CHARACTERIZATION OF THE HEPARIN-MEDIATED ACTIVATION OF PKR, THE INTERFERON-INDUCIBLE RNA-DEPENDENT PROTEIN-KINASE

Heparin can substitute for double-stranded (ds) RNA in the autophospho rylation and activation of the interferon-inducible, RNA-dependent eIF -2 alpha protein kinase (PKR). We have used heparin oligosaccharides o f defined lengths to examine the heparin-mediated activation of human PKR; Heparin oligosaccharide with 8 sugar residues was nearly as effic ient as 16-residue heparin (Hep-16) in mediating the activation of PKR autophosphorylation, whereas B-residue heparin was a poor activator. When examined in combination, Hep-16 and dsRNA did not act synergistic ally in activating PKR autophosphorylation. The RNA-binding activity o f recombinant PKR, measured with adenovirus VA RNA, was competed by po ly(rI):poly(rC) but not by Hep-16. When the catalytically inactive, hi stidine-tagged mutant PKR protein [His-PKR(K296R)] was examined as a s ubstrate for purified wild-type PKR, the intermolecular phosphorylatio n of His-PKR(K296R) was efficiently catalyzed by dsRNA-activated PKR b ut not by heparin-activated PKR. However, eIF-2 alpha phosphorylation was catalyzed by both heparin- and dsRNA-activated PKR. Preincubation of PKR with Hep-16 in the absence of ATP blocked subsequent autophosph orylation mediated either by Hep-16 or dsRNA, whereas preincubation wi th dsRNA either alone or in combination with Hep-16 did not impair sub sequent autophosphorylation. Neither Hep-16 nor dsRNA caused a detecta ble degradation of PKR during preincubation or subsequent autophosphor ylation of PKR. These results suggest that, while both dsRNA and hepar in are capable of activating PKR autophosphorylation, the structural a nd functional basis of PKR activation differs for these two classes of polyanionic biomolecules. (C) 1996 academic Press, Inc.