Site specificity of four pyruvate dehydrogenase kinase isoenzymes toward the three phosphorylation sites of human pyruvate dehydrogenase
Site specificity of four pyruvate dehydrogenase kinase isoenzymes toward the three phosphorylation sites of human pyruvate dehydrogenase
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DOI：
10.1074/jbc.m103069200
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发表时间：
2001-10-05
影响因子：
4.8
通讯作者：
Patel, MS
中科院分区：
文献类型：
--
作者：
Korotchkina, LG;Patel, MS
哺乳动物丙酮酸脱氢酶复合物的活性通过丙酮酸脱氢酶（EI）组分的α亚基的三个特定丝氨酸残基（位点1，Ser-264;位点2，Ser-271;位点3，Ser-203）的磷酸化-去磷酸化来调节。磷酸化由四种丙酮酸脱氢酶激酶（PDK）同工酶进行。四种哺乳动物PDKs对E1的三个磷酸化位点的特异性进行了研究，使用重组E1突变蛋白，只有一个功能磷酸化位点存在。然而，所有四种PDK在磷酸盐缓冲液中以不同的速率磷酸化位点1和位点2（对于位点1，PDK 2> PDK 4接近于PDK 1> PDK 3;对于位点2，PDK 3> PDK 4> PDK 2> PDK 1）。位点3仅被PDK 1磷酸化。PDK 3证明了二氢硫辛酰胺乙酰转移酶的最大激活作用。在游离形式中，所有PDK磷酸化位点1，并且PDK 4对位点2具有最高活性。四种PDK的活性通过二氢硫辛酰胺乙酰转移酶的硫辛酰基部分的还原和乙酰化状态在不同程度上刺激，其中PDK 2的刺激最大。取代位点I丝氨酸与谷氨酸，这模仿磷酸化依赖性失活的El，并没有影响磷酸化位点2的四个PDKs和位点3的PDK 1。具有独特组织分布的四种PDK磷酸化的位点特异性可能有助于丙酮酸脱氢酶复合物在正常和病理生理状态下的组织特异性调节。
Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation-dephosphorylation of three specific serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the alpha subunit of the pyruvate dehydrogenase (EI) component. Phosphorylation is carried out by four pyruvate dehydrogenase kinase (PDK) isoenzymes. Specificity of the four mammalian PDKs toward the three phosphorylation sites of E1 was investigated using the recombinant E1 mutant proteins with only one functional phosphorylation site present. All four PDKs phosphorylated site 1 and site 2, however, with different rates in phosphate buffer (for site 1, PDK2 > PDK4 approximate to PDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1). Site 3 was phosphorylated by PDK1 only. The maximum activation by dihydrolipoamide acetyltransferase was demonstrated by PDK3. In the free form, all PDKs phosphorylated site 1, and PDK4 had the highest activity toward site 2. The activity of the four PDKs was stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2. Substitution of the site I serine with glutamate, which mimics phosphorylation-dependent inactivation of El, did not affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1. Site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states.