CYTOSKELETAL ASSOCIATION OF PROTEIN KINASE C

蛋白激酶C(PKC)是一种参与细胞调控的关键酶 生长分化功能和致癌作用。我们已经开发出 一个模型系统，我们可以在其中定义与PKC相关的分子事件 刺激-反应耦合具有广泛的理解意义 各种疾病过程，包括癌症。我们已经研究了PKC 佛波醇对培养大鼠垂体细胞(GH4CI)的激活作用 二丁酸酯(PDBu)模拟内源性PKC激活剂二酰甘油 (DAG)，并刺激催乳素的合成和分泌。 促甲状腺激素释放激素(TRH)，刺激肌醇脂质 代谢，提高DAG和钙离子水平。使用免疫印迹分析 利用我们的阿尔法(3型)PKC特异性单抗(MAbs)，我们有 确定TRH和PDBu导致膜结合增加 Alpha-PKC。TRH和PDBU的重新分配也可以通过以下方式查看 免疫荧光。TRH和PDBu介导的膜结合是 螯合剂和洗涤剂的稳定性表明PKC与 不溶性细胞骨架(CSK)成分。免疫荧光实验 证实α-PKC保留在洗涤剂和螯合剂提取液中 从TRH和PDBu处理的制剂，但不是静息培养。这也 提示激活的PKC与CSK相关。为了支持…… 因此，我们发现PKC的激活引起了CSK组织的重大变化。 我们的工作假设是，在静息细胞中，α-PKC是不活跃的 可溶的。PDBu或TRH激活导致与CSK相关 组件。可能是因为钙离子，增强了这种联系。CSK协会 这是由于激活将PKC放在适当的位置 CSK蛋白的磷酸化。磷酸化导致CSK的变化 与观察到的生物反应相关的组织。我们的 目的是鉴定α-PKC结合的CSK蛋白，并鉴定 CSK相关底物。我们已经确定，除了 钙敏感的PKC同工酶α和β，这些细胞表达 钙非依赖性同工酶，epsilon-PKC。PKC异构性的目的 可能是修改同工酶的激活辅因子要求，底物 特异性，或亚细胞定位。因此，我们还设计了 区分α-PKC和非α-PKC的实验 磷酸化事件，以充分确定PKC在 催乳素合成和分泌的调节。

Protein kinase C (PKC) is a key enzyme involved in regulation of cell growth and differentiated function and in carcinogenesis. We have developed a model system in which we can define molecular events associated with PKC stimulus-response coupling which has broad implications in understanding a variety of disease processes, including cancer. We have studied PKC activation in cultured rat pituitary cells (GH4CI) in which phorbol dibutyrate (PDBu) mimics the endogenous PKC activator, diacylglycerol (DAG), and stimulates synthesis and secretion of prolactin. Thyrotropin-releasing hormone (TRH), which stimulates inositol lipid metabolism, increases both DAG and Ca2+ levels. Using immunoblot analysis with our alpha- (Type 3) PKC specific monoclonal antibodies (mAbs), we have determined that TRH and PDBu cause increased membrane association of alpha-PKC. Redistribution by TRH and PDBU can also be seen by immunofluorescence. The TRH- and PDBu-mediated membrane association is chelator and detergent stable indicating that PKC is associated with insoluble cytoskeletal (CSK) components. Immunofluorescence experiments verified that alpha-PKC was retained in detergent- and chelator-extracted preparations from TRH- and PDBu-treated, but not resting cultures.This also suggest that activated PKC is associated with the CSK. In support of this,we found that PKC activation causes major changes in CSK organization. Our working hypothesis is that in resting cells, alpha-PKC is inactive and soluble. Activation with PDBu or TRH leads to association with CSK components. Possibly, Ca2+, enhances this association. The CSK association that occurs with activation places PKC in the appropriate place for phosphorylation of CSK proteins. Phosphorylation leads to changes in CSK organization which are associated with observed biological responses. Our aims are to identify the CSK proteins to which alpha-PKC binds and identify the CSK-associated substrates. We have determined that in addition to the calcium-sensitive PKC isozymes alpha- and beta-, these cells express the calcium-independent isozyme, epsilon-PKC. The purpose of PKC heterogeneity may be to modify isozyme cofactor requirements for activation, substrate specificity, or subcellular localization. Therefore, we have also designed experiments to distinguish between alpha- and non-alpha-PKC mediated phosphorylation events in order to fully define the role of PKC in regulation of prolactin synthesis and secretion.