Vasilets L A, Postina R, Kirichenko S N
Max-Planck-Institute for Biophysics, Frankfurt/Main, Germany.
FEBS Lett. 1999 Jul 16;455(1-2):8-12. doi: 10.1016/s0014-5793(99)00851-0.
The Na+/K+-ATPase is a target protein for protein kinase C (PKC). The PKC-mediated phosphorylation of the rat alpha1 subunit at Ser-23 results in the inhibition of its transport function. To understand the molecular basis of the inhibition by PKC, the Ser-23 in the rat alpha1 subunit has been replaced by negatively (Asp, Glu) or positively (Lys) charged, or uncharged (Gln, Ala) residues, and the mutants were expressed in Xenopus oocytes. Ouabain-specific 86Rb uptake and pump-generated current as well as sensitivity to ouabain and to external K+ have been investigated. When Ser-23 was replaced by the negatively charged residues, transport function was inhibited, and simultaneously synthesis of the alpha subunits was enhanced. In addition, if Ser-23 was substituted by Glu, the K(I) value for inhibition of transport by ouabain was drastically increased from 46.5 microM to 1.05 mM. The data suggest that insertion of a negative charge within the N-terminus of alpha subunit of the Na+/K+-ATPase due to phosphorylation of Ser-23 plays an important role in the PKC-mediated inhibition of transport function.
钠钾ATP酶是蛋白激酶C（PKC）的靶蛋白。PKC介导的大鼠α1亚基在丝氨酸23位点的磷酸化会抑制其转运功能。为了解PKC抑制作用的分子基础，大鼠α1亚基中的丝氨酸23已被带负电荷（天冬氨酸、谷氨酸）、正电荷（赖氨酸）或不带电荷（谷氨酰胺、丙氨酸）的残基取代，并且这些突变体在非洲爪蟾卵母细胞中表达。已研究了哇巴因特异性的86Rb摄取、泵产生的电流以及对哇巴因和细胞外钾离子的敏感性。当丝氨酸23被带负电荷的残基取代时，转运功能受到抑制，同时α亚基的合成增强。此外，如果丝氨酸23被谷氨酸取代，哇巴因抑制转运的K（I）值从46.5微摩尔急剧增加到1.05毫摩尔。数据表明，由于丝氨酸23的磷酸化，在钠钾ATP酶α亚基的N端插入一个负电荷在PKC介导的转运功能抑制中起重要作用。
