PKG1-modified TSC2 regulates mTORC1 activity to counter adverse cardiac stress

Abstract

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.

CHIP phosphorylation by protein kinase G enhances protein quality control and attenuates cardiac ischemic injury

“…2a ). In a prior Mass Spec-derived database of proteins in adult rat myocytes subjected to 15 minutes PKG activation 25 , we found CHIP modified at S20 (S20 in rat and mouse, S19 human, Fig. 2b ), a highly conserved residue in the TPR domain (Fig.…”

“…While this study focused on the impact of PKG on CHIP, studies have also shown the kinase modifies many other pathways relevant to myocardial pathological signaling and remodeling 34 , and these could also have contributed to the net effects observed. Among these mechanisms are PKG-mediated mitochondrial protection and cytoprotection against ischemia/infarction 35 , activating the regulator of G-protein signaling proteins RGS2 and RGS4 to inhibit Gq-receptor coupled signaling, blocking transient receptor potential canonical channel 6 to blunt fibrosis and hypertrophy 36 , 37 , and activating tuberous sclerosis complex 2 (TSC2) to blunt the mechanistic target of rapamycin complex-1 (mTORC1) stimulating autophagy and reducing pathological growth 25 . Specific to the UPS, PKG activates the 26S proteasome enhancing degradation of damaged/misfolded proteins in models of heart disease and neurodegenerative 22 – 24 .…”

Translating Translation to Mechanisms of Cardiac Hypertrophy

“…Recently, it was demonstrated that TSC2 phosphorylation by protein kinase G1 (PKG1) can prevent cardiac hypertrophy in response to pressure overload by suppressing mTORC1 signaling. Importantly, phosphorylation of PKG1 target sites in TSC2 does not affect the basal level of mTOR activity [68]. Together, these studies indicate that targeting of the specific regulatory components of mTORC1 may provide effective therapies while minimizing off target effects associated with inhibition of the mTORC kinase itself.…”

Cardiac Cyclic Nucleotide Phosphodiesterases: Roles and Therapeutic Potential in Heart Failure

“…Hence, PKG is anti-adrenergic. PKG also phosphorylates tuberin (TSC2), restraining mammalian target of rapamycin complex 1 (mTORC1) activity [24]. In this way, PKG regulates basal cardiac contractility, lowering inotropy and raising lusitropy, and precludes cardiac hypertrophy, fibrosis and apoptosis.…”