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

1)诱导型和脑区特异性γ -PKC- gfp转基因小鼠的建立及神经元中PKC易位的检测我们已经建立了受基因调控的脑区特异性PKC- gfp转基因小鼠。在这些小鼠中，在神经元特异性烯醇酶（NSE）启动子控制下驱动转基因时，纹状体和小脑浦肯野细胞中可见γ - pkc - gfp的表达。而当目的基因在CaMKII启动子的控制下驱动时，在前脑包括海马、大脑皮层、纹状体和嗅球中观察到γ - pkc - gfp的表达。我们制备了小脑切片，观察了γ - pkc - gfp在体内和活体状态下的易位。γ - pkc - gfp的易位可由代谢性谷氨酸受体（mGluR）激动剂或局部电刺激引起。在电刺激下，γ - pkcgfp沿着浦肯野细胞的树突轴增殖。我们的转基因小鼠将成为研究神经兴奋性在蛋白水平上的传播的有益工具2)PKC振荡已知mGluRS的激活会导致Ca^<2+>振荡的产生。我们发现PKC是振荡易位的，伴随着细胞内Ca^<2+>。840 （T840）苏氨酸残基的磷酸化对于Ca^<2+>和PKC振荡的调节至关重要3)PDK1（磷酸肌苷依赖性激酶1）在PKC易位中的作用PDK1是一种磷酸化激活环苏氨酸残基的激酶，在AGC激酶家族中发现，包括PKC。为了阐明PDK1在PKC易位中的作用，我们构建了激活环中苏氨酸残基被丙氨酸取代的PKC突变体。我们在细胞中表达了这种PKC突变体，并检查了它的易位。我们发现PDK1不敏感的PKC突变体比野生PKC在质膜上保留的时间更长，这表明PDK1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