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Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis
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Published on: August 15, 2013

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M. tuberculosis Ser/Thr protein kinase D phosphorylates an anti-anti-sigma factor homolog

Andrew E Greenstein1, Jason A MacGurn, Christina E Baer

  • 1Department of Molecular and Cell Biology, University of California, Berkeley, California, United States of America.

Plos Pathogens
|April 7, 2007
PubMed

Insights

Mycobacterium tuberculosis protein kinase D (PknD) regulates gene transcription by phosphorylating Rv0516c, an anti-anti-sigma factor. This phosphorylation inhibits Rv0516c binding, altering the bacterium's transcriptional program.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • Receptor Ser/Thr protein kinases in Mycobacterium tuberculosis (Mtb) are implicated in regulating developmental changes and disease processes.
  • The specific functions of these kinases, particularly in transcriptional regulation, remain largely unestablished.

Purpose of the Study:

  • To investigate the role of Mtb protein kinase D (PknD) in regulating gene transcription.
  • To elucidate the mechanism by which PknD influences sigma factor regulatory pathways.

Main Methods:

  • Overexpression of Mtb PknD in Mycobacterium tuberculosis.
  • In vitro kinase assays using purified PknD, PknB, and PknE kinase domains.
  • Phosphorylation site analysis of Rv0516c.
  • In vitro binding assays between Rv0516c and Rv2638.

Main Results:

  • PknD overexpression altered the transcription of numerous Mtb genes, including Rv0516c and genes regulated by sigma factor F.
  • PknD directly phosphorylated Rv0516c at Thr2 in a unique N-terminal extension, a site distinct from consensus phosphorylation sites.
  • This phosphorylation event inhibited the binding of Rv0516c to its homologous anti-anti-sigma factor, Rv2638.
  • PknB and PknE phosphorylated different sigma regulators, indicating kinase-specific functions.

Conclusions:

  • PknD plays a crucial role in modulating Mtb's transcriptional program.
  • Phosphorylation of Rv0516c by PknD at an unconventional site serves as a regulatory mechanism to control gene expression.
  • This signaling pathway, involving PknD and Rv0516c, represents a novel mechanism for regulating Mtb virulence and adaptation.

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