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Molecular and Cellular Biology, January 2009, p. 254-265, Vol. 29, No. 1
0270-7306/09/$08.00+0     doi:10.1128/MCB.01030-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

TRBP Control of PACT-Induced Phosphorylation of Protein Kinase R Is Reversed by Stress{triangledown} ,{dagger}

Aïcha Daher,1 Ghislaine Laraki,1,2,|| Madhurima Singh,4 Carlos E. Melendez-Peña,1,2,{dagger}{dagger} Sylvie Bannwarth,1,2,{ddagger} Antoine H. F. M. Peters,5,¶ Eliane F. Meurs,6 Robert E. Braun,5,§ Rekha C. Patel,4 and Anne Gatignol1,2,3*

Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research,1 Departments of Microbiology and Immunology,2 Experimental Medicine, McGill University, Montréal, Québec, Canada,3 Department of Biological Sciences, University of South Carolina, Columbia, South Carolina,4 Department of Genome Sciences, University of Washington School of Medicine, Seattle, Washington,5 Department of Virology, Pasteur Institute, Paris, France6

Received 30 June 2008/ Returned for modification 31 July 2008/ Accepted 10 October 2008

The TAR RNA binding Protein, TRBP, inhibits the activity of the interferon-induced protein kinase R (PKR), whereas the PKR activator, PACT, activates its function. TRBP and PACT also bind to each other through their double-stranded RNA binding domains (dsRBDs) and their Medipal domains, which may influence their activity on PKR. In a human immunodeficiency virus (HIV) long terminal repeat-luciferase assay, PACT unexpectedly reversed PKR-mediated inhibition of gene expression. In a translation inhibition assay in HeLa cells, PACT lacking the 13 C-terminal amino acids (PACT{Delta}13), but not full-length PACT, activated PKR and enhanced interferon-mediated repression. In contrast, in the astrocytic U251MG cells that express low TRBP levels, both proteins activate PKR, but PACT{Delta}13 is stronger. Immunoprecipitation assays and yeast two-hybrid assays show that TRBP and PACT{Delta}13 interact very weakly due to a loss of binding in the Medipal domain. PACT-induced PKR phosphorylation was restored in Tarbp2–/– murine tail fibroblasts and in HEK293T or HeLa cells when TRBP expression was reduced by RNA interference. In HEK293T and HeLa cells, arsenite, peroxide, and serum starvation-mediated stresses dissociated the TRBP-PACT interaction and increased PACT-induced PKR activation, demonstrating the relevance of this control in a physiological context. Our results demonstrate that in cells, TRBP controls PACT activation of PKR, an activity that is reversed by stress.

* Corresponding author. Mailing address: Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, 3999 Côte Ste Catherine, Montréal, QC H3T1E2, Canada. Phone: (514) 340-8260, ext. 5284. Fax: (514) 340-7576. E mail: anne.gatignol{at}mcgill.ca

{triangledown} Published ahead of print on 20 October 2008.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

|| Present address: Boehringer-Ingelheim, Laval, Québec, Canada.

{dagger}{dagger} Present address: GSK-Biological, Laval, Québec, Canada.

{ddagger} Present address: Laboratoire de Génétique Moléculaire, Hôpital de l'Archet 2, Nice, France.

Present address: Friedrich Miescher Institute, Basel, Switzerland.

§ Present address: The Jackson Laboratory, Bar Harbor, ME 04609.

Molecular and Cellular Biology, January 2009, p. 254-265, Vol. 29, No. 1
0270-7306/09/$08.00+0     doi:10.1128/MCB.01030-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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