PACT-mediated enhancement of reporter gene expression at the translational level.
The cellular protein, PACT, can directly activate protein kinase (PKR) in vitro by the interaction of PACT domain 3 with PKR. In contrast, in vivo, PACT-mediated PKR activation and concomitant inhibition of protein synthesis require additional cellular stresses. We observed that without such stresses, cotransfection of a PACT expression vector with various reporter genes enhances their levels of expression. This effect was promoter and inducer-independent and PACT specific and mediated by PACT domains 1 and 2. PACT did not increase the level of the reporter mRNA but enhanced its translation by suppressing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) caused by the transfection process. To further examine the phenomenon, we generated cell lines expressing a PACT mutant containing only domains 1 and 2. Reporter gene expression was higher and eIF2alpha phosphorylation was lower in such cell lines compared with the corresponding control cells. Thus, different domains of PACT can either promote or inhibit translation by appropriately modulating the status of eIF2alpha phosphorylation.
Li
Shoudong
S
Department of Molecular Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Sen
Ganes C
GC
D001483:Base Sequence; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D016899:Interferon-beta / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C104647:PRKRA protein, human; D010446:Peptide Fragments; D016601:RNA-Binding Proteins; D016899:Interferon-beta