Figure 4.

Expression of WIPK under the Inducible Promoter Does Not Result in an Increase of Its Activity.

(A) Expression of WIPK under the same conditions used for SIPK fails to result in an increase of its activity. Tobacco leaves were infiltrated with Agrobacterium carrying WIPK or its inactive K73R mutant (WIPK^R). DEX (30 μM) was infiltrated 48 hr later, and samples were taken at the times indicated. Expression of transgenes was monitored by immunoblot (IB) analysis using anti-Flag antibody (top), and kinase activities in the leaf tissues were determined by in-gel kinase assay using MBP as a substrate (bottom).

(B) Recombinant WIPK is active in both autophosphorylation and cross-phosphorylation of MBP. Autophosphorylation activities of 0.5 μg of His-tagged wild-type SIPK and WIPK and their mutants were determined as described in Methods. Phosphorylated SIPK (P-SIPK) and WIPK (P-WIPK) were visualized by autoradiography after SDS-PAGE (top). Cross-phosphorylation (Cross-P) activities of SIPK and WIPK (0.1 μg) were determined using MBP as a substrate. Phosphorylated MBP (P-MBP) was visualized by autoradiography after SDS-PAGE (bottom).