To determine if the altered PM H ATPase activity in the double mutants correlates with seedling responses to salt in alkaline conditions, 5 d old j3 1, pks5 1, j3 1 pks5 1, pks5 3, j3 1 pks5 3, pks5 4, and j3 1 pks5 4 plants grown on medium at pH 5.8 were transferred to MS medium at pH 5.8, pH 7.7 with 75 mM NaCl, or pH 8.1 with 75 mM NaCl. Consistent with measurements of PM H ATPase activity, all of the double mutants showed phenotypes similar to their pks5 parent , suggesting that J3 regulates PM H ATPase activity and plant response to salt at alkaline pH by mediating PKS5 activity. J3 Represses PKS5 Kinase Activity Our results demonstrate that J3 interacts with and functions genetically upstream of PKS5. In addition, these proteins have opposite effects on the regulation of PM H ATPase activity and seedling sensitivity to salt at alkaline pH. One explanation for these observations is J3 represses PKS5 kinase activity. To test this hypothesis, a protein kinase assay was performed.
As predicted, J3 repressed PKS5 kinase activity , and the more J3 protein that was added to the reaction, the more PKS5 activity was inhibited. The specificity of this repression was shown based on the lack of J3 repression of the kinase activity of SOS2 , a PKS5 homolog. When we overexpressed Tivozanib molecular weight J3 in the pks5 1 and pks5 3 mutants, the pks5 3 salt sensitive phenotype in alkaline conditions was rescued, whereas the phenotype of pks5 1 was not significantly altered . These results further support the conclusion that J3 regulation of the response of the plant to salt in alkaline conditions takes place via repression of PKS5 kinase activity. DISCUSSION The PM H ATPase is a highly regulated enzyme with numerous physiological functions . Evidence exists for changes in phosphorylation status of the H ATPase leading to either activation or inhibition of enzyme activity . Activation of the enzyme requires phosphorylation of its C terminus leading to the binding of 14 3 3 proteins and the removal of an autoinhibition by the R domain.
Several phosphorylation sites have been identified in the C terminal region of the protein . While little is known about the protein kinases or phosphatases that are directly responsible for altering PM H ATPase phosphorylation status, several proteins have been implicated in the C terminal phosphorylation events leading to binding of the regulatory 14 3 3 protein . PKS5 was identified as a protein that negatively regulates PM H ATPase activity Patupilone and controls intracellular pH homeostasis in response to alkaline pH . PKS5 phosphorylates Ser 931 on the AHA2 isoform of the PM H ATPase, which, in turn, blocks interaction between AHA2 and an activating 14 3 3 protein. Phosphorylation of this residue is independent of Thr 947 phosphorylation of AHA2 .