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J Proteomics. 2019 Feb 10;192:321-333. doi: 10.1016/j.jprot.2018.09.013. Epub 2018 Sep 27.

New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach.

Author information

1
Unidad de Bioquímica y Proteómica Analíticas Institut Pasteur de Montevideo, Instituto de Investigaciones Biológicas Clemente Estable, Mataojo 2020, Montevideo 11400, Uruguay; Unit of Dynamics of Host-Pathogen Interactions, Institut Pasteur, Paris, France.
2
Unidad de Bioquímica y Proteómica Analíticas Institut Pasteur de Montevideo, Instituto de Investigaciones Biológicas Clemente Estable, Mataojo 2020, Montevideo 11400, Uruguay.
3
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
4
Integrated Mycobacterial Pathogenomics Unit, Institut Pasteur, Paris, France.
5
Institut Pasteur de Montevideo, Unidad de Biofísica de Proteínas, Uruguay.
6
Unité de Microbiologie Structurale, Institut Pasteur, CNRS URA 2185, Paris, France.
7
Laboratorio de Fisicoquímica Biológica, Facultad de Ciencias, Universidad de la República, Uruguay.
8
Departamento de Bioquímica, Facultad de Medicina, CEINBIO, Universidad de la República, Uruguay.
9
Laboratory for Proteomics and Protein Engineering, Carlos Chagas Institute, Paraná, Brazil.
10
Unité de Microbiologie Structurale, Institut Pasteur, CNRS URA 2185, Paris, France; Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Ocampo y Esmeralda, S2002LRK, Rosario, Argentina.
11
Unidad de Bioquímica y Proteómica Analíticas Institut Pasteur de Montevideo, Instituto de Investigaciones Biológicas Clemente Estable, Mataojo 2020, Montevideo 11400, Uruguay. Electronic address: duran@pasteur.edu.uy.

Abstract

PknG from Mycobacterium tuberculosis is a multidomain Serine/Threonine protein kinase that regulates bacterial metabolism as well as the pathogen's ability to survive inside the host by still uncertain mechanisms. To uncover PknG interactome we developed an affinity purification-mass spectrometry strategy to stepwise recover PknG substrates and interactors; and to identify those involving PknG autophosphorylated docking sites. We report a confident list of 7 new putative substrates and 66 direct or indirect partners indicating that PknG regulates many physiological processes, such as nitrogen and energy metabolism, cell wall synthesis and protein translation. GarA and the 50S ribosomal protein L13, two previously reported substrates of PknG, were recovered in our interactome. Comparative proteome analyses of wild type and pknG null mutant M. tuberculosis strains provided evidence that two kinase interactors, the FHA-domain containing protein GarA and the enzyme glutamine synthetase, are indeed endogenous substrates of PknG, stressing the role of this kinase in the regulation of nitrogen metabolism. Interestingly, a second FHA protein was identified as a PknG substrate. Our results show that PknG phosphorylates specific residues in both glutamine synthetase and FhaA in vitro, and suggest that these proteins are phosphorylated by PknG in living mycobacteria.

KEYWORDS:

Affinity purification-mass spectrometry; FhaA; Mycobacterium tuberculosis; PknG; Serine/Threonine protein kinase; glutamine synthetase

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