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 pre-fractionation, TiO2 enrichment and liquid chromatograpy–tandem mass spectrometry (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 (CpcBs) were found to be phosphorylated on Ser22, Ser49, Thr94 and Ser154. 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 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 CpcBs 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.
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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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    • "In addition to PknA and PknB, another group of STPKs comprised of PknG, PknL, and PknF appear to be involved in different aspects of M. tuberculosis growth regulation (Cowley et al., 2004; Deol et al., 2005; Canova et al., 2008). In support of the likely important role played by STPKs in M. tuberculosis, a mass spectrometry-based phosphoproteomic study identified more than 500 Ser/Thr phosphorylation sites on 300 proteins in M. tuberculosis (Prisic et al., 2010) and, more recently, a complementary study detected a number of Tyr phosphorylated proteins in M. tuberculosis (Kusebauch et al., 2014). "
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