title: The C-terminal, third conserved motif of the protein activator PACT plays an essential role in the activation of double-stranded-RNA-dependent protein kinase (PKR).
abstract: One of the key mediators of the antiviral and antiproliferative actions of interferon is double-stranded-RNA-dependent protein kinase (PKR). PKR activity is also involved in the regulation of cell proliferation, apoptosis and signal transduction. We have recently identified PACT, a novel protein activator of PKR, as an important modulator of PKR activity in cells in the absence of viral infection. PACT heterodimerizes with PKR and activates it by direct protein-protein interactions. Endogenous PACT acts as an activator of PKR in response to diverse stress signals, such as serum starvation and peroxide or arsenite treatment, and is therefore a novel, stress-modulated physiological activator of PKR. In this study, we have characterized the functional domains of PACT that are required for PKR activation. Our results have shown that, unlike the N-terminal conserved domains 1 and 2, the third conserved domain of PACT is dispensable for its binding of double-stranded RNA and inter action with PKR. However, a deletion of domain 3 results in a loss of PKR activation ability, in spite of a normal interaction with PKR, thereby indicating that domain 3 plays an essential role in PKR activation. Purified recombinant domain 3 could also activate PKR efficiently in vitro. Our results indicate that, although dispensable for PACT's high-affinity interaction with PKR, the third motif is essential for PKR activation. In addition, domain 3 and eukaryotic initiation factor 2alpha both interact with PKR through the same region within PKR, which we have mapped to lie between amino acid residues 318 and 551.
authors: Department of Biological Sciences, University of South Carolina, 700 Sumter Street, Columbia, SC 29208, U.S.A.
authors: Xu
authors: X
authors: Huang
authors: Brian
authors: B
authors: Hutchins
authors: Rekha C
authors: RC
authors: Patel
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017209:Apoptosis; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000378:metabolism; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D019892:eIF-2 Kinase / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; C104647:PRKRA protein, human; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D019892:eIF-2 Kinase