网格蛋白介导的甲状旁腺激素刺激下的钠钾ATP酶内吞作用需要α1亚基中丝氨酸11的ERK依赖性磷酸化。
Clathrin-mediated endocytosis of Na+,K+-ATPase in response to parathyroid hormone requires ERK-dependent phosphorylation of Ser-11 within the alpha1-subunit.
作者信息
Khundmiri Syed Jalal, Bertorello Alejandro M, Delamere Nicholas A, Lederer Eleanor D
机构信息
Department of Medicine, University of Louisville, Louisville, Kentucky, USA.
出版信息
J Biol Chem. 2004 Apr 23;279(17):17418-27. doi: 10.1074/jbc.M311715200. Epub 2004 Feb 19.
Parathyroid hormone (PTH) inhibits Na(+),K(+)-ATPase activity through protein kinase C- (PKC) and extracellular signal-regulated kinase- (ERK) dependent pathways and increases serine phosphorylation of the alpha(1)-subunit. To determine whether specific serine phosphorylation sites within the Na(+),K(+)-ATPase alpha(1)-subunit are involved in the Na(+),K(+)-ATPase responses to PTH, we examined the effect of PTH in opossum kidney cells stably transfected with wild type rat Na(+),K(+)-ATPase alpha(1)-subunit (WT), serine 11 to alanine mutant alpha(1)-subunit (S11A), or serine 18 to alanine mutant alpha(1)-subunit (S18A). PTH increased phosphorylation and endocytosis of the Na(+),K(+)-ATPase alpha(1)-subunit into clathrin-coated vesicles in cells transfected with WT and S18A rat Na(+),K(+)-ATPase alpha(1)-subunits. PTH did not increase the level of phosphorylation or stimulate translocation of Na(+),K(+)-ATPase alpha(1)-subunits into clathrin-coated vesicles in cells transfected with the S11A mutant. PTH inhibited ouabain-sensitive (86)Rb uptake and Na(+),K(+)-ATPase activity (ouabain-sensitive ATP hydrolysis) in WT- and S18A-transfected opossum kidney cells but not in S11A-transfected cells. Pretreatment of the cells with the PKC inhibitors and ERK inhibitor blocked PTH inhibition of (86)Rb uptake, Na(+),K(+)-ATPase activity, alpha(1)-subunit phosphorylation, and endocytosis in WT and S18A cells. Consistent with the notion that ERK phosphorylates Na(+),K(+)-ATPase alpha(1)-subunit, ERK was shown to be capable of causing phosphorylation of Na(+),K(+)-ATPase alpha(1)-subunit immunoprecipitated from WT and S18A but not from S11A-transfected cells. These results suggest that PTH regulates Na(+),K(+)-ATPase by PKC and ERK-dependent alpha(1)-subunit phosphorylation and that the phosphorylation requires the expression of a serine at the 11 position of the Na(+),K(+)-ATPase alpha(1)-subunit.
甲状旁腺激素（PTH）通过蛋白激酶C-（PKC）和细胞外信号调节激酶-（ERK）依赖的途径抑制Na(+)、K(+)-ATP酶活性，并增加α(1)-亚基的丝氨酸磷酸化。为了确定Na(+)、K(+)-ATP酶α(1)-亚基内的特定丝氨酸磷酸化位点是否参与Na(+)、K(+)-ATP酶对PTH的反应，我们研究了PTH对稳定转染野生型大鼠Na(+)、K(+)-ATP酶α(1)-亚基（WT）、丝氨酸11突变为丙氨酸的α(1)-亚基（S11A）或丝氨酸18突变为丙氨酸的α(1)-亚基（S18A）的负鼠肾细胞的影响。PTH增加了转染WT和S18A大鼠Na(+)、K(+)-ATP酶α(1)-亚基的细胞中Na(+)、K(+)-ATP酶α(1)-亚基的磷酸化和内吞作用，使其进入网格蛋白包被的小泡。PTH没有增加转染S11A突变体的细胞中Na(+)、K(+)-ATP酶α(1)-亚基的磷酸化水平，也没有刺激其向网格蛋白包被的小泡的转运。PTH抑制了WT和S18A转染的负鼠肾细胞中哇巴因敏感的(86)Rb摄取和Na(+)、K(+)-ATP酶活性（哇巴因敏感的ATP水解），但对S11A转染的细胞没有影响。用PKC抑制剂和ERK抑制剂预处理细胞可阻断PTH对WT和S18A细胞中(86)Rb摄取、Na(+)、K(+)-ATP酶活性、α(1)-亚基磷酸化和内吞作用的抑制。与ERK使Na(+)、K(+)-ATP酶α(1)-亚基磷酸化的观点一致，ERK能够使从WT和S18A转染细胞中免疫沉淀的Na(+)、K(+)-ATP酶α(1)-亚基发生磷酸化，但不能使从S11A转染细胞中免疫沉淀的该亚基发生磷酸化。这些结果表明，PTH通过PKC和ERK依赖的α(1)-亚基磷酸化来调节Na(+)、K(+)-ATP酶，并且这种磷酸化需要在Na(+)、K(+)-ATP酶α(1)-亚基的11位表达丝氨酸。
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