title: Target of Rapamycin Complex 2 Regulates Actin Polarization and Endocytosis via Multiple Pathways.
abstract: Target of rapamycin is a Ser/Thr kinase that operates in two conserved multiprotein complexes, TORC1 and TORC2. Unlike TORC1, TORC2 is insensitive to rapamycin, and its functional characterization is less advanced. Previous genetic studies demonstrated that TORC2 depletion leads to loss of actin polarization and loss of endocytosis. To determine how TORC2 regulates these readouts, we engineered a yeast strain in which TORC2 can be specifically and acutely inhibited by the imidazoquinoline NVP-BHS345. Kinetic analyses following inhibition of TORC2, supported with quantitative phosphoproteomics, revealed that TORC2 regulates these readouts via distinct pathways as follows: rapidly through direct protein phosphorylation cascades and slowly through indirect changes in the tensile properties of the plasma membrane. The rapid signaling events are mediated in large part through the phospholipid flippase kinases Fpk1 and Fpk2, whereas the slow signaling pathway involves increased plasma membrane tension resulting from a gradual depletion of sphingolipids. Additional hits in our phosphoproteomic screens highlight the intricate control TORC2 exerts over diverse aspects of eukaryote cell physiology.
authors.affiliation: From the Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, 1211 Geneva.
authors.forename: Delphine
authors.initials: D
authors.lastname: Rispal
authors.affiliation: From the Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, 1211 Geneva.
authors.forename: Sandra
authors.initials: S
authors.lastname: Eltschinger
authors.affiliation: From the Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, 1211 Geneva.
authors.forename: Michael
authors.initials: M
authors.lastname: Stahl
authors.affiliation: the Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, 8093 Zürich.
authors.forename: Stefania
authors.initials: S
authors.lastname: Vaga
authors.affiliation: the Institute of Molecular Life Sciences, University of Zürich, 8057 Zürich.
authors.forename: Bernd
authors.initials: B
authors.lastname: Bodenmiller
authors.affiliation: the Novartis Institutes for Biomedical Research, Novartis Campus, 4056 Basel.
authors.forename: Yann
authors.initials: Y
authors.lastname: Abraham
authors.affiliation: the Novartis Institutes for Biomedical Research, Novartis Campus, 4056 Basel.
authors.forename: Ireos
authors.initials: I
authors.lastname: Filipuzzi
authors.affiliation: the Novartis Institutes for Biomedical Research, Novartis Campus, 4056 Basel.
authors.forename: N Rao
authors.initials: NR
authors.lastname: Movva
authors.affiliation: the Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, 8093 Zürich, the Faculty of Science, University of Zürich, 8057 Zürich, and.
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
authors.affiliation: the Novartis Institutes for Biomedical Research, Novartis Campus, 4056 Basel, stephen.helliwell@novartis.com.
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Helliwell
authors.affiliation: From the Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, 1211 Geneva, the National Centre for Competence in Research Chemical Biology, 1211 Geneva, Switzerland robbie.loewith@unige.ch.
authors.forename: Robbie
authors.initials: R
authors.lastname: Loewith
mesh_terms: D000199:Actins / Q000378:metabolism*; D004705:Endocytosis; D005656:Fungal Proteins / Q000378:metabolism; D000076225:Mechanistic Target of Rapamycin Complex 2; D046912:Multiprotein Complexes / Q000502:physiology*; D010766:Phosphorylation; D025341:Principal Component Analysis; D011494:Protein Kinases / Q000378:metabolism; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000502:physiology*
chemical_list: D000199:Actins; D005656:Fungal Proteins; D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C533049:Fpk1 protein, S cerevisiae; C533050:Fpk2 protein, S cerevisiae; D000076225:Mechanistic Target of Rapamycin Complex 2; D058570:TOR Serine-Threonine Kinases
keywords: cell signaling; lipid signaling; phosphoproteome; plasma membrane; protein kinase; target of rapamycin (TOR); target of rapamycin complex 2, Ypk1, sphingolipids, actin polarization, endocytosis, flippase kinase