依赖的蛋白激酶，钙/钙调蛋白依赖的激酶， 和钙/磷脂依赖的激酶)将通过添加 脂溶性环核苷酸类似物，钙离子载体，或佛波醇 酯化到标记的培养基上。细胞外效应器 新陈代谢(即儿茶酚胺和晶状体营养因子)也将 检查它们对细胞骨架磷酸化的影响。这些研究 将允许对标记的磷蛋白进行可视化和定量 细胞骨架制剂经过凝胶电泳和 放射自显影。 一种利用细胞骨架制剂和 伽马-32-P(ATP)将揭示最佳离子条件、pH、 温度，以及磷酸化的时间进程。其他内容 实验将确定游离钙水平，钙调素， 磷脂部分和环核苷酸水平影响 特定细胞骨架底物的磷酸化。有限的蛋白质分解 还将检查某些标记蛋白质的可能性 细胞骨架蛋白是多种激酶系统的底物。 这项研究的结果将解决可能在以下方面运行的机制 正常晶状体纤维分化及其机制的改变 可能会导致白内障的发生。

I propose to characterize the protein kinase systems in the lens which phosphorylate cytoskeletal proteins. Phosphorylation of cytoskeletal proteins has been linked to the maintenance of cell shape and cell shape change and thus may be involved in the differentiation of lens fibers and the maintenance of lens clarity. A whole lens organ culture system supplemented with 32P-orthophosphate will be utilized to examine aspects of cytoskeletal phosphorylation. The development and aging aspects of lens cytoskeletal phosphoproteins will be examined in embryonic and post-hatching chicks and in epithelial cells induced to elongate. Intracellular effectors of phosphorylation (i.e. cAMP and cGMP - dependent protein kinases, calcium/calmodulin-dependent kinase, and calcium/phospholipid-dependent kinase) will be stimulated by adding lipid soluble cyclic nucleotide analogs, calcium ionophore, or phorbol ester to the labeled culture media. Extracellular effectors of cell metabolism (i.e. catecholamines and lens trophic factors) will also be examined for their effect on cytoskeletal phosphorylation. These studies will allow visualization and quantitation of labeled phosphoproteins after cytoskeletal preparations are subjected to gel electrophoresis and autoradiography. An in vitro assay system utilizing cytoskeletal preparations and Gamma-32-P(ATP) will reveal information about optimal ionic conditions, pH, temperature, and the time course for phosphorylation. Additional experiments will determine how free calcium levels, calmodulin, phospholipid moieties and cyclic nucleotide levels effect the phosphorylation of specific cytoskeletal substrates. Limited proteolysis of labeled protein will also examine the possibility that certain cytoskeletal proteins are substrates for multiple kinase systems. The results of this study will address mechanisms which may operate during normal lens fiber differentiation and how alterations in these mechanisms may contribute to a cataractogenic process.