An FHA Phosphoprotein Recognition Domain Mediates Protein EmbR Phosphorylation by PknH, a Ser/Thr Protein Kinase from <i>Mycobacterium tuberculosis</i>

Molle, Virginie; Kremer, Laurent; Girard-Blanc, Christine; Besra, Gurdyal S.; Cozzone, and Alain J.; Prost, Jean‐François

doi:10.1021/bi035150b

Cited by 137 publications

(177 citation statements)

References 51 publications

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“…Both point mutants were incapable of phosphorylating FHA-2 (Fig. 4C) (23). In order to determine if the Cpn0712 FHA-2 domain utilized similar conserved residues to mediate phosphorylation by PknD, we created point mutants GST-FHA-2[S441A] and GST-FHA-2[N464A] and tested them as substrates for PknD.…”

Section: Construction Of Expression Plasmids and Pknd-ap Fusion Constmentioning

confidence: 99%

Chlamydophila pneumoniae PknD Exhibits Dual Amino Acid Specificity and Phosphorylates Cpn0712, a Putative Type III Secretion YscD Homolog

Johnson

Mahony

2007

J Bacteriol

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Chlamydophila pneumoniae is an obligate intracellular bacterium that causes bronchitis, pharyngitis, and pneumonia and may be involved in atherogenesis and Alzheimer's disease. Genome sequencing has identified three eukaryote-type serine/threonine protein kinases, Pkn1, Pkn5, and PknD, that may be important signaling molecules in Chlamydia. Full-length PknD was cloned and expressed as a histidine-tagged protein in Escherichia coli. Differential centrifugation followed by sodium carbonate treatment of E. coli membranes demonstrated that His-PknD is an integral membrane protein. Fusions of overlapping PknD fragments to alkaline phosphatase revealed that PknD contains a single transmembrane domain and that the kinase domain is in the cytoplasm. To facilitate solubility, the kinase domain was cloned and expressed as a glutathione Stransferase (GST) fusion protein in E. coli. Purified GST-PknD kinase domain autophosphorylated, and catalytic mutants (K33G, D156G, and K33G-D156G mutants) and activation loop mutants (T185A and T193A) were inactive. PknD phosphorylated recombinant Cpn0712, a type III secretion YscD homolog that has two forkhead-associated domains. Thin-layer chromatography revealed that the PknD kinase domain autophosphorylated on threonine and tyrosine and phosphorylated the FHA-2 domain of Cpn0712 on serine and tyrosine. To our knowledge, this is the first demonstration of a bacterial protein kinase with amino acid specificity for both serine/threonine and tyrosine residues and this is the first study to show phosphorylation of a predicted type III secretion structural protein.

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Section: Construction Of Expression Plasmids and Pknd-ap Fusion Constmentioning

confidence: 99%

Chlamydophila pneumoniae PknD Exhibits Dual Amino Acid Specificity and Phosphorylates Cpn0712, a Putative Type III Secretion YscD Homolog

Johnson

Mahony

2007

J Bacteriol

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“…Experimental reports have suggested functional roles in cell morphology (PknA and -B) (5,6), stress response (PknH) (7), glucose transport (PknF) (8,9), and regulation of cellular Glu͞Gln levels (PknG) (10). However, endogenous substrates could be identified in only a few cases (6,8,11,12). Signaling pathways and mechanisms are largely uncharted territory.…”

mentioning

confidence: 99%

Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis

Alderwick

Molle

Kremer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Ser͞Thr phosphorylation has emerged as a critical regulatory mechanism in a number of bacteria, including Mycobacterium tuberculosis. This problematic pathogen encodes 11 eukaryoticlike Ser͞Thr kinases, yet few substrates or signaling targets have been characterized. Here, we report the structure of EmbR (2.0 Å), a putative transcriptional regulator of key arabinosyltransferases (EmbC, -A, and -B), and an endogenous substrate of the Ser͞Thr-kinase PknH. EmbR presents a unique domain architecture: the N-terminal winged-helix DNA-binding domain forms an extensive interface with the all-helical central bacterial transcriptional activation domain and is positioned adjacent to the regulatory Cterminal forkhead-associated (FHA) domain, which mediates binding to a Thr-phosphorylated site in PknH. The structure in complex with a phospho-peptide (1.9 Å) reveals a conserved mode of phospho-threonine recognition by the FHA domain and evidence for specific recognition of the cognate kinase. The present structures suggest hypotheses as to how EmbR might propagate the phospho-relay signal from its cognate kinase, while serving as a template for the structurally uncharacterized Streptomyces antibiotic regulatory protein family of transcription factors.PknH ͉ x-ray crystallography ͉ arabinosyltransferase ͉ transcriptional regulator ͉ ethambutol

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“…The STPK, having been autophosphorylated in response to a particular stimulus, would then phosphorylate the EBP and activate it to initiate transcription. A precedent is available in Mycobacterium tuberculosis where the FHA domain of the ToxR-like transcriptional regulator EmbR allows the protein to interact with an autophosphylated form of PknH, an STPK, which then catalyzes phosphorylation of EmbR (52). An FHA-EBP might also be activated indirectly by means of an STPK-phosphorylated coactivator protein.…”

Section: Discussionmentioning

confidence: 99%

Enhancer-binding proteins with a forkhead-associated domain and the σ ⁵⁴ regulon in Myxococcus xanthus fruiting body development

Jelsbak

Givskov

Kaiser

2005

Proc. Natl. Acad. Sci. U.S.A.

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An FHA Phosphoprotein Recognition Domain Mediates Protein EmbR Phosphorylation by PknH, a Ser/Thr Protein Kinase from Mycobacterium tuberculosis

Cited by 137 publications

References 51 publications

Chlamydophila pneumoniae PknD Exhibits Dual Amino Acid Specificity and Phosphorylates Cpn0712, a Putative Type III Secretion YscD Homolog

Chlamydophila pneumoniae PknD Exhibits Dual Amino Acid Specificity and Phosphorylates Cpn0712, a Putative Type III Secretion YscD Homolog

Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis

Enhancer-binding proteins with a forkhead-associated domain and the σ ⁵⁴ regulon in Myxococcus xanthus fruiting body development

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An FHA Phosphoprotein Recognition Domain Mediates Protein EmbR Phosphorylation by PknH, a Ser/Thr Protein Kinase from Mycobacterium tuberculosis

Cited by 137 publications

References 51 publications

Chlamydophila pneumoniae PknD Exhibits Dual Amino Acid Specificity and Phosphorylates Cpn0712, a Putative Type III Secretion YscD Homolog

Chlamydophila pneumoniae PknD Exhibits Dual Amino Acid Specificity and Phosphorylates Cpn0712, a Putative Type III Secretion YscD Homolog

Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis

Enhancer-binding proteins with a forkhead-associated domain and the σ 54 regulon in Myxococcus xanthus fruiting body development

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Enhancer-binding proteins with a forkhead-associated domain and the σ ⁵⁴ regulon in Myxococcus xanthus fruiting body development