Identification of functional domains of the interferon-induced enzyme PKR in cells lacking endogenous PKR.
The interferon (IFN)-induced, double-stranded RNA (dsRNA)-activated human protein kinase (PKR) has been shown to exert antiviral and antiproliferative effects. Activation of the enzyme in mammalian cells results in protein synthesis inhibition and cell death by apoptosis. Previous studies on the structure-function relationship of PKR have been based on vectors expressing the enzyme in mammalian cells containing endogenous PKR. As exogenously expressed PKR can form heterodimers with endogenous PKR, the results obtained on the functional characterization of mutant forms of PKR have been taken with caution. To address the natural consequences of heterodimer formation between endogenous and exogenous PKR, we have analyzed the structure-function relationship of PKR ectopically expressed from vaccinia virus (VV) recombinants in cells lacking the endogenous enzyme. We demonstrate that PKR-mediated inhibition of protein synthesis and induction of apoptosis is not dependent on the presence of endogenous PKR. Further, PKR activity is independent of the presence of dsRNA binding motifs (dsRBM). Moreover, single-point mutations of the third basic domain decreased PKR activation. Our findings demonstrate that PKR can be activated in the absence of its N-terminal domain (amino acids 1-232) and that the third basic domain is important for its biologic function.
Rivas
C
C
Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Gil
J
J
Esteban
M
M
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D020134:Catalytic Domain; D004790:Enzyme Induction; D006801:Humans; D007372:Interferons / Q000494:pharmacology*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009154:Mutation; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D013329:Structure-Activity Relationship; D014616:Vaccinia virus; D019892:eIF-2 Kinase / Q000096:biosynthesis* / Q000235:genetics
D000998:Antiviral Agents; D011500:Protein Synthesis Inhibitors; D007372:Interferons; D019892:eIF-2 Kinase