Figure 5.

Active 44-kDa WIPK required both threonine and tyrosine phosphorylation. Protein extracts were prepared in the absence of phosphatase inhibitors from TMV-infected leaves at 8 hr after shifting plants from 32°C to 22°C or 48 hr after infection at 22°C. Samples containing 20 μg of protein were treated with either the serine/threonine-specific phosphatase, PP-2A₁ (0.25 unit in 30 μl), or the tyrosine-specific protein phosphatase, YOP (2 units in 30 μl), for 20 min at 30°C in the presence or absence of a phosphatase inhibitor. The PP-2A₁ inhibitor, okadaic acid (OA), and YOP inhibitor, Na₃VO₄ (Van)_, were used at a concentration of 1 μM and 1 mM, respectively. After phosphatase treatment, kinase activity was detected by the in-gel kinase activity assay.