Identification of Novel Physiological Substrates of Mycobacterium bovis BCG Protein Kinase G (PknG) by Label-free Quantitative Phosphoproteomics

Abstract

Mycobacterial Ser/Thr kinases play a critical role in bacterial physiology and pathogenesis. Linking kinases to the substrates they phosphorylate in vivo, thereby elucidating their exact functions, is still a challenge. The aim of this work was to associate protein phosphorylation in mycobacteria with important subsequent macro cellular events by identifying the physiological substrates of PknG in Mycobacterium bovis BCG. The study compared the phosphoproteome dynamics during the batch growth of M. bovis BCG versus the respective PknG knock-out mutant (ΔPknG-BCG) strains. We employed TiO2 phosphopeptide enrichment techniques combined with label-free quantitative phosphoproteomics workflow on LC-MS/MS. The comprehensive analysis of label-free data identified 603 phosphopeptides on 307 phosphoproteins with high confidence. Fifty-five phosphopeptides were differentially phosphorylated, of these, 23 phosphopeptides were phosphorylated in M. bovis BCG wild-type only and not in the mutant. These were further validated through targeted mass spectrometry assays (PRMs). Kinase-peptide docking studies based on a published crystal structure of PknG in complex with GarA revealed that the majority of identified phosphosites presented docking scores close to that seen in previously described PknG substrates, GarA, and ribosomal protein L13. Six out of the 22 phosphoproteins had higher docking scores than GarA, consistent with the proteins identified here being true PknG substrates. Based on protein functional analysis of the PknG substrates identified, this study confirms that PknG plays an important regulatory role in mycobacterial metabolism, through phosphorylation of ATP binding proteins and enzymes in the TCA cycle. This work also reinforces PknG's regulation of protein translation and folding machinery.

New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach

“…Thus, based on the employed experimental approach, the high enrichment of proteins related to translation and the available data reporting ribosomal proteins as kinases substrates (including PknG), we predict that ribosomal proteins are genuine partners of PknG. This conclusion is also supported by a recent report that identified protein translation as one of the main processes regulated by PknG [23]. Nakedi et al used a label free phosphoproteomic approach to identify proteins that were differentially phosphorylated in wild type M. bovis BCG and a PknG knockout mutant strain, and their list of candidate PknG substrates include two ribosomal proteins (the 50S ribosomal protein L2 and the 30S ribosomal protein S16), both of which were also identified in the present work (Table S5 interactors among which 31 were exclusive of PknG while the remaining 91 were also interactors of at least one more protein kinase.…”

“…Very interestingly, the strategy designed here allowed us to recover the only two previously well-characterized substrates of PknG: GarA and the 50 ribosomal protein L13, pointing to a confident list of biologically relevant interactors. None of the other reports on substrates or direct interactors of PknG succeeded in the identification of these validated substrates [22][23][24].…”

“…In addition, it was shown that PknG can regulate the activity of the Nudix hydrolase RenU through the phosphorylation of the ribosomal protein L13 [21]. Besides these better characterized substrates, very recent phosphoproteomics and protein microarray analysis have expanded the list of putative PknG substrates and interactors [22][23][24], and few candidates were further selected to test its interaction with, or phosphorylation by PknG. These studies suggest that RmlA and MurC activities are also regulated by PknG mediated phosphorylation [22,24].…”

An Aspartate-Specific Solute-Binding Protein Regulates Protein Kinase G Activity To Control Glutamate Metabolism in Mycobacteria

“…GlnX is another candidate sensor for additional sensory inputs as it is homologous to the E. coli Tar receptor, which binds both protein and small molecule activators ( 19 , 20 ). Fitting with its activation by multiple stimuli, PknG also has multiple substrates and may regulate other processes in addition to regulation of the TCA cycle via GarA ( 5 , 35 ).…”

Role of post‐translational modifications in the acquisition of drug resistance in Mycobacterium tuberculosis

“…PknG is secreted during infection in macrophages and plays an important role in blocking the phagosome-lysosome fusion, thus ensuring the survival of bacteria in the host [38]. In mycobacteria, PknG regulates the central carbon and nitrogen metabolism through phosphorylation of its substrates, primarily GarA [39,40]. Proteins involved in metabolism have an important role in drug binding and are responsible for the development of resistance [41,42].…”