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Molecular and Cellular Biology, June 2006, p. 4642-4651, Vol. 26, No. 12
0270-7306/06/$08.00+0     doi:10.1128/MCB.01655-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Leukemia-Associated Mutations within the NOTCH1 Heterodimerization Domain Fall into at Least Two Distinct Mechanistic Classes{dagger}

Michael J. Malecki,{ddagger} Cheryll Sanchez-Irizarry,{ddagger} Jennifer L. Mitchell, Gavin Histen, Mina L. Xu, Jon C. Aster,* and Stephen C. Blacklow*

Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

Received 25 August 2005/ Returned for modification 4 October 2005/ Accepted 7 March 2006

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.

* Corresponding author. Mailing address: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115. Phone for Jon C. Aster: (617) 525-7329. Fax: (617) 264-5169. E-mail: jaster{at}rics.bwh.harvard.edu. Phone for Stephen C. Blacklow: (617) 525-4413. Fax: (617) 525-4414. E-mail: sblacklow{at}rics.bwh.harvard.edu.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

{ddagger} M.J.M. and C.S.-I. contributed equally to this work.

Molecular and Cellular Biology, June 2006, p. 4642-4651, Vol. 26, No. 12
0270-7306/06/$08.00+0     doi:10.1128/MCB.01655-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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