A Phosphothreonine Residue at the C-Terminal End of the Plasma Membrane H(+)-ATPase Is Protected by Fusicoccin-Induced 14–3–3 Binding

We have isolated the plasma membrane H(+)−ATPase in a phosphorylated form from spinach (Spinacia oleracea L.) leaf tissue incubated with fusicoccin, a fungal toxin that induces irreversible binding of 14–3–3 protein to the C terminus of the H(+)-ATPase, thus activating H(+) pumping. We have identified threonine-948, the second residue from the C-terminal end of the H(+)-ATPase, as the phosphorylated amino acid. Turnover of the phosphate group of phosphothreonine-948 was inhibited by 14–3–3 binding, suggesting that this residue may form part of a binding motif for 14–3–3. This is the first identification to our knowledge of an in vivo phosphorylation site in the plant plasma membrane H(+)-ATPase.