The early phosphorylation signaling cascade of the peptide hormone systemin was defined through comparing systemin-induced responses with non-specific responses induced by the inactive analogon systemin-A17 or mock treatment. Systemin-responsive phosphorylation of kinases and phosphatases was identified over a time course of 2 to 45 minutes, as well as typical substrate phosphorylation sites. We identified the C-terminal threonine of H+-ATPase LHA1 as a substrate of phosphatase PLL5 and MAP-Kinase MPK2, thereby defining a signaling circuit of rapid dephosphorylation and re-phosphorylation after 15 minutes.