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J Biol Chem. 2015 Jun 12;290(24):14963-78. doi: 10.1074/jbc.M114.627794. Epub 2015 Apr 16.

Target of Rapamycin Complex 2 Regulates Actin Polarization and Endocytosis via Multiple Pathways.

Author information

1
From the Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, 1211 Geneva.
2
the Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, 8093 Zürich.
3
the Institute of Molecular Life Sciences, University of Zürich, 8057 Zürich.
4
the Novartis Institutes for Biomedical Research, Novartis Campus, 4056 Basel.
5
the Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, 8093 Zürich, the Faculty of Science, University of Zürich, 8057 Zürich, and.
6
the Novartis Institutes for Biomedical Research, Novartis Campus, 4056 Basel, stephen.helliwell@novartis.com.
7
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.

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.

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

PMID:
25882841
PMCID:
PMC4463442
DOI:
10.1074/jbc.M114.627794
[Indexed for MEDLINE]
Free PMC Article

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