title: Analysis of monomeric and dimeric phosphorylated forms of protein kinase R.
abstract: PKR (protein kinase R) is induced by interferon and is a key component of the innate immunity antiviral pathway. Upon binding double-stranded RNA (dsRNA) or dimerization in the absence of dsRNA, PKR undergoes autophosphorylation at multiple serines and threonines that activate the kinase. Although it has previously been demonstrated that phosphorylation enhances PKR dimerization, gel filtration analysis reveals a second monomeric phosphorylated form. These forms are termed phosphorylated dimeric PKR (pPKRd) and phosphorylated monomeric PKR (pPKRm). These two forms do not reversibly interconvert. Sedimentation equilibrium measurements reveal that pPKRm dimerizes weakly with a K(d) similar to that of unphosphorylated PKR. Isoelectric focusing and mass spectrometry demonstrate that both pPKRm and pPKRd are heterogeneous in their phosphorylation states, with an average of 9 or 10 phosphates. Equilibrium chemical denaturation analysis indicates that phosphorylation destabilizes the kinase domain by approximately 1.5 kcal/mol in the dimeric form but not in the monomeric form. Limited proteolysis also reveals that phosphorylation induces a conformational change in pPKRd that is not detected in pPKRm. pPKRm binds dsRNA with an affinity similar to that of unphosphorylated PKR, whereas binding cannot be detected with pPKRd. Despite these substantial differences in biophysical properties, both pPKRm and pPKRd are catalytically competent and are activated to phosphorylate the PKR substrate eIF2alpha in the absence of dsRNA. Thus, both monomeric and dimeric forms of phosphorylated PKR may participate in the interferon antiviral pathway.
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Anderson
authors.forename: Christine
authors.initials: C
authors.lastname: Quartararo
authors.forename: Raymond S
authors.initials: RS
authors.lastname: Brown
authors.forename: Yu
authors.initials: Y
authors.lastname: Shi
authors.forename: Xudong
authors.initials: X
authors.lastname: Yao
authors.forename: James L
authors.initials: JL
authors.lastname: Cole
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry; D002384:Catalysis; D002850:Chromatography, Gel; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry / Q000378:metabolism; D007372:Interferons / Q000737:chemistry / Q000502:physiology; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D055503:Protein Multimerization; D015398:Signal Transduction / Q000502:physiology; D013379:Substrate Specificity; D019892:eIF-2 Kinase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; D015852:Eukaryotic Initiation Factor-2; D007527:Isoenzymes; D007372:Interferons; D019892:eIF-2 Kinase