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Copyright Ó 2006 by the Genetics Society of America DOI: 10.1534/genetics.106.061630 Structure–Function Analysis of Delta Trafficking, Receptor Binding and Signaling in Drosophila Annette L. Parks,1 Jane R. Stout,1,2 Scott B. Shepard,1,3 Kristin M. Klueg,4 Ana A. Dos Santos,5 Todd R. Parody,6 Martina Vaskova7 and Marc A. T. Muskavitch8 Biology Department, Boston College, Chestnut Hill, Massachusetts 02467 Manuscript received June 19, 2006 Accepted for publication September 18, 2006 ABSTRACT The transmembrane proteins Delta and Notch act as ligand and receptor in a conserved signaling pathway required for a variety of cell fate specification events in many organisms. Binding of Delta to Notch results in a proteolytic cascade that releases the Notch intracellular domain, allowing it to participate in transcriptional activation in the nucleus. Recent research has implicated the endocytic and ubiquitylation machinery as essential components of Delta–Notch signaling. Our analysis of chimeric and missense Delta variants has delineated a number of structural requirements for Delta trafficking, receptor binding, and signaling. We find that while the Delta N-terminal domain is necessary and sufficient for binding to Notch, the integrity of the epidermal-growth-factor-like repeat (ELR) 2 is also required for Notch binding. Screening of 117 Delta mutant lines for proteins that exhibit aberrant subcellular trafficking has led to the identification of 18 Delta alleles (DlTD alleles) that encode ‘‘trafficking-defective’’ Delta proteins. We find, unexpectedly, that many DlTD alleles contain missense mutations in ELRs within the Delta extracellular domain. Finally, we find that two DlTD alleles contain lysine missense mutations within the Delta intracellular domain (DeltaICD) that may identify residues important for DeltaICD mono-ubiquitylation and subsequent Delta endocytosis and signaling. HE Notch signaling pathway affects cell fate cluding lymphocytic leukemia, spondylocostal dysosto- T decisions throughout development in a vast array sis, and Alagille syndrome (Gridley 2003; Weng et al. of organisms. Notch (N) was originally classified as a 2004). neurogenic gene on the basis of the observation that In Drosophila, the extracellular domains of the Notch homozygosity and hemizygosity for N loss-of-function transmembrane ligands Delta and Serrate bind to the mutations result in hypertrophy of neural tissue at the Notch extracellular domain (NotchECD) in a manner expense of epidermal tissue in developing embryos that is dependent on NotchECD ELRs 11 and 12 (Rebay (Poulson 1937). The Notch pathway has since been et al. 1991). A subsequent proteolytic cascade results in shown to play key roles in cell specification within the release and translocation of the Notch intracellular many tissues, including the eye, wing, and notum domain (NotchICD) to the nucleus, where it forms a (Artavanis-Tsakonas et al. 1999; Portin 2002), and complex with the Suppressor of Hairless [Su(H)] pro- ongoing research continues to broaden the understood tein and regulates transcription of target genes, includ- spectrum of Notch activity. Dysfunction of this pathway ing those of the Enhancer of split-Complex (Greenwald has been implicated in several human diseases, in- 1998; Artavanis-Tsakonas et al. 1999; Baron 2003; Kadesch 2004). Regulation of the Notch pathway is 1These authors contributed equally to this work. varied and complex. Some proteins regulate signaling 2Present address: Medical Sciences, Indiana University, Bloomington, positively (Deltex) or negatively (Numb) by binding to IN 47405. the NotchICD. Others, such as Hairless, impede signal- 3Present address: Wyeth, Cambridge, MA 02140. ing by binding directly to Su(H); while yet another 4Present address: Drosophila Genomics Resource Center, Indiana regulatory mechanism involves Fringe-dependent gly- University, Bloomington, IN 47405. cosylation of the NotchECD (Artavanis-Tsakonaset al. 5Present address: Information Technology, Fred Hutchinson Cancer chweisguth e orgne Research Center, Seattle, WA 98109. 1999; S 2004; L B et al. 2005a). 6Present address: Department of Chemistry, Indiana University, A number of research findings have recently impli- Bloomington, IN 47405. cated components of the subcellular trafficking machin- 7Present address: Department of Biopharmaceutical Sciences, University ery in Delta signal activation (Le Borgne et al. 2005a; of Illinois, Chicago, IL 60612. Chitnis 2006; Le Borgne 2006). Delta signal activation 8Corresponding author: Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467. appears to depend on ubiquitylation of Delta by the E3 E-mail: [email protected] ubiquitin ligases Neuralized (Neur) or Mind bomb1 Genetics 174: 1947–1961 (December 2006) 1948 A. L. Parks et al. (Mib1), both of which can stimulate Delta endocytosis ELRs, and intracellular domain residues play specific and signaling (Deblandre et al. 2001; Lai et al. 2001; roles in Delta–Notch signaling. We demonstrate that the Pavlopoulos et al. 2001; Lai et al. 2005; Le Borgne Delta N-terminal domain, including the Delta-Serrate- et al. 2005b). Proteins encoded by the Bearded family of Lag-2 (DSL) domain, and Delta ELR 2 are required for genes (e.g., Tom, Brd-C) have recently been shown to Notch binding and signaling in vivo; that sequences bind Neur and inhibit Delta endocytosis (Bardin and within the Delta N-terminal domain, other than the DSL Schweisguth 2006; De Renzis et al. 2006). Epsin, an domain, are required for Delta–Notch binding and adapter for clathrin-mediated endocytosis, can activate homotypic Delta binding; that mutations in multiple Delta signaling in Drosophila and Caenorhabditis elegans DeltaELRs are implicated in Delta endocytosis and and also appears to regulate ligand endocytosis, possibly Notch signaling in vivo; and that alterations in lysine by targeting Delta to a recycling endosomal compart- residues within the DeltaICD, potential sites for ubiq- ment (Overstreet et al. 2004; Tian et al. 2004; Wang uitylation, are correlated with loss of Delta endocytosis and Struhl 2004, 2005). Other clathrin-coat compo- and signaling in vivo. nents such as the clathrin heavy chain and a-adaptin have also been implicated in Notch signaling on the MATERIALS AND METHODS basis of genetic interactions (Cadavid et al. 2000; K. M. Klueg and M. A. T. Muskavitch, unpublished ob- Sequences used for alignments: The following sequences servation). Recent work suggests that the recycling were used: Drosophila melanogaster Delta, NP_732412.1; endosome-associated protein Rab11, as well as the Homo sapiens Delta-like 1, NP_005609.2; Mus musculus Delta- like 1, NP_031891.2; Gallus gallus Delta-like 1, NP_990304.1; Rab11-binding proteins Sec15 and Nuclear fallout Danio rerio DeltaA, NP_571029.1; D. melanogaster Serrate, (Nuf), are necessary for Delta trafficking, and has NP_524527.3; H. sapiens Jagged 1, NP_000205.1. reinforced the idea that Delta must be transported Drosophila strains: Oregon-R, BER-1, ss e4 ro, and E(spl)D tx are through recycling endosomes as a prerequisite for maintained in our laboratory. The stock ru h th st cu sr es ca (ru cu Notch activation (Emery et al. 2005; Jafar-Nejad et al. ca) was obtained from the Bloomington Drosophila Stock Center. Stocks of e4 tx, Canton-S and ywNAx9B2/y w f:¼ were 2005). In what may be a second signaling-related obtained from S. Artavanis-Tsakonas (Massachusetts General endocytic event, Delta endocytosis and uptake of the Hospital Cancer Center, Boston). Dl trafficking-defective NotchECD into signal-sending cells has also been (DlTD) alleles were maintained over TM6C, cu Sb e Tb ca and correlated with active Delta–Notch signaling (Parks include DlBE21 and DlBX9 from a mutant screen using E(spl)D tx lton BE30 BE32 BX43 CE3 CE6 CE7 CE9 et al. 2000). This trans-endocytosis of the NotchECD is (A et al. 1989); Dl , Dl , Dl , Dl , Dl , Dl , Dl , DlCE15, and DlCE16 from a mutant screen using ss e4 ro (Alton dependent on the vesicle-scission enzyme dynamin et al. 1989); DlRF (Xu et al. 1990; Parody and Muskavitch (Parks et al. 2000), a protein essential for Notch sig- 1993) from parental strain ywNAx9B2/y w f:¼; Dl9Q76 (received naling in many contexts (Poodry 1990). Interestingly, from M. Mortin, National Institutes of Health, Bethesda, MD; dynamin activity is required in both signal-sending isolated by Tearle and Nusslein-Volhard 1987) from a mutant screen using ru cu ca; Dl48.7 (from S. Artavanis- and signal-receiving cells for active Notch signaling 4 90 eugnet Tsakonas) isolated from a screen using e tx; Dl /TM2 and (S et al. 1997a). Dl195/TM2 (from S. Artavanis-Tsakonas) from parental strain Delta protein is detected exclusively in intracellular Canton-S; and Dl87h/TM6C (parent unknown; obtained from endocytic vesicles in many Drosophila tissues at specific W. J. Welshons, Iowa State University, Ames, IA). times during development (Kra¨mer and Phistry 1996; The 1348-Gal4 (Huppert et al. 1997), 31-1-Gal4 (Brand and errimon taehling ampton Huppert et al. 1997; Kra¨mer and Phistry 1999; Parks P 1993), and dpp-Gal4 drivers (S -H et al. 1994) were employed to induce ectopic expression et al. 2000). In the developing retina, wild-type Delta in combination with responders including UAS-DeltaWT-1 protein is solely detected in vesicles at all stages and in (Jacobsen et al. 1998), UAS-DeltaWT-2 (designated UAS-Delta all cell types (Parks et al. 1995). We have previously in Seugnet et al. 1997b), or UAS-DeltaVariants (see below). described Delta (Dl) alleles that encode proteins that Molecular cloning and sequence analysis: Constructs used to direct expression of DeltaWT (pMTDl1) and Notch accumulate aberrantly on retinal cell surfaces and con- ehon arks (pMTNMg) have been described (F et al. 1990). In the stitute loss-of-function alleles (P et al. 1995, 2000), descriptions below, Delta nucleotides correspond to those consistent with the notion that Delta internalization is of the Dl1 cDNA (Kopczynski et al. 1988; EMBL accession critical for function. no. Y00222). pDl1 refers to the Dl1 cDNA cloned into In this report, we describe the functional analysis of a pBluescript1 (Kopczynski et al.