Mutations of Ser-23 of the alpha 1 subunit of the rat Na+/K+-ATPase to negatively charged amino acid residues mimic the functional effect of PKC-mediated phosphorylation

The Na+/K+-ATPase is a target protein for protein kinase C (PKC), The PKC-m ediated phosphorylation of the rat al subunit at Ser-23 results in the inhi bition of its transport function. To understand the molecular basis of the inhibition by PKC, the Ser-23 in the rat al submit has been replaced by neg atively (Asp, Glu) or positively (Lys) charged, or uncharged (Gln, Ala) res idues, and the mutants were expressed in Xenopus oocytes, Ouabain-specific Rb-86 uptake and pump-generated current as well as sensitivity to ouabain a nd to external K+ have been investigated. When Ser-23 was replaced by the n egatively charged residues, transport function was inhibited, and simultane ously synthesis of the a subunits was enhanced, In addition, if Ser-23 was substituted by Glu, the K-I value for inhibition of transport by ouabain wa s drastically increased from 46.5 mu M to 1.05 mM, The data suggest that in sertion 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 t he PKC-mediated inhibition of transport function. (C) 1999 Federation of Eu ropean Biochemical Societies.