PKG-Modified TSC2 Regulates mTORC1 Activity to Counter Adverse Cardiac Stress

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 activity3–9. 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 disease10–13. 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.Phosphorylation of one of two adjacent serine residues in TSC2 is both required and sufficient for PKG1-mediated cardiac protection against pressure overload in mice; these serine residues provide a genetic tool for the bidirectional regulation of stress-stimulated mTORC1 activity.

[1]  Thomas Danner,et al.  Phosphodiesterase 9A Controls Nitric-oxide Independent cGMP and Hypertrophic Heart Disease , 2015, Nature.

[2]  Michael L. Gatza,et al.  Proteogenomics connects somatic mutations to signaling in breast cancer , 2016, Nature.

[3]  L. Cantley,et al.  Spatial Control of the TSC Complex Integrates Insulin and Nutrient Regulation of mTORC1 at the Lysosome , 2014, Cell.

[4]  Steven P Gygi,et al.  Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[5]  David M. Sabatini,et al.  mTOR Signaling in Growth, Metabolism, and Disease , 2017, Cell.

[6]  David A. Scott,et al.  Genome engineering using the CRISPR-Cas9 system , 2013, Nature Protocols.

[7]  B. Turk,et al.  Cyclic GMP-dependent Stimulation of Serotonin Transport Does Not Involve Direct Transporter Phosphorylation by cGMP-dependent Protein Kinase* , 2012, Journal of Biological Chemistry.

[8]  D. Kuppuswamy,et al.  Regulation of mTOR and S6K1 activation by the nPKC isoforms, PKCepsilon and PKCdelta, in adult cardiac muscle cells. , 2007, Journal of Molecular and Cellular Cardiology.

[9]  M. Hall,et al.  Cardiac Raptor Ablation Impairs Adaptive Hypertrophy, Alters Metabolic Gene Expression, and Causes Heart Failure in Mice , 2011, Circulation.

[10]  T. Alain,et al.  Pharmacological and Genetic Evaluation of Proposed Roles of Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK), Extracellular Signal-regulated Kinase (ERK), and p90RSK in the Control of mTORC1 Protein Signaling by Phorbol Esters* , 2011, The Journal of Biological Chemistry.

[11]  J. Sadoshima,et al.  New Insights Into the Role of mTOR Signaling in the Cardiovascular System. , 2018, Circulation Research.

[12]  N. Hariharan,et al.  Oxidative stress stimulates autophagic flux during ischemia/reperfusion. , 2011, Antioxidants & redox signaling.

[13]  D. Kwiatkowski,et al.  Coordinated regulation of protein synthesis and degradation by mTORC1 , 2014, Nature.

[14]  D. Kass,et al.  Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy , 2005, Nature Medicine.

[15]  D. Kuppuswamy,et al.  Regulation of mTOR and S6K1 activation by the nPKC isoforms, PKCε and PKCδ, in adult cardiac muscle cells , 2007 .

[16]  Alma L Burlingame,et al.  A semisynthetic epitope for kinase substrates , 2007, Nature Methods.

[17]  B. Viollet,et al.  AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1 , 2011, Nature Cell Biology.

[18]  Hongbing Zhang,et al.  Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. , 2003, The Journal of clinical investigation.

[19]  K. Nakao,et al.  Inhibition of TRPC6 Channel Activity Contributes to the Antihypertrophic Effects of Natriuretic Peptides-Guanylyl Cyclase-A Signaling in the Heart , 2010, Circulation research.

[20]  Dong I. Lee,et al.  Prevention of PKG1α oxidation augments cardioprotection in the stressed heart. , 2015, The Journal of clinical investigation.

[21]  K. Inoki,et al.  TSC2 Mediates Cellular Energy Response to Control Cell Growth and Survival , 2003, Cell.

[22]  A. Shah,et al.  mTOR Hyperactivation by Ablation of Tuberous Sclerosis Complex 2 in the Mouse Heart Induces Cardiac Dysfunction with the Increased Number of Small Mitochondria Mediated through the Down-Regulation of Autophagy , 2016, PloS one.

[23]  Jenna Scotcher,et al.  Disulfide-activated protein kinase G Iα regulates cardiac diastolic relaxation and fine-tunes the Frank–Starling response , 2016, Nature Communications.

[24]  Ming You,et al.  TSC2 Integrates Wnt and Energy Signals via a Coordinated Phosphorylation by AMPK and GSK3 to Regulate Cell Growth , 2006, Cell.

[25]  M. Latronico,et al.  MTORC1 regulates cardiac function and myocyte survival through 4E-BP1 inhibition in mice. , 2010, The Journal of clinical investigation.

[26]  Donald J. Zack,et al.  Expansion of the CRISPR-Cas9 genome targeting space through the use of H1 promoter-expressed guide–RNAs , 2014, Nature Communications.

[27]  D. Kass,et al.  Cardiac Phosphodiesterases and Their Modulation for Treating Heart Disease. , 2016, Handbook of experimental pharmacology.

[28]  Steven P Gygi,et al.  Quantitative phosphorylation profiling of the ERK/p90 ribosomal S6 kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Tian Xu,et al.  Akt regulates growth by directly phosphorylating Tsc2 , 2002, Nature Cell Biology.

[30]  Paul Tempst,et al.  Phosphorylation and Functional Inactivation of TSC2 by Erk Implications for Tuberous Sclerosisand Cancer Pathogenesis , 2005, Cell.