Structural basis for species-specific differences in the phosphorylation of Na,K-ATPase by protein kinase C

Abstract

There is considerable evidence that protein kinases play a role in regulation of the activity of the Na,K-ATPase, but the characteristics of direct kinase phosphorylation of Na,K-ATPase subunits are still not well understood. There are 36 sites that could qualify as protein kinase C motifs in rat al. Here we have used protein fragmentation with trypsin to localize the site of phosphorylation of the rat Na,K-ATPase α1 subunit to within the first 32 amino acids of the N terminus and then used direct sequencing of the phosphorylated protein to determine which of two candidate serine residues was modified. The result was that at most 25% of the 32P was found on Ser- 11, a site that is well conserved in Na,K-ATPase α1 subunits. The remaining 75% or more of the 32P was found on Ser-18, a site that is absent in many Na,K-ATPase α subunit sequences. This accounts for the observation that dog and pig al subunits can be phosphorylated by protein kinase C only to much lower levels than can rat α1. It is also likely to be relevant to other known species-specific effects of protein kinase C on Na,K-ATPase.