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.

Figure 1

Gel filtration analysis of phosphorylated PKR. A) Overplot of initial reaction mixture (solid) and reapplication of purified pPKRd (dashed) and pPKRm (dotted) . The pPKRm and pPKRd fractions were isolated by preparative gel filtration, concentrated to about 5 mg/ml and equilibrated for 5 days at 4°C. The chromatograms are vertically offset for clarity. B) Overplot of pPKRm (solid) and pPKRm after re-reaction with ATP (dotted). pPKRm isolated from initial preparative gel filtration was concentrated to 80 μM and equilibrated into 50 mM Tris, 100 mM NaCl, 12 mM MgCl₂, 0.1 mM EDTA, 10 mM BME, pH 8.0. Half of the sample was incubated with ATP and MgCl for 4 hours at 20°C. Both samples were separately reapplied to the column. Analytical gel filtration chromatography was performed on a GE Healthcare Superdex 200 10/300 column in AU200 buffer at 0.5 ml/min and 4°C.

Figure 2

Analysis of pPKRm and pPKRd phosphorylation states. A) IEF gel of unphosphorylated and phosphorylated PKR forms. Samples were loaded at a concentration of 3 mg/ml and analyzed on a pH 3-7 IEF gel (Invitrogen ) with SERVA markers according to the manufacturers' instructions. Gels were stained with Coomassie blue. B) Deconvoluted electrospray ionization mass spectrum of pPKRm. C) Deconvoluted electrospray ionization mass spectrum of pPKRd. See Materials and Methods for experimental details.

Figure 3

Analysis of the stability of phosphorylated PKR. A) Limited proteolysis of pPKRm and pPKRd. Samples were incubated at 1 mg/ml at several ratios of PKR: trypsin at 20 °C for 30 minutes. Reactions were quenched by the addition of SDS sample loading buffer and heating to 90°C for 10 minutes. The samples were run on a 4-12% acrylamide bis-tris gel under denaturing conditions and visualized with Coomassie blue staining. Lane 1 contains unphosphorylated PKR that has not been digested. Lanes 2-5 contain pPKRm with increasing concentrations of trypsin. Lanes 6-9 contain pPKRd and increasing concentrations of trypsin. B) Analysis of the stability of pPKRm by equilibrium urea unfolding titration. pPKRm was prepared at 0.15 mg/ml and variable urea concentrations and equilibrated at 20°C for 3 hours. Unfolding was monitored by tryptophan fluorescence emission with excitation at 295 and emission at 340 nm. The data are shown as filled circles and the fit to a two-state model using SAVUKA is shown as a solid line. The best-fit parameters are ΔG° = 6.41 ± 0.31 kcal mol⁻¹ and m = 2.11 ± 0.01 kcal mol⁻¹ M⁻¹. Inset: residuals of the fit.

Figure 4

Sedimentation equilibrium analysis of pPKRm dimerization. The open symbols are the data and the solid lines are a global fit using a monomer-dimer equilibrium model incorporating incompetent dimer. For clarity, only every third data point is shown. The inset shows a superposition of the residuals from the global fit. The best-fit parameters are: K_d = 473 (377, 624) μM with 5% incompetent dimer and RMS = 0.0158 fringes.

Figure 5

Sedimentation velocity analysis of pPKRm binding to a 20 bp dsRNA. Samples were prepared at 1 μM dsRNA + 0.5 eq. pPKRm (A), dsRNA + 1 eq. pPKRm (B), dsRNA +2 eq. pPKRm (C) and dsRNA + 6 eq. pPKRm (D). The sedimentation velocity profiles were subtracted in pairs to remove time-invariant noise and the four data sets were globally fit to a 1:1 binding stoichiometry model using SEDANAL (36). The top panels show the data (points) and fit (solid lines) and the bottom panels show the residuals (points). The best-fit parameters are: K_d = 3.10 (1.67, 5.49) μM and RMS deviations = 0.0071 OD.

Figure 6

eIF2α kinase activity of pPKRm and pPKRd. A) Effects of dsRNA on eIF2α phosphorylation. Unphosphorylated PKR control () and pPKRm (). B) Comparison of unphosphorylated PKR (white), pPKRm (grey) and pPKRd (black). For both A and B, samples contained 400 μM ATP and were quenched after 20 minutes. C) Kinetics of eIF2α phosphorylation catalyzed by unphosphorylated PKR (), pPKRm () and pPKRd (). All samples contained 40 μM ATP and the unphosphorylated PKR sample contained 10 μg/ml dsRNA. The best fit line for a single exponential fit is shown for each reaction, with rate constants of 0.64 min⁻¹ (PKR), 0.22 min⁻¹ (pPKRm) and 2.2 min⁻¹ (pPKRd).