title: Forkhead-associated (FHA) domain containing ABC transporter Rv1747 is positively regulated by Ser/Thr phosphorylation in Mycobacterium tuberculosis.
abstract: One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tuberculosis, is through reversible phosphorylation of proteins mediated by protein kinases and phosphatases. This study concerns one of these enzymes, the serine/threonine protein kinase PknF, that is encoded in an operon with Rv1747, an ABC transporter that is necessary for growth of M. tuberculosis in vivo and contains two forkhead-associated (FHA) domains. FHA domains are phosphopeptide recognition motifs that specifically recognize phosphothreonine-containing epitopes. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues, Thr-150 and Thr-208. To determine the in vivo consequences of phosphorylation, infection experiments were performed in bone marrow-derived macrophages and in mice using threonine-to-alanine mutants of Rv1747 that prevent specific phosphorylation and revealed that phosphorylation positively modulates Rv1747 function in vivo. The role of the FHA domains in this regulation was further demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. FHA-1 domain mutation resulted in attenuation in macrophages highlighting the critical role of this domain in Rv1747 function. A mutant deleted for pknF did not, however, have a growth phenotype in an infection, suggesting that other kinases can fulfill its role when it is absent. This study provides the first information on the molecular mechanism(s) regulating Rv1747 through PknF-dependent phosphorylation but also indicates that phosphorylation activates Rv1747, which may have important consequences in regulating growth of M. tuberculosis.
authors.affiliation: Division of Mycobacterial Research, Medical Research Council National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
authors.forename: Vicky L
authors.initials: VL
authors.lastname: Spivey
authors.forename: Virginie
authors.initials: V
authors.lastname: Molle
authors.forename: Rachael H
authors.initials: RH
authors.lastname: Whalan
authors.forename: Angela
authors.initials: A
authors.lastname: Rodgers
authors.forename: Jade
authors.initials: J
authors.lastname: Leiba
authors.forename: Lasse
authors.initials: L
authors.lastname: Stach
authors.forename: K Barry
authors.initials: KB
authors.lastname: Walker
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Smerdon
authors.forename: Roger S
authors.initials: RS
authors.lastname: Buxton
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D009876:Operon / Q000235:genetics; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction; D013912:Threonine / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C487813:Rv1747 protein, Mycobacterium tuberculosis; D010769:Phosphothreonine; D010768:Phosphoserine; D013912:Threonine; D012694:Serine; C487814:PknF protein, Mycobacterium tuberculosis; D017346:Protein Serine-Threonine Kinases