Mutation of the protein kinase C phosphorylation site on rat alpha1 Na+,K+-ATPase alters regulation of intracellular Na+ and pH and influences cell shape and adhesiveness.
The enzyme Na+,K+-ATPase creates the transmembrane Na+ gradient that is of vital importance for functioning of all eukaryotic cells. Na+, K+-ATPase can be phosphorylated by protein kinase A (PKA) and protein kinase 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 alpha1, intracellular Na+ was higher and pH was lower than in cells transfected with rat Na+,K+-ATPase alpha1 in which the PKC phosphorylation site, Ser-23, had been mutated into 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 S23A mutant had a more rounded appearance and attached less well to fibronectin than did untransfected cells or cells transfected with wild type rat Na+, K+-ATPase alpha1. These results indicate a functional role for PKC-mediated phosphorylation of rat Na+,K+-ATPase alpha1 and suggest a connection between this enzyme and cell adhesion.
Belusa
R
R
Department of Woman and Child Health, Karolinska Institute, S-112 81 Stockholm, Sweden.
Wang
Z M
ZM
Matsubara
T
T
Sahlgren
B
B
Dulubova
I
I
Nairn
A C
AC
Ruoslahti
E
E
Greengard
P
P
Aperia
A
A
D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D048429:Cell Size; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*
D015240:Phorbol 12,13-Dibutyrate; D012964:Sodium; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase