The mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis and autophagy1. Its hyperactivation contributes to disease in numerous organs, including the heart1,2, although broad inhibition of mTORC1 risks interference with its homeostatic roles. Tuberin (TSC2) is a GTPase-activating protein and prominent intrinsic regulator of mTORC1 that acts through modulation of RHEB (Ras homologue enriched in brain). TSC2 constitutively inhibits mTORC1; however, this activity is modified by phosphorylation from multiple signalling kinases that in turn inhibits (AMPK and GSK-3β) or stimulates (AKT, ERK and RSK-1) mTORC1 activity3,4,5,6,7,8,9. Each kinase requires engagement of multiple serines, impeding analysis of their role in vivo. Here we show that phosphorylation or gain- or loss-of-function mutations at either of two adjacent serine residues in TSC2 (S1365 and S1366 in mice; S1364 and S1365 in humans) can bidirectionally control mTORC1 activity stimulated by growth factors or haemodynamic stress, and consequently modulate cell growth and autophagy. However, basal mTORC1 activity remains unchanged. In the heart, or in isolated cardiomyocytes or fibroblasts, protein kinase G1 (PKG1) phosphorylates these TSC2 sites. PKG1 is a primary effector of nitric oxide and natriuretic peptide signalling, and protects against heart disease10,11,12,13. Suppression of hypertrophy and stimulation of autophagy in cardiomyocytes by PKG1 requires TSC2 phosphorylation. Homozygous knock-in mice that express a phosphorylation-silencing mutation in TSC2 (TSC2(S1365A)) develop worse heart disease and have higher mortality after sustained pressure overload of the heart, owing to mTORC1 hyperactivity that cannot be rescued by PKG1 stimulation. However, cardiac disease is reduced and survival of heterozygote Tsc2S1365A knock-in mice subjected to the same stress is improved by PKG1 activation or expression of a phosphorylation-mimicking mutation (TSC2(S1365E)). Resting mTORC1 activity is not altered in either knock-in model. Therefore, TSC2 phosphorylation is both required and sufficient for PKG1-mediated cardiac protection against pressure overload. The serine residues identified here provide a genetic tool for bidirectional regulation of the amplitude of stress-stimulated mTORC1 activity.
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The authors declare that the data supporting the findings of this study are available within the paper and the Supplementary Information. Numerical values corresponding to figures that describe the results from in vivo model studies are provided as separate Source Data for Figs. 1f–h, 2a, 3d, 4c, e and Extended Data Fig. 1a. Other source data related to the study are available from the corresponding author upon reasonable request. Any reagents developed for this study, including novel plasmids, viral vectors and the Tsc2 knock-in mouse models can be made available upon direct request to the corresponding author.
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This study was supported by National Institutes of Health (NIH) National Heart Lung and Blood Institute grants HL-135827, HL-119012, HL089297, T32-HL-07227 (D.A.K.), HHSN268201000032C (J.E.V.E. and D.A.K.), F31-HL134196 (K.M.K.-S.), F31-HL143905 (B.L.D.-E.), American Heart Association Post-Doctoral Fellowships (M.J.R., D.I.L. and T.N.), Deutsche Forschungsgemeinschaft OE 688/1-1 (C.U.O.), Fondation Leducq TransAtlantic Network of Excellence, and an Abraham and Virginia Weiss Professorship (D.A.K.), an Erika J. Glazer Endowed Chair in Women’s Heart Health (J.E.V.E.) and the Barbra Streisand Women’s Heart Center (J.E.V.E.), R01AI077610 and R01AI091481 (J.D.P.), and the Bloomberg~Kimmel Institute for Cancer Immunotherapy (J.D.P.). We thank P. Eaton for providing plasmid constructs expressing PKG1α(WT) and PKG1α(M438G), J. Sadoshima for providing the LC3-II–GFP–RFP reporter-expressing adenovirus, B. Manning for the DNA construct of wild-type human TSC2 and J. T. Kass for assisting with protein kinase bioinformatics analyses.