Protein purification and in vitro kinase assay

For in vitro kinase assays, full-length coding sequence of SnRK2.6 and kinase domains of RAFs were cloned into either pGEX-4T-1, pET28a or pET-SUMO vectors and transformed into BL21 or ArecticExpression cells. The recombinant proteins were expressed and purified using standard protocols. For the phosphorylation assay, recombinant kinase domains of RAFs (aa541-821 for RAF1-KD, aa650-933 for RAF2-KD, aa600-880 for RAF3-KD, aa710-992 for RAF4-KD, aa733-1030 for RAF5-KD, aa645-956 for RAF6-KD, aa470-773 for RAF7-KD, aa424-671 for RAF8-KD, aa436-730 for RAF9-KD, aa466-767 for RAF10-KD, aa472-765 for RAF11-KD, aa457-735 for RAF12-KD) were incubated with “kinase-dead” forms of SnRK2.6 with or without Ser to Ala mutations at Ser171 and Ser175 in reaction buffer (25 mM Tris HCl, pH 7.4, 12 mM MgCl₂, 2 mM DTT), with 1 μM ATP plus 1 µCi of [γ-³²P] ATP for 30 min at 30 °C. Reactions were stopped by boiling in SDS sample buffer and proteins were separated by 10% SDS-PAGE.

For the dephosphorylation assay, SnRK2.6^M94G coated on Glutathione Sepharose (Cytiva) were dephosphorylated with Lambda Protein Phosphatase (λPP, NEB, P0753S) for 30 min and the λPP was removed by washing three times with protein buffer (25 mM Tris HCl, pH 7.4, 150 mM NaCl). To detect the effects of RAF3-KD and RAF10-KD on SnRK2.6^M94G, SnRK2.2^M96G, and SnRK2.3^M95G thiophosphorylation and activity, recombinant GST-RAF3/10-KD was incubated with pre-dephosphorylated SnRK2.6^M94G, SnRK2.2^M96G, or SnRK2.3^M95G for 30 min in reaction buffer (25 mM Tris HCl, pH 7.4, 12 mM MgCl₂, 2 mM MnCl₂, 0.5 mM DTT, 50 μM ATP, 50 μM N⁶-Benzyl-ATPγS). Then ABF2 was added to the reaction and incubated for an additional 30 min. This phosphorylation reaction was stopped by adding EDTA to a final concentration of 25 μM. A final concentration of 2.5 mM p-nitrobenzyl mesylate (Abcam, ab138910) was added to proceed the alkylating reaction for 1 h at room temperature. Samples with SDS sample buffer were boiled and separated by SDS-PAGE, transferred to Polyvinylidene fluoride (PVDF) membrane, and immunoblotted with antibodies against thiophosphate ester (Abcam, ab92570). To pre-activate SnRK2.6, HIS-SUMO-RAF-KD proteins were coated on the Ni-NTA beads and incubated with SnRK2.6 (in solution) in the presence of ATP. HIS-SUMO-RAF-KD were removed by centrifuging after the reaction.