Functional significance of phosphorylation of rat renal Na+, K+-ATPase by PKA and PKC

Abstract

The activity of Na+,K+-ATPase (NKA) is short term regulated by hormones as well as by signalling pathways that involve activation of PKA or PKC, but the mechanism(s) remains unclear. This study was performed to evaluate whether this regulation of NKA occurs via PKA- or PKC-mediated phosphorylation of the NKA a subunit.

Experiments in vitro showed that PKA and PKC phosphorylate purified rat renal NKA and that phosphorylation of NKA by PKC directly inhibits NKA activity. PKA phosphorylates the alpha subunit at the highly conserved seryl residue, Ser943. PKC phosphorylates at Ser23. cDNAs coding for wild type NKA and NKA in which the PKA site Ser943 was mutated to Ala were stably transfected into COS cells. It was shown, by using two site-selective phosphorylation state-specific antibodies, that activation of PKA by cAMP or forskolin results in phosphorylation of the wild type enzyme at Ser943 and that activation of PKC by the phorbol ester PDBu or the diacylglycerol analogue OAG results in phosphorylation at Ser23.

Phosphorylation of NKA is accompanied by an inhibition of enzyme activity, as reflected by a decrease in 86Rb+ transport and ATP hydrolysis. The PKA-mediated phosphorylation and inhibition of NKA also occurs in non-transfected COS cells. The inhibition of NKA activity by PKA in COS cells is not associated with internalization of the enzyme from the cell surface. Phosphorylation of NKA is implicated in regulation of enzyme activity by 1st messenger. The ß-adrenergic agonist isoproterenol induces phosphorylation of NKA on Ser943 and inhibits its activity. This effect was reproduced by forskolin, and was abolished by the specific PKA inhibitor, H89, which suggests that it is mediated via the cAMP-PKA pathway.

Okadaic acid, an inhibitor of Ser/Thr protein phosphatases, enhances phosphorylation and inhibition of NKA induced by isoproterenol. The change in activity of NKA linearly correlates with the magnitude of the a subunit phosphorylation. A direct link between modulation of NKA activity and phosphorylation of NKA a subunit is indicated by site-directed mutagenesis experiments. When Ser943 was converted to Ala943, stimulation of the phosphorylation and inhibition of the activity of NKA induced by isoproterenol or forskolin were not observed. The regulation of NKA activity by PKC is modulated by the state of phosphorylation of Ser943 by PKA. When Ser943 was mutated Ala943, the inhibition of enzyme activitv by PDBu was reduced. In contrast, when Ser943 was mutated to Asp943, the effect of PDBu was slightly enhanced. Sp-5,6-DCl-cBIMPS, a specific PKA activator, increased the phosphorylation of Ser943 and this was associated with an enhanced response to PDBu.

Finally, functional effects of PKA and PKC on NKA activity were deterrnined at various levels of [Ca2+]i, which were achieved with Ca2+ ionophore A23187. Activation of PKA or PKC caused inhibition of NKA at low [Ca2+]j (125 nM) and stimulation or no change at high [Ca2+]j (450 nM). Under both [Ca2+]j conditions, the a subunit of NKA was phosphorylated. The change in NKA activity was linearly dependent on the level of [Ca2+]j. In conclusion, PKA and PKC can modulate the activity of NKA by direct phosphorylation of its a subunit. The functional effect of phosphorylation is dependent on [Ca2+]i. When [Ca2+]i is low, it induces an inhibition of NKA activity; when [Ca2+]i is high, it induces a stimulation.