Since Pfnek3 is a ser/thr kinase, residue T290 is a potential phosphorylation site which Pfnek3 may phosphorylate and in turn regulate the activity of Pfmap2. To confirm this postulation, LC–MS/MS was employed to identify amino
Efavirenz modifications in Pfmap2. Prior to LC–MS/MS analyses, catalytically inactive GST-Pfmap2 was co-incubated with GST-Pfnek3 in a kinase assay and subsequently separated by SDS–PAGE. This excludes the possibility of autophosphorylation by Pfmap2. A neutral loss of phosphoric acid (98 Da) was detected on the peptide QLTSHVVTR and the phosphorylated residue was found to be T290 (Fig. 3A). To ascertain that the
phosphorylation was indeed mediated by Pfnek3, the experiment was repeated using the ‘kinase dead’ Pfnek3 (GST-ΔPfnek3), which abolished the phosphorylation on residue T290 (Fig. 3B). Therefore through LC–MS/MS analyses, it was confirmed that Pfnek3 phosphorylates Pfmap2 at residue T290. These data point towards Pfnek3 as the upstream kinase capable of activating Pfmap2 by phosphorylating at residue T290, confirming that the TSH motif is the activation site. Most importantly, the postulation that Pfnek3 functions as a “MAPKK” in the malarial parasite is now strengthened.
