Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases

Abstract

The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs) that are structurally related to eukaryotic kinases. To gain insight into the role of Ser/Thr phosphorylation in this major global pathogen, we used a phosphoproteomic approach to carry out an extensive analysis of protein phosphorylation in M. tuberculosis. We identified more than 500 phosphorylation events in 301 proteins that are involved in a broad range of functions. Bioinformatic analysis of quantitative in vitro kinase assays on peptides containing a subset of these phosphorylation sites revealed a dominant motif shared by six of the M. tuberculosis STPKs. Kinase assays on a second set of peptides incorporating targeted substitutions surrounding the phosphoacceptor validated this motif and identified additional residues preferred by individual kinases. Our data provide insight into processes regulated by STPKs in M. tuberculosis and create a resource for understanding how specific phosphorylation events modulate protein activity. The results further provide the potential to predict likely cognate STPKs for newly identified phosphoproteins.

Fig. 1.

Flow diagram of approach to Ser/Thr protein phosphorylation detection and phosphorylation site motif identification and verification in M. tuberculosis.

Fig. 2.

Phosphorylation site motif analysis. pLOGos generated using motif-x based on in vitro assays in which peptide phosphorylation was >3-fold above median are shown for PknA, PknB, PknD, PknE, PknF, and PknH. The pLOGos show the relative statistical significance (with respect to the M. tuberculosis proteomic background) of residues within 6 aa of the central Thr phosphorylation site. Residues above the midline are overrepresented, whereas those below the midline are underrepresented. The red horizontal line indicates the 0.01 significance level (after Bonferroni correction).

Fig. 3.

In vitro phosphorylation of ideal (WT) and substituted peptides by the six kinases that share a substrate phosphorylation site motif. A peptide from Rv0497 that has features of the shared phosphorylation site motif was synthesized, together with 31 additional peptides incorporating substitutions predicted to enhance or diminish phosphorylation. The phosphoacceptor is shown in bold. Substituted residues are underlined. Phosphorylation of each peptide is expressed as a ratio relative to phosphorylation of the original peptide (WT). Substitutions that increase or decrease phosphorylation by 50% or more are shaded in green or red, respectively. Mean values of two independent experiments are shown.

Fig. 4.

Model of PknB structure in a complex with an ideal peptide substrate. (A) The PknB kinase domain in complex with an ideal peptide substrate (AELTGEIPI) was modeled using x-ray crystal structures of the M. tuberculosis PknB kinase domain [Protein Data Bank (PDB) no. 1o6y] and of a Phk-peptide substrate complex (PDB no. 2phk). ATP is in red, and the peptide in blue. (B) Likely contacts between PknB active site residues and the peptide, within 4 Å, are shown. Colors of PknB residues correspond to highlighted amino acids shown in C. The peptide is in blue except for the phosphoacceptor Thr, which is yellow. Hydrogen atoms and the main chain atoms were omitted, except for hydrogen bond contacts. (C) Alignment of the kinase domains of all M. tuberculosis STPKs, highlighting residues that are predicted to be in close contact with the peptide. The numbers in the color code indicate the position in the peptide substrate with which the residues shaded in that color may interact. In addition to the interactions indicated by the color code, kinase residues labeled with “*” are predicted to interact with the −2 position, “#” with the +2 position, and “&” with +2 and +5 positions.

Fig. 5.

Kinase activity of WT and substituted forms of PknB. (A) Val176 of PknB was mutated to Asp or Arg, and in vitro phosphorylation of an ideal peptide (peptide 1 in Fig. 3) by WT and substituted PknB proteins was performed and quantified by ³³P incorporation at serial time points. (B) Autoradiography of WT and substituted PknB after incubation with γ-P³³-ATP followed by SDS/PAGE. (C) GelCode Blue (Pierce) stained gel showing approximately equal amounts of protein loaded on the gel.