Chibalin A V, Pedemonte C H, Katz A I, Féraille E, Berggren P O, Bertorello A M
Department of Molecular Medicine, Karolinska Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden.
J Biol Chem. 1998 Apr 10;273(15):8814-9. doi: 10.1074/jbc.273.15.8814.
Inhibition of Na+,K+-ATPase activity by dopamine is an important mechanism by which renal tubules modulate urine sodium excretion during a high salt diet. However, the molecular mechanisms of this regulation are not clearly understood. Inhibition of Na+,K+-ATPase activity in response to dopamine is associated with endocytosis of its alpha- and beta-subunits, an effect that is protein kinase C-dependent. In this study we used isolated proximal tubule cells and a cell line derived from opossum kidney and demonstrate that dopamine-induced endocytosis of Na+,K+-ATPase and inhibition of its activity were accompanied by phosphorylation of the alpha-subunit. Inhibition of both the enzyme activity and its phosphorylation were blocked by the protein kinase C inhibitor bisindolylmaleimide. The early time dependence of these processes suggests a causal link between phosphorylation and inhibition of enzyme activity. However, after 10 min of dopamine incubation, the alpha-subunit was no longer phosphorylated, whereas enzyme activity remained inhibited due to its removal from the plasma membrane. Dephosphorylation occurred in the late endosomal compartment. To further examine whether phosphorylation was a prerequisite for subunit endocytosis, we used the opossum kidney cell line transfected with the rodent alpha-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the alpha-subunit with a concomitant decrease in Na+,K+-ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent alpha-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser11 and Ser18). Our results support the hypothesis that protein kinase C-dependent phosphorylation of the alpha-subunit is essential for Na+,K+-ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.
多巴胺对钠钾ATP酶活性的抑制作用是肾小管在高盐饮食期间调节尿钠排泄的重要机制。然而，这种调节的分子机制尚不清楚。多巴胺诱导的钠钾ATP酶活性抑制与其α和β亚基的内吞作用有关，这一效应依赖于蛋白激酶C。在本研究中，我们使用分离的近端小管细胞和源自负鼠肾的细胞系，证明多巴胺诱导的钠钾ATP酶内吞作用及其活性抑制伴随着α亚基的磷酸化。蛋白激酶C抑制剂双吲哚马来酰胺可阻断酶活性及其磷酸化。这些过程的早期时间依赖性表明磷酸化与酶活性抑制之间存在因果关系。然而，多巴胺孵育10分钟后，α亚基不再磷酸化，而酶活性由于其从质膜上移除而仍然受到抑制。去磷酸化发生在内体晚期区室。为了进一步研究磷酸化是否是亚基内吞作用的先决条件，我们使用了转染了啮齿动物α亚基cDNA的负鼠肾细胞系。用多巴胺处理该细胞系导致α亚基的磷酸化和内吞作用，同时钠钾ATP酶活性降低。相比之下，在转染了缺乏假定蛋白激酶C磷酸化位点（Ser11和Ser18）的啮齿动物α亚基的细胞中未观察到这些效应。我们的结果支持以下假设：α亚基的蛋白激酶C依赖性磷酸化对于钠钾ATP酶内吞作用至关重要，并且这两个事件都导致了多巴胺引起的酶活性降低。
