Figure 2.

Constitutively active NtMEK2 mutant phosphorylates and activates SIPK and WIPK. (A) NtMEK2^DD, the T227D/S233D double mutant of NtMEK2, has elevated kinase activity. Autophosphorylation activities of 0.5 μg of His-tagged wild-type (WT) NtMEK2 and its mutants were determined as described in Materials and Methods. (B) NtMEK2^DD preferentially phosphorylates SIPK and WIPK. Phosphorylation activities of HisNtMEK2^WT and HisNtMEK2^DD (0.1 μg) were determined by using inactive mutant MAPKs (HisMAPK^R, 1 μg) as substrates. Reactions in the absence (−) of either MAPK or MAPKK were used as controls. (C) Phosphorylation by NtMEK2^DD activates SIPK and WIPK. Wild-type MAPKs (HisMAPK, 1 μg) were incubated in the absence (−) or with 0.1 μg of HisNtMEK2^WT or HisNtMEK2^DD in the presence of 50 μM unlabeled ATP. MBP (final concentration of 0.25 μg/μl) and [γ-³²P]ATP (1 μCi per reaction) were then added. Phosphorylated MBP (P-MBP) that reflects the activity of MAPK was visualized by autoradiography after SDS/PAGE.