MUTATION OF THE PROTEIN-KINASE-C PHOSPHORYLATION SITE ON RAT ALPHA-1 NA-ATPASE ALTERS REGULATION OF INTRACELLULAR NA+ AND PH AND INFLUENCESCELL-SHAPE AND ADHESIVENESS(,K+)

The enzyme Na+,K+-ATPase creates the transmembrane Na+ gradient that i s of vital importance for functioning of all eukaryotic cells. Na+,K+- ATPase can be phosphorylated by protein kinase A (PKA) and protein kin ase C (PKC), and these sites of phosphorylation have been identified, In the present study, we have examined the physiological significance of PKC phosphorylation of rat Na+,K+-ATPase. In COS cells transfected with wild type rat Na+,K+-ATPase alpha 1, intracellular Na+ was higher and pH was lower than in cells transfected with rat Na+,K+-ATPase alp ha 1 in which the PKC phosphorylation site, Ser-23, had been mutated i nto alanine. Phorbol dibutyrate inhibited Na+,K+-ATPase-dependent ATP hydrolysis and Rb+ uptake in cells expressing wild type Na+,K+-ATPase but not in cells expressing S23A Na+,K+-ATPase. Cells expressing the S 23A mutant had a more rounded appearance and attached less well to fib ronectin than did untransfected cells or cells transfected with wild t ype rat Na+,K+-ATPase alpha 1. These results indicate a functional rol e for PKC-mediated phosphorylation of rat Na+,K+-ATPase alpha 1 and su ggest a connection between this enzyme and cell adhesion.