Lin Dingbo, Takemoto Dolores J
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
J Biol Chem. 2005 Apr 8;280(14):13682-93. doi: 10.1074/jbc.M407762200. Epub 2005 Jan 10.
The accumulation of reactive oxygen species (ROS, for example H2O2) is linked to several chronic pathologies, including cancer and cardiovascular and neurodegenerative diseases (Gate, L., Paul, J., Ba, G. N., Tew, K. D., and Tapiero, H. (1999) Biomed. Pharmacother. 53, 169-180). Protein kinase C (PKC) gamma is a unique isoform of PKC that is found in neuronal cells and eye tissues. This isoform is activated by ROS such as H2O2. Mutations (H101Y, G118D, S119P, and G128D) in the PKCgamma Cys-rich C1B domain caused a form of dominant non-episodic cerebellar ataxia in humans (Chen, D.-H., Brkanac, Z., Verlinde, C. L. M. J., Tan, X.-J., Bylenok, L., Nochli, D., Matsushita, M., Lipe, H., Wolff, J., Fernandez, M., Cimino, P. J., Bird, T. D., and Raskind, W. H. (2003) Am. J. Hum. Genet. 72, 839-849; van de Warrenburg, B. P. C., Verbeek, D. S., Piersma, S. J., Hennekam, F. A. M., Pearson, P. L., Knoers, N. V. A. M., Kremer, H. P. H., and Sinke, R. J. (2003) Neurology 61, 1760-1765). This could be due to a failure of the mutant PKCgamma proteins to be activated by ROS and to subsequently inhibit gap junctions. The purpose of this study was to demonstrate the cellular mechanism of activation of PKCgamma by H2O2 and the resultant effects on gap junction activity. H2O2 stimulated PKCgamma enzyme activity independently of elevations in cellular diacylglycerol, the natural PKC activator. Okadaic acid, a phosphatase inhibitor, did not affect H2O2-stimulated PKCgamma activity, indicating that dephosphorylation was not involved. The reductant, dithiothreitol, abolished the effects of H2O2, suggesting a direct oxidation of PKCgamma at the Cys-rich C1 domain. H2O2 induced the C1 domain of PKCgamma to translocate to plasma membranes, whereas the C2 domain did not. Direct effects of H2O2 on PKCgamma were demonstrated using two-dimensional SDS-PAGE. Results demonstrated that PKCgamma formed disulfide bonds in response to H2O2. H2O2-activated PKCgamma was targeted into caveolin-1- and connexin 43-containing lipid rafts, and the PKCgamma phosphorylated the connexin 43 gap junction proteins on Ser-368. This resulted in disassembly of connexin 43 gap junction plaques and decreased gap junction activity. Results suggested that H2O2 caused oxidation of the C1 domain, activation of the PKCgamma, and inhibition of gap junctions. This inhibition of gap junctions could provide a protection to cells against oxidative stress.
活性氧物质（ROS，例如H2O2）的积累与多种慢性疾病相关，包括癌症、心血管疾病和神经退行性疾病（Gate, L., Paul, J., Ba, G. N., Tew, K. D., and Tapiero, H. (1999) Biomed. Pharmacother. 53, 169 - 180）。蛋白激酶C（PKC）γ是PKC的一种独特亚型，存在于神经元细胞和眼部组织中。这种亚型可被诸如H2O2的ROS激活。PKCγ富含半胱氨酸的C1B结构域中的突变（H101Y、G118D、S119P和G128D）在人类中导致了一种显性非发作性小脑共济失调（Chen, D.-H., Brkanac, Z., Verlinde, C. L. M. J., Tan, X.-J., Bylenok, L., Nochli, D., Matsushita, M., Lipe, H., Wolff, J., Fernandez, M., Cimino, P. J., Bird, T. D., and Raskind, W. H. (2003) Am. J. Hum. Genet. 72, 839 - 849; van de Warrenburg, B. P. C., Verbeek, D. S., Piersma, S. J., Hennekam, F. A. M., Pearson, P. L., Knoers, N. V. A. M., Kremer, H. P. H., and Sinke, R. J. (2003) Neurology 61, 1760 - 1765）。这可能是由于突变的PKCγ蛋白无法被ROS激活并随后抑制缝隙连接。本研究的目的是证明H2O2激活PKCγ的细胞机制以及对缝隙连接活性的后续影响。H2O2独立于细胞二酰甘油（天然PKC激活剂）的升高刺激PKCγ酶活性。磷酸酶抑制剂冈田酸不影响H2O2刺激的PKCγ活性，表明去磷酸化不参与其中。还原剂二硫苏糖醇消除了H2O2的作用，表明在富含半胱氨酸的C1结构域对PKCγ进行了直接氧化。H2O2诱导PKCγ的C1结构域转位至质膜，而C2结构域则不然。使用二维SDS - PAGE证明了H2O2对PKCγ的直接作用。结果表明PKCγ响应H2O2形成二硫键。H2O2激活的PKCγ靶向进入含有小窝蛋白-1和连接蛋白43的脂筏，并且PKCγ使连接蛋白43缝隙连接蛋白的Ser - 368位点磷酸化。这导致连接蛋白43缝隙连接斑块的解体并降低缝隙连接活性。结果表明H2O2导致C1结构域氧化、PKCγ激活以及缝隙连接抑制。这种对缝隙连接的抑制可为细胞提供针对氧化应激的保护。
