ALPHA-1 BUT NOT ALPHA-2 OR ALPHA-3 ISOFORMS OF NA,K-ATPASE ARE EFFICIENTLY PHOSPHORYLATED IN A NOVEL PROTEIN-KINASE-C MOTIF

Protein kinase C (PKC) phosphorylates the catalytic alpha 1 subunit of Na,K-ATPase in purified enzyme preparations and in intact cells. Litt le is known, however, whether all three known alpha isoforms are subst rates for PKC and whether direct phosphorylation is implicated in the modulation of the transport activity of the different Na,K-ATPase isoz ymes. In this study, we investigated the structural requirements for P KC phosphorylation of alpha 1, alpha 2, and alpha 3 isoforms of differ ent species after expression in Xenopus oocytes. By using a combinatio n of site-directed mutagenesis and computer-assisted protein modeling, we characterized a novel Ser-X-His motif which in concert with more d istantly located basic residues acts as an efficient substrate for PKC -mediated phosphorylation in the N-terminus of most Na,K-ATPase alpha 1 isoforms. As indicated by controlled proteolysis, alpha 2 isoforms a re also phosphorylated in the N-terminus but to a much lower extent th an al isoforms containing the Ser-X-His motif. Phosphorylation and pho sphoamino acid analysis of fusion proteins containing the wild-type or mutant N-terminus of alpha 2 reveal that Thr-Thr-Ser-X-Asn or Thr-Thr -Ala-X-Asn motifs represent weak targets for PKC phosphorylation. Fina lly, our data suggest that, with the exception of rat alpha 3, all alp ha 3 isoforms from other species are not substrates for PKC, On the ba sis of the phosphorylation efficiency, we may speculate that only al b ut not alpha 2 or alpha 3 isoforms of Na,K-ATPase are likely candidate s for regulatory PKC phosphorylation.