Phosphorylation of Na,K-ATPase by Protein Kinase C at Ser18 Occurs in Intact Cells but Does Not Result in Direct Inhibition of ATP Hydrolysis

Abstract

Na,K-ATPase activity has been demonstrated to be regulated by a variety of hormones in different tissues. It is known to be directly phosphorylated on its ␣-subunit, but the functional effects of protein kinases remain controversial. We have developed a sensitive, antibodybased assay for detection of the level of phosphorylation of the ␣1-isoform of rat Na,K-ATPase at the serine residue that is most readily phosphorylated by protein kinase C (PKC) in vitro, Ser 18 . By stimulation of endogenous PKC and inhibition of phosphatase activity, it was possible to consistently obtain a very high stoichiometry of phosphorylation (close to 0.9) in several types of intact cells. This demonstrates the accessibility and competency of the site for endogenous phosphorylation. The cells used were derived from rat (NRK 52E, C6, L6, and primary cultures of cerebellar granule cells, representing epithelial cells, glia, muscle cells, and neurons). In the presence of the phosphatase inhibitor calyculin A, full phosphorylation was preserved during subsequent assays of enzyme activity in vitro. Assay of the hydrolysis of ATP in NRK and C6 cells, however, indicated that there was no significant effect of phosphorylation on the V max of the Na,K-ATPase or on the apparent affinity for Na ؉ . Any regulatory effect of PKC on sodium pump activity thus must be lost upon disruption or permeabilization of the cells and is not a direct consequence of enzyme alteration by covalent phosphorylation of Ser 18 .

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?

“…This observation is in agreement with a growing number of studies, which report stimulation of Na,K-ATPase activity in response to PKC activation (Lynch et al, 1986;Hootman et al, 1987;Gupta et al, 1991;Féraille et al, 1995;Pedemonte et al, 1997) but contrasts with findings on rat ␣1 subunits in which PKC-dependent phosphorylation inhibits (Belusa et al, 1997) or does not alter (Feschenko and Sweadner, 1997) Na,K-ATPase activity. However, our study and these former studies (Belusa et al, 1997;Feschenko and Sweadner, 1997) cannot be directly compared, because we specifically studied the role of the ubiquitous Ser-16 phosphorylation site, whereas others focused on the role of the rat-specific Ser-23 phosphorylation site. Indeed, among higher vertebrates, the rat ␣1 subunit is the only one that exhibits two PKC phosphorylation sites: the ubiquitous site on Ser-16 and an additional site on Ser-23 (Feschenko and Sweadner, 1995;Béguin et al, 1996b), accounting for 80% of in vitro PKC phosphorylation in this species (Feschenko and Sweadner, 1995).…”

“…Indeed, stimulatory, inhibitory, or no effects have been attributed to PKC phosphorylation (Bertorello et al, 1991;Middleton et al, 1993;Fisone et al, 1995;Carranza et al, 1996;Feschenko and Sweadner, 1997;Pedemonte et al, 1997). In view of the multiple mechanisms that affect Na,K pump activity and the possible interplay between different signaling pathways, these conflicting results suggest that tissue-specific factors and uncontrolled experimental conditions may mask the basic function of PKC phosphorylation.…”

“…This rapid modulation of Na,K-ATPase activity by hormones may be linked to a direct phosphorylation of the Na,KATPase ␣1 subunit by serine/threonine kinases. For instance, in purified enzyme preparations (Bertorello et al, 1991;Feschenko and Sweadner, 1994), in homogenates of Xenopus oocytes (Chibalin et al, 1992), and in intact cells (Middleton et al, 1993;Béguin et al, 1994;Carranza et al, 1996;Feschenko and Sweadner, 1997), protein kinase C (PKC) can phosphorylate the ␣1 isoforms of Na,K-ATPase and to a much lesser extent the ␣2 and ␣3 isoforms (Béguin † These authors contributed equally to this work. et al, 1996b).…”

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.…”

C-Peptide stimulates Na+,K+-ATPase activity via PKC alpha in rat medullary thick ascending limb

“…However, previous studies have shown that PKC phosphorylation of the Na + ,K + -ATPase α1-subunit is associated with either inhibition or stimulation of Na + ,K + -ATPase activity [18,39,40,41,42]. These differential effects rely on metabolic factors [43], intracellular calcium concentrations [44], PKC isoforms [38], as well as cell specific factors [45,46]. Taken together, all these results suggest that PKC phosphorylation of the α1-subunit does not directly alter Na + ,K + -ATPase intrinsic properties but rather could recruit cell-specific regulatory factors that determine the functional effect of Na + ,K + -ATPase phosphorylation.…”