Figure 2.

MKP1 is phosphorylated by elf26-activated MPK6 on Thr-109 in vitro, but loss of MPK6 does not completely prevent elf26-induced phosphorylation in vivo. A, MPK6 was immunoprecipitated from Arabidopsis cells before and after elicitation with 100 nm elf26. The top gel is an autoradiograph of ³²P incorporated in MBP, recombinant 6×His-tagged MKP1 1-161, MKP1 1-161 (T109A), or MKP1 1-161 (T64A). The bottom gel is a duplicate gel stained with Coomassie Blue (CBB) to verify equal loading. The asterisk indicates likely partially degraded MKP1. B, Pyo-MKP1 was transiently expressed from the CaMV 35S promoter in Arabidopsis protoplasts isolated from 5-week-old mkp1 (Ws) and mkp1 mpk6 (Ws) adult plants. The protoplasts were treated with 1 µm elf26 for the indicated times. Protein extracts were separated with 8% mini-format (8.3 × 7.3 cm) SDS-PAGE and immunoblotted with anti-Glu-Glu antibody to detect the Pyo-MKP1 protein (top) or the anti-phospho-p42/44 MAPKs to detect activated MAPKs (middle). The membrane was stained with Coomassie Blue as a loading control (bottom). Mock control samples were from protoplasts isolated from mkp1 (Ws) without transfection with Pyo-MKP1 plasmid. Experiments were performed at least three times with similar results to those shown.