Serine 18 Phosphorylation of RAX, the PKR Activator, Is Required for PKR Activation and Consequent Translation Inhibition

• Published in: The Journal of biological chemistry 2004-10, Vol.279 (41), p.42687-42693
• Main Authors: Bennett, Richard L, Blalock, William L, May, W Stratford
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Annexin A5 - pharmacology; Apoptosis; Binding Sites; Blotting, Western; Cell Death; Cell Survival; eIF-2 Kinase - metabolism; Enzyme Activation; Eukaryotic Initiation Factor-2 - metabolism; Eye Proteins - metabolism; Flow Cytometry; Genes, Dominant; Homeodomain Proteins - metabolism; Humans; Immunoprecipitation; Interleukin-3 - metabolism; Mice; Models, Biological; Molecular Sequence Data; Phosphorylation; Protein Biosynthesis; Protein Processing, Post-Translational; RNA, Double-Stranded - chemistry; Sequence Homology, Amino Acid; Serine - chemistry; Signal Transduction; Time Factors; Transcription Factors - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M403321200

It is now apparent that the double-stranded (ds)RNA-dependent protein kinase, PKR, is a regulator of diverse cellular responses to stress. Recently, the murine dsRNA-binding protein RAX and its human ortholog PACT were identified as cellular activators of PKR. Previous reports demonstrate that following stress, RAX/PACT associates with and activates PKR resulting in eIF2α phosphorylation, consequent translation inhibition, and cell death via apoptosis. Although RAX/PACT is phosphorylated during stress, any regulatory role for this post-translational modification has been uncertain. Now we have discovered that RAX is phosphorylated on serine 18 in both human and mouse cells. The non-phosphorylatable form of RAX, RAX(S18A), although still able to bind dsRNA and associate with PKR, fails to activate PKR following stress. Furthermore, stable expression of RAX(S18A) results in a dominant-negative effect characterized by deficiency of eukaryotic initiation factor 2 α subunit phosphorylation, delay of translation inhibition, and failure to undergo rapid apoptosis following removal of interleukin-3. We propose that the ability of RAX to activate PKR is regulated by a sequential mechanism featuring RAX association with PKR, RAX phosphorylation at serine 18, and activation of PKR.