Survival of Pathogenic Mycobacteria in Macrophages Is Mediated through Autophosphorylation of Protein Kinase G

ABSTRACT Pathogenic mycobacteria survive within macrophages through the inhibition of phagosome-lysosome fusion. A crucial factor for avoiding lysosomal degradation is the mycobacterial serine/threonine protein kinase G (PknG). PknG is released into the macrophage cytosol upon mycobacterial infection, suggesting that PknG might exert its activity by interfering with host signaling cascades, but the mode of action of PknG remains unknown. Here, we show that PknG undergoes autophosphorylation on threonine residues located at the N terminus. In contrast to all other mycobacterial kinases investigated thus far, autophosphorylation of PknG was not involved in the regulation of its kinase activity. However, autophosphorylation was crucial for the capacity of PknG to promote mycobacterial survival within macrophages. These results will contribute to a better understanding of the virulence mechanisms of pathogenic mycobacteria and may help to design improved inhibitors of PknG to be developed as antimycobacterial compounds.

[1]  Y Av-Gay,et al.  The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis. , 2000, Trends in microbiology.

[2]  J. Pieters,et al.  A Coat Protein on Phagosomes Involved in the Intracellular Survival of Mycobacteria , 1999, Cell.

[3]  J. Pieters.  Manipulation of the Macrophage Response by Pathogenic Mycobacteria , 2008 .

[4]  Susan S. Taylor,et al.  Regulation of protein kinases; controlling activity through activation segment conformation. , 2004, Molecular cell.

[5]  Liem Nguyen,et al.  Protein Kinase G from Pathogenic Mycobacteria Promotes Survival Within Macrophages , 2004, Science.

[6]  M. Pallen,et al.  Bacterial FHA domains: neglected players in the phospho-threonine signalling game? , 2002, Trends in microbiology.

[7]  N. Srinivasan,et al.  EmbR, a regulatory protein with ATPase activity, is a substrate of multiple serine/threonine kinases and phosphatase in Mycobacterium tuberculosis , 2006, The FEBS journal.

[8]  Philip E. Bourne,et al.  Structural Evolution of the Protein Kinase–Like Superfamily , 2005, PLoS Comput. Biol..

[9]  V. Molle,et al.  Two FHA domains on an ABC transporter, Rv1747, mediate its phosphorylation by PknF, a Ser/Thr protein kinase from Mycobacterium tuberculosis. , 2004, FEMS microbiology letters.

[10]  David G. Russell,et al.  Mycobacterium tuberculosis: here today, and here tomorrow , 2001, Nature Reviews Molecular Cell Biology.

[11]  J. Pieters,et al.  Application of an improved density gradient electrophoresis apparatus to the separation of proteins, cells and subcellular organelles , 1993, Electrophoresis.

[12]  A. Ullrich,et al.  Serine/threonine protein kinases PknF and PknG of Mycobacterium tuberculosis: characterization and localization. , 2001, Microbiology.

[13]  J. Pieters,et al.  Subcellular fractionation by organelle electrophoresis: Separation of phagosomes containing heat‐killed yeast particles , 1998, Electrophoresis.

[14]  J. Corbin,et al.  Autophosphorylation: a salient feature of protein kinases , 1993, Molecular and Cellular Biochemistry.

[15]  Liem Nguyen,et al.  Role of Protein Kinase G in Growth and Glutamine Metabolism of Mycobacterium bovis BCG , 2005, Journal of bacteriology.

[16]  Daniel Durocher,et al.  The FHA domain , 2002, FEBS letters.

[17]  B. Barrell,et al.  Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence , 1998, Nature.

[18]  T. Parish,et al.  Mycobacterium : genomics and molecular biology , 2009 .

[19]  J. Pieters,et al.  Survival of Mycobacteria in Macrophages Is Mediated by Coronin 1-Dependent Activation of Calcineurin , 2007, Cell.

[20]  L. Johnson,et al.  Active and Inactive Protein Kinases: Structural Basis for Regulation , 1996, Cell.

[21]  S. Cole,et al.  Conserved autophosphorylation pattern in activation loops and juxtamembrane regions of Mycobacterium tuberculosis Ser/Thr protein kinases. , 2005, Biochemical and biophysical research communications.

[22]  S. Cole,et al.  PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho‐Ser/Thr phosphatase, in Mycobacterium tuberculosis , 2003, Molecular microbiology.

[23]  Tom Alber,et al.  Mycobacterium tuberculosis serine/threonine kinases PknB, PknD, PknE, and PknF phosphorylate multiple FHA domains , 2005, Protein science : a publication of the Protein Society.

[24]  M. Steinmetz,et al.  Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis , 2007, Proceedings of the National Academy of Sciences.

[25]  H. Ploegh,et al.  Isolation and characterization of the intracellular MHC class II compartment , 1994, Nature.

[26]  G. Besra,et al.  An FHA phosphoprotein recognition domain mediates protein EmbR phosphorylation by PknH, a Ser/Thr protein kinase from Mycobacterium tuberculosis. , 2003, Biochemistry.

[27]  S. Kaufmann,et al.  Handbook of tuberculosis : Molecular biology and biochemistry , 2008 .

[28]  J. Pieters,et al.  Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway , 1997, Molecular microbiology.

[29]  P. Alzari,et al.  Regulation of glutamate metabolism by protein kinases in mycobacteria , 2008, Molecular microbiology.

[30]  W. N. Burnette,et al.  "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. , 1981, Analytical biochemistry.

[31]  Carl A. Miecskowski,et al.  Structure/Function Studies of Ser/Thr and Tyr Protein Phosphorylation in Mycobacterium tuberculosis , 2006, Journal of Molecular Microbiology and Biotechnology.