Maintaining Basal B-RAF Kinase Activity and ABA signaling via Single Serine Dephosphorylation and Autophosphorylation

The phytohormone abscisic acid (ABA) regulates plant responses to environmental stresses, development, and immunity. Under unfavorable conditions, ABA forms a complex with its receptor proteins PYR1/PYLs/RCARs, inhibiting A clade PP2C phosphatases and releasing SnRK2s from PP2C-mediated inhibition. RAF kinases from the B1, B2, and B3 subgroups phosphorylate and reactivate SnRK2s, initiating ABA responses. While ABA does not significantly activate B-RAFs, their basal activity is essential for initiating ABA signaling. However, the mechanisms sustaining this basal B-RAF activity are not fully understood. In this study, we revealed that A clade PP2Cs interact with and dephosphorylate B3 subgroup RAFs at a conserved serine residue, corresponding to Ser619 in RAF3, within the phosphate-binding loop. A phosphomimicking mutation at this residue, RAF3S619D, failed to bind ATP and exhibited diminished kinase activity in vitro and in vivo.