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. 2010 Feb 23;285(17):12714–12725. doi: 10.1074/jbc.M110.105189

FIGURE 2.

FIGURE 2.

In vitro phosphorylation of MabA. A, phosphorylation of MabA by multiple STPK. Nine recombinant STPKs (PknA to PknL) encoded by the M. tuberculosis genome were expressed and purified as GST fusions and incubated with purified His-tagged MabA and [γ-33]ATP. The quantity of the different STPKs varied from 0.6 to 4.2 μg to obtain the optimal autophosphorylation activity for each kinase. Samples were separated by SDS-PAGE and stained with Coomassie Blue (upper panel) and visualized by autoradiography after overnight exposure to a film (lower panel). The upper bands illustrate the autokinase activity of each STPK, and the lower bands represent phosphorylated MabA. B, in vitro phosphorylation of the single and triple MabA mutants by PknB. Four μg of purified MabA_WT, MabA_T21A, MabA_T114A, MabA_T191A, and MabA_T21A/T114A/T191A were incubated individually with PknB and [γ-33]ATP. Samples were separated by SDS-PAGE and stained with Coomassie Blue (upper panel) and visualized by autoradiography after overnight exposure to a film (lower panel). Upper bands reflect the intense autokinase activity of PknB, and the lower bands illustrate the phosphorylation state of each MabA variant following in vitro phosphorylation by PknB.