Fig. 3.
BZR1 interacts with and is phosphorylated by components of the MAPK complex. A, MBP-BZR1, but not MBP-PRE1 (PRE1, a HLH protein used as a negative control) pulled down GST-MKK5 and GST-MAPK4. The blots were probed with anti-GST antibodies. B, GST-MAPK4, but not GST-CYP59, pulled down MBP-BZR1, MBP-BZR1N, and (weakly) MBP-BZR1C. The blots were probed with anti-MBP antibodies. Ponceau S staining shows the amount of bait protein on the beads. C and D, Co-immunoprecipitation (IP) of BZR1 with MKK5. Arabidopsis protoplasts transfected with 35S::MKK5-YFP and 35S::BZR1-MH were immunoprecipitated using anti-c-Myc antibody (C) or anti-YFP antibodies (D), and the immunoblot was probed with anti-YFP or anti-c-Myc antibodies. E, A yeast two-hybrid assay showed that MKK5 interacted with BZR1, amino acids 1–198 of BZR1, and bzr1-1D. Yeast grown on leucine-, tryptophan-, and histidine-deficient medium (-LTH) supplemented with 1 mm 3AT revealed the interaction. Empty ADT7 was used as a negative control. F, In vitro kinase assay of MBP-BZR1 phosphorylation by GST-MKK5 but not by kinase dead GST-MKK5K99M. Upper panel, autoradiograph; bottom panel, Coomassie blue staining of the protein gel.