REGULATION OF NA,K-ATPASE TRANSPORT ACTIVITY BY PROTEIN-KINASE-C

Considerable evidence indicates that the renal Na+,K+-ATPase is regula ted through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH2-terminal end of the Na+,K+-ATPase alpha-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na+,K+ ATPa se activity. To determine whether the alpha-subunit NH2-terminus is in volved in the regulation of Na+,K+-ATPase activity by PKC, we have exp ressed the wild-type rodent Na+,K+-ATPase alpha-subunit and a mutant o f this protein that lacks the first thirty-one amino acids at the NH2- terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant pheno-type characteristic of rodent kidney cell s. The presence of the alpha-subunit NH2-terminal segment was not nece ssary to express the maximal Na+,K+-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb+-tra nsport in intact cells were the same in cells transfected with the wil d-type rodent alpha 1 and the NH2-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na+,K+-ATPase me diated Rb+-uptake and reduced the intracellular Na+ concentration of c ells transfected with wild-type alpha 1 cDNA. In contrast, these effec ts were not observed in cells expressing the NH2-deletion mutant of th e alpha-subunit. Treatment with phorbol ester appears to affect specif ically the Na+,K+-ATPase activity and no evidence was observed that ot her proteins involved in Na+-transport were affected. These results in dicate that amino acid(s) located at the alpha-subunit NH2-terminus pa rticipate in the regulation of the Na+,K+-ATPase activity by PKC.