Characterization of PknQ phosphorylation.

<p>(<b>A</b>) Homology modeling showing the structure of the PknQ kinase domain (PknQ_kd). The structure shows a ribbon diagram (left) and surface-filled model (right) of PknQ_kd, highlighting the N- and C-terminal regions. (<b>B</b>) SDS-PAGE analysis of His₆-tagged PknQ_kd. PknQ_kd was purified to near homogeneity as observed on SDS-PAGE. (<b>C</b>) Time-dependent autophosphorylation of PknQ. PknQ was allowed to phosphorylate itself for 0–30 minutes. Relative phosphorylation (normalized to protein amounts) is plotted and corresponding autoradiogram (top) and SDS-PAGE images (bottom) are shown. The intensity of phosphorylation on protein bands was calculated using Personal Molecular Imager (Bio-Rad) using quantification software Quantity One (Bio-Rad). The phosphorylation after 30 min was taken as 100% (signal saturation) and relative phosphorylation was calculated. The experiment was repeated thrice and error bars show S.D. of three values. (<b>D</b>) Autophosphorylation of PknQ_kd and phosphorylation of Myelin basic protein (MyBP). Autoradiogram (top) and corresponding SDS-PAGE images (bottom) are shown. No phosphorylation was observed in the presence of the kinase-dead mutant PknQ^K41M. (<b>E</b>) Time-dependent phosphorylation of MyBP using PknQ_kd. Relative phosphorylation (normalized to protein amounts) is plotted and corresponding autoradiogram (top) and SDS-PAGE images (bottom) are shown. The quantification of phosphorylation intensity was done as described earlier (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003315#pntd-0003315-g001" target="_blank">Fig. 1C</a>). The experiment was repeated three times and error bars show S.D. of three values.