title: Identification of mycobacterial GarA as a substrate of protein kinase G from M. tuberculosis using a KESTREL-based proteome wide approach.
abstract: Signal transduction in bacteria is generally mediated via two-component systems. These systems depend on the transfer of a phosphate molecule from a donor to an acceptor by histidine kinases, thereby activating the acceptor to allow downstream signaling/activation. Several bacterial genomes, including the genome of M. tuberculosis, were shown to encode eukaryotic-like kinases. To better understand the function of these kinases and the regulatory networks within which they operate, identification of downstream targets is essential. We here present a straightforward approach for the identification of bacterial Ser/Thr-kinase substrates. This approach is based on the KESTREL (Kinase Tracking and Substrate Elucidation) procedure combined with reversed-phase chromatography and two-dimensional gel electrophoresis. Using this method, GarA was identified as one potential substrate for the mycobacterial Ser/Thr-protein kinase G (PknG). These results show that the modified KESTREL approach can be successfully employed for the identification of substrates for bacterial Ser/Thr-kinases.
authors.affiliation: Biozentrum, University of Basel, Klingelbergstrasse 50, CH 4056 Basel, Switzerland.
authors.forename: Philipp
authors.initials: P
authors.lastname: Mueller
authors.affiliation: Biozentrum, University of Basel, Klingelbergstrasse 50, CH 4056 Basel, Switzerland. Electronic address: jean.pieters@unibas.ch.
authors.forename: Jean
authors.initials: J
authors.lastname: Pieters
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002845:Chromatography / Q000379:methods; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D009163:Mycobacterium bovis / Q000201:enzymology / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D020543:Proteome; D040901:Proteomics / Q000379:methods; D015398:Signal Transduction; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D047908:Intracellular Signaling Peptides and Proteins; D020543:Proteome; D017346:Protein Serine-Threonine Kinases; D017869:Cyclic GMP-Dependent Protein Kinases
keywords: KESTREL; Mycobacterium tuberculosis; Serine; Threonine kinase