Fialkow L, Chan C K, Rotin D, Grinstein S, Downey G P
Department of Medicine, University of Toronto, Ontario, Canada.
J Biol Chem. 1994 Dec 9;269(49):31234-42.
In addition to their role in bacterial killing, reactive oxygen intermediates (ROI) produced by the NADPH oxidase may participate in the regulation of intracellular pathways. We have recently demonstrated that ROI produced by the oxidase regulate tyrosine phosphorylation in neutrophils, possibly by alterations in the cellular redox state. The purpose of the present study was to characterize the identities of certain of the redox-sensitive tyrosine-phosphorylated substrates and the significance of the increased phosphorylation. As a prominent 42-44-kDa phosphorylated band was noted in oxidant-treated cells, we investigated the possible phosphorylation and activation of mitogen-activated protein (MAP) kinase under these conditions. Immunoprecipitation of MAP kinase followed by immunoblotting with anti-phosphotyrosine antibodies indicated that a 42-44-kDa polypeptide was tyrosine-phosphorylated in response to treatment of cells, either with the oxidizing agent diamide or with H2O2 in cells where catalase was inhibited. Using an in vitro renaturation assay with myelin basic protein as the substrate, oxidant-induced stimulation of kinase activity of a 42-44-kDa band was observed in both whole cell extracts and in MAP kinase immunoprecipitates. The mechanism of redox-sensitive activation of MAP kinase was examined. First, exposure of cells to oxidants caused a significant increase in the activity of MEK (the putative activator of MAP kinase), as determined by an in vitro kinase assay using recombinant catalytically inactive glutathione S-transferase-MAP kinase as the substrate. Additionally, oxidant treatment of cells resulted in inhibition of the activity of CD45, a protein tyrosine phosphatase known to dephosphorylate and inactivate MAP kinase. We conclude that oxidant treatment of neutrophils can activate MAP kinase by stimulating its tyrosine and (presumably) threonine phosphorylation via MEK activation, a response that may be potentiated by inhibition of MAP kinase dephosphorylation by phosphatases such as CD45.
除了在细菌杀伤中的作用外，NADPH氧化酶产生的活性氧中间体（ROI）可能参与细胞内信号通路的调节。我们最近证明，氧化酶产生的ROI可能通过改变细胞氧化还原状态来调节中性粒细胞中的酪氨酸磷酸化。本研究的目的是确定某些氧化还原敏感的酪氨酸磷酸化底物的身份以及磷酸化增加的意义。由于在氧化剂处理的细胞中观察到一条突出的42 - 44 kDa磷酸化条带，我们研究了在这些条件下丝裂原活化蛋白（MAP）激酶的可能磷酸化和激活情况。用抗磷酸酪氨酸抗体进行免疫印迹，对MAP激酶进行免疫沉淀，结果表明，在用氧化剂二酰胺处理细胞或在过氧化氢酶被抑制的细胞中用H2O2处理后，一条42 - 44 kDa的多肽发生了酪氨酸磷酸化。使用髓鞘碱性蛋白作为底物的体外复性试验，在全细胞提取物和MAP激酶免疫沉淀物中均观察到氧化剂诱导的42 - 44 kDa条带激酶活性的刺激。我们研究了MAP激酶氧化还原敏感激活的机制。首先，通过使用重组无催化活性的谷胱甘肽S - 转移酶 - MAP激酶作为底物的体外激酶试验确定，细胞暴露于氧化剂会导致MEK（MAP激酶的假定激活剂）活性显著增加。此外，细胞的氧化剂处理导致CD45活性受到抑制，CD45是一种已知可使MAP激酶去磷酸化并使其失活的蛋白酪氨酸磷酸酶。我们得出结论，对中性粒细胞进行氧化剂处理可通过MEK激活刺激其酪氨酸和（可能）苏氨酸磷酸化来激活MAP激酶，这种反应可能因诸如CD45等磷酸酶对MAP激酶去磷酸化的抑制而增强。
