Figure 6
Analysis of CaWRKYa as a substrate of CaMK1 and CaMK2.
(a) MBP-CaMK1 and -CaMK2 had an autophosphorylation activity. In vitro kinase assay was performed with MBP-CaMK1 (1 μg) and -CaMK2 (1 μg) incubated with no substrate and MBP protein (1 μg) was used as a negative control. Proteins were incubated in the reaction buffer containing [γ-33P]-ATP for 1 h at RT, respectively. After heating for 5 min, the samples were run in SDS-PAGE gel. The gels were dried in gel dryer for 1 h and phosphorylation signals were detected by BAS reader. Arrows indicate phosphorylated CaMK1 and CaMK2. (b) MyBP protein (1 μg) was phosphorylated by MBP-CaMK1 and -CaMK2 in vitro kinase assay. Arrows indicate phosphorylated CaMK1, CaMK2 and MyBP. (c) A schematic illustration of CaWRKYa mutant constructs and confirmation of the purified proteins in SDS-PAGE gel. MBP-CaWRKYaN, MBP-CaWRKYaN-mSP (S85A, S97A, S104A, S116A) and MBP-CaWRKYaN-mD (K66A, K68A, L75A, M77A) proteins were made by mutagenesis. Purified proteins were checked via 15% SDS PAGE gel. Asterisk indicates specific band. (d) MBP-CaMK1 (1 μg) or -CaMK2 (1 μg) were incubated with CaWRKYaN (1 μg), CaWRKYaN-mSP (1 μg) and CaWRKYaN-mD (1 μg) proteins and then in vitro kinase assay was performed. MBP was used as a negative control. N.S indicates non-specific bands.
