PHOSPHORYLATION OF NA,K-ATPASE BY PROTEIN-KINASE-C AT SER(18) OCCURS IN INTACT-CELLS BUT DOES NOT RESULT IN DIRECT INHIBITION OF ATP HYDROLYSIS

Na,K-ATPase activity has been demonstrated to be regulated by a variet y of hormones in different tissues. It is known to be directly phospho rylated on its alpha-subunit, but the functional effects of protein ki nases remain controversial, We have developed a sensitive, antibody-ba sed assay for detection of the level of phosphorylation of the alpha 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 stimul ation of endogenous PKC and inhibition of phosphatase activity, it was possible to consistently obtain a very high stoichiometry of phosphor ylation (close to 0.9) in several types of intact cells, This demonstr ates the accessibility and competency of the site for endogenous phosp horylation. 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 phosp hatase inhibitor calyculin A, full phosphorylation was preserved durin g subsequent assays of enzyme activity in vitro. Assay of the hydrolys is of ATP in NRK and C6 cells, however, indicated that there was no si gnificant effect of phosphorylation on the V-max of the Na,K-ATPase or on the apparent affinity for Na+, Any regulatory effect of PHC on sod ium pump activity thus must be lost upon disruption or permeabilizatio n of the cells and is not a direct consequence of enzyme alteration by covalent phosphorylation of Ser(18).