Mind Bomb-2 Is an E3 Ligase for Notch Ligand

Abstract

The zebrafish gene, mind bomb (mib), encodes a protein that positively regulates of the Delta-mediated Notch signaling. It interacts with the intracellular domain of Delta to promote its ubiquitination and endocytosis. In our search for the mouse homologue of zebrafish mind bomb, we cloned two homologues in the mouse genome: a mouse orthologue (mouse mib1) and a paralogue, named mind bomb-2 (mib2), which is evolutionarily conserved from Drosophila to human. Both Mib1 and Mib2 have an E3 ubiquitin ligase activity in their C-terminal RING domain and interact with Xenopus Delta (XD) via their N-terminal region. Mib2 is also able to ligate ubiquitin to XD and shift the membrane localization of Delta to intracellular vesicles. Importantly, Mib2 rescues both the neuronal and vascular defects in the zebrafish mibta52b mutants. In contrast to the functional similarities between Mib1 and Mib2, mib2 is highly expressed in adult tissues, but almost not at all in embryos, whereas mib1 is abundantly expressed in both embryos and adult tissues. These data suggest that Mib2 has functional similarities to Mib1, but might have distinct roles in Notch signaling as an E3 ubiquitin ligase. The zebrafish gene, mind bomb (mib), encodes a protein that positively regulates of the Delta-mediated Notch signaling. It interacts with the intracellular domain of Delta to promote its ubiquitination and endocytosis. In our search for the mouse homologue of zebrafish mind bomb, we cloned two homologues in the mouse genome: a mouse orthologue (mouse mib1) and a paralogue, named mind bomb-2 (mib2), which is evolutionarily conserved from Drosophila to human. Both Mib1 and Mib2 have an E3 ubiquitin ligase activity in their C-terminal RING domain and interact with Xenopus Delta (XD) via their N-terminal region. Mib2 is also able to ligate ubiquitin to XD and shift the membrane localization of Delta to intracellular vesicles. Importantly, Mib2 rescues both the neuronal and vascular defects in the zebrafish mibta52b mutants. In contrast to the functional similarities between Mib1 and Mib2, mib2 is highly expressed in adult tissues, but almost not at all in embryos, whereas mib1 is abundantly expressed in both embryos and adult tissues. These data suggest that Mib2 has functional similarities to Mib1, but might have distinct roles in Notch signaling as an E3 ubiquitin ligase. The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism, in which both the ligands and receptors are type 1 transmembrane proteins, thus restricting the Notch pathway to the regulation of interactions between physically adjacent cells (1Sorkin A. Von Zastrow M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 600-614Crossref PubMed Scopus (704) Google Scholar, 2Kramer H. Science's STKE. 2000; (http://stke.sciencemag.org/cgi/content/full/sigtrans;2000/53/PE1)PubMed Google Scholar, 3Artavanis-Tsakonas S. Matsuno K. Fortini M.E. Science. 1995; 268: 225-232Crossref PubMed Scopus (1406) Google Scholar). To date, four Notch receptors and five ligands (Delta-like (Dll)-1, -3, and -4, and Jagged (Jag) 1 and 2) have been described in mammals. The interactions between these Notch receptors and ligands are essential for a wide range of developmental cell-to-cell interactions. When ligand-receptor interactions induce the sequential proteolytic cleavage events that release the intracellular domain of Notch receptor from the cell membrane, the Notch signaling pathway is activated (4Black R.A. White J.M. Curr. Opin. Cell Biol. 1998; 10: 654-659Crossref PubMed Scopus (428) Google Scholar, 5Annaert W. De Strooper B. Trends Neurosci. 1999; 22: 439-443Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 6Mumm 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). In the nucleus, the intracellular domain of Notch receptor forms a complex with CSL (CBF1/RBP-Jκ, Su(H), Lag-1). This complex is a transcriptional activator, which induces the expression of genes encoding Hairy/Enhancer-of-split (HES)-related proteins, which inhibit the basic helix-loop-helix transcription factors (7Iso T. Kedes L. Hamamori Y. J. Cell. Physiol. 2003; 194: 237-255Crossref PubMed Scopus (1015) Google Scholar, 8Nam Y. Weng A.P. Aster J.C. Blacklow S.C. J. Biol. Chem. 2003; 278: 21232-21239Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Many components of the Notch signaling pathway in Drosophila were originally identified by mutants that confer a similar neurogenic phenotype in early embryos. The mutations of these genes, such as notch, delta, E(SPL), mastermind, and neuralized, result in an increase of neural cells at the expense of epidermal structures (9Thomas J.B. Crews S.T. FASEB J. 1990; 4: 2476-2482Crossref PubMed Scopus (4) Google Scholar). The signaling by Notch receptors involves three successive cleavages: the S1 cleavage in the trans-Golgi network by Furin-like proteases, the ligand-dependent extracellular S2 cleavage by metalloproteinases, and the intramembraneous S3 cleavage by γ-secretase complexes to release the active intracellular domain of Notch receptor (10Baron M. Semin. Cell Dev. Biol. 2003; 14: 113-119Crossref PubMed Scopus (275) Google Scholar). In the signaling cell, Delta-Notch interactions result in the endocytosis of Delta, which carries along the bound Notch extracellular domain, and the endocytosis-defective Delta mutants have reduced signaling capacity. The ligand-dependent extracellular S2 cleavage of Notch receptor is coupled to the trans-endocytosis of the Notch extracellular domain into the ligand expressing/signal sending cell (2Kramer H. Science's STKE. 2000; (http://stke.sciencemag.org/cgi/content/full/sigtrans;2000/53/PE1)PubMed Google Scholar). To date, there are two candidate genes, Neuralized (Neur) and Mib, which promote the ubiquitination and endocytosis of Notch ligands. Recently, Itoh et al. (11Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar) reported that Mib promotes the ubiquitination and endocytosis of zebrafish DeltaD and DeltaB. Although Mind bomb and Neur might be key regulators in the endocytosis of Delta for the activation of Notch receptors, the molecular diversity of the vertebrate genes encoding five Notch ligands (Dll1, Dll3, Dll4, Jag1, and Jag2) and four Notch receptors (Notch1–4) in mammals suggests that there might be an additional E3 ubiquitin ligase that regulates Notch ligands. Indeed, all of these ligands have unique expression patterns, and the knock-out mice for each gene display a distinct phenotype. In parallel with the diversity of the ligands, more complex endocytic machinery might exist as well (12Hrabe de Angelis M. McIntyre 2nd, J. Gossler A. Nature. 1997; 386: 717-721Crossref PubMed Scopus (547) Google Scholar, 13Sidow A. Bulotsky M.S. Kerrebrock A.W. Bronson R.T. Daly M.J. Reeve M.P. Hawkins T.L. Birren B.W. Jaenisch R. Lander E.S. Nature. 1997; 389: 722-725Crossref PubMed Scopus (101) Google Scholar, 14Kusumi K. Sun E.S. Kerrebrock A.W. Bronson R.T. Chi D.C. Bulotsky M.S. 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Genes Dev. 1998; 12: 1046-1057Crossref PubMed Scopus (346) Google Scholar, 20Krebs L.T. Shutter J.R. Tanigaki K. Honjo T. Stark K.L. Gridley T. Genes Dev. 2004; 18: 2469-2473Crossref PubMed Scopus (438) Google Scholar). In our search for the mouse Mind bomb homologue, we found one Mind bomb orthologue (Mib1) and one Mind bomb paralogue, Mind bomb-2 Mib2 (AY974090) skeletrophin (21Takeuchi T. Heng H.H. Ye C.J. Liang S.B. Iwata J. Sonobe H. Ohtsuki Y. Am. J. Pathol. 2003; 163: 1395-1404Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Here we show that Mib2 is another E3 ubiquitin ligase that ubiquitinates the Notch ligand, Delta, and promotes its endocytosis. that Mib2 might be an E3 ubiquitin ligase for Delta in the Notch signaling of the mib2 and of in the Mib2 RING mouse as the and to the of the mouse mib2 The for mib2 a of which cloned into the ubiquitin mind the The mib2 mutants with mutations in the RING domain were of the cloned were by and were to the and C-terminal of The for the N-terminal were and The for the C-terminal were and the expression in the mouse adult tissues, the with both in of the in with zebrafish embryos were described for the C.H. E. H. Yeo S.Y. T.L. Neurosci. PubMed Scopus Google Kim C.H. Chitnis A.B. Development. PubMed Google and Kim C.H. Chitnis A.B. Development. PubMed Google Scholar). To the with the the embryos were in in at for 1 and in In for mouse mib1 and mib2 were from and expression by In for zebrafish mib1 and mib2 were cloned into and with by The for the zebrafish mib1 were and The for the zebrafish mib2 were and Cell and cells were in and were with of were in 1 of The were and the by of the of were a and to a The membrane with the of and a To the and were and The zebrafish mibta52b as the mib1 The mouse mib2 into the and the encoding mouse mib2 in the The mib2 into zebrafish embryos. The of into the embryos from the with of were in the cells were the were to the The were protein at for 1 The were by the of protein with an at for The complexes bound to the protein were with and in and the were a were by with an by a an In and in ubiquitination were from described (11Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). the N-terminal ubiquitin and The Mib1 and Mib2 RING and their mutants were to the at the were by to the To the ubiquitination activity in cells were with of and the cells were The were in and by The were and the were with an by an cells were with the cells were in and in with for at The cells were in and in at and with mouse and in in for 1 at the cells were with an with for at The cells were and with for three the cells were and with a were a Mib2 a Mib1 Mib1 is a E3 ubiquitin ligase that is an essential of Notch signaling in (11Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). the and we found that the and mouse one orthologue and one paralogue, mind bomb-2 To the of Mib2, its cloned and from mouse and we found that has To the the we the and the of we that there were two also the of these two to the in the tissues. of the two of the mib2 with in the tissues. also found that of the mib2 from the of skeletrophin in the of the that our is Mib1 and Mib2 have and in their and also have in their domain and Mib2 has two one one and two RING (11Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). of these are in a similar as with of Mib2 to be evolutionarily conserved from Drosophila to not and a mib2 which its not the which might be of a in the mib2 highly expressed in the and whereas mib1 highly expressed in not The expression of these two genes suggest a distinct of mib2 in as with of Mib2 an E3 Mib1 has an E3 ubiquitin ligase activity with Delta, both in and in (11Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). the and the similar domain we that Mib2 might have a similar To the ubiquitin ligase activity of the Mib2 RING in RING were with and and The of by a the RING domain from Mib1 in a of molecular whereas a similar of such activity When RING in we ubiquitination in an and that Mib2 RING an E3 ubiquitin ligase Mib2 two RING we which domain is for the E3 ubiquitin ligase three RING that have a in the RING domain the RING domain and both and In the a in the RING domain, the ubiquitination activity and whereas the in the RING domain not the that the Mib2 RING is for the ubiquitin in To the activity of Mib2 in cells were with encoding and cell were with an and with an Both Mib1 and Mib2 of ubiquitination activity as with the and ubiquitination in the of the and the of these Mib2 with Xenopus Delta zebrafish Mib1 and Drosophila Neur interact with Delta, and to to the endocytic machinery (11Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar, C. Dev. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, E. C. C. Dev. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). To the that Mib2 also interacts with and ubiquitinates Delta, Mib2 with Xenopus Delta Xenopus Delta its intracellular domain in with an and with an to the of ligands. The that both Mib1 and Mib2 interact with but not with These data that Mib2 to XD and that the intracellular domain of Delta ligand is essential for the with To the of Mib2 that interacts with Delta ligand, with forms of the N-terminal the and the C-terminal of the Mib2 forms were with an and with an to the of Both Mib2 and the protein that the N-terminal were with and these that Mib2 interacts with the intracellular domain of Delta its N-terminal region. Mib2 Xenopus Delta Mib1 interacts with Delta its N-terminal and and the Mib1 RING domain is for the ubiquitination of Delta (11Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). Mib2 also in of and with Delta, we promotes ubiquitination of and the candidate E3 ubiquitin were in by an and the of with an The ubiquitination of in the of both and and Mib2 which has mutations in the and RING and a of and the C-terminal of Mib2, which not Delta, a of ubiquitination and these we that Mib2 interacts with and ubiquitinates Delta its N-terminal and RING domain, Mib2 has two RING and the Mib2 RING is for To both RING are essential for the ubiquitination of Delta, Mib2 with mutations in their RING domain and as were with of these mutants in the activity of the Mib2 RING in These data that both RING in Mib2 are essential for ubiquitination of its Mib2 the of Xenopus Delta the localization of Mib2, we that the intracellular S. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). cells with were with for the early with the early but not with these that Mib2 is an protein in the early In the ubiquitination of membrane as a for endocytosis for B. Curr. Opin. Cell Biol. 2003; PubMed Scopus Google Scholar, L. FASEB J. 1997; PubMed Scopus Google Scholar). To Mib2 promotes the endocytosis of Delta, we along with in When in the as structures and expressed the membrane a and In both and were expression the cell and in the as a with and These suggest that Mib2 promotes of When with mutations in the RING domain and were with in of these mutants were able to promote endocytosis of and and and and endocytosis of in the of and which not interact with both the E3 ubiquitin ligase activity of Mib2 and the intracellular domain of XD are for Delta endocytosis. Mib2 the and of mibta52b Mib2 has similarities to Mib1, such as ligand the ubiquitin ligase and the endocytic activity for Delta, we the that Mib2 the defects in and of the zebrafish mib1 The zebrafish mibta52b mutants such as of neuronal cell cells in embryos from mibta52b were in one at the with encoding zebrafish Mib1 mouse Mib2, and their were by expression at the The of zebrafish mib1 the expression in mibta52b mutants with its mouse mib2 in the mibta52b expression also reduced as with that of the a of cells in the mib2 in the mibta52b that Mib2 is able to Notch signaling to inhibit the neuronal in the a and (11Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). The zebrafish mibta52b mutants also defects in such as the of and expression of the The is expressed the in mibta52b embryos Kim C.H. Chitnis A.B. Development. PubMed Google Scholar). When mouse mib2 in the of mibta52b embryos, to the the at the In mouse mib2 in zebrafish mibta52b the expression of the whereas embryos to in the and these data suggest that Mib2 has functional similarities to of mib1 and the and functional similarities between Mib1 and Mib2, the zebrafish mibta52b mutants display defects in the Notch signaling pathway (11Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). mouse mib2 into zebrafish mibta52b mutants the neurogenic and we that zebrafish mib2 might not be expressed is expressed at a in zebrafish embryos, as with zebrafish mib1 mouse mib2 expressed in the and of mouse embryos, and in the and of mouse embryos, whereas mouse mib1 highly expressed in the and of both and mouse embryos and in the and at 1 and the adult mib1 and mib2 were expressed in similar patterns, in the in the and and the In the mib1 highly expressed in the whereas the expression of mib2 mib1 highly expressed in both embryos and adult tissues, whereas mib2 highly expressed in and but in embryos, that mib1 might have a and mib1 and mib2 might in and of mouse mib1 and and in mib2 and mib1 in and embryos. that mib1 is expressed the and highly expressed in the and whereas expression of mib2 in the and in mib2 and and mib1 and in the and and the from 1 and and and the unique expression of mib1 and the expression of both mib1 and Many E3 ubiquitin are to Notch signaling Curr. Biol. 2002; 12: Full Text Full Text PDF PubMed Scopus Google Scholar). are in two one that ubiquitinates Notch receptor and the that regulates Notch ligands. ubiquitinates the intracellular domain of and with to the endocytosis of Notch receptors C.J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, G. S. I. J. M. A. I. J. Mol. Cell. Biol. PubMed Scopus Google Scholar). Both Mind bomb and Neur Delta and thus promote its endocytosis (11Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar, C. Dev. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, E. C. C. Dev. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, C. Dev. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In the we have cloned mouse Mib2, another protein of zebrafish Mib2 interacts with and ubiquitinates the localization of Delta to a the endocytosis of Importantly, rescues the defects in and of the zebrafish mibta52b mutants. we that Mib2 is another E3 ubiquitin ligase that the Notch ligand in the Notch signaling Both mouse Mib1 and Mib2 have similar domain which of a a and RING Mib homologues also exist in and (11Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). has RING whereas has RING not both have similar domain as with in and that Mib1 and Mib2 are evolutionarily conserved from to human. of the domain structures between Mib1 and Mib2 that the RING domain is one of the there are three in Mib1 and two in Mib2, whereas E3 ubiquitin have one RING Both Mib1 and Mib2 have RING at their C-terminal and the domain at their N-terminal RING domain is by a of and that are in which is for the of the domain and its RING are as the is a a S. Lorick K.L. Weissman A.M. Semin. Biol. 2003; PubMed Scopus Google Scholar). Both Mib1 and Mib2 are the type and have an E3 activity with in (11Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar, Y. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). in ubiquitination that the RING in Mib2 is not essential for its ubiquitination and endocytosis of XD by Mib2 both RING that both are essential in The activity of the two RING has not been the that ubiquitin from to the Notch ligands by Mib1 and Mib2 into the interactions. are five zebrafish mib1 mutants and with mutations in the mib1 The mutants display more the mutants C. Jiang Y.J. L. Lewis J. Development. 1998; PubMed Google Scholar). of and have a and the and have mutations in the RING domain and (11Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). These the that a in have which might be of the type Mib1 that is from the mib2 is expressed in the early of and mib1 is highly expressed is more that the activity of the Mib1 protein (11Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. Weissman A.M. Lewis J. Chandrasekharappa S.C. Chitnis A.B. Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). When mouse mib2 is in the of mibta52b embryos, the of Mib2 might the of and for the defects in and The expression of Mib1 and Mib2 in adult tissues, but is unique in Mib2 is highly expressed in adult tissues, such as and not Mib2 expression in the which is with a (21Takeuchi T. Heng H.H. Ye C.J. Liang S.B. Iwata J. Sonobe H. Ohtsuki Y. Am. J. Pathol. 2003; 163: 1395-1404Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). This might be of between mouse and human. Mib1, also as protein protein is expressed in adult but highly in and Y. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). we also expression of Mib1 in and but expression in a range of not These and unique expression of Mib1 and Mib2 suggest that these two E3 but in the regulation of Notch ligands. Notch signaling is for the and the of adult such as cells in the and neural cells S. T. V. D. A.J. J.S. Conlon R.A. A. D. Genes Dev. 2002; PubMed Scopus Google Scholar, Curr. Opin. 2004; PubMed Scopus Google Scholar, J. P. B. 2002; PubMed Scopus Google Scholar). Notch signaling is also in such as cell J. D.C. J. Cell. Full Text PDF PubMed Scopus Google Scholar) and and M. A. K. P. M. H. Nat. Genet. 2003; PubMed Scopus Google Scholar). Many and mouse and and are with in the Notch signaling Although the these suggest roles for Notch in and both Mib1 and Mib2 might be in Notch signaling by Notch ligands. are Notch ligands, such as Delta and in four Delta homologues and and three Jagged homologues in and three ligands -3, and and ligands and in mammals. the regulation of these Notch ligands, two E3 Mib1 and have been identified In the we have identified a E3 Mib2, that interacts with Delta and promotes its endocytosis the Notch signaling pathway is cells interact with Notch ligands expressed by adjacent we suggest that these three E3 might have roles in Notch S. M. for the ubiquitination and Y. J. for of RING with