Logvinenko N S, Dulubova I, Fedosova N, Larsson S H, Nairn A C, Esmann M, Greengard P, Aperia A
Department of Woman and Child Health, Karolinska Institute, Stockholm, Sweden.
Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):9132-7. doi: 10.1073/pnas.93.17.9132.
Phosphorylation of the alpha-1 subunit of rat Na+,K(+)-ATPase by protein kinase C has been shown previously to decrease the activity of the enzyme in vitro. We have now undertaken an investigation of the mechanism by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains of the protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosphorylated the alpha-1 subunit of the rat Na+,K(+)-ATPase within the extreme NH2-terminal domain, on serine-23. The phosphorylation of this residue resulted in a shift in the equilibrium toward the E1 form, as measured by eosin fluorescence studies, and this was associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays. The rate of transition from E2 to E1 was apparently unaffected by phosphorylation by protein kinase C. These results, together with previous studies that examined the effects of tryptic digestion of Na+,K(+)-ATPase, suggest that the NH2-terminal domain of the alpha-1 subunit, including serine-23, is involved in regulating the activity of the enzyme.
先前已表明，蛋白激酶C使大鼠Na⁺,K⁺-ATP酶的α-1亚基磷酸化会在体外降低该酶的活性。我们现在对这种抑制作用发生的机制进行了研究。对含有该蛋白假定胞质结构域的重组谷胱甘肽S-转移酶融合蛋白进行磷酸化分析、定点诱变及二维肽图分析表明，蛋白激酶C使大鼠Na⁺,K⁺-ATP酶的α-1亚基在最末端的NH₂-末端结构域内的丝氨酸-23处发生磷酸化。通过曙红荧光研究测定，该残基的磷酸化导致平衡向E1形式转移，并且通过ATP酶活性测定，这与该酶表观K⁺亲和力的降低相关。从E2到E1的转变速率显然不受蛋白激酶C磷酸化的影响。这些结果，连同先前研究Na⁺,K⁺-ATP酶胰蛋白酶消化作用影响的研究，表明α-1亚基的NH₂-末端结构域，包括丝氨酸-23，参与调节该酶的活性。
