Roles of protein kinase C (PKC) in the formation of neural plasticity. -Investigation using GFP-tagged PKC

1)可诱导的脑区特异性pKC-GFP转基因小鼠的建立及神经元内PKC转位的检测在这些小鼠中，在神经元特异烯醇化酶(NSE)启动子的控制下，转基因在纹状体和小脑浦肯野细胞中有表达。而在钙调素蛋白激酶II(CaMKII)启动子控制下的目的基因在前脑(包括海马区、大脑皮层、纹状体和嗅球)可观察到γ-PKC-GFP的表达。制备小脑脑片，观察γ-PKC-GFP在活体和活体状态下的移位。γ-PKC-GFP的易位是由代谢性谷氨酸受体激动剂或局部电刺激引起的。在电刺激条件下，γ-PKCGFP沿浦肯野细胞的树突状突起生长。我们的转基因小鼠将是在蛋白水平研究神经兴奋性传播的有益工具2)PKC振荡已经知道mGluRs的激活会导致Ca^&lt；2+&gt；振荡的产生。我们发现，PKC与细胞内的Ca~(2+)和Gt~(2+)同时发生振荡易位。840位苏氨酸残基(T840)的磷酸化对Ca~(2+)和Gt；和PKC振荡的调节至关重要。3)PDK1在PKC转位中的作用。PDK1是一种磷酸化激活环苏氨酸残基的激酶，发现于AGC激酶家族，包括PKC。为了阐明PDK1在PKC易位中的作用，我们构建了突变的PKC，其激活环中的苏氨酸残基被丙氨酸取代。我们在细胞中表达了这个突变的PKC，并检测了它的易位。我们发现，与野生型PKC相比，PDK1不敏感的PKC突变体在质膜上保留的时间更长，表明PDK1影响PKC靶向机制

1) Development of inducible and brain region specific gamma-PKC-GFP transgenic mice and examination of PKC translocation in neuronsWe have developed the tet-regulated and brain region specific PKC-GFP transgenic mice. In these mice, the expression of gamma-PKC-GFP was seen in striatum and cerebellar Purkinje cells when transgene was driven under the control of neuron specific enolase (NSE) promoter. Whereas, when the target gene was driven under the control of calcium calmodulin kinase II (CaMKII) promoter, gamma-PKC-GFP expression was observed in forebrain including hippocampus, cerebral cortex, striatum and olfactory bulb. We prepared the cerebellar brain slices and observed the translocation of gamma-PKC-GFP in vivo and living states. The translocation of gamma-PKC-GFP was elicited by agonists of metabotropic glutamate receptors (mGluR) or local electronic stimulation. In case of electronic stimulation, gamma-PKCGFP was propagated along the dendritic shaft of Purkinje cells. Our transgenic mice would be a beneficial tool for investigating the propagation of neural excitability at protein levels2) PKC oscillationsIt has bee known that activation of mGluRS results in the generation of Ca^<2+> oscillation. We found that PKC was oscillatory translocated, concomitantly with intracellular Ca^<2+>. Phosphorylation of threonine residue at 840 (T840) was crucial for the regulation of Ca^<2+> and PKC oscillation3) Role of PDK1 (phosphoinositide-dependent kinase 1) in PKC translocationPDK1 is a kinase that phosphorylates the threonine residues of activation loop, found in AGC kinase family, including PKC. To elucidate the role of PDK1 in PKC translocation, we constructed mutant PKC whose threonine residue in activation loop was substituted to alanine. We expressed this mutant PKC in cells and examined its translocation. We found that PDK1-insesitive PKC mutant was retained at the plasma membrane longer than wild PKC, indicating that PDK1 affect PKC targeting mechanism