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...

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.

The abundance of mRNA of alpha 1-, alpha 2-, alpha 3-, beta 1-, and beta 2-isoforms of Na(+)-K(+)-ATPase was examined in several renal structures of normal and adrenalectomized (ADX) rats. In situ hybridization with 35S-labeled cRNA probes was performed on kidney sections from adult rats. The number of silver grains per unit surface area was quantified over cells of the glomerulus, proximal convoluted tubules (PCT), early distal tubules (EDT), and cortical collecting ducts (CCD). In normal rat kidney, alpha 1- and beta 1-mRNA was detected in PCT, EDT, and CCD, with the following range of magnitude: EDT > CCD > PCT > glomerulus. The amount of alpha 1- and beta 1-mRNA was equivalent. A large abundance of these two mRNA species was also found in the medullary thick ascending limb of the loop of Henle. Expression of alpha 2, alpha 3, and beta 2 was very low and evenly distributed over any cell type. In ADX, a significant decrease in alpha 1-mRNA (30%) was observed in EDT and CCD, with no change in PCT. beta 1-mRNA abundance was unaffected by adrenalectomy. These results indicate that 1) in the rat kidney alpha 1- and beta 1-mRNA are coexpressed at a similar level that varies along the renal tubule according to the cell type, 2) minute expression of alpha 2-, alpha 3-, and beta 2-mRNA is present in the kidney, and 3) corticosteroid depletion reduces the expression of alpha 1- and not beta 1-mRNA in the corticosteroid-sensitive tubular cells.

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