In the tobacco PMA2 isoform, the corresponding residue is phosphorylated in vivo , although it is not known whether a SOS2 like protein kinase is involved in this event. In this study, we identified a component in the PKS5 signaling pathway. J3 shares similar patterns of tissue specific expression and subcellular localization with PKS5 and interacts with PKS5 in planta . However, j3 knockout mutants display the opposite phenotype from pks5 loss of function mutants, with the j3 mutants displaying increased sensitivity to NaCl at alkaline pH and decreased PM H ATPase activity . Double mutant analysis suggests that J3 relies on and functions upstream of PKS5. Overexpression of J3 rescues the pks5 3 salt sensitive phenotype in alkaline conditions but does not alter pks5 1 phenotype . These findings are consistent with the observation that J3 represses PKS5 kinase activity . Interestingly, j3 1 pks5 3 and j3 1 pks5 4 double mutants have similar levels of PM H ATPase activity and similar sensitivity to growth in media with NaCl at alkaline pH to what is seen for their pks5 parent .
The fact that the phenotypes in these double mutants are not more severe suggests that other, as yet unidentified, components may also be involved in the regulation of phosphorylation dephosphorylation of Ser 931 and that there is a threshold effect of PKS5 kinase activity on the regulation of PM H ATPase activity. PM H ATPase activity is stimulated by many environmental changes, known to be regulated by a calcium dependent SCaBP1 PKS5 pathway. When the Arabidopsis y27632 proteins are expressed in yeast, repression of PM H ATPase activity by PKS5 is dependent on the presence of SCaBP1 ; however, it is currently not known how SCaBP1 regulates the PKS5 protein. The predicted functions of the SCaBP proteins are to perceive changes in intracellular calcium levels and then to interact with, activate, and recruit PKS kinase proteins to cell membranes to activate their targets . It has been shown that ANJ1, a DnaJ like protein in Atriplex nummularia, is farnesylated and geranylgeranylated in planta .
These modifications Orotic acid rely on a Cys in the CAQQ motif at the C terminus of the ANJ1 protein and increase the association of ANJ1 with the cell membrane . This CAQQ motif is absolutely conserved in the J3 C terminus, suggesting that, during growth in NaCl at alkaline pH, J3 is prenylated and this leads to the localization of J3 to the plasma membrane and to activation of PM H ATPase via inhibition of PKS5 or, alternatively, by modifying the affinity of PKS5 for the H ATPase. In summary, we identified a new component of the PKS5 signal transduction pathway that positively regulates PM H ATPase activity in Arabidopsis.