Double-Stranded-RNA-Dependent Protein Kinase and TAR RNA-Binding Protein Form Homo- and Heterodimers in vivo

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1995-10, Vol.92 (21), p.9445-9449
Main Authors: Cosentino, Gregory P., Venkatesan, Sundararajan, Serluca, Farbrizio C., Green, Simon R., Mathews, Michael B., Sonenberg, Nahum
Format: Article
Language:English
Subjects:
AIDS/HIV
Amino Acid Sequence
Biochemistry
Dimerization
Double stranded RNA
eIF-2 Kinase
Genes
human immunodeficiency virus
Humans
Molecular Sequence Data
Open reading frames
Phosphorylation
Protein Binding
Protein Conformation
Protein-Serine-Threonine Kinases - genetics
Protein-Serine-Threonine Kinases - metabolism
Proteins
Recombinant Fusion Proteins - metabolism
Ribonucleic acid
RNA
RNA-Binding Proteins - genetics
RNA-Binding Proteins - metabolism
Sequence Deletion
Structure-Activity Relationship
Transactivation
Transcriptional Activation
Truncation
Two hybrid system techniques
Yeasts
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Summary:The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.92.21.9445