Lai, E. C., Roegiers, F., Qin, X., Jan, Y. N. & Rubin, G. M. The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta. Development 132, 2319-2332
The receptor Notch and its ligands of the Delta/Serrate/LAG2 (DSL) family are the central components in the Notch pathway, a fundamental cell signaling system that regulates pattern formation during animal development. Delta is directly ubiquitinated by Drosophila and Xenopus Neuralized, and by zebrafish Mind bomb, two unrelated RING-type E3 ubiquitin ligases with common abilities to promote Delta endocytosis and signaling activity. Although orthologs of both Neuralized and Mind bomb are found in most metazoan organisms, their relative contributions to Notch signaling in any single organism have not yet been assessed. We show here that a Drosophila ortholog of Mind bomb (D-mib) is a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, we show that D-mib associates physically and functionally with both Serrate and Delta. We find that D-mib uses its ubiquitin ligase activity to promote DSL ligand activity, an activity that is correlated with its ability to induce the endocytosis and degradation of both Delta and Serrate (see also Le Borgne et al., 2005). We further demonstrate that D-mib can functionally replace Neuralized in multiple cell fate decisions that absolutely require endogenous Neuralized, a testament to the highly similar activities of these two unrelated ubiquitin ligases in regulating Notch signaling. We conclude that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophila development.
"An evolutionarily conserved F-box protein Fbw7 (also known as Sel-10) has been shown to ubiquitinate NICD, resulting in proteasomal degradation of NICD in Caenorhabditis elegans and mammals, although a direct demonstration of its role in Drosophila is missing (Hubbard et al. 1997; Oberg et al. 2001; Wu et al. 2001; Tetzlaff et al. 2004; Tsunematsu et al. 2004; Matsumoto et al. 2011; Nicholson et al. 2011). In addition, work from several laboratories indicated that two distinct E3 ligases, Neuralized (Neur) and Mind bomb (Mib1), directly promote mono-ubiquitination of the ligand proteins Delta and Serrate to facilitate their endocytosis (Deblandre et al. 2001; Lai et al. 2001; Pavlopoulos et al. 2001; Yeh et al. 2001; Itoh et al. 2003; Chen and Corliss 2004; Lai et al. 2005; Le Borgne et al. 2005). Furthermore, clonal analysis in Drosophila suggested that neur and mib1 function in the signal-sending cell and are required for most Notch-mediated processes (Le Borgne and Schweisguth 2003; Li and Baker 2004; Pitsouli and Delidakis 2005; Wang and Struhl 2005). "
[Show abstract][Hide abstract] ABSTRACT: Notch signaling is highly conserved in all metazoan animals and plays critical roles in cell fate specification, cell proliferation, apoptosis, and stem cell maintenance. Although core components of the Notch signaling cascade have been identified, many gaps in the understanding of the Notch signaling pathway remain to be filled. One form of posttranslational regulation, which is controlled by the ubiquitin-proteasome system, is known to modulate Notch signaling. The ubiquitination pathway is a highly coordinated process in which the ubiquitin moiety is either conjugated to or removed from target proteins by opposing E3 ubiquitin ligases and deubiquitinases (DUBs). Several E3 ubiquitin ligases have been implicated in ubiquitin conjugation to the receptors and the ligands of the Notch signaling cascade. In contrast, little is known about a direct role of DUBs in Notch signaling in vivo. Here, we report an in vivo RNA interference screen in Drosophila melanogaster targeting all 45 DUBs that we annotated in the fly genome. We show that at least four DUBs function specifically in the formation of the fly wing margin and/or the specification of the scutellar sensory organ precursors, two processes that are strictly dependent on the balanced Notch signaling activity. Furthermore, we provide genetic evidence suggesting that these DUBs are necessary to positively modulate Notch signaling activity. Our study reveals a conserved molecular mechanism by which protein deubiquitination process contributes to the complex posttranslational regulation of Notch signaling in vivo.
"Previous work has demonstrated that Mib1 is absolutely required for the activity of Ser and Dl –. In the absence of mib1 function, the expression of Wg along the D/V boundary is absent –; (Fig. 7F). Dorsally restricted expression of UAS mib1 resulted in the establishment of Wg expression along the D/V boundary and wing development (Fig. 7G, H). "
[Show abstract][Hide abstract] ABSTRACT: Wing development in Drosophila requires the activation of Wingless (Wg) in a small stripe along the boundary of Fringe (Fng) expressing and non-expressing cells (FB), which coincides with the dorso-ventral (D/V) boundary of the wing imaginal disc. The expression of Wg is induced by interactions between dorsal and ventral cells mediated by the Notch signalling pathway. It appears that mutual signalling from dorsal to ventral and ventral to dorsal cells by the Notch ligands Serrate (Ser) and Delta (Dl) respectively establishes a symmetric domain of Wg that straddles the D/V boundary. The directional signalling of these ligands requires the modification of Notch in dorsal cells by the glycosyltransferase Fng and is based on the restricted expression of the ligands with Ser expression to the dorsal and that of Dl to the ventral side of the wing anlage. In order to further investigate the mechanism of Notch signalling at the FB, we analysed the function of Fng, Ser and Dl during wing development at an ectopic FB and at the D/V boundary. We find that Notch signalling is initiated in an asymmetric fashion on only one side of the FB. During this initial asymmetric phase, only one ligand is required, with Ser initiating Notch-signalling at the D/V and Dl at the ectopic FB. Furthermore, our analysis suggests that Fng has also a positive effect on Ser signalling. Because of these additional properties, differential expression of the ligands, which has been a prerequisite to restrict Notch activation to the FB in the current model, is not required to restrict Notch signalling to the FB.
PLoS ONE 11/2012; 7(11):e49007. DOI:10.1371/journal.pone.0049007 · 3.23 Impact Factor
"In the first, we asked whether ectopic expression of Dl in the larval wing epithelium can induce wingless (wg), a Notch target gene normally found in the DV boundary (de Celis et al., 1996; Doherty et al., 1996; Neumann and Cohen, 1996). This event is known to be dependent on mib1 (Lai et al., 2005; Le Borgne et al., 2005; Pitsouli and Delidakis, 2005; Wang and Struhl, 2005), which is ubiquitously expressed in the wing disk. "
[Show abstract][Hide abstract] ABSTRACT: DSL proteins are transmembrane ligands of the Notch receptor. They associate with a RING (really interesting new gene) family E3 ubiquitin ligase, either Neuralized (Neur) or Mindbomb 1 (Mib1), as a prerequisite to signaling. Although Neur and Mib1 stimulate internalization of DSL ligands, it is not known how ubiquitylation contributes to signaling. We present a molecular dissection of the intracellular domain (ICD) of Drosophila melanogaster Delta (Dl), a prototype DSL protein. Using a cell-based assay, we detected ubiquitylation of Dl by both Neur and Mib1. The two enzymes use distinct docking sites and displayed different acceptor lysine preferences on the Dl ICD. We generated Dl variants that selectively perturb its interactions with Neur or Mib1 and analyzed their signaling activity in two in vivo contexts. We found an excellent correlation between the ability to undergo ubiquitylation and signaling. Therefore, ubiquitylation of the DSL ICD seems to be a necessary step in the activation of Notch.
The Journal of Cell Biology 12/2011; 195(6):1017-31. DOI:10.1083/jcb.201105166 · 9.83 Impact Factor
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