Phosphoproteomic Approaches to Discover Novel Substrates of Mycobacterial Ser/Thr Protein Kinases

Mycobacterial STPKs are responsible for orchestrating phosphorylation-dependent signaling cascades that mediate bacterial growth and environmental adaptation. Recent advances in MS-based phosphoproteomics have significantly expanded the candidate substrate lists for individual mycobacterial STPKs. Integration of the available phosphoproteomic datasets provide new insights into the functional roles of specific STPKs in cell physiology. Future research should focus on in vivo phosphorylation network reconstruction to expose the fundamental signaling pathways in mycobacteria. Linking STPKs with their physiological substrates may reveal novel antimycobacterial agents. [Display omitted] Highlights •Mapping kinase-substrate relationships is vital in discovering new tuberculosis drug targets.•LC-MS/MS-based phosphoproteomics expand mycobacterial STPK substrate catalogues.•We review and integrate MS-generated datasets on novel candidate substrates.•Validation studies are necessary to confirm true physiological substrates of STPKs. Mycobacterial Ser/Thr protein kinases (STPKs) play a critical role in signal transduction pathways that ultimately determine mycobacterial growth and metabolic adaptation. Identification of key physiological substrates of these protein kinases is, therefore, crucial to better understand how Ser/Thr phosphorylation contributes to mycobacterial environmental adaptation, including response to stress, cell division, and host-pathogen interactions. Various substrate detection methods have been employed with limited success, with direct targets of STPKs remaining elusive. Recently developed mass spectrometry (MS)-based phosphoproteomic approaches have expanded the list of potential STPK substrate identifications, yet further investigation is required to define the most functionally significant phosphosites and their physiological importance. Prior to the application of MS workflows, for instance, GarA was the only known and validated physiological substrate for protein kinase G (PknG) from pathogenic mycobacteria. A subsequent list of at least 28 candidate PknG substrates has since been reported with the use of MS-based analyses. Herein, we integrate and critically review MS-generated datasets available on novel STPK substrates and report new functional and subcellular localization enrichment analyses on novel candidate protein kinase A (PknA), protein kinase B (PknB) and PknG substrates to deduce the possible physiological roles of these kina