Abstract
Clade A PP2C phosphatases are central components of the ABA-receptor coupled core signaling pathway, and are involved in multiple stress responses and developmental processes. However, the direct targets or partner proteins of A clade PP2Cs that participate in these biological processes are largely unknown. Here, we used a TurboID-based proximity labeling method to identify putative associated proteins of one A clade PP2C phosphatase, ABI1. By combining the results from affinity purification or proximity labeling of biotinylated proteins, we identified more than four hundred putative ABI1-associated proteins, including dozens of known ABI1-intreacting proteins, as well as proteins involved in TOR signaling, phospho-regulation, and other biological processes. We found that RAFs, a group of protein kinases that phosphorylate and activate SnRK2s in ABA and osmotic stress signaling, are direct substrates of ABI1. A conserved serine residue located in the P-loop of the kinase domain, corresponding to Ser619 in RAF3, is a major functional ABI1 target site. ABI1-mediated dephosphorylation on this site strongly promotes the kinase activity of most B2 and B3 RAFs. Thus, ABI1 has dual functions in ABA signaling by dephosphorylating and inhibiting SnRK2 to prevent SnRK2 activation in unstressed conditions, while dephosphorylating B2 and B3 subgroup RAFs to maintain their basal kinase activity. PP2C-mediated dephosphorylation at the conserved serine residue may be a mechanism for RAF activation in both plants and animals, with potential implications for tumorigenesis in humans.