Using genetic means to dissect homologous and heterologous protein-protein interactions of PKR, the interferon-induced protein kinase.
The interferon-induced protein kinase, PKR, is a pivotal component of interferon (IFN)-induced cellular antiviral and antiproliferative response. The identification and characterization of proteins, of both viral and cellular origins, that interact with PKR have proven to be a valuable probe for unraveling the cellular regulation and function of PKR. Several studies have demonstrated that PKR forms dimers and that dimerization is likely to be required for activation and/or catalytic function. It is therefore important to elucidate the mechanism of PKR dimer formation and the role of PKR effectors in modulating kinase dimerization. Herein we describe the use of the two genetic approaches, the lambda repressor fusion and the yeast two-hybrid systems, to detect and analyze homo- and heterotypic interactions with PKR. We also describe several biochemical methodologies commonly used in our laboratory to validate the genetic results. Although the examples in this article focus on PKR, the techniques can easily be adapted to investigate protein-protein associations in a variety of experimental systems. Finally, given the important role of PKR as a mediator of IFN-induced antiviral and antiproliferative effects, these studies may provide clues to the development of reagents that target PKR to enhance the therapeutic use of IFN in the treatment of disease.
Tan
S L
SL
School of Medicine, University of Washington, Seattle, Washington, 98195, USA.
Katze
M G
MG
D010582:Bacteriophage lambda / Q000235:genetics; D015153:Blotting, Western; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000378:metabolism; D010957:Plasmids; D011233:Precipitin Tests; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D005982:Glutathione Transferase; D019892:eIF-2 Kinase