Figure 2

Analysis of autophosphorylation of MHCK B and its truncations and the effect on kinase activity. (A) Autophosphorylation reactions were performed at 200 nM of each fusion protein, and level of autophosphorylation was determined as described previously [15] by subjecting aliquots of autophosphorylation reactions at 2 min, 5 min, and 20 min to SDS-PAGE, Coomassie Blue staining, and then scintillation counting of excised kinase bands. Visual analysis of autophosphorylation (above bar graph) was achieved via autoradiography of dried, Coomassie-stained SDS-polyacrylamide gels of autophosphorylation time points. The bars represent the average values from at least three separate experiments and the vertical lines are the standard errors of those means. (B) Pre-autophosphorylated GST-B-Δ-WD (50 nM) was compared with control (not pre-autophosphorylated) fusion protein for its ability to phosphorylate MHC. Kinase assays were performed as described previously (Figure 1B) and the activities of pre-autophosphorylated GST-B-Δ-WD (◆, "B-Δ-WD/15'AutoP") and control GST-B-Δ-WD (■, "B-Δ-WD/No AutoP") were measured over time. Each plotted point represents the average value from three separate experiments and the vertical lines are the standard errors of those means.