Identification and Characterization of Presenilin-independent Notch Signaling

Abstract

Presenilin (PS) proteins control the proteolytic cleavage that precedes nuclear access of the Notch intracellular domain. Here we observe that a partial activation of the HES1 promoter can be detected in PS1/PS2 (PS1/2) double null cells using Notch1⌬E constructs or following Delta1 stimulation, despite an apparent abolition of the production and nuclear accumulation of the Notch intracellular domain. PS1/2-independent Notch activation is sensitive to Numblike, a physiological inhibitor of Notch. PS1/2-independent Notch signaling is also inhibited by an active ␥-secretase inhibitor in the low micromolar range and is not inhibited by an inactive analogue, similar to PS-dependent Notch signaling. However, experiments using a Notch1-Gal4-VP16 fusion protein indicate that the PS1/2-independent activity does not release Gal4-VP16 and is therefore unlikely to proceed via an intramembranous cleavage. These data reveal that a novel PS1/2-independent mechanism plays a partial role in Notch signal transduction.

Processing of Notch and amyloid precursor protein by γ-secretase is spatially distinct

“…The Notch-Hes1 reporter assay was modified from procedures as described in ref. 41. HEK293 cells were plated in a six-well dish (Ϸ5 ϫ 10 5 cells) and transiently cotransfected with the following plasmids: luciferase reporter construct Hes1-Luc (0.3 g), Notch construct pCS2-⌬EMV-6MT (also named Notch ⌬E) (3.0 g), which contains the transmembrane and intracellular domains of Notch1 or control vector pCS2 vector (3.0 g), and pCH110 (0.1 g), which expresses ␤-galactosidase (Amersham Pharmacia) for normalization of the transfection efficiency and luciferase activity.…”

“…After inhibitor treatment, the cells were lysed and assayed for luciferase assay. The background luciferase activity is defined as the activity in the presence of 1 M L-685,458, which has been shown to inhibit ␥-secretase-mediated Notch signaling (41,43). MRL631 and MRL505 at 1 M block Notch⌬E cleavage-mediated luciferase activity and are as potent as L-685,458 (Fig.…”

The γ-secretase-generated intracellular domain of β-amyloid precursor protein binds Numb and inhibits Notch signaling

“…As shown, Nbl and Numbs inhibit the transactivation of a CBF1-dependent luciferase reporter construct by NICD and N⌬E (Fig. 4A) (22,24,27). N⌬E contains the membrane-spanning region of Notch1, and the release of NICD from N⌬E depends on ␥-secretase cleavage (29).…”

“…The inhibition of CBF1-luciferase by AID was dosedependent ( Fig. 4F) and correlated with the ability of AID molecules to interact with Nbl, such that AID 39 -55 and AID [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] did not inhibit CBF1-luciferase activation by NICD (Fig. 4G).…”

“…In response to binding the Delta-Serrate-Lag2 family of ligands, Notch undergoes regulated proteolysis in two sequential steps, first on the extracellular side by tumor necrosis factor-converting enzyme (3,4), followed by an intramembranous cleavage mediated by the Presenilindependent ␥-secretase that releases the Notch intracellular domain (NICD) (19)(20)(21)(22). The functionally active NICD translocates to the nucleus where it interacts with the CBF1-SuHLag1 (CSL ) family of transcription factors and activates the transcription of genes that regulate the ability of cells to respond to various proliferation, differentiation, or apoptotic cues (18,23).…”