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A Spatiotemporal Notch Interaction Map from Membrane to Nucleus

ABSTRACT: Notch signaling relies on ligand-induced proteolysis to liberate a nuclear effector that drives cell fate decisions. The location and timing of individual steps required for proteolysis and movement of Notch from membrane to nucleus, however, remain unclear. Here, we use proximity labeling with quantitative multiplexed mass spectrometry to monitor the microenvironment of endogenous Notch2 after ligand stimulation in the presence of a gamma secretase inhibitor and then as a function of time after inhibitor removal. Our studies show that gamma secretase cleavage of Notch2 occurs in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurs within 45 minutes of inhibitor washout. This work provides a spatiotemporal map of unprecedented detail tracking the itinerary of Notch from membrane to nucleus after activation and identifies molecular events in signal transmission that are potential targets for modulating Notch signaling activity.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Embryo

SUBMITTER: Marian Kalocsay

LAB HEAD: Steve Blacklow

PROVIDER: PXD039010 | Pride | 2023-07-03

REPOSITORIES: Pride

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			Action	DRS
	541307_AP_APEX_JAG_16plx_Jan2022_Fr1_13106_psc_20ppm.mzIdentML	Mzid
	541309_AP_APEX_JAG_16plx_Jan2022_Fr2_13107_psc_20ppm.mzIdentML	Mzid
	541311_AP_APEX_JAG_16plx_Jan2022_Fr3_13108_psc_20ppm.mzIdentML	Mzid
	541313_AP_APEX_JAG_16plx_Jan2022_Fr4_13109_psc_20ppm.mzIdentML	Mzid
	541315_AP_APEX_JAG_16plx_Jan2022_Fr5_13110_psc_20ppm.mzIdentML	Mzid

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A Spatiotemporal Notch Interaction Map from Membrane to Nucleus

Project description:Notch signaling relies on ligand-induced proteolysis to liberate a nuclear effector that drives cell fate decisions. The location and timing of individual steps required for proteolysis and movement of Notch from membrane to nucleus, however, remain unclear. Here, we use proximity labeling with quantitative multiplexed mass spectrometry to monitor the microenvironment of endogenous Notch2 after ligand stimulation in the presence of a gamma secretase inhibitor and then as a function of time after inhibitor removal. Our studies show that gamma secretase cleavage of Notch2 occurs in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurs within 45 minutes of inhibitor washout. This work provides a spatiotemporal map of unprecedented detail tracking the itinerary of Notch from membrane to nucleus after activation and identifies molecular events in signal transmission that are potential targets for modulating Notch signaling activity.

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