FIGURE 2.

M. tuberculosis STPKs display specific intermolecular phosphorylation patterns. A, autoradiograms showing the reactions of each active STPK construct (top) with the 11 inactivated substrate kinases (left). Each inactive Asp-to-Asn mutant kinase construct was incubated with each of the 11 M. tuberculosis STPKs in [γ-³²P]ATP transfer assays. His-MBP tags on the active kinases and tagless inactive kinases enable separation of the proteins in each reaction by SDS-PAGE. Assays were imaged by autoradiography in parallel. Products on the diagonal reflect efficient autophosphorylation. Off-diagonal bands in each column indicate cross-phosphorylation. B, PknA ICD was tested against the 11 Asp-to-Asn mutant kinases. No additional inter-kinase interactions were observed. C, PknG and PknI KDs were not phosphorylated by any of the other kinases. The full-length PknG and PknI constructs are cross-phosphorylated by multiple kinases, presumably on sites outside of the catalytic domain. D, autoradiographs were quantified in ImageJ and normalized to the autophosphorylation signal observed for each kinase. PknA and PknL do not efficiently autophosphorylate; therefore, these two kinases are not included in this graph. The PknH-PknE interaction was the lowest detected by LC-MS and was used as the lower boundary. Low intensity interactions at or below this cutoff are depicted by gray columns.