PHOSPHORYLATION BY PROTEIN-KINASE-C OF SERINE-23 OF THE ALPHA-1 SUBUNIT OF RAT NA-ATPASE AFFECTS ITS CONFORMATIONAL EQUILIBRIUM(,K+)

Phosphorylation of the alpha-1 subunit of rat Na+,K+-ATPase by protein kinase C has been shown previously to decrease the activity of the en zyme in vitro. We have now undertaken an investigation of the mechanis m by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putat ive cytoplasmic domains of the protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosp horylated the ru-l subunit of the rat Na+,K+-ATPase within the extreme NH2-terminal domain, on serine-23, The phosphorylation of this residu e resulted in a shift in the equilibrium toward the El form, as measur ed by eosin fluorescence studies, and this was associated with a decre ase in the apparent K+ affinity of the enzyme, as measured by ATPase a ctivity assays, The rate of transition from E2 to El was apparently un affected by phosphorylation by protein kinase C, These results, togeth er with previous studies that examined the effects of tryptic digestio n of Na+,K+-ATPase, suggest that the NH2-terminal domain of the alpha- 1 subunit, including serine-23, is involved in regulating the activity of the enzyme.