EPRS is a critical mTORC1–S6K1 effector that influences adiposity in mice

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Abstract

Metabolic pathways that contribute to adiposity and ageing are activated by the mammalian target of rapamycin complex 1 (mTORC1) and p70 ribosomal protein S6 kinase 1 (S6K1) axis1,2,3. However, known mTORC1–S6K1 targets do not account for observed loss-of-function phenotypes, suggesting that there are additional downstream effectors of this pathway4,5,6. Here we identify glutamyl-prolyl-tRNA synthetase (EPRS) as an mTORC1–S6K1 target that contributes to adiposity and ageing. Phosphorylation of EPRS at Ser999 by mTORC1–S6K1 induces its release from the aminoacyl tRNA multisynthetase complex, which is required for execution of noncanonical functions of EPRS beyond protein synthesis7,8. To investigate the physiological function of EPRS phosphorylation, we generated Eprs knock-in mice bearing phospho-deficient Ser999-to-Ala (S999A) and phospho-mimetic (S999D) mutations. Homozygous S999A mice exhibited low body weight, reduced adipose tissue mass, and increased lifespan, similar to S6K1-deficient mice9,10,11 and mice with adipocyte-specific deficiency of raptor, an mTORC1 constituent12. Substitution of the EprsS999D allele in S6K1-deficient mice normalized body mass and adiposity, indicating that EPRS phosphorylation mediates S6K1-dependent metabolic responses. In adipocytes, insulin stimulated S6K1-dependent EPRS phosphorylation and release from the multisynthetase complex. Interaction screening revealed that phospho-EPRS binds SLC27A1 (that is, fatty acid transport protein 1, FATP1)13,14,15, inducing its translocation to the plasma membrane and long-chain fatty acid uptake. Thus, EPRS and FATP1 are terminal mTORC1–S6K1 axis effectors that are critical for metabolic phenotypes.

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Figure 1: mTORC1–S6K1 phosphorylates EPRS at Ser999.
Figure 2: Reduced adiposity and extended lifespan of EprsA/A mice.
Figure 3: Insulin induces EPRS-FATP1 interaction and LCFA uptake.
Figure 4: EPRS linker binds FATP1 and induces membrane translocation.

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Acknowledgements

This work was supported by NIH grants P01HL029582, P01HL076491, R01GM086430, R01GM115476, and P50CA150964 (to P.L.F.), by AHA SDG 10SDG3930003 (to A.A), by CIHR fellowship (to D.H), by AHA fellowship (to K.V.), by NIH R01AR048914 and R01GM089771 (to J.C.), by Spanish Ministry BFU2012-38867 and Fundacio La Marato de TV3 #174/U/2016 grants (to S.C.K.), and by NIH R01CA158768, Spanish Ministry SAF2011-24967, and CIG European Commission PCIG10-GA-2011-304160 grants (to G.T.). We thank P. Bhattaram for helpful discussion.

Author information

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Authors

Contributions

A.A. and P.L.F. conceived and interpreted most experiments with contributions from J.M.B., J.C., S.C.K., G.T. and X.L. A.A. performed most experiments with contributions from J.J., F.T., J.S., A.C., D.H. and K.V. A.A.P. performed longevity analysis. A.A. and P.L.F. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Paul L. Fox.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks A. Stahl and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Identification of S6K1 as EPRS Ser999 kinase.

a, Screening of AGC kinase group members for phosphorylation of EPRS Ser999 by immunocomplex kinase assay and [γ-32P]ATP-labelling with S886A linker target in U937 cells. Kinase activity using kinase-specific substrate is shown (bottom; mean ± s.e.m., n = 3). b, Specificity of S6K1 for Ser999 phosphorylation, determined by 32P incorporation in wild-type (WT), S999A and S886 linker. c, siRNA targeting S6K1 inhibits IFNγ-stimulated EPRS phosphorylation in U937 cells determined by 32P-labelling (mean ± s.e.m., n = 3). d, Active recombinant kinases used for in vitro phosphorylation of linker bearing S886A (Ser999 P-acceptor) mutation shows site-specific phosphorylation by S6K1. e, Raptor not rictor is required for Ser999 phosphorylation. f, siRNA targeting the S6K1 3′UTR inhibits S6K1 expression and phosphorylation of EPRS Ser999, but not Ser886 (mean ± s.e.m., n = 3). g, Phosphorylation of S6K1 Thr389 is required for phosphorylation of EPRS. Cells were co-transfected with siRNA targeting the 3′-UTR to knock down endogenous S6K1, and with myc-tagged wild-type or mutant S6K1 cDNA; IFNγ-stimulated EPRS phosphorylation determined by 32P-labelling (mean ± s.e.m., n = 3). h, Cells treated as in e but followed by reciprocal co-immunoprecipitation.

Extended Data Figure 2 Gene targeting and generation of EprsA/A and EprsD/D knock-in mice, and their body weight phenotypes.

a, Knock-in mice bearing Ser999-to-Ala and -Asp mutations were generated by homologous recombination. b, Validation of wild-type (EprsS/S) and EPRS knock-in mice by PCR genotyping (top) and sequence analysis (bottom). c, Genotype analysis of littermates. Total of 1,410 and 644 progeny from interbreeding EprsS/A and EprsS/D mice, respectively, were used at postnatal days P0, P4, and P21. d, Growth curves of EprsA/A (mean ± s.e.m., n = 10 per group; P < 0.0001, two-way ANOVA) and EprsD/D (n = 10 per group) female mice. e, Representative images (left) and weights (right) of wild-type (EprsS/S; S/S) and EprsA/A (A/A) mice on embryonic day E16.5 and post-embryonic development stages. Data shown are mean ± s.e.m.; n = 11 for E16.5 embryos, n = 10 for P0 and P10 mice, and n = 14 for 3, 12, 20, 30, and 50-week mice. f, Representative images (left) and weights (right) of 50-week EprsS/S and EprsD/D (D/D) mice (mean ± s.e.m.; n = 10 per group).

Extended Data Figure 3 Lifespan analysis of EprsS/S, EprsA/A and EprsD/D mice monitored from weaning (>21 days).

a, Youngest and oldest 10% are the mean lifespan of the shortest- and longest-living 10% mice. Numbers of days are represented to nearest full day. Median and mean ± s.e.m.; are shown. b, Cox proportional hazard (CPH) regression analyses of EprsS/S and EprsA/A mice shows genotype as the most significant predictor of increased longevity. Longevity relative to survival days (that is, age at death) was analysed in pooled mice by CPH regression model. The four independent variables genotype, date of birth (DOB), parental ID (PID), and gender were replaced with a set of category variables. In case of genotype and gender, category represents their presence or absence. DOB and PID data were divided into multiple categories as described in the supplementary methods. Independent variables were fitted into the CPH model individually (univariate) or simultaneously (multivariate). Shown are: β (the unstandardized regression coefficient) with standard error (s.e.), the degrees of freedom (df), and the significance for each model fit. Exp(β) for the covariate of interest is the predicted change in hazard ratio for a unit increase in the predictor, and its 95% confidence interval (CI). c, Kaplan–Meier survival curves show no change in lifespan of male, female, or combined EprsD/D mice. Male (MC χ2 = 0.003, P = 0.956; GBW χ2 = 0.001, P = 0.972), female (MC χ2 = 0.158, P = 0. 0.691; GBW χ2 = 0.206, P = 0.650), and gender-combined (MC χ2 = 0.079, P = 0. 0.779; GBW χ2 = 0.076, P = 0.783). d, e, Survival and CPH regression analyses of EprsS/S and EprsD/D mice as described above in a and b.

Extended Data Figure 4 Adipose tissue deposition and lipolysis in EPRSA/A and EPRSD/D mice.

a, Length of mice was measured from head to beginning of tail using a digital caliper (Fisherbrand Traceable). Data shown are mean ± s.e.m., n = 15 for 20-week-old male mice. b, Ventral view of wild-type and EprsA/A mice abdominal cavity. c, Weights of adipose and non-adipose tissues from 20-week-old male EprsD/D and control mice (mean ± s.e.m., n = 14 per group, P value from unpaired t-test). d, Scanning electron micrographs of EWAT in 20-week-old male EprsS/S, EprsA/A, and EprsD/D mice. e, Total adipocyte cell number in EWAT of EprsA/A knock-in and wild-type mice. Data represent mean ± s.e.m., n = 5 per group. f, Elevated lipolysis in adipocytes from EprsA/A, but not EprsD/D, mice (mean ± s.e.m.; n = 6 per group). g, Elevated β-oxidation in WAT explants from EprsA/A mice as determined by release of 14CO2 from 14C-oleic acid (mean ± s.e.m.; n = 5 per group). h, Serum levels of insulin, glucose, triglycerides (TG) and free fatty acids (FFA) in 12-h fasted and 1-h post-prandial (fed) 16-week-old male mice (mean ± s.e.m., n = 10 per group, *P < 0.05, unpaired t-test). i, Growth curves of EprsA/A and EprsD/D mice (males, n = 12 per group, mean ± s.e.m., P < 0.001, two-way ANOVA) started at 6 weeks on an unrestricted high-fat diet (Harlan Teklad TD.06414) deriving 18, 60, and 21 kcal% from protein, fat, and carbohydrate, respectively. j, Phosphorylation of EPRS Ser999 in WAT from EprsA/A and EprsD/D mice after high-fat diet feeding for 24 weeks.

Extended Data Figure 5 Adiposity and lifespan of S6K1−/− mice.

a, PCR genotyping of wild-type (S6K1+/+), heterozygous (S6K1+/-) and homozygous (S6K1−/−) mice. b, Immunoblot analysis of S6K1, EPRS, and FATP1 in S6K1−/− mice. c, Weight of S6K1−/− mice at embryonic day E16.5, and at postnatal days P0 and P10. d, Kaplan–Meier survival curves shows increased longevity in male, female or combined S6K1−/− mice. Male (n = 29 per group; MC χ2 = 4.919, P = 0.027; GBW χ2 = 4.660, P = 0.031), female (n = 28 for S6K1+/+ and n = 26 for S6K1−/−; MC χ2 = 7.927, P = 0.005; GBW χ2 = 7.277, P = 0.007), and gender-combined (n = 26 for S6K1+/+ and n = 55 for S6K1−/−; MC χ2 = 11.78, P = 0.0006; GBW χ2 = 11.01, P = 0.0009). e, f, Lifespan and CPH regression analyses of S6K1+/+ and S6K1−/− mice as described above in Extended Data Fig. 3a, b.

Extended Data Figure 6 Glucose metabolism, food intake and faecal lipid excretion in EprsA/A and EprsD/D mice.

a, Glucose tolerance test (GTT) in 112-day-old EprsS/S, EprsA/A, and EprsD/D mice (mean ± s.e.m., n = 10 per group). b, Insulin tolerance test (ITT) on mice as in a (mean ± s.e.m., n = 10 per group). c, d, Same as a and b but using ~600-day-old mice (mean ± s.e.m., n = 9 per group). eg, Determination of food intake as g per mouse per day (left) or g per body weight (g) per day (right in male (e) and female (f) EprsA/A mice, and in male EprsD/D mice (g). All values represent mean ± s.e.m., n = 14 per group. h, Faecal lipid excretion in EprsS/S, EprsA/A, and EprsD/D mice (mean ± s.e.m., n = 6 per group). i, Serum ketone body (β-hydroxybutyrate) level in 6-h fasted mice.

Extended Data Figure 7 Energy metabolism in EprsA/A and EprsD/D mice.

a, b, Determination of VO2 (left) and VCO2 (right) in EprsS/S and EprsA/A male mice over a 24-h period. c, d, Respiratory exchange ratio (RER) (left) and heat generation (right) in 12-h light and dark cycles were determined (mean ± s.e.m., n = 6 per group). eh, Same as ad but comparing EprsS/S and EprsD/D male mice (mean ± s.e.m., n = 6 per group). i, Determination of VO2 in female EprsS/S, EprsA/A, and EprsD/D mice (mean ± s.e.m., n = 3 per group).

Extended Data Figure 8 Absence of GAIT pathway in adipocytes and inflammatory response in EPRSA/A mice.

a, Total protein synthesis determined by [35S]Met/Cys labelling (left), and by incorporation of [14C]Glu and [14C]Pro into TCA-precipitated proteins in adipocytes from EprsS/S, EprsA/A, and EprsD/D mice. b, Effect of siRNA-mediated knockdown of S6K1, raptor, and rictor on phosphorylation of EPRS Ser999 in differentiated mouse 3T3-L1 adipocytes in presence of 100 nM insulin. c, Effect of IFNγ and insulin on phosphorylation of EPRS Ser999 in differentiated 3T3-L1 adipocytes or mouse primary adipocytes determined using phospho-specific EPRS antibody. d, GAIT complex formation in IFNγ-stimulated U937 cells and insulin-stimulated 3T3-L1 adipocytes by immunoprecipitation with anti-EPRS antibody and immunoblot with antibodies against GAIT complex constituents. Cytosolic lysates from IFNγ-treated U937 cells served as positive control. e, Determination of GAIT pathway activity in IFNγ-stimulated U937 cells and insulin-stimulated 3T3-L1 adipocytes by in vitro translation of a control (T7 gene 10) and GAIT element bearing (Luc-ceruloplasmin (Cp) GAIT element) reporter RNAs. f, White blood cells counts in blood from EprsS/S and EprsA/A mice by Advia hematology system (LUC, large unstained cells). g, Determination of cytokine levels in serum from EprsS/S and EprsA/A mice. Mouse cytokine antibody arrays were incubated with serum (100 μg, protein, right). Pixel intensity was determined by densitometry (right; mean ± s.e.m., 2 mice per group). h, Immunoblot analysis of selected cytokines in serum from EprsS/S and EprsA/A mice.

Extended Data Figure 9 Tissue-specificity of insulin-stimulated LCFA uptake and EPRS-FATP1 interaction.

a, [14C]oleate uptake determined in insulin-stimulated hepatocytes, cardiac cells, soleus muscle strips, and BMDM from EprsS/S, EprsA/A, and EprsD/D mice (mean ± s.e.m., n = 6 mice per group). b, Insulin-stimulated EPRS Ser999 phosphorylation (top), [14C]oleate uptake (middle), and [14C]2-deoxy-d-glucose (DG) uptake (bottom) in adipocytes from white adipose tissue of S6K1+/+ and S6K1−/− mice (mean ± s.e.m., n = 5 mice per group). c, Efficiency of EPRS and FATP1 knockdown in 3T3-L1 adipocytes by siRNA targeting each mRNA alone and in combination, as determined by densitometry (NIH image J) of immunoblots shown in Fig. 3g (mean ± s.e.m., n = 4 experiments). d, Insulin-induced EPRS Ser999 phosphorylation, interaction with FATP1, and [14C]oleate uptake in human adipocytes (mean ± s.e.m., n = 3 experiments in duplicate). e, Co-immunoprecipitation experiment to determine FATP1 binding to EPRS in lysates from multiple tissues as indicated. f, EPRS and FATP1 expression in male and female S6K1−/− mice (mean ± s.e.m., n = 3 mice per group). g, Lack of interaction of EPRS and FATP1 in insulin-stimulated adipocytes of S6K1−/− mice.

Extended Data Figure 10 Hepatic lipids and FATP1/EPRS membrane localization in EprsA/A and EprsD/D mice.

a, Optimum cutting temperature (OCT) compound-fixed liver slices from EprsS/S, EprsA/A, and EprsD/D mice were stained with H&E (top) or oil red O (bottom), and the latter quantitated by densitometry (right; mean ± s.e.m., n = 3 mice per group). b, Determination of liver triglycerides in wild-type and mutant mice (mean ± s.e.m., n = 5 mice per group). c, Insulin-inducible membrane localization of EPRS and FATP1 in adipocytes from wild-type and mutant mice. d, Membrane fractionation shows EPRS and FATP1 co-localizing in plasma membrane (mean ± s.e.m., n = 3 experiments).

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Arif, A., Terenzi, F., Potdar, A. et al. EPRS is a critical mTORC1–S6K1 effector that influences adiposity in mice. Nature 542, 357–361 (2017). https://doi.org/10.1038/nature21380

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