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J. Biol. Chem., Vol. 282, Issue 1, 436-444, January 5, 2007

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Proteolytic Processing of Delta-like 1 by ADAM Proteases^*

Emilia Dyczynska¹, Danqiong Sun¹, Haiqing Yi, Atsuko Sehara-Fujisawa, Carl P. Blobel^¶, and Anna Zolkiewska²

From the Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606 8507, Japan, and ^¶Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, Weill Medical College of Cornell University, New York, New York 10021

Delta-like 1 (Dll1) is a mammalian ligand for Notch receptors. Interactions between Dll1 and Notch in trans activate the Notch pathway, whereas Dll1 binding to Notch in cis inhibits Notch signaling. Dll1 undergoes proteolytic processing in its extracellular domain by ADAM10. In this work we demonstrate that Dll1 represents a substrate for several other members of the ADAM family. In co-transfected cells, Dll1 is constitutively cleaved by ADAM12, and the N-terminal fragment of Dll1 is released to medium. ADAM12-mediated cleavage of Dll1 is cell density-dependent, takes place in cis orientation, and does not require the presence of the cytoplasmic domain of ADAM12. Full-length Dll1, but not its N- or C-terminal proteolytic fragment, co-immunoprecipitates with ADAM12. By using a Notch reporter construct, we show that Dll1 processing by ADAM12 increases Notch signaling in a cell-autonomous manner. Furthermore, ADAM9 and ADAM17 have the ability to process Dll1. In contrast, ADAM15 does not cleave Dll1, although the two proteins still co-immunoprecipitate with each other. Asn-353 present in the catalytic motif of ADAM12 and other Dll1-processing ADAMs, but absent in ADAM15, is necessary for Dll1 cleavage. Dll1 cleavage is reduced in ADAM9/12/15^-/- mouse embryonic fibroblasts (MEFs), suggesting that the endogenous ADAM9 and/or ADAM12 present in wild type MEFs contribute to Dll1 processing. Finally, the endogenous Dll1 present in primary mouse myoblasts undergoes cleavage in confluent, differentiating myoblast cultures, and this cleavage is decreased by ADAM12 small interfering RNAs. Our findings expand the role of ADAM proteins in the regulation of Notch signaling.

Received for publication, June 7, 2006 , and in revised form, November 14, 2006.

^* This work was supported by National Institutes of Health Grants GM065528 (to A. Z.) and GM64750 (to C. P. B.). This is contribution 06-334-J from the Kansas Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Biochemistry, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506. Tel.: 785-532-3082; Fax: 785-532-7278; E-mail: zolkiea{at}ksu.edu.

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