Proteolytic Processing of Delta-like 1 by ADAM Proteases

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. 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. Notch signaling regulates cell fate decisions during development and in the adult (1Lai E.C. Development. 2004; 131: 965-973Crossref PubMed Scopus (862) Google Scholar, 2Kadesch T. Curr. Opin. Genet. Dev. 2004; 14: 506-512Crossref PubMed Scopus (150) Google Scholar, 3Louvi A. Artavanis-Tsakonas S. Nat. Rev. Neurosci. 2006; 7: 93-102Crossref PubMed Scopus (706) Google Scholar). The signaling pathway is activated by direct interactions between Notch receptor, a transmembrane protein present at the surface of a signal-receiving cell, and a DSL (Delta/Serrate/Lag2) ligand, a transmembrane protein at the surface of a signal-sending cell. In mammals, there are four different Notch receptors (Notch 1-4), and five DSL ligands (Delta-like 1, 3, and 4 and Jagged 1 and 2). Ligand-bound Notch undergoes proteolytic cleavage at the S2 site in the extracellular domain, which is mediated by ADAM10 or ADAM17 (4Brou C. Logeat F. Gupta N. Bessia C. LeBail O. Doedens J.R. Cumano A. Roux P. Black R.A. Israël A. Mol. Cell. 2000; 5: 207-216Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar, 5Mumm J.S. Schroeter E.H. Saxena M.T. Griesemer A. Tian X. Pan D.J. Ray W.J. Kopan R. Mol. Cell. 2000; 5: 197-206Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar, 6Hartmann D. de Strooper B. Serneels L. Craessaerts K. Herreman A. Annaert W. Umans L. Lubke T. Lena I.A. von Figura K. Saftig P. Hum. Mol. Genet. 2002; 11: 2615-2624Crossref PubMed Google Scholar), members the ADAM family of metalloprotease-disintegrins (7Blobel C.P. Nat. Rev. Mol. Cell Biol. 2005; 6: 32-43Crossref PubMed Scopus (922) Google Scholar, 8Huovila A.P. Turner A.J. Pelto-Huikko M. Karkkainen I. Ortiz R.M. Trends Biochem. Sci. 2005; 30: 413-422Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). This is followed by the cleavage at the S3 site in the transmembrane domain of Notch by a γ-secretase complex (9Mumm J.S. Kopan R. Dev. Biol. 2000; 228: 151-165Crossref PubMed Scopus (848) Google Scholar, 10Selkoe D. Kopan R. Annu. Rev. Neurosci. 2003; 26: 565-597Crossref PubMed Scopus (560) Google Scholar). The intracellular domain of Notch translocates to the nucleus, where it interacts with CSL (CBF1, Su(H), Lag-1) transcription factors and activates expression of target genes (11Hayward S.D. Semin. Cancer Biol. 2004; 14: 387-396Crossref PubMed Scopus (85) Google Scholar). Similar to their receptors, Notch ligands also undergo ADAM-mediated cleavage in their extracellular domains, which is then followed by processing by γ-secretase (12Ikeuchi T. Sisodia S.S. J. Biol. Chem. 2003; 278: 7751-7754Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 13Six E. Ndiaye D. Laabi Y. Brou C. Gupta-Rossi N. Israël A. Logeat F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7638-7643Crossref PubMed Scopus (221) Google Scholar, 14LaVoie M.J. Selkoe D.J. J. Biol. Chem. 2003; 278: 34427-34437Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 15Bland C.E. Kimberly P. Rand M.D. J. Biol. Chem. 2003; 278: 13607-13610Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Two ADAMs were postulated to cleave Notch ligands in mammalian cells, ADAM10 and -17. ADAM10 was implicated in the processing of mouse (13Six E. Ndiaye D. Laabi Y. Brou C. Gupta-Rossi N. Israël A. Logeat F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7638-7643Crossref PubMed Scopus (221) Google Scholar) and rat Delta-like 1 (Dll1) 3The abbreviations used are: Dll1, Delta-like 1; ICD, intracellular domain of Dll1; PMA, phorbol myristate acetate; IP, immunoprecipitate; siRNA, small interfering RNA; MEF, mouse embryonic fibroblast; DMEM, Dulbecco's modified Eagle's medium; CTF, C-terminal fragment; NTF, N-terminal fragment; CHO, wild M.J. Selkoe D.J. J. Biol. Chem. 2003; 278: 34427-34437Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar), whereas ADAM17 was to cleave rat Jagged 1 M.J. Selkoe D.J. J. Biol. Chem. 2003; 278: 34427-34437Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). cleavage of Dll1 in mouse embryonic fibroblasts still to of the processing in MEFs (13Six E. Ndiaye D. Laabi Y. Brou C. Gupta-Rossi N. Israël A. Logeat F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7638-7643Crossref PubMed Scopus (221) Google Scholar), suggesting that ADAM10 is for Dll1 cleavage and other ADAMs for the processing in MEFs several ADAM proteins with domains, and U. K. S. J. D. Saftig P. C.P. J. Cell Biol. 2004; PubMed Scopus Google Scholar). to of ADAMs to of Dll1. Furthermore, although it is that proteolytic processing of Notch ligands Notch signaling in Rand M.D. X. N. W. P. T. Artavanis-Tsakonas S. PubMed Scopus Google Scholar, K. Rand M.D. B. Artavanis-Tsakonas S. J. Cell Biol. 2002; PubMed Scopus Google Scholar), the of ligand cleavage the Notch pathway the cell is Notch receptors with their ligands in a cell-autonomous K. O. M. S. K. Dev. Biol. 2002; PubMed Scopus Google Scholar, K. K. J. Dev. Biol. 2005; PubMed Scopus Google Scholar, E. W. A. C. J. C. J. Cell Biol. 2005; PubMed Scopus (221) Google Scholar). between receptors and ligands in cis Notch and the Notch pathway K. O. M. S. K. Dev. Biol. 2002; PubMed Scopus Google Scholar, K. K. J. Dev. Biol. 2005; PubMed Scopus Google Scholar, E. W. A. C. J. C. J. Cell Biol. 2005; PubMed Scopus (221) Google Scholar, A. A. J.S. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In proteolytic processing of by the of Notch in the cell A. E. R. E. Development. 2005; PubMed Scopus Google Scholar). In contrast, processing of Jagged 1 in mammalian by ADAM17 was postulated to Notch signaling in cis to of the C-terminal of Jagged 1 and Notch for γ-secretase M.J. Selkoe D.J. J. Biol. Chem. 2003; 278: 34427-34437Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). In this work we have the ability of several ADAMs other ADAM10 to cleave Dll1 and the of Dll1 cleavage Notch signaling in the cell. show that ADAM12, which not implicated in Notch process Dll1 but not demonstrate that Dll1 cleavage by ADAM12 activates Notch in a cell-autonomous manner. In we show that two other ADAMs, ADAM9 and are of Dll1 and we a the ADAM catalytic motif that necessary for the cleavage of Dll1. The of processing of Dll1 ADAM9/12/15-/- MEFs is reduced with the processing in wild type suggesting that the endogenous ADAM9 and/or ADAM12 present in wild type MEFs contribute to Dll1 cleavage. Finally, we show that the endogenous Dll1 present in primary mouse myoblasts undergoes cleavage in confluent, differentiating myoblast cultures, and this cleavage is decreased by ADAM12 small interfering Dll1 was by using a a and expression between and the site in Dll1 was of and were ADAMs and at their ADAM12 was by a the C-terminal The and ADAM12 and the ADAM15 were by using extracellular domain and in which C-terminal were with of was by R. Kopan four wild type or binding and were by D. of Notch reporter binding was by P. D. of Cell and and were in Dulbecco's modified Eagle's and or with at in the presence of a myoblasts were to and J. Cell Biol. PubMed Scopus Google Scholar) and in with and each and MEFs ADAM9/12/15-/- U. K. S. J. D. Saftig P. C.P. J. Cell Biol. 2004; PubMed Scopus Google Scholar) or wild type and with were in and each and were using In the of was 1 in a for reporter of was used expression of in cells, were for in the presence of a for expression was with the expression of were by cell using different for mouse ADAM12 and a were The of primary myoblasts were and with using to were to with The of expression of ADAM12 and the of Dll1 cleavage were Cell were for with for with 1 γ-secretase for 1 with 1 or for 1 with phorbol myristate were for and during the proteins were with 1 in a Cell were at for and were by and to a The was with and in then with primary in followed by with and using the the endogenous ADAM12 in primary cell were for using cell and Y. A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The primary were cytoplasmic mouse 1 were or and or used in the in of the in were by and using of Dll1 cleavage was at and the N-terminal fragment of Dll1 with and ADAM12, were to protein was cell was by and was used for with ADAMs or ADAM12, were with cell were at for and were used for with or with cytoplasmic Dll1 MEFs or the endogenous Dll1 primary was were with and protein 4 the were with and were with and by and or in were at with of of of ADAM12 or and of Dll1 or with or with were and for and were using the reporter The of was used for we ADAM12 is of processing Dll1. Dll1 was cells, for the of Dll1 the protein of and a of the C-terminal Dll1 fragment of in cell of wild type mouse ADAM12, but not the the of suggesting that ADAM12 cleaved Dll1. the fate of the N-terminal of Dll1, we between and of Dll1 of with we the N-terminal fragment of Dll1 The of was were co-transfected with wild type ADAM12, that Dll1 cleavage place in and that was released to medium. processing of Dll1 the processing mediated by endogenous ADAMs present in was at cell at the of at cell This that the cleavage have place in trans of ADAM12 and Dll1 of that were with Dll1 or with ADAM12 that the cleavage in cell of Dll1 cleavage that interactions of Dll1 or ADAM12 with proteins present the surface of for the processing of Dll1. was that of the extracellular domain of Dll1 was followed by cleavage of by γ-secretase (12Ikeuchi T. Sisodia S.S. J. Biol. Chem. 2003; 278: 7751-7754Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 13Six E. Ndiaye D. Laabi Y. Brou C. Gupta-Rossi N. Israël A. Logeat F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7638-7643Crossref PubMed Scopus (221) Google Scholar, 14LaVoie M.J. Selkoe D.J. J. Biol. Chem. 2003; 278: 34427-34437Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). In the of γ-secretase the intracellular domain of Dll1, was in but it was in with a of with a γ-secretase in of that at of Dll1 is by γ-secretase to Dll1, and the fragment is then to or cleavage of several substrate proteins is by or phorbol U. K. S. J. D. Saftig P. C.P. J. Cell Biol. 2004; PubMed Scopus Google Scholar, K. T. A. T. de Strooper B. D. Saftig P. J. 2005; PubMed Scopus Google Scholar, T. K. A. J. F. E. de Strooper B. D. Saftig P. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar, O. D. D. de Strooper B. Saftig P. T. M. M. J. Cell Biol. 2004; PubMed Scopus Google Scholar). ADAM12-mediated processing of Dll1 was not by and was by This that ADAM12 is of cleavage of Dll1. ADAM12 also cleave a for Dll1, we co-transfected with ADAM12 and and at a we used which for Notch ligand Notch at the S2 site (4Brou C. Logeat F. Gupta N. Bessia C. LeBail O. Doedens J.R. Cumano A. Roux P. Black R.A. Israël A. Mol. Cell. 2000; 5: 207-216Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar). of with PMA, the S2 cleavage was in In contrast, the S2 was not in cells, suggesting that ADAM12 ADAM12-mediated Dll1 processing not require cytoplasmic of ADAM12, the ADAM12 and the fragment a at the J. D. A. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) Dll1 Dll1 and ADAM12 by to the of ADAM12 the the Dll1 but not the The N-terminal extracellular fragment of Dll1 ADAM12 cleavage also the suggesting that the was of with ADAM12. The of Dll1 in ADAM12 is with the in in which the N-terminal fragment of Dll1 was released to and was not in with that Dll1, a Notch ligand but not Notch is cleaved by ADAM12, we the of Dll1 cleavage Notch signaling. the of the Notch pathway, we four T. P. E. S.D. Mol. Cell Biol. PubMed Scopus Google Scholar) or binding R. F. J. J. J. 2000; PubMed Scopus Google Scholar) for a CSL transcription activated by Notch (11Hayward S.D. Semin. Cancer Biol. 2004; 14: 387-396Crossref PubMed Scopus (85) Google Scholar). were co-transfected with mouse and a Notch reporter and with with Dll1 or with with to of the reporter with suggesting that the Dll1 in activated The of this was in several other in which or mouse fibroblasts were to present Notch ligands to E. W. A. C. J. C. J. Cell Biol. 2005; PubMed Scopus (221) Google Scholar, S. O. E. O. Logeat F. Brou C. Israël A. Scholar). of the of a in Notch in that with Notch with the are not This was to expression of the endogenous Notch ligands in that were of Notch In of the of ligand by Dll1 have a not have in Notch of the Notch reporter by with was for the reporter but not binding which to Notch Dll1 was with Notch in cells, the of Notch was to of in cis K. O. M. S. K. Dev. Biol. 2002; PubMed Scopus Google Scholar, K. K. J. Dev. Biol. 2005; PubMed Scopus Google Scholar, E. W. A. C. J. C. J. Cell Biol. 2005; PubMed Scopus (221) Google Scholar, A. A. J.S. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of Dll1-processing ADAM12 in of the Notch reporter The of ADAM12, which not process Dll1 not activate Notch This that Notch and Dll1 were in the cell, proteolytic processing of Dll1 the ability of Notch to and to activate its signaling Dll1 a substrate for other ADAMs that have not implicated in Dll1 and -17. ADAMs ADAM12 were to a C-terminal for a with with Dll1 that ADAM9 and to ADAM12, catalytic Dll1, but ADAM15 was not to process Dll1 the of the fragment of Dll1 and the of of each we that the cleavage of Dll1 with the ADAM17 ADAM12 ADAM proteins used in this with Dll1, by with ADAM15 with Dll1 but was to cleave ADAM15 a of the catalytic site of and J. M. R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the that are for Dll1 we the of the catalytic site of Dll1-processing ADAMs and of In to the Dll1-processing and we the of the catalytic site of a Dll1 (13Six E. Ndiaye D. Laabi Y. Brou C. Gupta-Rossi N. Israël A. Logeat F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7638-7643Crossref PubMed Scopus (221) Google Scholar, 14LaVoie M.J. Selkoe D.J. J. Biol. Chem. 2003; 278: 34427-34437Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). The motif for ADAM is W. F. U. P. W. Sci. PubMed Scopus Google Scholar), with binding a at the site and the catalytic that ADAMs the In contrast, ADAM15 a at this the of the the was for Dll1 cleavage. two ADAM12 and in cells, were and their and the were the ADAM12 its ability to process Dll1, the ADAM15 not catalytic Dll1 The of Dll1 processing by ADAM12 was not to its to with Dll1, in the that Asn-353 in ADAM12 is necessary for Dll1 but the presence of at the in ADAM15 is not for the cleavage. a of the domain of ADAM12, by using the of a is that the of Asn-353 the catalytic where it in the interactions with Dll1. ADAM in this work process Dll1 are at the endogenous we the of Dll1 cleavage in wild type and ADAM9/12/15-/- Dll1 was or and the of Dll1 cleavage was by cell to with or by of Dll1 and Dll1 The of Dll1 cleavage mediated by endogenous in MEFs was the cleavage in and in the of the to Dll1 and Dll1 in of the of cleavage Dll1 were in the of and in Dll1 is in Dll1 (13Six E. Ndiaye D. Laabi Y. Brou C. Gupta-Rossi N. 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Notch ligands undergo proteolytic processing by ADAMs, but the of ADAMs that cleavage the of processing are not In the Notch ligand is in S2 that ADAMs cleave of and of Rand M.D. X. N. W. P. T. Artavanis-Tsakonas S. PubMed Scopus Google Scholar, A. E. R. E. Development. 2005; PubMed Scopus Google Scholar). a of ADAM12, the ability to process A. E. R. E. Development. 2005; PubMed Scopus Google Scholar). Dll1 in CHO, or was cleaved by endogenous ADAMs present in (12Ikeuchi T. Sisodia S.S. J. Biol. Chem. 2003; 278: 7751-7754Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 13Six E. Ndiaye D. Laabi Y. Brou C. Gupta-Rossi N. Israël A. Logeat F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7638-7643Crossref PubMed Scopus (221) Google Scholar, 14LaVoie M.J. Selkoe D.J. J. Biol. Chem. 2003; 278: 34427-34437Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). of Dll1 or MEFs that the cleavage was reduced by in the of ADAM10 (13Six E. Ndiaye D. Laabi Y. Brou C. Gupta-Rossi N. Israël A. Logeat F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7638-7643Crossref PubMed Scopus (221) Google Scholar). This that ADAM10 was for Dll1 cleavage and that other ADAMs the cleavage of Dll1 in MEFs several ADAMs, and U. K. S. J. D. Saftig P. C.P. J. Cell Biol. 2004; PubMed Scopus Google Scholar), and we ADAM17 is the of and its Dll1 (13Six E. Ndiaye D. Laabi Y. Brou C. Gupta-Rossi N. Israël A. Logeat F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7638-7643Crossref PubMed Scopus (221) Google Scholar). Dll1 processing in was not by the ADAM17 or the ability of ADAM17 to cleave Dll1. of Dll1 by or not we show that ADAM and but not ADAM15, co-transfected with Dll1 cells, are of Dll1 cleavage. we have not the cleavage in Dll1, each of ADAMs a of a suggesting that the cleavage were or to each other. In the of ADAM12, we that Dll1 processing was by the of the N-terminal fragment of Dll1 to it was cell and in and it was by Furthermore, processing of Dll1 ADAM9/12/15-/- MEFs was with processing in wild type This that Dll1 was also to processing by the endogenous ADAM9 or ADAM12 or ADAM15 was not of Dll1 in cells, it not contribute to Dll1 processing in MEFs at the endogenous the of Dll1 cleavage in ADAM9/12/15-/- MEFs was it was with of Dll1 processing in MEFs (13Six E. Ndiaye D. Laabi Y. Brou C. Gupta-Rossi N. Israël A. Logeat F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7638-7643Crossref PubMed Scopus (221) Google Scholar) and with the of which have also to the processing. Our show that Asn-353 in ADAM12 is for Dll1 cleavage. of Asn-353 with a in ADAM15 ADAM12 Dll1. other ADAMs of Dll1, ADAM9 and and ADAM10 (13Six E. Ndiaye D. Laabi Y. Brou C. Gupta-Rossi N. Israël A. Logeat F. Proc. Natl. Acad. Sci. U. S. 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