权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

PHOSPHORYLATION OF CYTOSKELETAL PROTEINS IN LENS

晶状体中细胞骨架蛋白的磷酸化

基本信息

批准号：
3465462
负责人：
MARK E IRELAND
金额：
$ 9.86万
依托单位：
WAYNE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-09-01 至 1992-08-31
项目状态：
已结题

项目摘要

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.

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。