CYTOSKELETAL ASSOCIATION OF PROTEIN KINASE C

蛋白激酶C（PKC）是参与细胞增殖和凋亡调控的关键酶， 生长和分化功能以及致癌作用。我们已经开发 一个模型系统，我们可以定义与PKC相关的分子事件， 刺激-反应耦合，这对理解 包括癌症在内的各种疾病过程。我们研究了PKC 激活培养的大鼠垂体细胞（GH4 CI），其中佛波醇 二丁酸酯（PDBu）模拟内源性PKC激活剂二酰基甘油 (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，增加DAG和Ca 2+水平。使用免疫印迹分析 使用我们的α-（3型）PKC特异性单克隆抗体（mAb），我们 确定TRH和PDBu导致细胞膜结合增加， α-PKC TRH和PDBU的再分布也可以通过以下方式观察到： 免疫荧光TRH和PDBu介导的膜结合是 螯合剂和去污剂稳定，表明PKC与 不溶性细胞骨架（CSK）成分。免疫荧光实验 证实了α-PKC保留在去污剂和螯合剂提取的 制备TRH和PDBu-treated，但不是休息培养。这也 提示激活的PKC与CSK有关。支持 因此，我们发现PKC激活引起CSK组织的重大变化。 我们的工作假设是，在静息细胞中，α-PKC是无活性的， 可溶的PDBu或TRH激活导致与CSK相关 件. Ca 2+可能增强了这种关联。CSK协会 激活时发生的这种情况将PKC置于适当的位置， 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 目的是鉴定与α-PKC结合的CSK蛋白， CSK相关底物。我们已经确定，除了 钙敏感性PKC同工酶α和β，这些细胞表达 钙非依赖性同工酶，ε-PKC。PKC异质性的目的 可能是为了修饰同工酶辅因子的活化要求，底物 特异性或亚细胞定位。因此，我们还设计了 区分α-和非α-PKC介导的 磷酸化事件，以充分确定PKC在 调节催乳素的合成和分泌。

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.