The activation signal from tyrosine kinase receptors, such as the epidermal growth factor receptor (EGFR), is relayed via a highly conserved intracellular pathway involving Ras, Raf, and MAPK. In Drosophila, the EGFR and components of the intracellular pathway are broadly expressed, yet receptor activation evokes tissue-specific cell responses. Extracellular events that lead to receptor activation are one mechanism by which signaling is modulated. Here we show molecular and genetic evidence that Drosophila vein (vn) encodes a candidate EGFR ligand and that vn expression is spatially restricted. Consequently, vn may promote tissue-specific receptor activation. Unlike two other ligands, Gurken (Grk) and Spitz (Spi), which are transforming growth factor oMike proteins, Vn has both an immunoglobulin-like and an EGF-like domain. This combination of domains mirrors those in the vertebrate neuregulins that bind EGFR relatives. [Key Words: vein; Drosophila; EGF receptor; EGF; neuregulin]Received April 24, 1996; revised version accepted August 7, 1996.The Drosophila epidermal growth factor receptor (DER) is a receptor tyrosine kinase of the epidermal growth factor receptor (EGFR) subfamily that currently also includes four vertebrate proteins {EGFR/erbB1, neu/erbB2, erbB3, and erbB4) and Caenorhabditis elegans These receptors have important roles in development: EGFR mutant mice have epithelial defects that cause a number of phenotypes and early death (Miettinen et al. 1995;Sibilia and Wagner 1995;Threadgill et al. 1995); neu and erbB4 mutant embryos die in utero with heart and neural defects (Gassmann et al. 1995;Lee et al. 1995); let-23 is involved in determining vulval cell fate (Aroian et al. 1990; Hill and Sternberg 19921; and DER has multiple roles in Drosophila development involving cell survival, proliferation, and differentiation in embryos, imaginal discs, and the ovary (Shilo and

Substrate specificity of SCF E3 ubiquitin ligases is thought to be determined by the F box protein subunit. Another component of SCF complexes is provided by members of the Roc1/Rbx1/Hrt1 gene family, which encode RING-H2 proteins. Drosophila contains three members of this gene family. We show that Roc1a mutant cells fail to proliferate. Further, while the F box protein Slimb is required for Cubitus interruptus (Ci) and Armadillo/beta-catenin (Arm) proteolysis, Roc1a mutant cells hyperaccumulate Ci but not Arm. This suggests that Slimb and Roc1a function in the same SCF complex to target Ci but that a different RING-H2 protein acts with Slimb to target Arm. Consequently, the identity of the Roc subunit may contribute to the selection of substrates by metazoan SCF complexes.

In Drosophila the function of the epidermal growth factor (EGF) receptor is modulated zygotically by three EGF-like proteins: Spitz (Spi), which is a potent activator; Vein (Vn), which is a moderate activator; and Argos (Aos), which is an inhibitor. Chimeric molecules were constructed in which the EGF domain of Vn was swapped with the EGF domain from each factor. The modified Vn proteins behaved both in vitro and in vivo with properties characteristic of the factor from which the EGF domain was derived. These results demonstrate that the EGF domain is the key determinant that gives DER inhibitors and activators their distinct properties. Received January 13, 1998; revised version accepted February 10, 1998. The Drosophila epidermal growth factor (EGF) receptor (DER) is a member of the ErbB family of receptor tyrosine kinases (RTKs) and, like its vertebrate counterparts, controls cell differentiation, survival, and proliferation in many tissues throughout development (Perrimon and Perkins 1997;Schweitzer and Shilo 1997). DER function is modulated by four candidate ligands each of which possesses a predicted EGF-like domain. Gurken (Grk) and Spitz (Spi) are TGF␣-like proteins (Rutledge et al. 1992;Neuman-Silberberg and Schü pbach 1993). grk is a maternally active gene involved in establishing egg polarity (Neuman-Silberberg and Schü pbach 1993; Gonzá-lez-Reyes et al. 1995), whereas spi is zygotically active and functions in the embryo, adult eye, and wing (Rutledge et al. 1992;Freeman 1994b). Vn resembles the vertebrate neuregulins in that both possess an Ig-C2 domain in addition to the EGF-like domain . Vein (Vn) functions zygotically in the embryo and the adult wing Simcox et al. 1996;Simcox 1997;Yarnitzky et al. 1997). Here we show that Vn is a moderate activator of DER signaling in comparison with Spi, which is a potent DER activator. Argos (Aos) has been recognized recently as an inhibitor of the DER pathway and was the first extracellular factor shown to inhibit an RTK in vivo (Schweitzer et al. 1995a). Aos functions in the embryo, adult eye, and wing (Freeman et al. 1992;Sawamoto et al. 1994;Golembo et al. 1996).Considerable effort has been made to understand the structure-function relationships of vertebrate EGF-like mitogens to aid in the development of ErbB receptor superagonists or antagonists; however, to date these studies have not led to the design of effective factors (Groenen et al. 1994). The fly system offers a unique opportunity to define the molecular basis for the distinct properties of three natural ligands with different effects on a receptor and could facilitate the development of vertebrate factors with similar relative properties.Spi, Vn, and Aos are structurally unrelated except within the EGF domain (Fig. 1A). The EGF domain contains a series of six cysteines, which form three disulfide bonds to generate a looped structure, and a number of other highly conserved residues that are known to be required for binding and activating members of the vertebrate ErbB receptor family (Groenen ...

Cullin-dependent ubiquitin ligases regulate a variety of cellular and developmental processes by recruiting specific proteins for ubiquitin-mediated degradation. Cullin proteins form a scaffold for two functional modules: a catalytic module comprised of a small RING domain protein Roc1/Rbx1 and a ubiquitin-conjugating enzyme (E2), and a substrate recruitment module containing one or more proteins that bind to and bring the substrate in proximity to the catalytic module. Here, we present evidence that the three Drosophila Roc proteins are not functionally equivalent. Mutation of Roc1a causes lethality that cannot be rescued by expression of Roc1b or Roc2 by using the Roc1a promoter. Roc1a mutant cells hyperaccumulate Cubitus interruptus, a transcription factor that mediates Hedgehog signaling. This phenotype is not rescued by expression of Roc2 and only partially by expression of Roc1b. Targeted disruption of Roc1b causes male sterility that is partially rescued by expression of Roc1a by using the Roc1b promoter, but not by similar expression of Roc2. These data indicate that Roc proteins play nonredundant roles during development. Coimmunoprecipitation followed by Western or mass spectrometric analysis indicate that the three Roc proteins preferentially bind certain Cullins, providing a possible explanation for the distinct biological activities of each Drosophila Roc/Rbx. INTRODUCTIONProtein modification by ubiquitin is widely used by eukaryotic organisms to regulate many important cellular and developmental processes (Ciechanover et al., 2000;Ben-Neriah, 2002;Conaway et al., 2002;Wojcik, 2002). Three enzymatic activities, ubiquitin activation (E1), conjugation (E2), and ligation (E3), lead to the covalent attachment of ubiquitin, a 76-amino acid protein, to specific target proteins (Hershko and Ciechanover, 1998). Monoubiquitylation can influence the activity or subcellular localization of the target protein (Pickart, 2001;Raiborg et al., 2003). Repeated rounds of the E2-E3 reaction result in the formation of polyubiquitin chains that generally serve as a signal for destruction by the 26S proteosome, but they can also have nonproteolytic effects on protein function (Hershko and Ciechanover, 1998;Kaiser et al., 2000;Pickart, 2000Pickart, , 2001. The E3 plays a crucial role in this pathway not only because it mediates the transfer of ubiquitin to the target protein but also because it provides substrate specificity (Jackson et al., 2000).One well characterized E3 is the multisubunit SCF complex (Deshaies, 1999;Jackson and Eldridge, 2002). Consisting of four members (Skp1, CUL-1/Cdc53, an F-box-containing protein, and Roc1/Rbx1/Hrt1), the SCF regulates many developmental processes such as the cell cycle, signaling pathways, circadian rhythms, and apoptosis (Koepp et al., 1999;Maniatis, 1999;DeSalle and Pagano, 2001;Grima et al., 2002;Ko et al., 2002;Nateri et al., 2004). The specificity of SCF complexes is conferred by the F-box subunit, which binds to the target protein through a protein-protein interaction moti...

Signaling by the Drosophila EGF receptor (DER) is modulated by four known EGF-like proteins: the agonists Vein (Vn), Spitz (Spi), and Gurken (Grk) and the antagonist Argos (Aos). DER is broadly expressed and thus tissue-specific regulation of ligand expression and activity is an important mechanism for controlling signaling. Here we investigate the tissue-specific regulation of Vn signaling by examining vn transcriptional control and Vn target gene activation in the embryo and the wing. The results show a complex temporal and spatial regulation of vn transcription involving multiple signaling pathways and tissue-specific activation of Vn target genes. In the embryo, vn is a target of Spi/DER signaling mediated by the ETS transcription factor PointedP1 (PntP1). This establishes a positive feedback loop in addition to the negative feedback loop involving Aos. The simultaneous production of Vn provides a mechanism for dampening Aos inhibition and thus fine-tunes signaling. In the larval wing pouch, vn is not a target of Spi/DER signaling but is expressed along the anterior-posterior boundary in response to Hedgehog (Hh) signaling. Repression by Wingless (Wg) signaling further refines the vn expression pattern by causing a discontinuity at the dorsal-ventral boundary. The potential for vn to activate DER target genes correlates with its roles in development: vn has a minor role in embryogenesis and does not induce DER target genes such as aos and pntP1 in the embryo. Conversely, vn has a major role in wing development and Vn/DER signaling is a potent inducer of DER target genes in the wing disc. Spi also has the potential to induce DER target genes in the wing disc. However, the ligands appear to evoke specific responses that result in different patterns of target gene expression. Finally, we show that other factors modulate the potential of Vn so that induction of Vn/DER target genes in the wing pouch is cell specific.