Phosphorylation by protein kinase C of serine-23 of the alpha-1 subunit of rat Na+,K(+)-ATPase affects its conformational equilibrium.

Abstract

Phosphorylation of the a-i subunit of rat Na+,K+-ATPase by protein kinase C has been shown previously to decrease the activity of the enzyme in vitro. We have now undertaken an investigation of the mechanism by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains of the protein, sitedirected mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosphorylated the a-1 subunit of the rat Na+,K+-ATPase within the extreme NH2-terminal domain, on serine-23. The phosphorylation of this residue resulted in a shift in the equilibrium toward the El form, as measured by eosin fluorescence studies, and this was associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays. The rate of transition from E2 to El was apparently unaffected by phosphorylation by protein kinase C. These results, together with previous studies that examined the effects of tryptic digestion of Na+,K+-ATPase, suggest that the NH2-terminal domain of the a-i subunit, including serine-23, is involved in regulating the activity of the enzyme. Na+,K+-ATPase is an integral membrane protein believed to be present in all animal cells. The enzyme transports three Na+ out of the cell and two K+ into the cell for each ATP molecule hydrolyzed. Thus the enzyme contributes to the creation of transmembrane potential, cell volume control, and the Na+-dependent transport of protons, other ions, sugars, and amino acids (1). Na+,K+-ATPase activity has been shown to be dynamically regulated in a number of tissues by hormones and neurotransmitters through activation of second messengerdependent protein kinases (2-3). It has also been shown that the a-1 subunit of Na+,K+-ATPase is a substrate for both cAMP-dependent protein kinase and protein kinase C (PKC) in vitro and that the a-1 subunit is also phosphorylated by PKC in intact cells (4-10).cAMP-dependent protein kinase phosphorylates the rat a-1 subunit of Na+,K+-ATPase at Ser-943, and this is associated with a decreased apparent affinity for Na+ and a decreased Vm.x (11). Phosphorylation of the enzyme by PKC is associated with inhibition of ATPase activity in vitro (4), in the OK cell line (6), and in the the choroid plexus (9), but the mechanism by which this inhibition occurs is unknown. To elucidate the mechanism of enzyme inhibition by PKC, we have carried out studies to identify the residue(s) phosphorylated by this kinase. In agreement with recent studies (10), we found that PKC phosphorylates the rat a-1 subunit of Na+,K+-ATPase at Ser-23 in vitro. Furthermore, we have examined the effect of phosphorylation of this NH2-terminal residue on certain conformational transitions that occur during the Na+,K+-ATPase enzymatic cycle. The operational cycle of Na+,K+-ATPase is a sequence of transitions between two major conformations (termed El and E2) of the enzyme with different affinities for the translocated cations and ATP (12). The...

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 PDBu-induced increase in apparent Na affinity of Na,K-ATPase most likely relies on Ser-16 phosphorylation, because this effect is reproduced by ␣1-mutants mimicking constitutive Ser-16 phosphorylation (see Figure 7A). This effect of Ser-16 phosphorylation on the apparent Na affinity of Na,K-ATPase is in agreement with the results of Logvinenko et al (1996), who showed that in vitro phosphorylation of purified Na,K-ATPase by PKC shifts the conformational equilibrium of the Na,K pump toward E1, i.e., the Na conformation. These observations are consistent with earlier studies showing that the ␣1 subunit NH 2 -terminal domain is involved in conformational changes of the enzyme.…”

20-Hydroxyeicosa-tetraenoic acid (20 HETE) activates protein kinase C. Role in regulation of rat renal Na+,K+-ATPase.

“…Gels were stained with Coomassie brillant blue, destained, dried, and analyzed by radioautography or phosphorimaging. In accordance with what had been found in previous studies (13), phosphate was only incorporated into the catalytic ␣ subunit of Na ϩ ,K ϩ -ATPase. Radioactivity was quantified by scanning the phosphorimages using a Molecular Analyst program (Bio-Rad Laboratories, Hercules, CA).…”

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.…”