Abstract
The regulation of cellular metabolism and survival by trophic factors is not completely understood. Here, we describe a signaling cascade activated by the developmental regulator Notch, which inhibits apoptosis triggered by neglect in mammalian cells. In this pathway, the Notch intracellular domain (NIC), which is released after interaction with ligand, converges on the kinase mammalian target of rapamycin (mTOR) and the substrate-defining protein rapamycin independent companion of mTOR (Rictor), culminating in the activation of the kinase Akt/PKB. Biochemical and molecular approaches using site-directed mutants identified AktS473 as a key downstream target in the antiapoptotic pathway activated by NIC. Despite the demonstrated requirement for Notch processing and its predominant nuclear localization, NIC function was independent of CBF1/RBP-J, an essential DNA-binding component required for canonical signaling. In experiments that placed spatial constraints on NIC, enforced nuclear retention abrogated antiapoptotic activity and a membrane-anchored form of NIC-blocked apoptosis through mTOR, Rictor and Akt-dependent signaling. We show that the NIC-mTORC2-Akt cascade blocks the apoptotic response triggered by removal of medium or serum deprivation. Consistently, membrane-tethered NIC, and AktS473 inhibited apoptosis triggered by cytokine deprivation in activated T cells. Thus, this study identifies a non-canonical signaling cascade wherein NIC integrates with multiple pathways to regulate cell survival.
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Abbreviations
- CBF1:
-
Epstein–Barr virus (EBV) C-promoter-binding factor 1
- GFP:
-
green fluorescent protein
- FCS:
-
fluorescence correlation spectroscopy
- mTOR:
-
mammalian target of rapamycin
- NLS:
-
nuclear localization sequence
- NES:
-
nuclear export sequence
- NFL:
-
full-length Notch1
- NIC:
-
Notch intracellular domain
- PI3K:
-
phosphatidylinositol-3OH Kinase
- RBP-J:
-
recombination-binding protein Jk
- Rictor:
-
rapamycin insensitive companion of TOR
- Raptor:
-
regulatory-associated protein of mTOR
- sJag1:
-
soluble jagged1
- TUNEL:
-
terminal deoxynucleotidyl transferase-mediated nick-end labeling
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Acknowledgements
We thank the following for generously gifting plasmids used in the study: Jon Aster (Harvard Medical School, Boston, MA, USA) for the DN-CBF1 plasmid, Raphael Kopan (Washington University, St. Louis, MO, USA) for the Notch1LNG and Notch1LNG CC≫SS plasmids, and Barbara Osborne (University of Massachusetts/Amherst, Amherst, MA, USA) for the GFP-N1IC-NLS and GFP-N1IC-NES constructs. The human fibroblast cell line, the pMIG and pMIG-NIC constructs, and S6K antibody were obtained from A Rangarajan (IISc, Bangalore, India) and the CaSki cell line from S Krishna (NCBS, Bangalore, India). We are grateful to GV Shivashankar (NCBS, Bangalore, India), Satyajit Mayor (NCBS, Bangalore, India), and Veronica Rodrigues (DBS, Mumbai and NCBS, Bangalore, India) for comments and discussion. We thank GVS and members of his laboratory for their inputs for the FCS analysis; D Vaigundan for making the site-directed mutants of Akt and the NCBS central imaging and flow-cytometry facility funded by core funds and grants from the Department of Science and Technology (DST), Government of India – (Grant no. SR/S5/NM-36/2005 to the Centre of Nanotechnology and Grant no. 43/2003-SF), and the Wellcome Trust, UK. LRP was funded by a student fellowship from the Council of Scientific and Industrial Research, India, and MN was funded by a postdoctoral fellowship from the Department of Biotechnology, India. This study was funded by an International Senior Research Fellowship in Biomedical Sciences in India awarded by the Wellcome Trust, UK, and a grant awarded by the Department of Science and Technology, India, to AS.
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Perumalsamy, L., Nagala, M., Banerjee, P. et al. A hierarchical cascade activated by non-canonical Notch signaling and the mTOR–Rictor complex regulates neglect-induced death in mammalian cells. Cell Death Differ 16, 879–889 (2009). https://doi.org/10.1038/cdd.2009.20
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DOI: https://doi.org/10.1038/cdd.2009.20
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