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Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE
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A diminished activation capacity of the interferon-inducible protein kinase PKR in human T lymphocytes

S Li1, K Nagai, A E Koromilas

  • 1Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montréal, Québec, Canada.

European Journal of Biochemistry
|March 11, 2000
PubMed

Insights

Double-stranded RNA activated protein kinase (PKR) activation is impaired in human T cells due to a 60-kDa phosphoprotein (p60) that inhibits PKR. This impairment occurs post-translationally and is independent of cell transformation or viral infection.

Area of Science:

  • Immunology
  • Molecular Biology
  • Cell Signaling

Background:

  • The double-stranded RNA activated protein kinase (PKR) is an interferon-inducible enzyme crucial for regulating protein synthesis.
  • PKR activation is triggered by viral infection or double-stranded RNA and is modulated by various factors.
  • Understanding PKR activation mechanisms is vital for comprehending cellular responses to pathogens and immune signaling.

Purpose of the Study:

  • To investigate and compare the mechanisms of PKR activation across different human cell lines.
  • To elucidate the reasons behind diminished PKR activation observed in human T cells.

Main Methods:

  • Comparative analysis of PKR activation in various human cell lines, including T cells and non-T cells.
  • In vitro kinase assays to assess PKR activity and autophosphorylation.
  • Co-immunoprecipitation to identify interacting proteins with PKR.
  • Treatment with proteasome and phosphatase inhibitors to explore regulatory mechanisms.

Main Results:

  • PKR activation and eIF-2alpha phosphorylation are significantly reduced in both virus-transformed and non-transformed human T cells.
  • A 60-kDa phosphoprotein (p60) was found to co-immunoprecipitate with PKR in T cells but not in non-T cells.
  • T cell extracts containing phosphorylated p60 inhibited PKR autophosphorylation in vitro.
  • Phosphorylation of p60 was enhanced by phosphatase inhibitors, suggesting a role for dephosphorylation in its regulation.

Conclusions:

  • Impaired PKR activation in human T cells is associated with a p60 phosphoprotein that inhibits PKR activity.
  • This inhibition mechanism operates at the post-translational level and is independent of cell transformation or viral infection.
  • The findings reveal a novel regulatory pathway for PKR in T cells, impacting protein synthesis and cellular responses.

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