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. 2015 Apr 14;11(2):295-307.
doi: 10.1016/j.celrep.2015.03.021. Epub 2015 Apr 2.

A critical role for PKR complexes with TRBP in Immunometabolic regulation and eIF2α phosphorylation in obesity

Takahisa Nakamura  1 , Ryan C Kunz  2 , Cai Zhang  3 , Taishi Kimura  3 , Celvie L Yuan  3 , Brenna Baccaro  4 , Yuka Namiki  5 , Steven P Gygi  2 , Gökhan S Hotamisligil  6
Affiliations

A critical role for PKR complexes with TRBP in Immunometabolic regulation and eIF2α phosphorylation in obesity

Takahisa Nakamura et al. Cell Rep. .

Abstract

Aberrant stress and inflammatory responses are key factors in the pathogenesis of obesity and metabolic dysfunction, and the double-stranded RNA-dependent kinase (PKR) has been proposed to play an important role in integrating these pathways. Here, we report the formation of a complex between PKR and TAR RNA-binding protein (TRBP) during metabolic and obesity-induced stress, which is critical for the regulation of eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation and c-Jun N-terminal kinase (JNK) activation. We show that TRBP phosphorylation is induced in the setting of metabolic stress, leading to PKR activation. Suppression of hepatic TRBP reduced inflammation, JNK activity, and eIF2α phosphorylation and improved systemic insulin resistance and glucose metabolism, while TRBP overexpression exacerbated the impairment in glucose homeostasis in obese mice. These data indicate that the association between PKR and TRBP integrates metabolism with translational control and inflammatory signaling and plays important roles in metabolic homeostasis and disease.

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Figures

Figure 1
Figure 1. Identification of hepatic PKR protein complex in lean and obese mice
(A) Silver staining of PKR-associated proteins following immunoprecipitation from lean and obese liver. (B and C) Results of mass-spectrometry analyses for identification of PKR protein complexes in lean and obese (ob/ob) mice. Vertical hashed lines indicate a fold change of 10 (or -10), horizontal hashed lines indicate a corrected p-value of 0.05. Proteins are distributed into two distinct populations based on exclusivity, i.e., molecules found only in the PKR but not in the control samples (or vice versa) center at Log2(PKR/GFP) ratio of >35. See also Table S1 and S2. (D) Gene ontology analysis of statistically significant PKR interactors in liver of lean and obese mice. (E) Physical interaction between PKR and components of RLC, TRBP and Dicer, in liver of lean and obese mice. The graph on the right shows the quantification of the TRBP-PKR interaction and PKR-Dicer interaction. Data are shown as the mean ± SEM (n=3). *p<0.05. See also Figure S1A.
Figure 2
Figure 2. The role of RLC in regulation of PKR kinase activity and induction of eIF2α phosphorylation
(A) Physical interaction between endogenous PKR and Dicer in MEFs in the presence of PolyI:C. Pkr+/+ and Pkr−/− MEFs were treated with 1 g/ml of PolyI:C for one hour, followed by immunoprecipitation and western blots with antibodies as indicated. The graph on the right shows the quantification of the TRBP-PKR interaction. Data are shown as the mean ± SEM (n=3). **p<0.01. See also Figure S1B. (B) Expression level PKR in MEFs. Flag-tagged PKR was introduced by retrovirus-mediated gene transfer in Pkr−/− MEFs. (C) Physical interaction between PKR, Dicer, and TRBP in MEF cells. Cell lysates were prepared from Pkr−/− MEFs reconstituted with retrovirally expressed Flag-tagged wild type (WT) PKR and HA-tagged TRBP in the absence or presence of 1 g/ml of PolyI:C. SE: Short exposure. LE: Long exposure. (D) Effects of mutations in PKR's RNA-binding domains and kinase domain on formation with the PKR-RLC. Cell lysates were prepared from Pkr−/− MEFs reconstituted with vector (V), Flag-tagged WT PKR (WT), RNA-binding defective PKR (RD), and kinase-dead mutant (KD) in the absence or presence of 1 g/ml of PolyI:C for one hour. (E) Effects of mutations of PKR on PKR kinase activity. PKR activity was assessed with cell lysates used in Figure 2D using recombinant eIF2α protein. (F) Interaction of RLC with PKR. Cell lysates were prepared from Pkr−/− MEFs reconstituted with WT PKR and HA-tagged TRBP in the absence or presence of 1 g/ml of PolyI:C for one hour. G) Ability of TRBP to induce eIF2α phosphorylation through recruitment of PKR. In vitro kinase assay was performed with cell lysates used in Figure 2F using recombinant eIF2α protein.
Figure 3
Figure 3. Association of TRBP with PKR in palmitate-induced metabolic stress
(A) Physical interaction between endogenous PKR and Dicer in MEFs in the presence of palmitate. Pkr+/+ and Pkr−/− MEFs were treated with 100 μM of palmitate for one hour, followed by immunoprecipitation and immunoblotting with antibodies as indicated. The graph on the right shows the quantification of PKR-TRBP interaction. Data are shown as the mean ± SEM (n=4). *p<0.05. (B) Physical interaction between PKR, Dicer, and TRBP in MEF cells after palmitate exposure. Pkr−/− MEFs reconstituted with retrovirally expressed Flag-tagged WT or RD PKR were treated with 100 μM palmitate, followed by immunoprecipitation with anti-Flag antibody. (C) Palmitate-induced physical interaction between PKR and TRBP in MEF cells pretreated with 100 μM EPA for 14 hours. Pkr−/− MEFs reconstituted with Flag-tagged WT PKR (WT) were cultured in the presence of 100 μM EPA for 14 hours. These cells were treated with 100 μM palmitate for 2 hours at the end of EPA culture period. See also Figure S2A, S2B, and S2C. (D and E) Effects of Dicer or TRBP deficiency on palmitate-induced PKR activation. Dicer (D) and TRBP (E) knockout fibroblasts and their control cells were treated with 100 μM palmitate for 2 hours, followed by in vitro PKR kinase assay. Blots shown are representative of three independent experiments; graphs are mean ± SEM (n=3). *p<0.05. (F) TRBP deficiency results in reduced palmitate-induced JNK and eIF2α phosphorylation levels. TRBP knockout fibroblasts and control cells were treated with 100 μM palmitate for 2 hours, followed by western blot analysis. Blots shown are representative of four independent experiments; graphs are mean ± SEM (n=4). *p<0.05.
Figure 4
Figure 4. Role of TRBP phosphorylation on PKR activity
(A) Schematic domain structure of TRBP with arrows indicating the positions of four serine (S) phosphorylation residues as shown previously (Paroo et al., 2009). Abbreviations of TRBP mutants we use in this proposal are indicated below. (B) Effect of co-expression of constitutively active JNK1 (MKK7-JNK1) with WT or TRBP SA mutants on their mobility shifts detected by anti-TRBP antibody. (C) Interaction between PKR and TRBP variants in Pkr−/− MEFs. PKR and TRBP variants were examined by immunoprecipitaion followed by immunoblotting. (D) PKR activity in Pkr−/− MEFs reconstituted with Flag-tagged WT PKR retrovirally expressing WT, 4SA, or 4SD TRBP. The graph on the right shows the quantification of the results. Data are shown as the mean ± SEM (n=3). *p<0.05. (E) Palmitate-induced TRBP phosphorylation levels. WT MEFs and TRBP-deficient fibroblasts and were treated with 100 μM palmitate for 2 hours, followed by immunoprecipitation with anti-TRBP and antibody and western blot analysis with anti-phospho TRBP (ser152) antibody. Blots shown are representative of three independent experiments; graphs are mean ± SEM (n=3). *p<0.05. See also Figure S3A and S3B. (F) TRBP phosphorylation levels in liver of WT and leptin-deficient obese mice. The graph on the right shows the quantification of the results. Data are shown as the mean ± SEM (n=5). *p<0.05.
Figure 5
Figure 5. Improved glucose metabolism and insulin sensitivity in Pkr-deficient ob/ob mice
(A) Effects of Pkr-deficiency on body weight in ob/ob mice. (B) Glucose tolerance tests, performed in Pkr+/+ob/ob (n=12) and Pkr−/−ob/ob (n=7) at 7 weeks of age. Data are shown as the mean ± SEM (n=5). *p<0.05. (C) Insulin tolerance tests, performed on Pkr+/+ob/ob (n=12) and Pkr−/−ob/ob (n=7) at 9 weeks of age. Data are shown as the mean ± SEM (n=5). *p<0.05. (D and E) Blood glucose (D) and serum insulin (E) levels after 14 hours food withdrawal in Pkr+/+ (n=8), Pkr−/− (n=8), Pkr+/+ob/ob (n=8) and Pkr−/−ob/ob (n=8) at 10 weeks of age. See also Figure S4A, S4B, and S4C. (F and G) Phosphorylation level of eIF2α on serine 52 (F), which was detected by anti-phospho-eIF2α antibody, and JNK1 kinase activity (G), which was measured by a kinase assay using immunopurified JNK1, ATP [γ-32P] and recombinant c-Jun protein as substrate, in liver of Pkr+/+, Pkr−/−, Pkr+/+ob/ob, and Pkr−/−ob/ob mice at 10 weeks of age. The graph on the right shows the quantification of the results. Data are shown as the mean ± SEM. *p<0.05. See also Figure S4D. (H) Phosphorylation levels of IRS1 on serine 307 detected by anti-phospho IRS1 antibody. The graph on the right shows the quantification of the results. Data are shown as the mean ± SEM. *p<0.05.
Figure 6
Figure 6. Effects of TRBP expression on glucose metabolism and eIF2α phosphorylation in a genetic model of obesity
(A and B) Effects of hepatic TRBP expression in ob/ob mice on body weight (A) and liver weight (B). Data are shown as the mean ± SEM (n=8). (C) Effect of hepatic TRBP expression in ob/ob mice on fasting blood glucose. Data are shown as the mean ± SEM(n=8). *p<0.05. (D) Effects of hepatic TRBP expression on systemic glucose metabolism assessed by glucose tolerance tests. Data are shown as the mean ± SEM (n=8). *p<0.05 (E and F) Effects of hepatic TRBP expression on JNK and eIF2α phosphorylation. Phosphorylation levels of eIF2α and JNK were detected by anti-phospho-antibodies in liver lysates of Pkr+/+ob/ob (E) and Pkr−/−ob/ob (F) mice exogenously expressing a control gene (GUS) or TRBP. The graph on the right shows the quantification of the results. Data are shown as the mean ± SEM. *p<0.05.
Figure 7
Figure 7. Physiological roles of hepatic TRBP in obesity
(A) Effects of TRBP suppression on palmitate-induced PKR activation. TRBP expression in Pkr−/− MEFs reconstituted with Flag-tagged WT PKR were reduced by adenovirus-mediated shRNAs. These cells were treated with 100 mM palmitate for 2 hours, followed by immunoprecipitation with anti-Flag antibody, and in vitro kinase assay with recombinant eIF2α protein. The graph on the right shows the quantification of the results. Data are shown as the mean ± SEM. *p<0.05. See also Table S3. (B) Effects of hepatic TRBP suppression in ob/ob mice on TRBP, PKR, and Dicer expression following transduction of adenovirus-mediated control shRNA to LacZ. Liver tissue lysates were analyzed by immunoblotting with antibodies as indicated. To analyze the levels of PKR and Dicer interacting TRBP, the tissue lysates were immunoprecipitated with anti-TRBP antibody, followed by immunoblotting with antibodies as indicated. (C and D) Effects of hepatic TRBP reduction in ob/ob mice on liver weight (C) and body weight (D). Data are shown as the mean ± SEM (n=8). *p<0.05. (E) Effect of hepatic TRBP knockdown in ob/ob mice on fasting blood glucose. Data are shown as the mean ± SEM (n=8). *p<0.05. (F) Effects of hepatic TRBP suppression on systemic glucose metabolism assessed by glucose tolerance tests. Hepatic TRBP was reduced using two different shRNA constructs introduced by adenovirus-mediated gene transfer in ob/ob liver. Data are shown as the mean ± SEM (n=8). *p<0.05. (G) Effects of hepatic TRBP suppression in ob/ob mice on expression of key regulators of liver function. Expression levels of the genes were detected by quantitative PCR method. Data are shown as the mean ± SEM (n=8). *p<0.05. See also Table S4. (H) Hepatic TRBP knockdown results in reduced PKR expression, eIF2α phosphorylation, and JNK phosphorylation levels in obese liver. (I) Serine phosphorylation level of IRS1 was detected by an anti-phospho IRS1 antibody recognizing residue 307. The graph on the right shows the quantification of the results. Data are shown as the mean ± SEM. *p<0.05. (J) Enhanced insulin signaling, which was assessed by anti-phospho specific AKT antibody, observed in TRBP knocked-down liver of ob/ob mice. The graph on the right shows the quantification of insulin-induced phosphorylation levels of Akt. Data are shown as the mean ± SEM. *p<0.05.
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