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首页> 外文期刊>Biochemistry >Intrinsic Protein Kinase Activity in Mitochondrial Oxidative Phosphorylation Complexes
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Intrinsic Protein Kinase Activity in Mitochondrial Oxidative Phosphorylation Complexes

机译：线粒体氧化磷酸化复合物中的内在蛋白激酶活性

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Mitochondrial protein phosphorylation is a well-recognizedmetabolic controlmechanism,nwith the classical example of pyruvate dehydrogenase (PDH) regulation by specific kinases andnphosphatases of bacterial origin. However, despite the growing number of reported mitochondrialnphosphoproteins, the identity of the protein kinases mediating these phosphorylation events remainsnlargely unknown. The detection of mitochondrial protein kinases is complicated by the low concentra-ntion of kinase relative to that of the target protein, the lack of specific antibodies, and contamination fromnassociated, but nonmatrix, proteins. In this study, we use blue native gel electrophoresis (BN-PAGE) tonisolate rat and porcine heart mitochondrial complexes for screening of protein kinase activity. To detectnkinase activity, one-dimensional BN-PAGE gels were exposed to [γ-n32nP]ATP and then followed bynsodium dodecyl sulfate gel electrophoresis. Dozens of mitochondrial proteins were labeled with 32nPinnthis setting, including all five complexes of oxidative phosphorylation and several citric acid cyclenenzymes. The nearly ubiquitousn32nP protein labeling demonstrates protein kinase activity within eachnmitochondrial protein complex. The validity of this two-dimensional BN-PAGE method was demon-nstrated by detecting the known PDH kinases and phosphatases within the PDH complex band using Western blots and massnspectrometry. Surprisingly, these same approaches detected only a few additional conventional protein kinases, suggesting a majornrole for autophosphorylation in mitochondrial proteins. Studies on purified Complex V and creatine kinase confirmed that thesenproteins undergo autophosphorylation and, to a lesser degree, tenaciousn32nP-metabolite association. In-gel Complex IV activity wasnshown to be inhibited by ATP, and partially reversed by phosphatase activity, consistent with an inhibitory role for proteinnphosphorylation in this complex. Collectively, this study proposes that many of the mitochondrial complexes contain annautophosphorylation mechanism, which may play a functional role in the regulation of these multiprotein units
机译：线粒体蛋白磷酸化是公认的代谢控制机制，而经典的例子是丙酮酸脱氢酶（PDH）通过特定的激酶和细菌来源的磷酸酶调节。然而，尽管报道的线粒体磷酸蛋白的数量不断增加，但是介导这些磷酸化事件的蛋白激酶的身份仍然未知。线粒体蛋白激酶的检测相对于靶蛋白而言浓度低，缺乏特异性抗体以及游离的但非基质的蛋白造成的污染使该过程变得复杂。在这项研究中，我们使用蓝色天然凝胶电泳（BN-PAGE）分离大鼠和猪心脏线粒体复合物，以筛选蛋白激酶活性。为了检测激酶活性，将一维BN-PAGE凝胶暴露于[γ-n32nP] ATP，然后进行十二烷基硫酸钠硫酸钠凝胶电泳。用32nPinn此设置标记了数十个线粒体蛋白，包括所有五个氧化磷酸化复合物和几种柠檬酸循环酶。几乎普遍存在的n32nP蛋白标记证明了每个线粒体蛋白复合物中的蛋白激酶活性。这种二维BN-PAGE方法的有效性通过使用Western印迹和质谱法检测PDH复合带中已知的PDH激酶和磷酸酶来证明。令人惊讶的是，这些相同的方法仅检测到一些其他常规蛋白激酶，这表明线粒体蛋白中自磷酸化的主要成分。对纯化的复合物V和肌酸激酶的研究证实，这些蛋白进行自磷酸化，并在较小程度上发生了tenaciousn32nP-代谢物缔合。凝胶内复合物IV活性未显示受ATP抑制，而磷酸酶活性部分逆转，这与该复合物中蛋白磷酸化的抑制作用一致。总的来说，这项研究表明，许多线粒体复合物中都含有非自磷酸化机制，这可能在这些多蛋白单元的调控中发挥功能性作用。

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  • 来源
    《Biochemistry》 |2011年第13期|p.2515-2529|共15页
  • 作者

    Darci Phillips Angel M. Aponte Raul Covian and Robert S. Balaban;

  • 作者单位

    †Laboratory of Cardiac Energetics and ‡Proteomics Core Facility, National Heart, Lung and Blood Institute, National Institutes ofHealth, Department of Health and Human Services, Bethesda, Maryland 20892, United States;

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