Protein Kinase C Gamma in The Lens

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DESCRIPTION (provided by applicant): Protein kinase C gamma (PKC gamma) is a major isoform of PKC in the lens epithelium and cortex. This PKC phosphorylates the lens gap junction protein, Cx43, causing inhibition of gap junction activity. PKC gamma is specifically decreased during galactosemia, in streptozotocin diabetic rats, and in human diabetic lens. This decrease in PKC gamma would result in an increase in gap junction activity. The specific aims of this proposal are: 1) To determine how PKC gamma is controlled in normal lens epithelial cells by EGF, and by translocation, downregulation, turnover, and expression in response to lipid and calcium. 2) To determine how the control of PKC gamma alters gap junction activity through changes in Cx43 phosphorylation, gap junction activity, and gap junction assembly. Specific aims 1 and 2 will utilize lens cells in culture, overexpression systems, green fluorescent protein-PKC gamma (gfp-PKC gamma), gfp-cys-1 and 2 domains (lipid binding domains of PKC gamma), confocal microscopy, and biochemical studies of PKC gamma regulation. 3) To determine how PKC gamma is decreased during galactosemia and diabetes, and how this decrease affects the gap junctions. These studies will be done in galactosemic and streptozotocin diabetic rats, will include studies on Cx43 and Cx46, and will focus on the lens cortical swelling region which is observed in the rat diabetic model. Changes in gap junction activity have been reported to occur in numerous tissues during diabetes. We have identified PKC gamma as a control point for lens gap junction activity and have determined that this isoform is decreased during diabetes and galactosemia and in human diabetic lens. PKC gamma levels can be restored to normal with an aldose reductase inhibitor, suggesting the presence of osmotic response elements in the PKC gamma gene. When PKC gamma is decreased, the control of lens gap junctions may be lost, resulting in damage to the diabetic lens cell. The control of PKC gamma in normal cells and the loss of control in diabetes are the subject of this proposal. This information will provide direction for the design of drugs to prevent the loss of PKC gamma during diabetes. The restoration of normal PKC gamma levels may prevent diabetic cataracts.