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Volume 272, Number 28, Issue of July 11, 1997 pp. 17726-17733
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, January 23, 1997, and in revised form, April 16, 1997)

Marina S. Feschenko and Kathleen J. Sweadner

From the  Laboratory of Membrane Biology, Neuroscience Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129

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, antibody-based 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, Ser¹⁸. 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 Ser¹⁸.

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