Role of p38 protein kinase in the ligand-independent ubiquitination and down-regulation of the IFNAR1 chain of type I interferon receptor

Abstract

Phosphorylation-dependent ubiquitination and degradation of the IFNAR1 chain of type I interferon (IFN) receptor is a robust and specific mechanism that limits the magnitude and duration of IFNα/β signaling. Besides the ligand-inducible IFNAR1 degradation, the existence of an "inside-out" signaling that accelerates IFNAR1 turnover in the cells undergoing the endoplasmic reticulum (ER) stress and activated unfolded protein responses has been recently described. The latter pathway does not require either presence of ligands (IFNα/β) or catalytic activity of Janus kinases (JAK). Instead, this pathway relies on activation of the PKR-like ER kinase (PERK) and ensuing specific priming phosphorylation of IFNAR1. Here, we describe studies that identify the stress activated p38 protein kinase as an important regulator of IFNAR1 that acts downstream of PERK. Results of the experiments using pharmacologic p38 kinase inhibitors, RNA interference approach, and cells from p38α knock-out mice suggest that p38 kinase activity is required for priming phosphorylation of IFNAR1 in cells undergoing unfolded protein response. We further demonstrate an important role of p38 kinase in the ligand-independent stimulation of IFNAR1 ubiquitination and degradation and ensuing attenuation of IFNα/β signaling and anti-viral defenses. We discuss the distinct importance of p38 kinase in regulating the overall responses to type I IFN in cells that have been already exposed to IFNα/β versus those cells that are yet to encounter these cytokines.

FIGURE 1.

Pharmacologic inhibitors of p38 kinase attenuate the UPR-induced phosphorylation of IFNAR1. A, KR2 cells were infected with VSV (at 0.01 MOI for 1 h) and then washed and incubated for 8 more hours. After that, cells were treated with either p38 inhibitor, VX702 (1 μm), PI3K inhibitor LY294002 (20 μm), or JNK inhibitor SP600125 (20 μm) and harvested 6 h later (altogether 14 h post-infection). IFNAR1 was immunoprecipitated from the whole cell lysates using EA12 antibody, and phosphorylation of IFNAR1 was detected by immunoblotting using the indicated antibodies. Levels of VSV-M protein in whole cell lysates were analyzed by immunoblotting to assess infection of the cells, and phosphorylation of eIF2α was determined to detect the induction of UPR. B, HeLa cells were either infected VSV (as outlined in A) and incubated with VX702 (VX; 1 μm) or SB203580 (SB; 10 μm) and harvested 14 h post-infection. Other plates of HeLa cells were pretreated with these p38 kinase inhibitors for 1 h and then treated with either TG (1 μm) or IFNα (5,000 international units/ml) for 30 min. After that, the cells were harvested, and IFNAR1 was immunoprecipitated and analyzed as in A. Analyses of phosphorylation and levels of eIF2α in whole cell lysates are also shown.

FIGURE 2.

Expression of p38α kinase is required for UPR-induced phosphorylation of IFNAR1. A, 2fTGH cells stably transduced with small hairpin RNA against p38α or against GFP (shCON) were treated with TG (1 μm for 30 min), or IFNα (5,000 international units/ml for 30 min) or infected with VSV (MOI of 0.01 for 14 h). Analyses of phosphorylation and levels of IFNAR1, p38 kinase, and eIF2α were carried out as described in the legend to Fig. 1. B, analyses of IFNAR1 phosphorylation in HeLa cells were carried out as outlined in A. C, MEFs obtained from p38α^−/− or wild type mice were infected with VSV (MOI of 0.01 for 14 h) or treated with TG (1 μm) or mouse IFNβ (5000 international units/ml) for 30 min. Whole cell lysates (WCL) were used for immunoprecipitating IFNAR1. Analyses of phosphorylation and levels of IFNAR1, p38α kinase, and eIF2α were carried out as described in A. D, whole cell lysates were obtained from wild type or p38α knock-out MEFs that were either infected with VSV (MOI of 0.01 for 14 h) or not. These lysates were used as a source of kinase activity to phosphorylate on the recombinant GST-IFNAR1 (1 μg) in an in vitro kinase reaction as described under “Experimental Procedures.” Phosphorylation of GST-IFNAR1 on Ser-532 and levels of this protein were analyzed by immunoblotting using indicated antibodies.

FIGURE 3.

Role of p38α kinase in IFNAR1 ubiquitination induced by UPR. A, 2fTGH cells stably transduced with control shRNA or shRNA against p38α were infected with 0.01 MOI of VSV or treated with 1 μm of TG or 5000 international units/ml of IFNα as described in the legend to Fig. 2. Cell lysates obtained under denaturing conditions were subjected to IFNAR1 immunoprecipitation and analysis by immunoblotting using the indicated antibodies. Phosphorylation and levels of p38 kinase in the lysates is also shown. B, experiment described in A was carried out on MEFs from wild type or p38α knock-out mice. Mouse IFNβ (5000 international units/ml) was used instead of human IFNα. Analyses of ubiquitination and levels of IFNAR1 and of phosphorylation and levels of p38α are shown.

FIGURE 4.

p38α kinase regulates IFNAR1 stability. A, HeLa cells were exposed (or not) to VSV (MOI of 0.01) for 1 h and then incubated for 8 h. After that, cells were treated or not with or not with VX702 (1 μm) for 6 h before being treated with cycloheximide (CHX; 50 μg/ml) for the indicated times and harvested. Levels of IFNAR1 were analyzed by immunoprecipitation followed by immunoblotting. Levels of β-actin in the supernatants of the immunoprecipitation reactions were determined to control equal loading. B, 2fTGH cells that harbor control shRNA or shRNA against p38α were infected, treated, and analyzed as described in A. Levels of p38α kinase in the whole cell lysates was determined by immunoblotting using anti-p38α antibody to determine the efficacy of knockdown. C, MEFs obtained from p38α^−/− mice or wild type mice were processed and analyzed as described in B.

FIGURE 5.

Role of p38 kinase in down-regulation of IFNAR1. A, HeLa cells were infected with VSV (MOI of 0.01 for 1 h), washed, and harvested at the indicated time points post-infection. Whole cell lysates were analyzed by immunoblotting using the indicated antibodies. Levels of IFNAR1 were also assessed by immunoprecipitation and immunoblotting. B, HeLa cells were infected with VSV (MOI of 0.01 for 1 h), washed, and incubated for 8 h. Then, cells were treated (or not) with VX702 (1 μm) and harvested at indicated times post-infection. Levels of IFNAR1 and β-actin were determined as described in Fig. 4A. C, 2fTGH cells that received indicated shRNA were infected with VSV as described in B and harvested at the indicated times post-infection. Levels of IFNAR1 and β-actin were assessed as described in B. D, down-regulation of IFNAR1 in indicated 2fTGH cells treated with TG (1 μm, 3 h) was analyzed as in C. E, wild type or p38^−/− MEFs were infected with VSV (MOI of 0.01) and harvested 0, 16, and 20 h following infection. IFNAR1 was immunoprecipitated, and levels were detected by immunoblotting. F, down-regulation of murine IFNAR1 in indicated MEFs treated with TG (1 μm, 3 h) was analyzed as described in E.

FIGURE 6.

p38α kinase regulates the extent of IFNα/β signaling. A, HeLa cells were infected with MOI of 0.01 of VSV for 1 h, washed, and incubated with 1 μm VX702 as described above. Eighteen hours post-infection, cells were treated with IFNα (200 international units/ml for 30 min) as indicated. Whole cell lysates were analyzed for STAT1 levels and tyrosine phosphorylation using the indicated antibodies. B, 2fTGH cells stably transduced with shp38α or control cells (shCON) were treated and analyzed as described in A. C, MEFs obtained from wild type mice or from p38α knock-out mice were exposed to VSV (0.01 MOI) as indicated. Twenty hours following infection, cells were treated with mouse IFNβ (50 international units/ml for 30 min) and harvested. Analyses of STAT1 phosphorylation and levels are shown. D, analyses of STAT1 levels and phosphorylation in lysates from 2fTGH cells that received indicated shRNA were pretreated or not with TG (1 μm, 3 h) and then treated with human IFNα (200 international units/ml for 30 min) as indicated. E, analyses of STAT1 levels and phosphorylation in lysates from MEFs pretreated or not with TG (1 μm, 3 h) and then treated with mouse IFNβ (50 international units/ml for 30 min) as indicated.

FIGURE 7.

Role of p38α in cell resistance to VSV infection. A, titer of VSV produced in 2fTGH cells stably transfected with control shRNA or shRNA against p38α 14 h after infection with VSV at a MOI of 1.0 is shown on the left. The levels of VSV-M protein indicative of viral load (assessed by immunoblotting) are shown on the right. B, VSV titer and VSV-M expression in MEFs from indicated mice was measured 14 h post-infection as outlined in A.