Phosphorylation of MupFHA by PknQ.

<p>(<b>A</b>) <i>In vitro</i> kinase assay showing phosphorylation of MupFHA by PknQ and PknQ^K41M. The upper panel shows a coomassie-stained SDS-PAGE and the lower panel shows the corresponding autoradiogram. MupFHA is phosphorylated by PknQ while no phosphorylation was observed with PknQ^K41M. Surprisingly, in the presence of MupFHA, the level of PknQ phosphorylation was reduced (lanes 1 and 3). (<b>B</b>) Phosphorylation status of MupFHA, co-expressed with PknQ or PknQ^K41M in <i>E. coli</i>, was estimated using ProQ Diamond phosphoprotein staining (upper panel). MupFHA co-expressed with PknQ was found to be phosphorylated (MupFHA-P), while no phosphorylation was observed when it was co-expressed with PknQ^K41M (MupFHA-UP). The same gel was stained with Sypro Ruby stain (lower panel) to show equal loading of both samples. (<b>C</b>) The mutants of PknQ, which showed loss in autophosphorylation potential, were used to assess their phosphotransfer ability on MupFHA. Phosphorylation by wild-type PknQ was taken as 100% and relative phosphorylation was calculated. The experiments were repeated three times and error bars show S.D. of three values. Representative autoradiograms with MupFHA bands are shown above the histograms (left and right panels).