ROLE OF PKCGAMMA AND BINDING PARTNERS IN EXCITABLE CELLS

PKC γ是传统PKC家族的四个成员之一，受钙和二酰甘油调节。与该家族的任何其他成员不同，PKC γ具有独特的表达模式，主要在脑和脊髓中表达。最近在心脏中也发现了PKC γ。这里提出的实验旨在回答两个主要问题。第一种是允许PKC γ对某些信号做出反应，而不是通过磷酸化独特的底物来响应每个信号。我们的工作模型是PKC γ在空间上被限制在其两种状态，活性和非活性。空间定位是通过与特定蛋白质结合来实现的，并且结合的亲和力由上游信号调节。为了验证这一假设，我将首先确定结合蛋白/底物的活性和非活性PKC γ通过使用酵母双杂交方法。然后，我将在天然组织中测试这种相互作用，并确定调节这种相互作用的因素。第二个问题是激活可兴奋细胞中PKC γ的生理信号是什么。在神经元电活动升高时，它是由离子通道的钙内流激活的，还是仅由后向屈光性受体调节？为了回答这些问题，我将首先设计新的荧光指示剂，这将使我能够在单细胞测定中检测PKC γ的激活。使用这些指标，我将确定PKC γ激活的空间和时间方面的可兴奋细胞在各种刺激。

PKC gamma is one of the four members of the conventional PKC family, which are regulated by calcium and diacylglycerol. Unlike any other members of the family, PKC gamma has an unique expression pattern, being expressed predominantly in brain and spinal cord. Recently PKC gamma was also found in heart. The experiments proposed here are designed to answer two main questions. The first is what allows PKC gamma to respond to some signals but not others by phosphorylating unique substrates in response to each signal. Our working model is that PKC gamma is spatially confined in both its' states, active and inactive. Spatial localization is achieved by binding to particular proteins and the affinity of the binding is regulated by upstream signals. To test this hypothesis I will first identify binding proteins/substrates for the active and inactive PKC gamma by using the yeast two hybrid approach. I will then test the interaction in the native tissue and determine the factors that modulate the interaction. The second question is what are the physiological signals that activate PKC gamma in excitable cells. Is it activated by calcium influx through ion channels during elevated electrical activity of the neurons or is it exclusively regulated through metahotropic receptors? To answer these questions I will first design novel fluorescent indicators which will allow me to detect the activation of PKC gamma in a single cell assay. Using these indicators I will determine the spatial and temporal aspects of PKC gamma activation in excitable cells in response to a variety of stimuli.