Characterization of the phosphorylation sites of Mycobacterium tuberculosis serine/threonine protein kinases, PknA, PknD, PknE, and PknH by mass spectrometry.
In Mycobacterium tuberculosis (Mtb), regulatory phosphorylation of proteins at serine and/or threonine residues by serine/threonine protein kinases (STPKs) is an emerging theme connected with the involvement of these enzymes in virulence mechanisms. The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to identify the corresponding interaction networks. Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in STPKs is a major challenge in proteomics since some of these enzymes might be interesting therapeutical targets. Using different strategies to identify phosphorylated residues, we report, in the present work, MS studies of the entire intracellular regions of recombinant protein kinases PknA, PknD, PknE, and PknH from Mtb. The 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, seven and nine phosphorylated serine and/or threonine residues were identified as phosphorylation sites in the recombinant intracellular regions of PknA and PknH, respectively. The same technique led also to the identification of seven phosphorylation sites in each of the two recombinant kinases, PknD and PknE.
Molle
Virginie
V
Institute of Biology and Chemistry of Proteins, CNRS/University of Lyon, IFR128, 7 passage du Vercors, 69367 Lyon Cedex 07, France.
Zanella-Cleon
Isabelle
I
Robin
Jean-Philippe
JP
Mallejac
Souen
S
Cozzone
Alain J
AJ
Becchi
Michel
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D000596:Amino Acids; D017931:DNA Primers; D011994:Recombinant Proteins; D011494:Protein Kinases