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

1)诱导型脑区特异性γ-PKC-GFP转基因小鼠的建立及神经元PKC转位的检测我们建立了tet调控的脑区特异性γ-PKC-GFP转基因小鼠。在这些小鼠中，当在神经元特异性烯醇化酶（NSE）启动子的控制下驱动转基因时，在纹状体和小脑浦肯野细胞中观察到γ-PKC-GFP的表达。而当目的基因在钙调蛋白激酶II（CaMK II）启动子的调控下表达时，γ-PKC-GFP在前脑（包括海马、大脑皮质、纹状体和嗅球）中均有表达。制备小脑脑片，观察γ-PKC-GFP在体和活体的转位情况。γ-PKC-GFP的易位由代谢型谷氨酸受体（mGluR）的激动剂或局部电刺激引起。在电刺激的情况下，γ-PKCGFP沿着浦肯野细胞的树突状轴增殖。我们的转基因小鼠将是在蛋白质水平上研究神经兴奋性传播的有益工具。2）PKC振荡已知mGluRS的激活导致Ca^2+振荡的产生。我们发现，PKC是振荡移位的，伴随着细胞内Ca^<2+>。磷酸肌醇依赖性激酶1（phosphoinositide-dependent kinase 1，PDK 1）在蛋白激酶C（PKC）转位中的作用磷酸肌醇依赖性激酶1（phosphoinositide-dependent kinase 1，PDK 1）是一种磷酸化激活环中苏氨酸残基的激酶，存在于AGC激酶家族，包括蛋白激酶C（PKC）。为了阐明PDK 1在PKC转位中的作用，我们构建了突变型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种突变的PKC，并检测了它的易位。我们发现PDK 1不敏感的突变型PKC在细胞膜上的滞留时间比野生型PKC长，表明PDK 1影响PKC的靶向机制

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