Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and

environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling

components that mediate this regulation. Here, we report that an Arabidopsis thaliana Ser/Thr protein kinase, PKS5, is a

negative regulator of the plasma membrane proton pump (PM Hþ-ATPase). Loss-of-function pks5 mutant plants are more

tolerant of high external pH due to extrusion of protons to the extracellular space. PKS5 phosphorylates the PM Hþ-ATPase

AHA2 at a novel site, Ser-931, in the C-terminal regulatory domain. Phosphorylation at this site inhibits interaction between

the PM Hþ-ATPase and an activating 14-3-3 protein in a yeast expression system. We show that PKS5 interacts with the

calcium binding protein SCaBP1 and that high external pH can trigger an increase in the concentration of cytosolic-free

calcium. These results suggest that PKS5 is part of a calcium-signaling pathway mediating PM Hþ-ATPase regulation.