Conserved autophosphorylation pattern in activation loops and juxtamembrane regions of Mycobacterium tuberculosis Ser/Thr protein kinases

• Published in: Biochemical and biophysical research communications 2005-08, Vol.333 (3), p.858-867
• Main Authors: Durán, Rosario, Villarino, Andrea, Bellinzoni, Marco, Wehenkel, Annemarie, Fernandez, Pablo, Boitel, Brigitte, Cole, Stewart T., Alzari, Pedro M., Cerveñansky, Carlos
• Format: Article
• Language: English
• Subjects: Activation loop; Amino Acid Sequence; Bacterial signaling; Base Sequence; Biochemistry, Molecular Biology; Bioinformatics; Biological Physics; Cell Membrane; Cell Membrane - enzymology; Cellular Biology; Chemical Sciences; Computer Science; Cristallography; DNA Primers; Juxtamembrane region; Life Sciences; Mass spectrometry; Molecular Sequence Data; Mutagenesis, Site-Directed; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; Phosphopeptide identification; Phosphorylation; Physics; Protein-Serine-Threonine Kinases; Protein-Serine-Threonine Kinases - chemistry; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Recombinant Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Ser/Thr kinase autophosphorylation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structural Biology
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2005.05.173

The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to establish interaction networks involving signaling elements. Using different strategies to identify phosphorylated residues, we report here mass spectrometry studies of the entire intracellular regions of four ‘receptor-like’ protein kinases from Mycobacterium tuberculosis (PknB, PknD, PknE, and PknF), each consisting of an N-terminal kinase domain and a juxtamembrane region of varying length (26–100 residues). The enzymes were observed to incorporate different numbers of phosphates, from five in PknB up to 11 in PknD or PknE, and all detected sites were dephosphorylated by the cognate mycobacterial phosphatase PstP. Comparison of the phosphorylation patterns reveals two recurrent clusters of pThr/pSer residues, respectively, in their activation loops and juxtamembrane regions, which have a distinct effect on kinase activity. All studied kinases have at least two conserved phosphorylated residues in their activation loop and mutations of these residues in PknB significantly decreased the kinase activity, whereas deletion of the entire juxtamembrane regions in PknB and PknF had little effect on their activities. These results reinforce the hypothesis that mycobacterial kinase regulation includes a conserved activation loop mechanism, and suggest that phosphorylation sites in the juxtamembrane region might be involved in putative kinase-mediated signaling cascades.