PHOSPHYORYLATION OF CYTOSKELETAL PROTEINS IN CHICK LENS

我建议描述透镜中的蛋白激酶系统， 使细胞骨架蛋白磷酸化。 细胞骨架磷酸化 蛋白质与维持细胞形状和细胞形状 因此可能涉及透镜纤维的分化， 保持透镜的清晰度。 补充有32 P-正磷酸盐的整个透镜器官培养系统将 用于检查细胞骨架磷酸化的各个方面。 的 透镜细胞骨架磷蛋白的发育和老化方面将是 在胚胎和孵化后的小鸡和上皮细胞中检查 被诱导伸长。 磷酸化的细胞内效应物（即cAMP 和cGMP依赖性蛋白激酶，钙/钙调蛋白依赖性激酶， 和钙/磷脂依赖性激酶）将通过加入 脂溶性环核苷酸类似物、钙离子载体或佛波醇 酯添加到标记的培养基中。 细胞外效应物 代谢（即，儿茶酚胺和透镜营养因子）也将被 检查它们对细胞骨架磷酸化的影响。 这些研究 将允许标记的磷蛋白的可视化和定量， 对细胞骨架制备物进行凝胶电泳， 放射自显影 一种利用细胞骨架制备物的体外测定系统， γ-32-P（ATP）将揭示有关最佳离子条件，pH值， 温度和磷酸化的时间过程。 额外 实验将确定游离钙水平，钙调蛋白， 磷脂部分和环核苷酸水平影响 特异性细胞骨架底物的磷酸化。 限制性蛋白水解 标记蛋白质的研究也将研究某些 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机制，可能运作期间， 正常的透镜纤维分化以及这些机制的改变 可能会导致白内障

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.