K depletion increases protein tyrosine kinase-mediated phosphorylation of ROMK.
K depletion increases protein tyrosine kinase-mediated phosphorylation of ROMK.
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K 耗尽会增加蛋白酪氨酸激酶介导的 ROMK 磷酸化。
DOI：
10.1152/ajprenal.00160.2002
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Wang,Wen-Hui
中科院分区：
文献类型：
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作者：
Lin,Dao-Hong;Sterling,Hyacinth;Lerea,KennethM;Welling,Paul;Jin,Lianhong;Giebisch,Gerhard;Wang,Wen-Hui
我们纯化了His标记的ROMK1，并用32P标记的ATP进行了体外磷酸化实验，以确定ROMK1蛋白是否是PTK的底物。在纯化的ROMK1蛋白中加入活性c-Src和[32P]ATP可导致ROMK1蛋白的磷酸化。然而，c-Src不能磷酸化R1Y337A，在R1Y337A中，酪氨酸残基337位突变为丙氨酸。此外，磷酸肽图谱从胰酶消化的ROMK1蛋白中鉴定出两个磷酸肽。相反，在胰酶消化的R1Y337A蛋白中没有发现磷酸化肽。这表明两个磷酸化的多肽可能含有相同的酪氨酸残基。此外，c-Src和[32P]ATP的加入使与ROMK1的COOH末端的氨基酸序列333-362相对应的合成肽磷酸化。然后，我们研究了饮食中钾摄入量对酪氨酸磷酸化ROMK水平的影响。虽然用ROMK抗体免疫沉淀的ROMK通道与缺K和高K饲料大鼠的ROMK通道相同，但低K饲料大鼠被PTK磷酸化的ROMK通道多于高K饲料大鼠。我们的结论是，ROMK1可以被PTK磷酸化，酪氨酸残基337是磷酸化的关键位点。此外，ROMK的酪氨酸磷酸化也受到饮食中钾摄入量的调节。这有力地表明，PTK是调节肾脏K分泌的不依赖于醛固酮的信号转导通路的重要成员。
We purified His-tagged ROMK1 and carried out in vitro phosphorylation assays with32P-radiolabeled ATP to determine whether ROMK1 protein is a substrate for PTK. Addition of active c-Src and [32P]ATP to the purified ROMK1 protein resulted in the phosphorylation of the ROMK1 protein. However, c-Src did not phosphorylate R1Y337A in which tyrosine residue 337 was mutated to alanine. Furthermore, phosphopeptide mapping identified two phosphopeptides from the trypsin-digested ROMK1 protein. In contrast, no phosphorylated peptide has been found in the trypsin-digested R1Y337A protein. This suggested that two phosphorylated peptides might contain the same tyrosine residue. Also, addition of c-Src and [32P]ATP phosphorylated the synthesized peptide corresponding to amino acid sequence 333–362 of the COOH terminus of ROMK1. We then examined the effect of dietary K intake on the tyrosine-phosphorylated ROMK level. Although the ROMK channels pulled down by immunoprecipitation with ROMK antibody were the same from rats on a K-deficient diet or on a high-K diet, more ROMK channels were phosphorylated by PTK in rats on a K-deficient diet than those on a high-K diet. We conclude that ROMK1 can be phosphorylated by PTK and that tyrosine residue 337 is the key site for the phosphorylation. Also, the tyrosine phosphorylation of ROMK is modulated by dietary K intake. This strongly suggests that PTK is an important member of the aldosterone-independent signal transduction pathway for regulating renal K secretion.