Phosphorylation of specific serine residues in the PKR activation domain of PACT is essential for its ability to mediate apoptosis.
Activation of the latent protein kinase, PKR, by extracellular stresses and triggering of resultant cellular apoptosis are mediated by the protein, PACT, which itself gets phosphorylated in stressed cells. We have analyzed the underlying biochemical mechanism by carrying out alanine-scanning mutagenesis of the PKR activation domain of PACT. Among the indispensable residues identified were two serine residues, whose phosphorylation was essential for the cellular actions of PACT. Two-dimensional gel analysis, Western analysis using phosphoamino acid-specific antiserum, and in vivo 32P labeling of PACT demonstrated that constitutive phosphorylation of one of the two residues, Ser246, was required for stress-induced phosphorylation of the other, Ser287. Substitution of either of them by threonine or aspartic acid, but not alanine, was tolerated. Substitution of both residues with the phosphoserine mimetic, aspartic acid, produced a mutant PACT that, unlike the wild-type protein, caused PKR activation and apoptosis, even in unstressed cells. These results indicate that phosphorylation of specific serine residues in the activation domain of PACT is the major mode of transmission of cellular stress response to PKR.
Peters
Gregory A
GA
Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Li
Shoudong
S
Sen
Ganes C
GC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017209:Apoptosis / Q000502:physiology*; D001665:Binding Sites; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012694:Serine; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
C104647:PRKRA protein, human; D016601:RNA-Binding Proteins; D012694:Serine; D019892:eIF-2 Kinase