New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach
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 spectromet...
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todo:paper_18743919_v192_n_p321_Gil2023-10-03T16:34:06Z New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach Gil, M. Lima, A. Rivera, B. Rossello, J. Urdániz, E. Cascioferro, A. Carrión, F. Wehenkel, A. Bellinzoni, M. Batthyány, C. Pritsch, O. Denicola, A. Alvarez, M.N. Carvalho, P.C. Lisa, M.-N. Brosch, R. Piuri, M. Alzari, P.M. Durán, R. Affinity purification-mass spectrometry FhaA glutamine synthetase Mycobacterium tuberculosis PknG Serine/Threonine protein kinase 50s ribosomal protein L13 60 kda chaperonin 1 acetyl propionyl coenzyme A carboxylase alpha chain alcohol dehydrogenase iron containing protein bacterial enzyme fhaa protein gara protein glutamate ammonia ligase inorganic pyrophosphatase pkng protein protein DnaK protein serine threonine kinase unclassified drug affinity chromatography Article autophosphorylation bacterial cell wall bacterial strain controlled study enzyme analysis enzyme phosphorylation enzyme purification enzyme substrate ferredoxin iron sulfur binding protein in vitro study mass spectrometry molecular docking Mycobacterium tuberculosis nitrogen fixation nonhuman priority journal translation regulation 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. © 2018 Elsevier B.V. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_18743919_v192_n_p321_Gil |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Affinity purification-mass spectrometry FhaA glutamine synthetase Mycobacterium tuberculosis PknG Serine/Threonine protein kinase 50s ribosomal protein L13 60 kda chaperonin 1 acetyl propionyl coenzyme A carboxylase alpha chain alcohol dehydrogenase iron containing protein bacterial enzyme fhaa protein gara protein glutamate ammonia ligase inorganic pyrophosphatase pkng protein protein DnaK protein serine threonine kinase unclassified drug affinity chromatography Article autophosphorylation bacterial cell wall bacterial strain controlled study enzyme analysis enzyme phosphorylation enzyme purification enzyme substrate ferredoxin iron sulfur binding protein in vitro study mass spectrometry molecular docking Mycobacterium tuberculosis nitrogen fixation nonhuman priority journal translation regulation |
| spellingShingle |
Affinity purification-mass spectrometry FhaA glutamine synthetase Mycobacterium tuberculosis PknG Serine/Threonine protein kinase 50s ribosomal protein L13 60 kda chaperonin 1 acetyl propionyl coenzyme A carboxylase alpha chain alcohol dehydrogenase iron containing protein bacterial enzyme fhaa protein gara protein glutamate ammonia ligase inorganic pyrophosphatase pkng protein protein DnaK protein serine threonine kinase unclassified drug affinity chromatography Article autophosphorylation bacterial cell wall bacterial strain controlled study enzyme analysis enzyme phosphorylation enzyme purification enzyme substrate ferredoxin iron sulfur binding protein in vitro study mass spectrometry molecular docking Mycobacterium tuberculosis nitrogen fixation nonhuman priority journal translation regulation Gil, M. Lima, A. Rivera, B. Rossello, J. Urdániz, E. Cascioferro, A. Carrión, F. Wehenkel, A. Bellinzoni, M. Batthyány, C. Pritsch, O. Denicola, A. Alvarez, M.N. Carvalho, P.C. Lisa, M.-N. Brosch, R. Piuri, M. Alzari, P.M. Durán, R. New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach |
| topic_facet |
Affinity purification-mass spectrometry FhaA glutamine synthetase Mycobacterium tuberculosis PknG Serine/Threonine protein kinase 50s ribosomal protein L13 60 kda chaperonin 1 acetyl propionyl coenzyme A carboxylase alpha chain alcohol dehydrogenase iron containing protein bacterial enzyme fhaa protein gara protein glutamate ammonia ligase inorganic pyrophosphatase pkng protein protein DnaK protein serine threonine kinase unclassified drug affinity chromatography Article autophosphorylation bacterial cell wall bacterial strain controlled study enzyme analysis enzyme phosphorylation enzyme purification enzyme substrate ferredoxin iron sulfur binding protein in vitro study mass spectrometry molecular docking Mycobacterium tuberculosis nitrogen fixation nonhuman priority journal translation regulation |
| description |
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. © 2018 Elsevier B.V. |
| format |
JOUR |
| author |
Gil, M. Lima, A. Rivera, B. Rossello, J. Urdániz, E. Cascioferro, A. Carrión, F. Wehenkel, A. Bellinzoni, M. Batthyány, C. Pritsch, O. Denicola, A. Alvarez, M.N. Carvalho, P.C. Lisa, M.-N. Brosch, R. Piuri, M. Alzari, P.M. Durán, R. |
| author_facet |
Gil, M. Lima, A. Rivera, B. Rossello, J. Urdániz, E. Cascioferro, A. Carrión, F. Wehenkel, A. Bellinzoni, M. Batthyány, C. Pritsch, O. Denicola, A. Alvarez, M.N. Carvalho, P.C. Lisa, M.-N. Brosch, R. Piuri, M. Alzari, P.M. Durán, R. |
| author_sort |
Gil, M. |
| title |
New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach |
| title_short |
New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach |
| title_full |
New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach |
| title_fullStr |
New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach |
| title_full_unstemmed |
New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach |
| title_sort |
new substrates and interactors of the mycobacterial serine/threonine protein kinase pkng identified by a tailored interactomic approach |
| url |
http://hdl.handle.net/20.500.12110/paper_18743919_v192_n_p321_Gil |
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