Fig. 1: SnRK2.6 activation requires transphosphorylation by B2 and B3 RAFs in vitro.
From: Initiation and amplification of SnRK2 activation in abscisic acid signaling
a Recombinant RAF kinase domains (KDs) were used to phosphorylate SnRK2.6KR (SnRK2.6K50R, a kinase-dead form of SnRK2.6), SnRK2.6KR with Ser171Ala mutation (SnRK2.6KR-S171A), SnRK2.6KR with Ser175Ala mutation (SnRK2.6KR-S175A), or SnRK2.6KR proteins with Ser171AlaSer175Ala mutations (SnRK2.6KR-AA), in the presence of [γ-32p]ATP. Autoradiograph (left) and Coomassie staining (right) show phosphorylation and loading, respectively, of purified GST-RAF-KD and HIS-SnRK2.6KR. b GST-SnRK2.6, HIS-SUMO-RAF3-KD, and HIS-SUMO-RAF10-KD phosphorylate HIS-SnRK2.6KR in vitro. Recombinant undephosphorylated GST-SnRK2.6 was used to phosphorylate HIS-SnRK2.6KR in the presence of [γ-32p]ATP. Autoradiograph (left) and Coomassie staining (right) show phosphorylation and loading, respectively, of purified GST-SnRK2.6, HIS-SUMO-RAF3-KD, HIS-SUMO-RAF10-KD, and HIS-SnRK2.6KR. c SnRK2.6M94G but not wild type SnRK2.6 can use N6-Benzyl-ATPγS to thiophosphorylate substrate. d RAF3-KD and RAF10-KD trigger the autophosphorylation of pre-dephosphorylated GST-SnRK2.6M94G (de-SnRK2.6M94G). e RAF3-KD activates pre-dephosphorylated GST-SnRK2.6M94G (de-SnRK2.6M94G) and the reactivated SnRK2.6M94G phosphorylates itself and ABF2. f RAF3-KD and RAF10-KD activate HIS-SUMO-SnRK2.2M96G and HIS-SUMO-SnRK2.3M95G. For d to f, Anti-γ-S immunoblot (upper) and Coomassie staining (lower) show thiophosphorylation and loading, respectively, of recombinant GST-RAF3-KD, GST-RAF10-KD, GST-SnRK2.6M94G, HIS-SUMO-SnRK2.2M96G, HIS-SUMO-SnRK2.3M95G, and GST-ABF2. Asterisks indicate preincubation of SnRK2.6M94G with N6-Benzyl-ATPγS for 30 min before further reaction. Arrows indicate degraded fragments co-purified with GST-SnRK2.6M94G. Images shown are representative of at least two independent experiments. Source data are provided in Source Data.
