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 t he 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 gen es, HIS3 and lacZ. Expression of full-length wild-type mouse PKR(1-515 )WT as a Gal4 fusion protein did not exhibit the growth suppression ph enotype in yeast characteristic of wild-type human PKR(1-551)WT Coexpr ession 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-ty pe mouse PKR(1-515)WT as full-length PKR-Gal4 activation domain fusion s resulted in activation of the HIS3 and lacZ reporters. The N-termina l RNA-binding region of human PKR, both WT and the K64E RNA-binding-de ficient mutant, also interacted with mouse PKR(1-515)WT sufficiently t o activate the reporters but the human catalytic region did not. Mouse and human full-length PKR proteins expressed as glutathione S-transfe rase (GST) fusions in Escherichia coil were purified on Sepharose bead s. Using GST-PKR fusion chromatography, direct physical interaction be tween the mouse and human PKR homologs was established. Intraspecies P KR interactions were more efficient than interspecies PKR interactions , and interactions between RNA-binding-sufficient PKR proteins were mo re 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 PK R. 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 o f human PKR. (C) 1997 Academic Press.