title: Localization of mTORC2 activity inside cells.
abstract: Activation of protein kinase Akt via its direct phosphorylation by mammalian target of rapamycin (mTOR) complex 2 (mTORC2) couples extracellular growth and survival cues with pathways controlling cell growth and proliferation, yet how growth factors target the activity of mTORC2 toward Akt is unknown. In this study, we examine the localization of the obligate mTORC2 component, mSin1, inside cells and report the development of a reporter to examine intracellular localization and regulation by growth factors of the endogenous mTORC2 activity. Using a combination of imaging and biochemical approaches, we demonstrate that inside cells, mTORC2 activity localizes to the plasma membrane, mitochondria, and a subpopulation of endosomal vesicles. We show that unlike the endosomal pool, the activity and localization of mTORC2 via the Sin1 pleckstrin homology domain at the plasma membrane is PI3K and growth factor independent. Furthermore, we show that membrane recruitment is sufficient for Akt phosphorylation in response to growth factors. Our results indicate the existence of spatially separated mTORC2 populations with distinct sensitivity to PI3K inside cells and suggest that intracellular localization could contribute to regulation of mTORC2 activity toward Akt.
authors.affiliation: Max F. Perutz Laboratories, Department of Structural and Computational Biology, Vienna BioCenter, 1030 Vienna, Austria.|Department of Medical Biochemistry, Medical University of Vienna, 1030 Vienna, Austria.
authors.forename: Michael
authors.identifier: http://orcid.org/0000-0003-4234-7187
authors.initials: M
authors.lastname: Ebner
authors.affiliation: Department of Medical Biochemistry, Medical University of Vienna, 1030 Vienna, Austria.|Center for Molecular Biology, Student Services, University of Vienna, 1030 Vienna, Austria.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Sinkovics
authors.affiliation: Vienna BioCenter Summer School Program, Division of Systems Biology of Signal Transduction, 69120 Heidelberg, Germany.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Szczygieł
authors.affiliation: Center for Molecular Biology, Student Services, University of Vienna, 1030 Vienna, Austria.
authors.forename: Daniela Wolfschoon
authors.initials: DW
authors.lastname: Ribeiro
authors.affiliation: Max F. Perutz Laboratories, Department of Structural and Computational Biology, Vienna BioCenter, 1030 Vienna, Austria ivan.yudushkin@mfpl.ac.at.|Department of Medical Biochemistry, Medical University of Vienna, 1030 Vienna, Austria.
authors.forename: Ivan
authors.identifier: http://orcid.org/0000-0002-6637-027X
authors.initials: I
authors.lastname: Yudushkin
mesh_terms: D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D011992:Endosomes / Q000201:enzymology; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D000076225:Mechanistic Target of Rapamycin Complex 2; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000201:enzymology; D020559:Monomeric GTP-Binding Proteins; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D059008:Time-Lapse Imaging; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; C000603431:mSin1 protein, human; D000076225:Mechanistic Target of Rapamycin Complex 2; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D020559:Monomeric GTP-Binding Proteins