Mutations in the double-stranded RNA-activated protein kinase insert region that uncouple catalysis from eIF2alpha binding.
The interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase, PKR, inhibits protein synthesis via phosphorylation of the alpha subunit of the translation initiation factor eIF2. A kinase insert region N-terminal of PKR kinase subdomain V, which is conserved among eIF2alpha kinases, has been proposed to determine substrate specificity of these kinases. To investigate the function of this kinase insert region, selective PKR mutants were generated, and kinase activities and eIF2alpha affinities were analyzed in vitro. The in vivo function was investigated by growth inhibitory assays in yeast and translational assays in COS cells. Among the 13 mutations, 5 lost kinase activity and 3 exhibited less than 30% of wild-type eIF2alpha binding activity. The deletion of the conserved sequence (amino acids 362-370) resulted in a protein that had no kinase activity and only about 25% of wild-type eIF2alpha binding, suggesting that this sequence is not only required for PKR kinase activity but also is important for substrate interaction. It was determined that the hydrophobicity of the conserved sequence of PKR is required for kinase activity but is not crucial for eIF2alpha binding. The amino acid residue Glu-367 in the conserved motif was shown to be directly involved in substrate binding but was not important for kinase activity. These results suggest that the activation of PKR is not a prerequisite for its binding to the substrate and that the conserved motif in subdomain V contributes to the interaction of PKR and eIF2alpha.
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Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Williams
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D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D003001:Cloning, Molecular; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
D015852:Eukaryotic Initiation Factor-2; D019892:eIF-2 Kinase