Figure 1

Comparison of the kinase activities of MHCK-B and its truncations toward myosin II and MH-1 peptide substrate. (A) Illustrations of the domain organization of MHCK-B and its truncations used in these studies. (B) Kinase assays containing 50 nM purified GST-MHCK-B (◆), GST-B-Δ-WD (▲), or GST-B-Δ-N-WD (■) and 1.0 μM purified Dictyostelium myosin II were performed as described previously [4]. (C) The same proteins (50 nM) were also assessed for MH-1 peptide (50 μM) phosphorylation over time [4]. (D) Coomassie stained SDS-PAGE gel showing MHC in pellet (P) and supernatant (S) fractions. The assembly of Dictyostelium myosin II (800 nM) was assessed as described previously [6] either after mock phosphorylation (untreated) or after phosphorylation for 15 min with 80 nM kinase. After phosphorylation, samples were adjusted to 40 mM NaCl to optimize filament assembly and then subjected to centrifugation to pellet assembled myosin. For graphs (B) and (C), each plotted point represents the average value from three separate experiments and the vertical lines are the standard errors of those means.