Protein Kinase C Gamma in The Lens

描述（由申请人提供）：蛋白激酶C γ（PKC γ）是一种 透镜上皮和皮质中PKC的主要亚型。PKC磷酸化 透镜间隙连接蛋白，Cx43，引起间隙连接的抑制 活动PKC γ在半乳糖血症期间特异性降低， 链脲佐菌素糖尿病大鼠和人糖尿病透镜中。这种PKC的减少 γ将导致间隙连接活性的增加。具体目标 本研究的主要目的是：1）确定PKC γ是如何在正常人中被控制的， 透镜上皮细胞通过EGF，并通过易位，下调，周转， 以及响应于脂质和钙的表达。2)以确定如何 PKC γ的控制通过Cx43的变化改变间隙连接活性 磷酸化、间隙连接活性和间隙连接组装。具体 目标1和2将利用培养物中的透镜细胞，过表达系统，绿色 荧光蛋白-PKC γ（gfp-PKC γ），gfp-cys-1和2结构域（脂质 PKC γ的结合结构域）、共聚焦显微镜和生化研究 PKC γ调节。3)为了确定PKC γ是如何在 半乳糖血症和糖尿病，以及这种减少如何影响差距连接。 这些研究将在半乳糖血症和链脲佐菌素糖尿病大鼠中进行， 将包括对Cx43和Cx46的研究，并将重点放在透镜皮质上 在大鼠糖尿病模型中观察到的肿胀区域。差距变化 据报道，连接活性发生在许多组织中， 糖尿病我们已经确定PKC γ作为透镜间隙连接的控制点 活性，并已确定这种亚型在糖尿病期间减少， 半乳糖血症和人糖尿病透镜中。PKC γ水平可以恢复到 正常与醛糖还原酶抑制剂，表明存在渗透压 PKC γ基因中的反应元件。当PKC γ降低时， 透镜缝隙连接的控制可能会丢失，导致糖尿病患者的损害 透镜盒。PKC γ在正常细胞中的控制和PKC γ在正常细胞中的失控， 糖尿病是这项提案的主题。这些信息将提供 药物设计的方向，以防止PKC γ的损失， 糖尿病恢复正常的PKC γ水平可能会防止糖尿病 白内障

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.