title: Leukemia-associated mutations within the NOTCH1 heterodimerization domain fall into at least two distinct mechanistic classes.
abstract: The NOTCH1 receptor is cleaved within its extracellular domain by furin during its maturation, yielding two subunits that are held together noncovalently by a juxtamembrane heterodimerization (HD) domain. Normal NOTCH1 signaling is initiated by the binding of ligand to the extracellular subunit, which renders the transmembrane subunit susceptible to two successive cleavages within and C terminal to the heterodimerization domain, catalyzed by metalloproteases and gamma-secretase, respectively. Because mutations in the heterodimerization domain of NOTCH1 occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL), we assessed the effect of 16 putative tumor-associated mutations on Notch1 signaling and HD domain stability. We show here that 15 of the 16 mutations activate canonical NOTCH1 signaling. Increases in signaling occur in a ligand-independent fashion, require gamma-secretase activity, and correlate with an increased susceptibility to cleavage by metalloproteases. The activating mutations cause soluble NOTCH1 heterodimers to dissociate more readily, either under native conditions (n = 3) or in the presence of urea (n = 11). One mutation, an insertion of 14 residues immediately N terminal to the metalloprotease cleavage site, increases metalloprotease sensitivity more than all others, despite a negligible effect on heterodimer stability by comparison, suggesting that the insertion may expose the S2 site by repositioning it relative to protective NOTCH1 ectodomain residues. Together, these studies show that leukemia-associated HD domain mutations render NOTCH1 sensitive to ligand-independent proteolytic activation through two distinct mechanisms.
authors.affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Malecki
authors.forename: Cheryll
authors.initials: C
authors.lastname: Sanchez-Irizarry
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Mitchell
authors.forename: Gavin
authors.initials: G
authors.lastname: Histen
authors.forename: Mina L
authors.initials: ML
authors.lastname: Xu
authors.forename: Jon C
authors.initials: JC
authors.lastname: Aster
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Blacklow
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D019281:Dimerization; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000235:genetics* / Q000378:metabolism*; D008024:Ligands; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D051881:Receptor, Notch1 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
chemical_list: D008024:Ligands; C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D011994:Recombinant Proteins