title: The Mycobacterium tuberculosis serine/threonine kinase PknL phosphorylates Rv2175c: mass spectrometric profiling of the activation loop phosphorylation sites and their role in the recruitment of Rv2175c.
abstract: Although Mycobacterium tuberculosis (M. tb) comprises 11 serine/threonine protein kinases, the mechanisms of regulation of these kinases and the nature of their endogenous substrates remain largely unknown. Herein, we characterized the M. tb kinase PknL by demonstrating that it expresses autophosphorylation activity and phosphorylates Rv2175c. On-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, five phosphorylated threonine residues were identified in PknL. Among them, we showed that the activation loop phosphorylated residues Thr173 and Thr175 were essential for the autophosphorylation activity of PknL. Phosphorylation of the activation loop Thr173 residue is also required for optimal PknL-mediated phosphorylation of Rv2175c. Together, our results indicate that phosphorylation of the PknL activation loop Thr residues not only controls PknL kinase activity but is also required for recruitment and phosphorylation of its substrate. Rv2175c was found to be phosphorylated when overexpressed and purified from Mycobacterium smegmatis as 2-DE indicated the presence of different phosphorylated isoforms. Given the presence of the dcw gene cluster in the close vicinity of the pknL/Rv2175c locus, and its conservation in all mycobacterial species, we propose that PknL/Rv2175c may represent a functional pair in the regulation of mycobacterial cell division and cell envelope biosynthesis.
authors.affiliation: Institut de Biologie et Chimie des Protéines, CNRS UMR 5086, Université Lyon 1, IFR128 BioSciences, Lyon-Gerland, Lyon, France.
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Canova
authors.forename: Romain
authors.initials: R
authors.lastname: Veyron-Churlet
authors.forename: Isabelle
authors.initials: I
authors.lastname: Zanella-Cleon
authors.forename: Martin
authors.initials: M
authors.lastname: Cohen-Gonsaud
authors.forename: Alain J
authors.initials: AJ
authors.lastname: Cozzone
authors.forename: Michel
authors.initials: M
authors.lastname: Becchi
authors.forename: Laurent
authors.initials: L
authors.lastname: Kremer
authors.forename: Virginie
authors.initials: V
authors.lastname: Molle
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004789:Enzyme Activation / Q000502:physiology; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases