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 alpha 1. Here we have used protein fragmentation with trypsin to localize the site of phosphorylation of the rat Na,K-ATPase alpha 1 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 alpha 1 subunits. The remaining 75% or more of the 32P was found on Ser-18, a site that is absent in many Na,K-ATPase alpha subunit sequences. This accounts for the observation that dog and pig alpha 1 subunits can be phosphorylated by protein kinase C only to much lower levels than can rat alpha 1. It is also likely to be relevant to other known species-specific effects of protein kinase C on Na,K-ATPase.

Stress-induced Expression of the γ Subunit (FXYD2) Modulates Na,K-ATPase Activity and Cell Growth

“…No significant difference was noticed in the levels of the ␣1 subunit in the purified preparations. Further, we did not observe any significant difference in the phosphorylation level of ␣1 at the protein kinase C site (Ser-18) (34,35). Staining with McK1 antibody, which recognizes only non-phosphorylated ␣1, was practically identical in preparations from control and hypertonicity-treated cultures (500 mosM, NaCl) (Fig.…”

“…Polyclonal antiserum K3 raised against rat kidney enzyme was used to detect the ␣1 and ␤1 subunits (29). The protein kinase C phosphorylation level of ␣1 (C, ␣1*) at the Ser-18 was monitored with McK1 antibody (34). Na,K-ATPase activity was determined as a function of Na ϩ (B and D), and the curves were normalized to the amount of ␣ as detected on blots.…”

Is Phosphorylation of the α1 Subunit at Ser-16 Involved in the Control of Na,K-ATPase Activity by Phorbol Ester–activated Protein Kinase C?

“…The first PKC phosphorylation site identified is present in all ␣1 subunits and was mapped to Ser-16 in an unusual (Ser-GluHis) PKC motif (Béguin et al, 1994(Béguin et al, , 1996b. A second ratspecific ␣1 subunit PKC phosphorylation site was mapped to Ser-23 (Feschenko and Sweadner, 1995;Béguin et al, 1996b). It should be mentioned that Ser-16 and Ser-23 numbered from the initial methionine residue are also named Ser-11 and Ser-18 by other authors according to the removal of the NH 2 -terminal 5 amino acids during the processing of the ␣1 chains.…”

Mutation of the Protein Kinase C Phosphorylation Site on Rat α1 Na+,K+-ATPase Alters Regulation of Intracellular Na+ and pH and Influences Cell Shape and Adhesiveness

“…Serine 23 has recently been identified as the major PKC phosphorylation site in rat Na ϩ ,K ϩ -ATPase ␣1 (9,10). In this study Ser-23 was mutated to alanine and the functional consequences examined in intact cells.…”