Abstract
Notch signaling is a cellular pathway regulating cell-fate determination and adult tissue homeostasis. Little is known about how canonical Notch ligands or Fringe enzymes differentially affect NOTCH1 and NOTCH2. Using cell-based Notch signaling and ligand-binding assays, we evaluated differences in NOTCH1 and NOTCH2 responses to Delta-like (DLL) and Jagged (JAG) family members and the extent to which Fringe enzymes modulate their activity. In the absence of Fringes, DLL4-NOTCH1 activation was more than twice that of DLL4-NOTCH2, whereas all other ligands activated NOTCH2 similarly or slightly more than NOTCH1. However, NOTCH2 showed less sensitivity to the Fringes. Lunatic fringe (LFNG) enhanced NOTCH2 activation by DLL1 and -4, and Manic fringe (MFNG) inhibited NOTCH2 activation by JAG1 and -2. Mass spectral analysis showed that O-fucose occurred at high stoichiometry at most consensus sequences of NOTCH2 and that the Fringe enzymes modified more O-fucose sites of NOTCH2 compared with NOTCH1. Mutagenesis studies showed that LFNG modification of O-fucose on EGF8 and -12 of NOTCH2 was responsible for enhancement of DLL1-NOTCH2 activation, similar to previous reports for NOTCH1. In contrast to NOTCH1, a single O-fucose site mutant that substantially blocked the ability of MFNG to inhibit NOTCH2 activation by JAG1 could not be identified. Interestingly, elimination of the O-fucose site on EGF12 allowed LFNG to inhibit JAG1-NOTCH2 activation, and O-fucosylation on EGF9 was important for trafficking of both NOTCH1 and NOTCH2. Together, these studies provide new insights into the differential regulation of NOTCH1 and NOTCH2 by Notch ligands and Fringe enzymes.
Highlights
The Notch signaling pathway plays essential roles in development of metazoans, and defects in the Notch pathway result in a wide variety of congenital disorders and cancers [1,2,3]
Using mass spectral glycoproteomics methods, we demonstrated that the majority of predicted O-fucose consensus sequences are modified by protein O-fucosyltransferase 1 (POFUT1) at high stoichiometry, that Lunatic fringe (LFNG) modifies O-fucose on a subset of these epidermal growth factor–like (EGF) repeats, and that Manic fringe (MFNG) and Radical fringe (RFNG) modify a subset of those EGF repeats modified by LFNG
Because the effects of LFNG on DLL1-NOTCH2 binding were so much lower than on DLL1-NOTCH1, and NOTCH2 had a higher affinity for DLL1 than NOTCH1 (Fig. 1C), we examined which regions of the NOTCH2 extracellular domain (ECD) are responsible for binding to DLL1
Summary
The Notch signaling pathway plays essential roles in development of metazoans, and defects in the Notch pathway result in a wide variety of congenital disorders and cancers [1,2,3]. Using cell-based NOTCH1 activation and ligand-binding assays, we showed that O-fucose on EGF8 and -12 of NOTCH1 are the major sites responsible for Fringe-mediated enhancement of NOTCH1 activation by DLL1. We showed that LFNG and MFNG modification of O-fucose on EGF6 and -36 are mainly responsible for inhibition of NOTCH1 activation by JAG1 [22].
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