PROTEIN AND LIPID PHOSPHORYLATION AND THE CORNEA

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This proposal is a continuation of our ongoing studies of lipid and protein phosphorylation in cornea, focusing on the events of cell signal transduction during wound closure and epithelial cell proliferation. We will test the following hypothesis: Protein kinase C (PKC) subspecies and phosphatidylinositol kinases (PIK) are central components of the growth-control pathway in corneal epithelial cells. These cell-signaling pathways are activated in response to growth factors released during injury, and their sustained activation ultimately results in the synthesis of proteins necessary for injury repair. Two models of wound healing will be used: a mild alkali burn model and a complete debridement of the epithelial layer. A major PKC subspecies in the cornea is alphaPKC, which promotes the transcriptional activation of immediate early genes and leads to gene cascades that modulate the cell cycle in cellular proliferation and wound healing. We propose to identify the modulators of PKC subspecies as well as the effect of growth factors and wound healing on PIKs. Also, we intend to define how growth-regulated genes are transcriptionally activated. Correlation with wound closure and cellular proliferation will be established. Powerful analytical procedures such as high performance liquid chromatography (HPLC), capillary gas liquid chromatography (CGLC), and fast performance liquid chromatography, (FPLC), will be used. The rate of wound closure and 3H thymidine incorporation in DNA will be measured as an indication of cellular proliferation and wound healing. Quantification of specific mRNAs will be accomplished after norther blots with storage phosphor imaging. The results obtained will define the involvement of PKCs and PIKs in corneal wound healing.