title: 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, PKCε (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 PKCε phosphorylation. Furthermore, phosphorylation of PKCε 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-PKCε significantly lowered the insulin-stimulated Akt S473 phosphorylation, indicating an upstream role for PKCε in the Akt activation. Biochemical analyses also revealed that PKCε 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 PKCε functions downstream of mTORC2 leading to Akt activation.
authors.affiliation: Cardiology Division of the Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, SC 29425-2221, United States.
authors.forename: Phillip C
authors.initials: PC
authors.lastname: Moschella
authors.forename: John
authors.initials: J
authors.lastname: McKillop
authors.forename: Dorea L
authors.initials: DL
authors.lastname: Pleasant
authors.forename: Rebecca K
authors.initials: RK
authors.lastname: Harston
authors.forename: Sundaravadivel
authors.initials: S
authors.lastname: Balasubramanian
authors.forename: Dhandapani
authors.initials: D
authors.lastname: Kuppuswamy
mesh_terms: D000818:Animals; D002415:Cats; D002478:Cells, Cultured; D000076225:Mechanistic Target of Rapamycin Complex 2; D046912:Multiprotein Complexes / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010766:Phosphorylation; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D000076225:Mechanistic Target of Rapamycin Complex 2; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D051744:Protein Kinase C-epsilon