ROLE OF PKCGAMMA AND BINDING PARTNERS IN EXCITABLE CELLS

PKC-γ是传统的PKC家族的四个成员之一，受钙和二酰甘油的调节。与该家族的任何其他成员不同，PKC Gamma有一种独特的表达模式，主要在大脑和脊髓中表达。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。第一个是让PKC伽马对某些信号作出反应，而不是对其他信号作出反应，方法是使独特的底物磷酸化以响应每个信号。我们的工作模型是PKC伽马在空间上被限制在两种状态下，即活动和非活动状态。空间定位是通过与特定蛋白质结合来实现的，结合的亲和力受上游信号的调节。为了验证这一假设，我将首先使用酵母双杂交方法鉴定活性和非活性PKC伽马的结合蛋白/底物。然后，我将测试天然组织中的相互作用，并确定调节这种相互作用的因素。第二个问题是，是什么生理信号激活了可兴奋细胞中的PKC-γ。它是由神经元电活动升高时通过离子通道的钙内流激活的，还是仅通过趋化受体调节的？为了回答这些问题，我将首先设计新的荧光指示剂，使我能够在单细胞检测中检测PKC伽马的激活。使用这些指标，我将确定可兴奋细胞对各种刺激反应的PKC伽马激活的空间和时间方面。

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.