Interaction of the human protein kinase PKR with the mouse PKR homolog occurs via the N-terminal region of PKR and does not inactivate autophosphorylation activity of mouse PKR.
The RNA-dependent protein kinase (PKR) is implicated in the antiviral and antiproliferative actions of interferon. Mutant forms of human PKR display a transdominant behavior when expressed in transfected cells. The potential for the human PKR protein to physically interact with the mouse PKR homolog has therefore been examined. The yeast two-hybrid system was used to probe the association between mouse and human PKR proteins as measured by activation of two Gal4-responsive reporter genes, HIS3 and IacZ. Expression of full-length wild-type mouse PKR(1-515)WT as a Gal4 fusion protein did not exhibit the growth suppression phenotype in yeast characteristic of wild-type human PKR(1-551)WT. Coexpression of mouse PKR(1-515)WT as a Gal4 DNA-binding domain fusion with either the catalytic-deficient human PKR(1-551) K296R mutant, the RNA-binding-deficient human PKR(1-551)K64E/K296R double mutant, or wild-type mouse PKR(1-515)WT as full-length PKR-Gal4 activation domain fusions resulted in activation of the HIS3 and lacZ reporters. The N-terminal RNA-binding region of human PKR, both WT and the K64E RNA-binding-deficient mutant, also interacted with mouse PKR(1-515)WT sufficiently to activate the reporters but the human catalytic region did not. Mouse and human full-length PKR proteins expressed as glutathione S-transferase (GST) fusions in Escherichia coli were purified on Sepharose beads. Using GST-PKR fusion chromatography, direct physical interaction between the mouse and human PKR homologs was established. Intraspecies PKR interactions were more efficient than interspecies PKR interactions, and interactions between RNA-binding-sufficient PKR proteins were more efficient than those involving an RNA-binding mutant as measured by binding to GST-PKR protein Sepharose beads. The N-terminal region of human PKR within amino acids 1-184 was sufficient for binding mouse PKR. Purified mouse full-length PKR(1-515)WT GST fusion protein retained kinase activity on Sepharose beads, but the activity was not impaired by association with either the full-length or the N-terminal region of human PKR.
Rende-Fournier
R
R
Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara 93106, USA.
Ortega
L G
LG
George
C X
CX
Samuel
C E
CE
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D019281:Dimerization; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D012313:RNA; D005982:Glutathione Transferase; D019892:eIF-2 Kinase