Figure 2
Phosphorylation activity of EhAK1.
(A) Autophosphorylation and substrate phosphorylation activities of His-EhAK1. Each reaction was set with 2 μg of recombinant tagged EhAK1 or K85A in the presence of γ-32P-ATP, MgCl2 at 30 °C for 1 h in kinase assay buffer and for substrate 2 μg of histone type (IIIS) or myelin basic protein (MBP) was used. K85A mutant of EhAK1 was used as a control. The kinase reaction was stopped by adding Laemmli buffer with 1 mM EDTA. The products were analysed on SDS-PAGE and visualized using a phosphorimager or gel was stained by coomassie brilliant blue and bands were cut and counts were taken in a scintillation counter. (B) Time kinetics of kinase activity. The autophosphorylation activity of His tagged EhAK1 was carried out as described in panel 1A and reaction was incubated for indicated period of time in kinase assay buffer. (C) Optimisation of Mg2+ concentration. The autophosphorylation activity of His tagged EhAK1 was carried out as described in panel 1A, in presence of indicated concentration of MgCl2. (D) Michelis-Menten parameters for wild-type EhAK1. Vmax for ATP was determined to be 9.4 nmol/min/mg protein and Km was 28 nM. The cold ATP was varied from 0.4 pmol to 8 μmol in presence of 10 μCi [32P-γ]ATP. The protein was phosphorylated for 1 h at 30 °C. The amount of radioactivity incorporated was determined. All the points were in duplicates. The experiment was repeated three times. (E) Role of zinc binding residues in activity of EhAK1: Upper panel shows the schematic representation of Zn2+ and coordination with residues (Black) in TRPM7 (PDB-1IAJ). Conserved residues present in EhAK1 are shown in red. Lower panel shows the histone phosphorylation activity describe in panel 1A, of wild type, H195A , H253A, C255, C259A and all four mutants (2H & 2C: 4A) of EhAK1 were checked.
