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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29EY006129-02
Application #
3465459
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1986-09-01
Project End
1991-08-31
Budget Start
1987-09-01
Budget End
1988-08-31
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Wayne State University
Department
Type
Schools of Medicine
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Ireland, M E; Richiert, D M; Tran, K (1994) Regulation of lens beta-adrenergic receptors by receptor occupancy and dexamethasone. J Ocul Pharmacol 10:543-51
Ireland, M E; Klettner, C; Nunlee, W (1993) Cyclic AMP-mediated phosphorylation and insolubilization of a 49-kDa cytoskeletal marker protein of lens fiber terminal differentiation. Exp Eye Res 56:453-61
Ireland, M E; Braunsteiner, A; Mrock, L (1993) Cell-cell interactions affect the accumulation of a cytokeratin-like protein during lens fiber development. Dev Biol 160:494-503
Ireland, M E; Tran, K; Mrock, L (1993) Beta-adrenergic mechanisms affect cell division and differentiation in cultured chick lens epithelial cells. Exp Eye Res 57:325-33
Jacobs, D B; Ireland, M; Pickett, T et al. (1992) Functional characterization of insulin and IGF-I receptors in chicken lens epithelial and fiber cells. Curr Eye Res 11:1137-45
Ireland, M E; Shanbom, S (1991) Lens beta-adrenergic receptors. Functional coupling to adenylate cyclase and photoaffinity labeling. Invest Ophthalmol Vis Sci 32:541-8
Brown, H G; Pappas, G D; Ireland, M E et al. (1990) Ultrastructural, biochemical, and immunologic evidence of receptor-mediated endocytosis in the crystalline lens. Invest Ophthalmol Vis Sci 31:2579-92
Ireland, M E; Jacks, L A (1989) Initial characterization of lens beta-adrenergic receptors. Invest Ophthalmol Vis Sci 30:2190-4
Ireland, M; Maisel, H (1989) A family of lens fiber cell specific proteins. Lens Eye Toxic Res 6:623-38
Ireland, M E; Maisel, H (1988) Isoproterenol treatment causes cytoskeletal reorganization in chicken lens fiber cells. Invest Ophthalmol Vis Sci 29:1356-60

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