Figure 2: Phosphorylation of ABI4 by MPK3 and MPK6.

(a) Putative MAPK docking motif and three potential phosphorylation sites in the N terminus of ABI4. The MAPK docking motif is highlighted in red, and the potential phosphorylation sites are highlighted in green. (b,c) Phosphorylation of recombinant ABI4 by the activated MPK3 and MPK6 in vitro. Recombinant MPK3/MPK6 were first activated by MKK9^DD and subsequently incubated with ABI4 in the presence of [γ-³²P]ATP. Autoradiography was used to visualize phosphorylated proteins (upper panel) and equal input of MKK9^DD, MPK3/MPK6 and was confirmed by immunoblot analysis (lower panel). Reactions with various components omitted (−) were used as controls. (d,e) Identification of the MPK3/MPK6 phosphorylation sites in ABI4 based on in vitro phosphorylation assays. Recombinant wild type and mutant ABI4 proteins with single, double and triple mutations were subjected to phosphorylation assays with preactivated recombinant MPK3/MPK6 in the presence of [γ-³²P]ATP. The phosphorylation level was visualized by autoradiography (upper panel) and the loading of MKK9^DD, MPK3/MPK6 and ABI4 proteins were determined by immunoblot analysis. (f) Phosphorylation of ABI4 is induced in MKK5^DD/ABI4^WT but not in MKK5^KR/ABI4^WT transgenic plants after DEX treatment. Total protein was prepared at 3 h after DEX treatment, separated in a Phos-tag SDS–PAGE and immunodetected by an anti-GFP antibody (top). A second immunoblot was performed to detect ABI4 (middle) and Ponceau S staining was used to demonstrate equal loading (bottom). (g) Characterization of up-shifted band of ABI4 by dephosphorylation. The protein extracts from DEX-treated MKK5^DD/ABI4^WT seedlings were incubated with or without calf intestinal alkaline phosphatase (CIAP) and then subjected to Phos-tag SDS–PAGE (top) and SDS–PAGE analyses (middle). The ABI4^WT-GFP protein was detected by immunoblot analysis using anti-GFP antibody. Ponceau S staining was used to demonstrate equal loading (bottom). (h) Effect of mutation of MAPK-phosphorylation sites on ABI4 phosphorylation status in the MKK5^DD background after DEX treatment. Protein extracts from MKK5^DD/ABI4^AAA were prepared at 3 h after DEX treatment, separated in a Phos-tag SDS–PAGE and immunodetected by an anti-GFP antibody (top). Another immunoblot analysis was done at the same time to detect the ABI4 protein (middle). Ponceau S staining was used to demonstrate equal loading (bottom).