Ca2+-dependent phosphorylation regulates the plasma membrane H+-ATPase activity of maize (Zea mays L.) roots

Phosphorylation/dephosphorylation of the plasma-membrane H+-ATPase (EC 3.6.1.35) could act as a regulatory mechanism to control its activity. In this work, a plasmalemma-enriched fraction from maize roots and a partially purified H+-ATPase were used to investigate the effects of Ca2+ and calmodulin on the H+-ATPase activity and on its phosphorylation status. Both the hydrolytic and the proton-pumping activities were reduced approximately 50% by micromolar Ca2+ concentrations while calmodulin did not show any effect either alone or in the presence of Ca2+. The lack of effect of calmodulin antagonists indicated that calmodulin was not involved in this response. The addition of staurosporine, a kinase inhibitor, abolished the inhibitory effect of Ca2+. Phosphorylation of plasma membrane and partially purified H+-ATPase showed the same behavior. In the presence of Ca2+ a polypeptide of 100 kDa was phosphorylated. This polypeptide cross-reacted with antibodies raised against the H+-ATPase of maize roots. The autoradiogram of the immunodetected protein clearly showed that this polypeptide, which corresponds to the H+-ATPase, was phosphorylated. Additional clear evidence comes from the immunoprecipitation experiments: the data obtained show that the H+-ATPase activity is indeed influenced by its state of phosphorylation.