Conserved autophosphorylation pattern in activation loops and juxtamembrane regions of Mycobacterium tuberculosis Ser/Thr protein kinases.
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.
Durán
Rosario
R
Laboratorio de Bioquímica Analítica, Instituto de Investigaciones Biológicas Clemente Estable and Facultad de Ciencias, Montevideo, Uruguay.
Villarino
Andrea
A
Bellinzoni
Marco
M
Wehenkel
Annemarie
A
Fernandez
Pablo
P
Boitel
Brigitte
B
Cole
Stewart T
ST
Alzari
Pedro M
PM
Cerveñansky
Carlos
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D017931:DNA Primers; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases