mTOR complex 2 mediates Akt phosphorylation that requires PKCε in adult cardiac muscle cells

Abstract

Our earlier work showed that mammalian target of rapamycin (mTOR) is essential to the development of various hypertrophic responses, including cardiomyocyte survival. mTOR forms two independent complexes, mTORC1 and mTORC2, by associating with common and distinct cellular proteins. Both complexes are sensitive to a pharmacological inhibitor, torin1, although only mTORC1 is inhibited by rapamycin. Since mTORC2 is known to mediate the activation of a prosurvival kinase, Akt, we analyzed whether mTORC2 directly mediates Akt activation or whether it requires the participation of another prosurvival kinase, PKC ε (epsilon isoform of protein kinase-C). Our studies reveal that treatment of adult feline cardiomyocytes in vitro with insulin results in Akt phosphorylation at S473 for its activation which could be augmented with rapamycin but blocked by torin1. Silencing the expression of Rictor (rapamycin-insensitive companion of mTOR), an mTORC2 component, with a sh-RNA in cardiomyocytes lowers both insulin-stimulated Akt and PKC ε phosphorylation. Furthermore, phosphorylation of PKC ε and Akt at the critical S729 and S473 sites respectively was blocked by torin1 or Rictor knockdown but not by rapamycin, indicating that the phosphorylation at these specific sites occurs downstream of mTORC2. Additionally, expression of DN-PKC ε significantly lowered the insulin-stimulated Akt S473 phosphorylation, indicating an upstream role for PKC ε in the Akt activation. Biochemical analyses also revealed that PKC ε was part of Rictor but not Raptor (a binding partner and component of mTORC1). Together, these studies demonstrate that mTORC2 mediates prosurvival signaling in adult cardiomyocytes where PKC ε functions downstream of mTORC2 leading to Akt activation.

Qiliqiangxin Protects Against Cardiac Ischemia-Reperfusion Injury via Activation of the mTOR Pathway

“…mTORC1 activates p70S6 kinase that phosphorylates the ribosomal protein S6, and inhibits the binding of 4E-binding protein 1 (4EBP) to eukaryotic translation initiation factor 4E, resulting in the promotion of translation [9]. mTORC2 activates Akt, a key regulator of cardiomyocyte survival, by phosphorylation at Ser473 [10,11]. Previous studies using pharmacological mTOR inhibitors, including rapamycin, in both in vivo and ex vivo models of MI have found discrepant effects.…”

Ypk1 and Ypk2 kinases maintain Rho1 at the plasma membrane by flippase-dependent lipid remodeling after membrane stresses

“…Because crucial roles of TORC2 and Rho-PKC pathways in cardiac function and stress response have been demonstrated (Kajimoto et al, 2011;Volkers et al, 2013;Yano et al, 2014;Sciarretta et al, 2015;Zhao et al, 2014;Moschella et al, 2013), flippases and phosphatidylserine might also be key signaling mediators in these cell types.…”

mTOR Complexes Repress Hypertrophic Agonist–Stimulated Expression of Connective Tissue Growth Factor in Adult Cardiac Muscle Cells

“…Our study shows that blocking mTORC1 with acute rapamycin treatment results in an increased mTORC2 activity in both PO myocardium in vivo and in agoniststimulated adult CM in vitro 33 and that distinct PKC isoforms are involved in the signaling mechanisms by both these complexes. 26,34 Several studies implicate a link between the delta isoform of protein kinase C (PKC d ) and mTOR, 35,36 and our earlier work has shown that it plays an upstream role in mTOR phosphorylation in hypertrophic agonist-stimulated adult CM. 34 Because mTOR complexes have been shown to regulate both gene expression and protein translation, we explored the role of both of these complexes on the expression of CTGF during hypertrophic stimulation of isolated adult CM.…”

“…Because mTOR is reported to regulate hypertrophic agonist-induced signaling, [23][24][25][26] we hypothesized that mTOR might play a critical role for CTGF expression in CM during hypertrophic agonist stimulation.…”