Serine 18 phosphorylation of RAX, the PKR activator, is required for PKR activation and consequent translation inhibition.
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 eIF2alpha 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 alpha 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.
University of Florida College of Medicine, Shands Cancer Center, Gainesville, Florida 32610, USA.
Richard L
RL
Bennett
William L
WL
Blalock
W Stratford
WS
May
D000595:Amino Acid Sequence; D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D017209:Apoptosis; D001665:Binding Sites; D015153:Blotting, Western; D016923:Cell Death; D002470:Cell Survival; D004789:Enzyme Activation; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism*; D005434:Flow Cytometry; D005799:Genes, Dominant; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D007377:Interleukin-3 / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012330:RNA, Double-Stranded / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry*; D015398:Signal Transduction; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000378:metabolism*
D017304:Annexin A5; D015852:Eukaryotic Initiation Factor-2; D005136:Eye Proteins; D018398:Homeodomain Proteins; D007377:Interleukin-3; C486393:RAX protein, human; D012330:RNA, Double-Stranded; D014157:Transcription Factors; D012694:Serine; D019892:eIF-2 Kinase