Functional regulation of reconstituted Na, K‐ATPase by protein kinase A phosphorylation

Abstract

Reconstituted Na+,K+-ATPase from either pig kidney or shark rectal glands was phosphorylated by cAMP dependent protein kinase, PKA. The stoichiometry was ~0.9 mole Pilmole tx-subunit in the pig kidney enzyme and ,-~ 0.2 tool Pilmol a-subunit in the shark enzyme. In shark Na+,K+-ATPase PKA phosphorylation increased the maximum hydro!~tic activity for cytoplasmic Na ÷ activation and extracellular K activation without affecting the apparent Km values. In contrast, no significant functional effect after PKA phosphorylation was observed in pig kidney Na+,K+-ATPase.

Stimulation of ouabain‐sensitive 86Rb+ uptake and Na+,K+‐ATPase α‐subunit phosphorylation by a cAMP‐dependent signalling pathway in intact cells from rat kidney cortex

“…The increases in ion transport activity and in the degree of phosphorylation of the Na+,K+-ATPase suggest that cAMP modulates the enzyme activity by promoting the phosphorylation of its c~-subunit. In agreement with the present results, it has recently been demonstrated using a reconstituted enzyme preparation that PKA phosphorylation either does not affect or leads to an activation of Na+,K+-ATPase depending on the animal species origin of the enzyme preparation [11].…”

“…They are strengthened by the fact that several groups recently identified a single highly conserved PKA phosphorylation site on the a-subunit of Na+,K+-ATPase (Ser-943) [8,9]. Moreover, direct phosphorylation by PKA has been demonstrated in in vitro studies with purified [7] and reconstituted [11] Na+,K+-ATPase. However, in vivo direct cc-subunit Na+,K+-ATPase phosphorylation by PKA remains to be established.…”

“…The physiological link between an increased phosphorylation level and the activity of the Na+,K+-ATPase is another controversial point. Several studies reported that the increase in phosphorylation is accompanied by stimulation of the enzyme activity [10,11], but others showed an inhibition of Na+,K +-ATPase activity after its phosphorylation [7]. These discrepancies can be explained, at least in part, by the experimental conditions, i.e.…”

Interaction of Protein Kinase C and cAMP-dependent Pathways in the Phosphorylation of the Na,K-ATPase

“…Instead, Ser-938 may become accessible to the kinase only when the Na,K-ATPase is structurally disturbed, either by Triton X-100 or other detergents (13), by heat denaturation (54), and hypothetically, by the misfolding of some fraction of ␣ subunits when exogenously overexpressed. Epitope additions near this site disrupt folding to the extent that the site appears on the extracellular surface (55,56) or result in poor cell growth and cloning efficiency (57), consistent with the partially buried location of the analogous segment in the Ca 2ϩ -ATPase.…”

“…The molecular mechanism of regulation is an open question, however, because seemingly inconsistent functional effects have been reported with different experimental systems, including either inhibition or activation ostensibly through the same kinase. A variety of mechanisms have been proposed, including direct inhibition or stimulation by kinases (11)(12)(13), regulation of enzyme activity through pathways that include lipid metabolites (8,14), and regulation by recruitment to, and internalization from, the plasma membrane (10,15). Only in some cases has it been shown whether (and which) sites on the Na,K-ATPase are actually used by protein kinases in intact cells, however (5, 16 -20).…”