Waldron Richard T, Rey Osvaldo, Zhukova Elena, Rozengurt Enrique
Division of Digestive Diseases, Department of Medicine, UCLA David Geffen School of Medicine, UCLA-CURE Digestive Diseases Research Center and Molecular Biology Institute, UCLA, Los Angeles, California 90095-1786, USA.
J Biol Chem. 2004 Jun 25;279(26):27482-93. doi: 10.1074/jbc.M402875200. Epub 2004 Apr 14.
Oxidative stress induced by cell treatments with H(2)O(2) activates protein kinase D (PKD) via a protein kinase C (PKC)-dependent signal transduction pathway (Waldron, R. T., and Rozengurt, E. (2000) J. Biol. Chem. 275, 17114-17121). Here we show that oxidative stress induces PKC-dependent activation loop Ser(744) and Ser(748) phosphorylation to mediate dose- and time-dependent activation of PKD, both endogenously expressed in Swiss 3T3 cells and stably overexpressed in Swiss 3T3-GFP.PKD cells. Although oxidative stress induced PKD activation loop phosphorylation and activation with identical kinetics, both were dose-dependently blocked by preincubation of cells with selective inhibitors of PKC (GF109203X and Gö6983) or c-Src (PP2). Inhibition of Src tyrosine kinase activity eliminated oxidative stress-induced direct PKD tyrosine phosphorylation, but only partially attenuated activation loop phosphorylation and activation. Mutation of a putative tyrosine phosphorylation site on PKD, Tyr(469) to phenylalanine, had no effect on its activation by oxidative stress in transfected COS-7 cells. Similarly, a mutant with Tyr(469) replaced by aspartic acid had increased basal activity but was also further activated by oxidative stress. Thus, PKD tyrosine phosphorylation at this site neither produced full activation by itself nor was required for oxidative stress-induced activation mediated by activation loop phosphorylation. In addition to PKD activation, activation loop phosphorylation in response to oxidative stress also redistributed activated PKD to cell nuclei, as revealed by PKD indirect immunofluorescence, imaging of a PKD-green fluorescent protein fusion construct (GFP-PKD), and analysis of nuclear pellets. Cell preincubation with Gö6983 strongly diminished H(2)O(2)-induced nuclear relocalization of GFP-PKD. Taken together, these results indicate that PKC-mediated PKD Ser(744) and Ser(748) phosphorylation induced by oxidative stress integrates PKD activation with redistribution to the nucleus.
用H₂O₂处理细胞所诱导的氧化应激通过蛋白激酶C（PKC）依赖性信号转导途径激活蛋白激酶D（PKD）（Waldron，R.T.，和Rozengurt，E.（2000）《生物化学杂志》275，17114 - 17121）。在此我们表明，氧化应激诱导PKC依赖性的激活环Ser⁷⁴⁴和Ser⁷⁴⁸磷酸化，以介导PKD的剂量和时间依赖性激活，PKD在瑞士3T3细胞中内源性表达，并在瑞士3T3 - GFP.PKD细胞中稳定过表达。尽管氧化应激以相同的动力学诱导PKD激活环磷酸化和激活，但两者均被用PKC（GF109203X和Gö6983）或c - Src（PP2）的选择性抑制剂预孵育细胞而剂量依赖性地阻断。Src酪氨酸激酶活性的抑制消除了氧化应激诱导的直接PKD酪氨酸磷酸化，但仅部分减弱激活环磷酸化和激活。在转染的COS - 7细胞中，将PKD上一个假定的酪氨酸磷酸化位点Tyr⁴⁶⁹突变为苯丙氨酸对其由氧化应激诱导的激活没有影响。同样，一个将Tyr⁴⁶⁹替换为天冬氨酸的突变体具有增加的基础活性，但也被氧化应激进一步激活。因此，该位点的PKD酪氨酸磷酸化自身既不产生完全激活，也不是氧化应激诱导的由激活环磷酸化介导的激活所必需的。除了PKD激活外，氧化应激诱导的激活环磷酸化还将激活的PKD重新分布到细胞核，这通过PKD间接免疫荧光、PKD - 绿色荧光蛋白融合构建体（GFP - PKD）的成像以及核沉淀分析得以揭示。用Gö6983预孵育细胞强烈减少了H₂O₂诱导的GFP - PKD的核重新定位。综上所述，这些结果表明氧化应激诱导的PKC介导的PKD Ser⁷⁴⁴和Ser⁷⁴⁸磷酸化将PKD激活与重新分布到细胞核整合在一起。
