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

Division of Infectious Diseases, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2010; 107(16):7521-6. DOI: 10.1073/pnas.0913482107
Source: PubMed


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.

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    • "To study the conservation of phosphoproteins among bacterial prokaryotes, we compared the Synechocystis phosphoproteome with other bacterial phosphoproteomes reported previously (Macek et al. 2007, Voisin et al. 2007, Macek et al. 2008, Soufi et al. 2008, Aivaliotis et al. 2009, Lin et al. 2009, Ravichandran et al. 2009, Parker et al. 2010, Prisic et al. 2010, Schmidl et al. 2010, Sun et al. 2010, Ge et al. 2011, Manteca et al. 2011, Misra et al. 2011, Bai and Ji 2012, Yang et al. 2013). Eleven phosphoproteins have been detected using a 2D gel electrophoresis-based protocol in Synechocystis (Mikkat et al. 2014). "
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    ABSTRACT: Synechocystis sp. PCC 6803 (hereafter Synechocystis) is a model cyanobacterium and has been used extensively for studies concerned with photosynthesis and environmental adaptation. Although dozens of protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues have been predicted, only a few substrate proteins are known in Synechocystis. In this study, we report 194 in vivo phosphorylation sites from 149 proteins in Synechocystis, which were identified using a combination of peptide prefractionation, TiO2 enrichment, and LC-MS/MS analysis. These phosphorylated proteins are implicated in diverse biological processes, such as photosynthesis. Among all identified phosphoproteins involved in photosynthesis, the β subunits of phycocyanins (CpcB) were found to be phosphorylated on Ser-22, Ser-49, Thr-94 and Ser-154. Four non-phosphorylated mutants were constructed by using site-directed mutagenesis. The in vivo characterization of the cpcB mutants showed a slower growth under high light irradiance and displayed fluorescence quenching to a lower lying level and less efficient energy transfer inside the phycobilisome (PBS). Notably, the non-phosphorylated mutants exhibited a slower state transition than the wild type. The current results demonstrated that the phosphorylation status of CpcB affects the energy transfer and state transition of photosynthesis in Synechocystis. This study provides novel insights into the molecular mechanisms of protein phosphorylation in the regulation of photosynthesis in cyanobacteria and may facilitate the elucidation of the entire regulatory network by linking kinases to their physiological substrates.
    Plant and Cell Physiology 08/2015; DOI:10.1093/pcp/pcv118 · 4.93 Impact Factor
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    • "Whilst TCS TRs are regulated by reversible phosphorylation, it is widely presumed that other bacterial TRs are regulated by reversible ligand binding and not by phosphorylation. However, recent phosphoproteomics analyses have revealed that many bacterial TRs can be phosphorylated on serine, threonine and tyrosine residues (Macek et al., 2007; Prisic et al., 2010; Soufi et al., 2010; Derouiche et al., 2013). Moreover, some recent studies suggest that non-TCS bacterial TRs can also be phosphorylated on arginine (Schmidt et al., 2014), histidine (Hammerstrom et al., 2015) and cysteine residues (Sun et al., 2012). "
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    ABSTRACT: Reversible phosphorylation of bacterial transcriptional regulators belonging to the family of two-component systems is a well-established mechanism for regulating gene expression. Recent evidence points to the fact that reversible phosphorylation of bacterial transcriptional regulators on other types of residues, namely serine, threonine, tyrosine and cysteine, is also quite common. The phosphorylation of the ester type (phospho-serine/threonine/tyrosine) is more stable than the aspartate phosphorylation of two-component systems. The kinases which catalyze these phosphorylation events (Hanks-type serine/threonine-protein kinases and bacterial protein-tyrosine kinases) are also much more promiscuous than the two-component system kinases, i.e. each of them can phosphorylate several substrate proteins. By consequence, the dynamics and the topology of the signal transduction networks depending on these kinases differ significantly from the two component systems. Here we present an overview of different classes of bacterial transcriptional regulators phosphorylated and regulated by serine/threonine- and tyrosine-kinases. Particular attention is given to examples when serine/threonine- and tyrosine-kinases interact with two component systems, phosphorylating either the histidine kinases or the response regulators. We argue that these promiscuous kinases connect several signal transduction pathways, and serve the role of signal integration.
    Microbiology 07/2015; 161(9). DOI:10.1099/mic.0.000148 · 2.56 Impact Factor
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    • "Prisic et al. described the Serine/Threonine (S/T) phosphorylation profiles of the laboratory strain M. tuberculosis H 37 Rv under 6 different culture conditions (Prisic et al., 2010). This study identified 301 phosphorylated proteins after combining data from six different culture conditions (Prisic et al., 2010) and identified four phosphorylated STPKs, ribosomal and ribosome-associated proteins as well as phosphorylated substrates which suggest that protein phosphorylation provides a mechanism for regulating key physiological process during infection. A more recent study of H 37 Rv further expanded the knowledge of the phosphoproteome by identifying novel tyrosine (Y) phosphorylated proteins in M. tuberculosis further supporting the broad regulation of its physiology by phosphorylation (Kusebauch et al., 2014). "
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    ABSTRACT: Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.
    Frontiers in Microbiology 02/2015; 6. DOI:10.3389/fmicb.2015.00006 · 3.99 Impact Factor
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