Stress granules regulate double-stranded RNA-dependent protein kinase activation through a complex containing G3BP1 and Caprin1

Abstract

Stress granules (SGs) are dynamic cytoplasmic repositories containing translationally silenced mRNAs that assemble upon cellular stress. We recently reported that the SG nucleating protein G3BP1 promotes antiviral activity and is essential in double-stranded RNA-dependent protein kinase (PKR) recruitment to stress granules, thereby driving phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we delineate the mechanism for SG-dependent PKR activation. We show that G3BP1 and inactive PKR directly interact with each other, dependent on both the NTF2-like and PXXP domains of G3BP1. The G3BP1-interacting protein Caprin1 also directly interacts with PKR, regulates efficient PKR activation at the stress granule, and is also integral for the release of active PKR into the cytoplasm to engage in substrate recognition. The G3BP1-Caprin1-PKR complex represents a new mode of PKR activation and is important for antiviral activity of G3BP1 and PKR during infection with mengovirus. Our data links stress responses and their resultant SGs with innate immune activation through PKR without a requirement for foreign double-stranded RNA (dsRNA) pattern recognition.

Importance: Our previous work indicates that stress granules have antiviral activity and mediate innate immunity through functions of G3BP1; however, the mechanistic details of these functions were not resolved. We show that much of the antiviral activity of stress granules is contingent on the function of PKR in a complex with G3BP1 and Caprin1. The PKR-G3BP1-Caprin1 complex undergoes dynamic transitioning within and outside stress granules to accomplish PKR activation and translational repression. This mechanism appears to function distinctly from canonical pattern recognition of double-stranded RNA by PKR. Therefore, this mechanism bridges the stress response with innate immunity, allowing the cell to respond to many cellular stressors and amplify the pathogen pattern recognition systems of innate immunity.

FIG 1

G3BP1 deletions modulate PKR activity. (A) A domain map of G3BP1-GFP fusion proteins used throughout this study, indicating the borders of each domain. (B) HeLa, G3BP1-KO MEF, and U2OS cells were transfected with G3BP1 constructs and stained for PKR. Images were captured using deconvolution microscopy. PKR is represented in red, and G3BP1 (expressed from pG3BP1-GFP) is shown in green. DAPI (4′,6-diamidino-2-phenylindole)-stained nuclei are visible in blue. (C) The indicated GFP-tagged G3BP1 mutants were expressed in HeLa cells, and PKR was depleted with siRNAs. Total PKR, T446 phosphorylated PKR, eIF3C, and GFP were examined by Western blotting, as indicated. (D) Immunofluorescence was used to correlate P-eIF2α with PKR activation in HeLa cells. Previously published methodology was used to quantify P-eIF2α and is described in Materials and Methods (6).

FIG 2

G3BP1 and Caprin1 directly interact with PKR. (A) Purified inactive PKR (PKR) or active PKR (P-PKR) were incubated in the presence of MBP-tagged MS2 or MBP-tagged G3BP1 deletion mutants, as indicated, and precipitated with amylose resin. Precipitated material was examined with Western blotting for total PKR. (B) MBP precipitations were performed as described for panel A with either MBP-tagged MS2 or G3BP1. Reactions were performed in the presence of ATP with or without poly(I:C) to induce a conformational change in PKR protein. (C) Purified Caprin1 and either inactive PKR (PKR) or active PKR (P-PKR) were incubated together. IPs were performed with protein A-Sepharose and either nonspecific IgG or Caprin1 antibodies, as indicated. Precipitates were analyzed by Western blotting for Caprin1 or total PKR. Relative amounts of PKR and P-PKR inputs are shown with different Western blot exposures (separated by a black line).

FIG 3

G3BP1 outcompetes Caprin1 for binding PKR. (A) Competition experiments were performed with constant amounts of MBP-G3BP1 and inactive PKR and increasing concentrations of Caprin1 as indicated. Precipitates were Western blotted for Caprin1, total PKR, and G3BP1. (B) Competition experiments were conducted as described for panel A, except in the presence of increasing amounts of MBP-G3BP1, as indicated. Inputs displayed are for both panels A and B. Panels A and B are from the same gel and exposure conditions. Bands were quantified and normalized to intensity values for lane 2. (C) Caprin1 (siCap1) was depleted from HeLa cells, and cells were subsequently transfected with either GFP alone or G3BP1-GFP. GFP-tagged protein was immunoprecipitated, and precipitates were analyzed by Western blotting with antibodies against GFP, Caprin1, and total PKR. Bands for total PKR were quantified and normalized against lanes 5 and 7 for siCon and siCap1, respectively. (D) G3BP1 was depleted from HeLa cells (sig 1), and cells were transfected with either GFP alone or GFP-Caprin1 followed by IP of GFP-tagged protein as described for panel C. Immunoprecipitates were analyzed by Western blotting with antibodies against GFP, total PKR, t446 P-PKR, and endogenous G3BP1, as indicated. ImageJ was used to quantify total PKR and P-PKR, which were normalized to the respective GFP control, and values for each condition are indicated. siCon indicates cells treated with nontargeting siRNAs for panels C and D. Results for all panels are representative of experiments repeated in triplicate.

FIG 4

G3BP1 dimerization is not required for PKR activation. (A) The indicated GFP alone, GFP-G3BP1, the NTF2 domain (amino acids 1 to 135), the GFP-G3BP1 F33W mutant, GFP-G3BP1 Δ1-11aa (lacking the first 11 amino acids of G3BP1), or GFP-G3BP2a was transfected into HeLa cells and precipitated with anti-GFP Sepharose. Precipitated material was analyzed by Western blotting against GFP, Caprin1, total PKR, P-PKR, and endogenous G3BP1. The arrow beside the endogenous G3BP1 blot indicates the correct band, while the asterisk highlights an unknown product originating from expression of the GFP-G3BP1 Δ1-11aa protein. ImageJ was used to quantify induction of P-PKR in the input, total PKR immunoprecipitated, and endogenous G3BP1 precipitated with the indicated GFP-tagged proteins. Band intensity for GFP alone was set to 1 in both cases. Nonessential lanes were removed from the gels in panel A (indicated by white space). (B) HeLa cells were transfected with GFP alone or G3BP1-GFP (green), and cells were stained with antibodies against t446 P-PKR (red). Untransfected (white “U”) and transfected (yellow “T”) cells are indicated. Results for all panels are representative of experiments repeated in triplicate.

FIG 5

G3BP1 restricts Mengo-Zn infection. (A) HeLa cells were infected with Mengo-Zn at an MOI of 10 for the indicated times, and cells were harvested for Western blotting against total and t446 P-PKR, as indicated. (B) HeLa cells were infected as described for panel A, and cells were harvested at 10 hpi for IF analysis. Cells were stained with antibodies that recognize either P-eIF2α (red) or endogenous G3BP1 (green), as indicated. (C) GFP alone or GFP-tagged G3BP1 mutants, as indicated, were transfected into HeLa cells, and cells were subsequently infected by Mengo-Zn for 14 h prior to the harvesting of viral supernatants and cells for analysis. Lysates were Western blotted for either GFP, t446 P-PKR, total PKR, or eIF3C, as indicated. (D) Plaque assays were performed on viral supernatants from panel C. Results are presented as PFU × 10⁷ per ml. The lower band present in total PKR blots (panels A and C, *) is a nonspecific band appearing with this antibody. Results for all panels are representative of experiments repeated in triplicate.

FIG 6

G3BP1 and Caprin1 regulate PKR recruitment to antiviral SGs. (A) Western blots for Caprin1, G3BP1, and GAPDH were performed on control, G3BP1, or Caprin1 siRNA-treated HeLa cells, as indicated. (B) PKR localization was detected by IF in HeLa cells treated with siControl, siG3BP1, and siCaprin1 and infected for 14 h with Mengo-Zn. PKR intensity was normalized to Tia1 intensity for each granule and presented as a relative value. (C) HeLa cells treated with control (Con), G3BP1, or Caprin1 (Cap1) siRNAs and infected as described for panel B were stained and quantified for P-eIF2α intensity and are presented in a box and whisker plot. (D) HeLa cells were treated with either control or Caprin1 (Cap1)-specific siRNAs and then transfected with GFP-tagged G3BP1 (green) to induce P-PKR. Cells were fixed and stained for P-eIF2α (red) and DAPI (blue). (E) Levels of P-eIF2α were quantified from panel D as previously described (6). All plots with statistics were analyzed with a Student t test (**, P < 0.01; ***, P < 0.001).

FIG 7

Model for G3BP1-Caprin1 regulation of PKR and eIF2α phosphorylation. Caprin1, G3BP1, and PKR exist in a complex in the cell when PKR is inactive and SGs are not induced. The NTF2-like and PXXP domains are pictured in contact with PKR based on our data. The PXXP domain is involved in interactions with mRNPs containing translation initiation factors. Upon encountering a stress and activation of some stress signaling pathways, G3BP1 mediates aggregation and the appearance of small SGs containing PKR and Caprin1. Our data indicate that these small SGs do not activate PKR, and eIF2α phosphorylation is not observed (6). When small SGs coalesce into large SGs, cellular dsRNA or an unknown factor(s) (X?) activates PKR, causing reorganization of the G3BP1-Caprin1-PKR complex. Subsequently, active PKR and Caprin1 are ejected from the stress granule, where PKR seeks out the PKR substrate eIF2α. It is unclear from our data whether active PKR released from SGs is a monomer (depicted) or dimer (not depicted).