"Scanning mutagenesis" of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex
"Scanning mutagenesis" of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex
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DOI：
10.3389/fpls.2012.00153
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发表时间：
2012-01-01
影响因子：
5.6
通讯作者：
Thelen, Jay J.
中科院分区：
文献类型：
--
作者：
Ahsan, Nagib;Swatek, Kirby N.;Thelen, Jay J.
线粒体丙酮酸脱氢酶复合物（mtPDC）由E1 α亚基的可逆丝氨酸磷酸化通过专用的内在激酶调节。磷酸复合物在被内源性PP 2C型蛋白磷酸酶去磷酸化时被重新激活。磷酸化的Set残基的位置和侧翼氨基酸的序列都是高度保守的。我们已经使用了合成肽为基础的激酶客户端（KiC）测定加上重组丙酮酸脱氢酶热和E1 α-激酶进行“扫描诱变”的残基侧翼的磷酸化位点。与来自侧翼序列的“系统发育分析”的结果一致，基于直接肽的激酶测定耐受非常少的变化。甚至保守的改变，如Met的Leu、Ile或瓦尔，或Asp的Glu，都能使磷酸化水平显著降低。总的来说，结果表明，通过可逆磷酸化调节mtPDC是多个相互依赖的共同进化实例的一个极端例子。
The mitochondrial pyruvate dehydrogenase complex (mtPDC) is regulated by reversible seryl-phosphorylation of the E1 alpha subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated Set residue and the sequences of the flanking amino acids are highly conserved. We have used the synthetic peptide-based kinase client (KiC) assay plus recombinant pyruvate dehydrogenase Hot and E1 alpha-kinase to perform "scanning mutagenesis" of the residues flanking the site of phosphorylation. Consistent with the results from "phylogenetic analysis" of the flanking sequences, the direct peptide based kinase assays tolerated very few changes. Even conservative changes such as Leu, Ile, or Val for Met, or Glu for Asp, gave very marked reductions in phosphorylation. Overall the results indicate that regulation of the mtPDC by reversible phosphorylation is an extreme example of multiple, interdependent instances of co-evolution.