| Co-Investigator(Kenkyū-buntansha) |
SEKI Takahiro Hiroshima University, Graduate School of Biomedical Sciences, Research Associate, 大学院・医歯薬学総合研究科, 助手 (50335650)
AMANO Taku Hiroshima University, Graduate School of Biomedical Sciences, Assistant Professor, 大学院・医歯薬学総合研究科, 講師 (10294547)
MATSUBAYASHI Hiroaki Hiroshima University, Graduate School of Biomedical Sciences, Associate Professor, 大学院・医歯薬学総合研究科, 助教授 (60165850)
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| Research Abstract |
1) Development of inducible and brain region specific gamma-PKC-GFP transgenic mice and examination of PKC translocation in neurons
We 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 levels
2) PKC oscillations
It 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 oscillation
3) Role of PDK1 (phosphoinositide-dependent kinase 1) in PKC translocation
PDK1 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
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