Foley Timothy D, Armstrong John J, Kupchak Brian R
Department of Chemistry, University of Scranton, Scranton, PA 18510, USA.
Biochem Biophys Res Commun. 2004 Mar 12;315(3):568-74. doi: 10.1016/j.bbrc.2004.01.096.
The present study examined in subcellular fractions from rat brain the nature and sensitivity to hydrogen peroxide of constitutively expressed mitogen-activated protein kinase (MAPK) phosphatase activity. MAPK phosphatase activity was defined as the activity directed towards a dual-phosphorylated (pT/pY) peptide corresponding to the activation domain of the extracellular-regulated kinase (ERK) subtype of the MAPKs. The use of phosphatase inhibitors and biochemical analyses demonstrate that the MAPK phosphatase activity, which was highest in the microsomal membrane and soluble fractions, was attributable mainly, if not entirely, to protein phosphatase 2A (PP2A). Moreover, hydrogen peroxide (in the absence and presence of reduced glutathione) and glutathione disulfide inhibited the MAPK phosphatase activity by a dithiothreitol-reversible mechanism. These results provide direct support for mounting evidence that PP2A is a major regulator of MAPK phosphorylation in brain and suggest that inhibition of PP2A activity via reversible oxidation of a cysteine thiol(s) may underlie at least in part the activation of MAPKs occurring in response to hydrogen peroxide and oxidative stress.
本研究在大鼠脑亚细胞组分中检测了组成型表达的丝裂原活化蛋白激酶（MAPK）磷酸酶活性的性质及其对过氧化氢的敏感性。MAPK磷酸酶活性定义为针对与MAPKs的细胞外调节激酶（ERK）亚型激活域相对应的双磷酸化（pT/pY）肽的活性。磷酸酶抑制剂的使用和生化分析表明，在微粒体膜和可溶性组分中活性最高的MAPK磷酸酶活性主要（如果不是完全）归因于蛋白磷酸酶2A（PP2A）。此外，过氧化氢（在存在和不存在还原型谷胱甘肽的情况下）和谷胱甘肽二硫化物通过二硫苏糖醇可逆机制抑制MAPK磷酸酶活性。这些结果为越来越多的证据提供了直接支持，即PP2A是脑中MAPK磷酸化的主要调节因子，并表明通过半胱氨酸硫醇的可逆氧化抑制PP2A活性可能至少部分是响应过氧化氢和氧化应激而发生的MAPKs激活的基础。
