Phosphorylation of the plasma-membrane H(+)-ATPase of oat roots by a calcium-stimulated protein kinase.

When plasma-membrane vesicles isolated from oat (Avena sativa L.) root cells were incubated with [γ-(32)P]ATP, the H(+)-ATPase was found to be phosphorylated at serine and threonine residues. Phosphotyrosine was not detected. Endogenous ATPase kinase activity was also observed in plasma-membrane vesicles isolated from potato (Solanum tuberosum L.) root cells as well as from yeast (Saccharomyces cerevisiae). Identity of the phosphorylated oat root Mr=100 000 polypeptide as the ATPase was confirmed using conventional glycerol density-gradient centrifugation to purify the native enzyme and by a new procedure for purifying the denatured polypeptide using reversephase high-performance liquid chromatography. Kinase-mediated phosphorylation of the oat root plasma-membrane H(+)-ATPase was stimulated by the addition of low concentrations of Ca(2+) and by a decrease in pH, from 7.2 to 6.2. These results demonstrate that kinase-mediated phosphorylation of the H(+)-ATPase is a plausible mechanism for regulating activity. They further indicate that changes in the cytoplasmic [Ca(2+)] and pH are potentially important elements in modulating the kinase-mediated phosphorylation.