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.
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