SUMMARYThe mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis, and autophagy1. Its hyper-activation contributes to disease in many organs including the heart1,2, though broad mTORC1 inhibition risks interference with its homeostatic roles. Tuberin (TSC2) is a GTPase-activating protein and prominent intrinsic regulator of mTORC1 by modulating Rheb (Ras homolog enriched in brain). TSC2 constitutively inhibits mTORC1, but this activity is modified by phosphorylation from multiple signaling kinases to in turn inhibit (AMPK, GSK3β) or stimulate (Akt, ERK, RSK-1) mTORC1 activity3–9. Each kinase requires engagement of multiple serines, impeding analysis of their role in vivo. Here, we reveal phosphorylation or gain-or-loss of function mutations at either of two adjacent serines in TSC2 (S1365/1366 mouse; 1364/1365 human), with no prior known function, is sufficient to bi-directionally potently control growth-factor and hemodynamic-stress stimulated mTORC1 activity and consequent cell growth and autophagy. Basal mTORC1 activity, however, is unchanged. In heart, myocytes, and fibroblasts, phosphorylation occurs by protein kinase G (PKG), a primary effector of nitric oxide and natriuretic peptide signaling whose activation is protective against heart disease10–13. PKG suppression of hypertrophy and stimulation of autophagy in myocytes requires TSC2 phosphorylation. Homozygous knock-in (KI) mice expressing a phospho-silenced TSC2 (S1365A) mutation develop far worse heart disease and mortality from sustained pressure-overload (PO) due to hyperactive mTORC1 that cannot be rescued by PKG stimulation. Heterozygote SA-KI are rescued, and KI-mice expressing a phospho-mimetic (S1365E) mutation are protected. Neither KI model alters resting mTORC1 activity. Thus, TSC2 phosphorylation is both required and sufficient for PKG-mediated cardiac protection against pressure-overload. These newly identified serines provide a genetic tool to bi-directionally regulate the amplitude of stress-stimulated mTORC1 activity.
Rationale: Stimulated protein kinase G-1α (PKG1α) phosphorylates tuberous sclerosis complex 2 (TSC2) at serine 1365, potently suppressing mTORC1 activation by neuro-hormonal and hemodynamic stress. This reduces pathological hypertrophy and dysfunction and increases autophagy. PKG1α oxidation at cysteine 42 is also induced by these stressors, which blunts its cardioprotective effects. Objective: We tested the dependence of mTORC1 activation on PKG1α C42-oxidation, and its capacity to suppress such activation by soluble guanylyl cyclase-1 (GC-1) activation. Methods and Results: Cardiomyocytes expressing WT PKG1α (PKG1α WT ) or C42S redox-dead PKG1α CS/CS were exposed to endothelin-1 (ET-1). Cells expressing PKG1α WT exhibited substantial mTORC1 activation (p70 S6K, 4EBP1, and Ulk1 phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1α CS/CS expression. Mice with global knock-in of PKG1α CS/CS subjected to pressure-overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1α WT mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2 S1365 phosphorylation increased in PKG1α CS/CS more than PKG1α WT myocardium following PO. TSC2 S1365A/S1365A KI mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1α CS/CS mice, protection against PO-induced mTORC1 activation, cardio-depression, and mortality in PKG1α CS/CS mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1αWT and PKG1α CS/CS after PO, and blocked ET-1 stimulated mTORC1 in TSC2 S1365A expressing myocytes. Conclusions: Oxidation of PKG1α at C42 reduces its phosphorylation of TSC2, resulting in amplified PO-stimulated mTORC1 activity and associated hypertrophy, dysfunction, and depressed autophagy. This is ameliorated by direct GC-1 stimulation.
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