sevenless

Promoter Structure

There is a non consensus TATA box and several regions nearby with repeated sequence motifs, including an ATC/GGT/C motif that is repeated 12 times and a TCGGT motif repeated 5 and 6 times in two separate blocks . Downstream of the start site are other repeated elements, including one motif, ATCCAG, repeated 5 times. A 950 bp proximal promoter region is sufficient for normal expression (Bowtell, 1988).

The upstream region of sevenless is devoid of any detectable regulatory elements; sequences located 3' to the transcription start site are sufficient to promote the sevenless expression pattern. These gene-internal sequences function in both orientations on heterologouseven when placed at the 3' end of a lacZ reporter gene (Basler, 1989b).

Transcriptional Regulation

Ras1 plays a critical role in receptor tyrosine kinase (RTK) signal transduction pathways that function during Drosophila development. Mis-expression of constitutively active forms of Ras1 (Ras1V12) and the Sevenless (Sev) RTK (SevS11) during embryogenesis causes lethality due to inappropriate activation of RTK/Ras1 signaling pathways. Genetic and molecular data indicate that the rate of SevS11/sev-Ras1V12 lethality is sensitive to the expression level of both transgenes. To identify genes that encode components of RTK/Ras1 signaling pathways or modulators of RNA polymerase II transcription, advantage was taken of the dose-sensitivity of the system and a screen was carried out for second site mutations that would dominantly suppress the lethality. The collection of identified suppressors includes the PR55 subunit of Protein Phosphatase 2A, indicating that downstream of Sev and Ras1 this subunit acts as a negative regulator of phosphatase activity. The isolation of mutations in the histone deacetylase Rpd3 suggests that it functions as a positive regulator of sev enhancer-driven transcription. Finally, the isolation of mutations in the Trithorax group gene devenir and the characterized allelism with the Breathless RTK encoding gene provides evidence for Ras1-mediated regulation of homeotic genes (Maixner, 1998).

Targets of Activity

The ETS domain protein encoded by the P2 transcript of the pointed (pnt) gene is a nuclear target of the ras pathway signaling cascade acting downstream of MAP kinase. The PntP2 protein is phosphorylated by Rl/MAP kinase in vitro at a single site; this site is required for the protein to function in vivo. MAP kinase controls neural development through phosphorylation of two antagonizing transcription factors of the ETS family: Yan and PntP2 (Brunner, 1994).

Interactions with the ligand BOSS

A direct interaction between Bride of sevenless and Sevenless was demonstrated by the heterotypic aggregation of cell lines expressing these proteins. In the developing eye the Sevenless-dependent internalization of Bride of sevenless by the R7 precursor cell provides evidence for a direct interaction between these two proteins in vivo (Kramer, 1991).

For a ligand of a tyrosine-kinase receptor, the structure of BOSS is unique. It contains a large extracellular domain, seven transmembrane segments, and a carboxy-terminal cytoplasmic tail. BOSS activates tyrosine phosphorylation of the SEV receptor. The seven transmembrane domain of BOSS is necessary for its function; and a soluble form of BOSS, consisting of the extracellular domain, acts as an antagonist of the SEV receptor both in vivo and in vitro (Hart, 1993a).

The entire Boss protein from its extreme N-terminus to its extreme C-terminus is internalized by sev-expressing tissue culture cells and by the R7 precursor cell in the developing eye imaginal disc. The receptor-mediated transfer of a transmembrane ligand represents a novel mechanism for protein transfer between developing cells (Cagan, 1992).

The BOSS protein from D. virilis (Bossvir) retains strong amino acid identity with BOSS from D. melanogaster (Bossmel): 73% identity in the N-terminal extracellular domain and 91% identity in the seven-transmembrane domain, including the cytoplasmic tail. The Bossvir genes are able rescue the DM boss1 mutation, and the expression of Bossvir protein in DM is indistinguishable from that of Bossmel protein. The predicted SEV protein from DV (Sevvir) is 63% identical to SEV from DM (Sevmel). A chimeric gene, (sevvir/mel), encoding the extracellular domain of Sevvir and the cytoplasmic domain of Sevmel rescues the DM sevd2 mutation through interaction with either Bossvir or Bossmel (Hart, 1993b).

Regulated transcription of the prospero gene in the Drosophila eye provides a model for how gene expression is specifically controlled by signals from receptor tyrosine kinases. prospero is controlled by signals from the Egfr receptor and the Sevenless receptor. A direct link is established between Egfr activation of a transcription enhancer in prospero and binding of two transcription factors that are targets of Egfr signaling. Binding of the cell-specific Lozenge protein is also required for activation, and overlapping Lozenge protein distribution and Egfr signaling establishes expression in a subset of equivalent cells competent to respond to Sevenless. Sevenless activates prospero independent of the enhancer and involves targeted degradation of Tramtrack, a transcription repressor (Xu, 2000).

Thus, Egfr signaling is required to activate pros expression in the R7 equivalence group but is restricted from activating pros expression in other cells by the distribution of the transcription factor Lz. The transcriptional effectors of the Egfr pathway combinatorially interact with Lz at an eye-specific pros enhancer to restrict enhancer activity to the R7 equivalence group. It is suggested that this mechanism is a primary means by which pros transcription is restricted to the R7 equivalence group. This combinatorial mechanism supposes that Egfr signaling inactivates Yan and activates Pnt, but modification of these transcription factors is not sufficient to activate the enhancer. Lz is also required to activate the enhancer. The only cells that contain Lz, activated Pnt, and inactivated Yan are R1, R6, R7, and cone cells. Thus, the enhancer is activated in a subset of Egfr-responsive cells. Thus, differential expression of genes in response to an RTK/Ras signal appears to be controlled by each gene's capacity to bind and be regulated by different combinations of transcription factors (Xu, 2000).

A model is presented for the regulatory inputs into prospero. (1) In eye progenitor cells, the presence of Yan represses pros transcription through its binding to the enhancer and competitively excluding Pnt from binding to the same sites. (2) Lz begins to be produced in progenitor cells after the first wave of photoreceptor differentiation. However, Lz alone cannot activate the enhancer in progenitor cells that have not received a Spitz signal. (3) When a progenitor cell receives a Spitz signal, Egfr is activated. This inactivates Yan, allowing activated Pnt to bind to the enhancer. At the morphogenetic furrow, the enhancer is inactive despite Egfr-stimulated cells containing inactive Yan and active Pnt since progenitor cells in this region do not contain Lz, which is also required for enhancer activity. Hence, photoreceptors R2, R3, R4, R5, and R8 do not express pros. It is only in cells that receive a Spitz signal and contain Lz that the combination of Lz and Pnt bound to the enhancer activate the enhancer. (4) Ttk88 reduces the level of pros transcription through a mechanism independent of the eye enhancer. This repression may not be strong enough to block the eye enhancer in the R7 equivalence group but acts to limit its level of transcription. (5) When a progenitor cell receives both a Spitz and Boss signal, stronger or longer signal transduction induces Ttk88 inactivation. This Egf represses pros transcription and leads to a specific increase of Pros in R7 cells (Xu, 2000).

It is proposed that two RTKs, Egfr and Sev, regulate pros by activating the Ras1 intracellular pathway in R7 cells, but these RTKs regulate pros differentially. Egfr regulates pros by modifying Yan and Pnt, which act directly through the eye-specific enhancer. The Egfr signal in R7 cells appears to occur before Sev, and it sufficiently inactivates Yan and activates Pnt to switch on the enhancer before the Sev signal. This sufficiency is demonstrated in sev mutants where enhancer activity in R7 cells is no different from wild-type. In contrast, the Sev signal in R7 cells is not sufficient to switch on the enhancer in the absence of the Egfr signal since the enhancer is inactive in Egfr mutant R7 cells (Xu, 2000).

Sev regulates pros in R7 cells by inactivating Ttk88, which otherwise represses pros through sequence elements distinct from the eye-specific enhancer. This is demonstrated by finding that overproduced Ttk88 blocks Sev from activating pros, and Sev can regulate the eye-specific enhancer only if it is linked with Ttk88 binding sites. It is not clear if the Sev signal is sufficient to inactivate Ttk88 without an Egfr input since the assay for Ttk88 activity is a reporter gene that includes the eye-specific enhancer. It is quite possible that Ttk88 inactivation in R7 cells requires both Sev and Egfr signals, since Ebi, acting downstream of Egfr to promote Ttk88 degradation, and Phyl/Sina, acting downstream of Sev, are both required to inactivate Ttk88 in R7 cells (Xu, 2000).

How do these RTKs selectively regulate particular transcription factors and thereby regulate different aspects of pros transcription? The most attractive model is that RTK selection reflects the timing or intensity of each signal. If it is timing, then there must be a time period of competence during which a factor is sensitive to any RTK signal, and the time period is different for each factor. Alternatively, the intensity of a signal may dictate which transcription factor activities are sensitive. For example, Yan and Pnt activities may be insensitive to signal strength that is less than or equal to the level achieved by Sev but not Egfr within R7 cells. Ttk88 activity may be insensitive to signal strength that is less than or equal to the level achieved by Sev or Egfr alone but not the combination of the two within R7 cells. Signal 'strength' may be determined by the level of Ras pathway activity or the length of time that the Ras pathway is active. Sensitivity of transcription factors might be set either by the affinities of these factors for binding sites in a gene such as pros, or by the ability of factors to be substrates for RTK-stimulated modification. Given that Yan and Pnt are modified by a very different mechanism from Ttk88, substrate sensitivity is a possible determinant. In summary, RTK signals may provide specificity to gene regulation based on quantitative variation in which threshold transcription responses are set by transcription factors that have different sensitivities to RTK signal strength (Xu, 2000).

Interactions with the RAS pathway

drk (downstream of receptor kinases) encodes a widely expressed protein with a single SH2 domain and two flanking SH3 domains, homologous to the Sem-5 protein of C. elegans and mammalian GRB2. Genetic analysis suggests that drk function is essential for signaling by the Sevenless receptor tyrosine kinase. DRK biological activity correlates with binding of its SH2 domain to activated receptor tyrosine kinases and concomitant localization of DRK to the plasma membrane. In vitro, DRK also binds directly to the C-terminal tail of SOS, a RAS guanine nucleotide-releasing protein (GNRP), which, like RAS1 and DRK, is required for Sevenless signaling. DRK appears to bind autophosphorylated receptor tyrosine kinases with its SH2 domain and the SOS GNRP through its SH3 domains, thereby coupling receptor tyrosine kinases to RAS activation. The conservation of these signaling proteins during evolution indicates that this is a general mechanism for linking tyrosine kinases to RAS (Olivier, 1993).

drk, required for proper signaling by Sevenless, encodes a protein of the structure SH3-SH2-SH3. DRK protein is required for activation of p21Ras1 but not for any subsequent events. DRK protein can bind in vitro to Sevenless and to Son of sevenless (SOS), a putative guanine nucleotide exchange factor for p21Ras1. These results suggest that DRK acts to stimulate the ability of SOS to catalyze p21Ras1 activation by linking Sevenless and SOS in a signaling complex (Simon, 1993).

A highly conserved signal cascade functions subsequent to receptor tyrosine kinase activation. Signaling by the Sevenless receptor, required for differentiation of the R7 photoreceptor neuron in Drosophila, is reduced by mutations in E(sev)3A and E(sev)3B. E(sev)3A is a member of the Hsp90 family of stress proteins and E(sev)3B encodes a homolog of the cell cycle control protein Cdc37 from S. cerevisiae. Mutations in E(sev)3B also dominantly enhance mutations in Dmcdc2, the gene encoding the p34 protein kinase that regulates the G2/M transition. Together, these data support a role for Hsp90 proteins in tyrosine kinase regulation and suggest that signals promoting neuronal differentiation may involve cell cycle control (Cutforth, 1994).

The DRK SH3-SH2-SH3 adaptor protein provides a link between the activated SEV receptor and SOS, a guanine nucleotide release factor that activates Ras1. Tyr2546 in the cytoplasmic tail of SEV is required for DRK binding, probably because it provides a recognition site for the DRK SH2 domain. A mutation at this site does not completely block SEV function in vivo. This suggests that SEV can signal in a DRK-independent, parallel pathway or that DRK can also bind to an intermediate docking protein. Analysis of the DRK-SOS interaction has identified a high affinity binding site for DRK SH3 domains in the SOS tail. The N-terminal DRK SH3 domain is primarily responsible for binding to the tail of SOS in vitro, and for signalling to Ras in vivo (Raabe, 1995).

Drk, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2, is required during signaling by the Sevenless receptor tyrosine kinase (Sev). One role of Drk is to provide a link between activated Sev and the Ras1 activator Sos. The ability of activated Ras1 to bypass the requirement for Sev function during R7 development suggests that the primary function of Sev is to activate Ras. However, the model suggesting that the sole function of activated Sev is to bind Drk-Sos has been questioned by genetic studies that suggest the existence of multiple intracellular signaling pathways downstream of Sev. For example, although the association of Drk and Sos does not depend on the carboxy (C)-terminal SH3 domain of Drk, mutations that affect this domain partially compromise Sev signaling. Furthermore, a C-terminal SH3 domain-truncated Drk cannot rescue the lethality associated with homozygous drk mutations. These data suggest that Drk-binding proteins other than Sos may play important roles in signaling by Sev and other RTKs. Biochemical studies performed with mammalian systems have provided evidence that such Grb2-binding partners do exist. These include Cbl, a proto-oncogene product, and GAB1, a downstream component of the insulin and epidermal growth factor receptors. The possibility that Drk performs functions other than binding to Sos has been been investigated by identification of additional Drk-binding proteins. The phosphotyrosine-binding (PTB) domain-containing protein Disabled (Dab) binds to the Drk SH3 domains (Le, 1998).

To characterize the nature of the in vitro Dab-Drk interaction, it was necessary to determine which domains of Drk are required for binding to Dab. To answer this question, well-characterized mutations were used that had been shown to inactivate the function of either the SH2 or SH3 domain of Grb2. For example, changing the proline 49 residue to leucine (P49L) inactivates the N-terminal SH3 domain, while the arginine 86-to-lysine (R86K) mutation disrupts the SH2 domain and the glycine 203-to-arginine (G203R) mutation affects the C-terminal SH3 domain. The corresponding mutations were introduced, individually or in combination (P49L, R85K, G199R, P49L/G199R), into the [32P]GTK-DRK fusion protein and the ability of the mutant proteins to interact with the lambda gt11-encoded beta-galactosidase-DAB fusion protein was tested. Mutation of the SH2 domain does not affect binding, indicating that the in vitro Dab-Drk interaction does not require a functional Drk SH2 domain. However, the Dab-Drk interaction is dependent on the function of the SH3 domains because simultaneous mutations of both SH3 domains abolish binding. Moreover, while Dab binds to both SH3 domains, it appears to interact more strongly with the C-terminal domain. In addition, in vitro interaction between Drk and Dab requires the presence of the proline-rich region of Dav and suggests that the SH3 domains of Drk bind directly to sequences within the Dab proline-rich core (Le, 1998).

Dab is expressed in the ommatidial clusters, and loss of Dab function disrupts ommatidial development. Intense anti-Dab staining is observed both in the morphogenetic furrow and in developing ommatidial clusters posterior to the furrow. An apical-to-basal cross section revealed that Dab is localized to a small region just below the apical surface of the retinal epithelium. To determine which cells express Dab, the discs were costained with an antibody to ELAV, a neuronal marker present in the nuclei of developing and mature photoreceptors. The results from these experiments showed that Dab accumulates at the apical membrane of the developing photoreceptor cells. However, it was not possible to assign Dab expression to particular photoreceptors due to the apical constriction of these cells. The subcellular localization of Dab is similar to that of Drk, consistent with its role as a Drk-binding partner (Le, 1998).

Numerous abnormalities are observed in dab homozygous mutant clones. The most common defects are the absence of the R7 cell and the lack of one or more outer photoreceptors (R1 to R6) in mosaic ommatidia. In addition, large dab mutant clones show extensive ommatidial disorganization. including regions in which no photoreceptors are present. This phenotype is observed with three different alleles of dab and resembles those observed in clones of cells homozygous for weak alleles of either Sos or Ras1. These results indicate that Dab has an important function during photoreceptor and ommatidial development. Reduction of Dab function attenuates signaling by a constitutively activated Sev. Biochemical analysis suggests that Dab binds Sev directly via its PTB domain, becomes tyrosine phosphorylated upon Sev activation, and then serves as an adaptor protein for SH2 domain-containing proteins. Taken together, these results indicate that Dab is a novel component of the Sev signaling pathway (Le, 1998).

Disabled has been implicated in other RTK signaling pathways. A murine DAB-related protein, mDAB1, has been identified as a tyrosine-phosphorylated protein that binds to the non-receptor protein tyrosine kinase Src. Recently, several reports have shown that mice lacking mDAB1 function have neuronal defects similar to those seen in reeler mice, including abnormal cortical lamination resulting from disruptions of neuronal migration processes. These results suggest that mDAB1 might participate in a signaling pathway triggered by REELIN, a secreted protein released near the targets of migrating neurons. The neuronal defects associated with Drosophila and mouse dab mutations and the identification of DAB as a putative adaptor protein acting downstream of the receptor tyrosine kinase Sev suggest that Dab may function downstream of many RTKs, including ones required for proper development of the Drosophila central nervous system (Le, 1998).

The SH2 domain-containing phosphotyrosine phosphatase Corkscrew (Csw) is an essential component of the signaling pathway initiated by the activation of the sevenless receptor tyrosine kinase (Sev) during Drosophila eye development. Genetic and biochemical approaches have been used to identify a substrate for Csw. Expression of a catalytically inactive Csw was used to trap Csw in a complex with a 115 kDa tyrosine-phosphorylated substrate. This substrate was purified and identified as the product of the daughter of sevenless (dos) gene. Mutations of dos were identified in a screen for dominant mutations that enhance the phenotype caused by overexpression of inactive Csw during photoreceptor development. Analysis of dos mutations indicates that Dos is a positive component of the Sev signaling pathway and suggests that Dos dephosphorylation by Csw may be a key event during signaling by Sev (Herbst, 1996).

By screening for mutations that suppress signaling via a constitutively activated Sevenless protein, a novel gene has been identified: daughter of sevenless (dos). DOS is required not only for signal transduction via SEV but also in other receptor tyrosine kinase signaling pathways throughout development. The presence of an amino-terminally located pleckstrin homology domain and many potential tyrosine phosphorylation sites suggests that DOS functions as an adaptor protein able to interact with multiple signaling molecules. Genetic analysis demonstrates that DOS functions upstream of Ras1 and defines a signaling pathway that is independent of direct binding of the DRK SH2/SH3 adaptor protein to the SEV receptor tyrosine kinase (Raabe, 1996).

DFGF-R1 (breathless), a Drosophila FGF receptor homolog, is required for the migration of tracheal cells and the posterior midline glial cells during embryonic development. Deregulated receptors containing the cytoplasmic domains of DFGF-R2, DER, Torso, and Sevenless are all able to partially rescue the migration defects. Consistent with the notion that these RTKs share a common signaling pathway, constructs containing the activated downstream elements Dras1 and Draf are also able to rescue tracheal migration, demonstrating that these two proteins are key players in the DFGF-R1 signaling pathway (Reichman-Fried, 1994).

Mutations in Drosophila Cbl have not yet been identified. Despite this lack of information, studies have been made of possible developmental roles for Cbl. In the developing eye, the differentiation of R7 photoreceptor neurons depends on signaling through the Sevenless and EGF-receptor tyrosine kinases. Transformant flies were constructed that carry Drosophila Cbl cDNA under the transcriptional control of a promotor that causes the expression of Cbl in all cells that express Sevenless, including the R7 photoreceptor precursor. To assess the role of D-Cbl in R7 development, these transgenic flies were tested in a sensitized genetic assay. In this assay, signaling through Sevenless is compromised using a partially disabled Sevenless kinase. In such flies the dosage of genes participating in receptor tyrosine kinase signaling, and the fraction of ommatidia developing R7 cells provide an accurate readout of the strength of the transduction signal. A copy of Cbl transgene was introduced into the sensitized background. In such flies, the development of R7 cells is essentially eliminated. This result strongly argues that in the Drosophila eye, Cbl acts as a negative regulator of one or more receptor tyrosine kinase pathways that are essential for photoreceptor differention (Meisner, 1997).

The SRC homology 2 (SH2) domain protein-tyrosine phosphatase, Corkscrew (Csw) is required for signaling by receptor tyrosine kinases, including the Sevenless receptor tyrosine kinase (Sev), which directs Drosophila R7 photoreceptor cell development. To investigate the role of the different domains of Csw, domain-specific csw mutations were constructed and their effects on Csw function were assayed. Csw SH2 domain function is essential, but either Csw SH2 domain can fulfill this requirement. Csw and activated Sev are associated in vivo in a manner that does not require either Csw SH2 domain function or tyrosine phosphorylation of Sev. Thus, Sev is unlikely to be a binding partner for the Csw SH2 domains. Evaluation cannot presently be made as to whether the observed phosphotyrosine-independent association of Csw and Sev actually occurs in the developing R7 cell or is important for Sev signaling. In contrast, the interaction between Csw and Daughter of Sevenless, a Csw substrate, is dependent on SH2 domain function. These results suggest that the role of the Csw SH2 domains during Sev signaling is to bind Daughter of Sevenless rather than activated Sev. Although Csw protein-tyrosine phosphatase activity is required for full Csw function, a catalytically inactive Csw is capable of providing partial function. In addition, deletion of either the Csw protein-tyrosine phosphatase insert or the entire Csw carboxyl terminus, which includes a conserved Drk/Grb2 SH2 domain binding sequence, does not abolish Csw function (Allard, 1998).

Sprouty was identified in a genetic screen as an inhibitor of Drosophila EGF receptor signaling. The Egfr triggers cell recruitment in the eye, and sprouty minus eyes have excess photoreceptors, cone cells, and pigment cells. Tests provide evidence that Sprouty interacts specifically with the Egfr pathway. Halving the dose of sprouty (1) strongly enhances the rough eye caused by the misexpression of rhomboid, a specific activator of Egfr signaling; suppresses the rough eye caused by underrecruitment of photoreceptors in a hypomorphic allele of spitz, the TGF-like ligand of the Egfr; (3) suppresses the phenotypes of Egfr hypomorphic mutations both in the eye and the wing and (4) flies heterozygous for both sprouty and argos have mildly rough eyes, caused by a slight overrecruitment of all types of cell, although heterozygosity for either mutation alone causes no phenotype. Other genetic interactions between sprouty and the Egfr pathway are also detailed. All point to the same conclusion: Sprouty inhibits Egfr signaling (Casci, 1999).

Both full-length Sprouty and a truncated Sprouty containing residues 1-369 (i.e., without the cys-rich domain and C-terminal residues) were assayed for their ability to bind in vitro translated members of the Ras pathway. Strong interactions are detected between Sprouty and Drk, an SH2-SH3 containing adaptor protein homologous to mammalian Grb2, and between Sprouty and GTPase-activating protein 1 (Gap1), a Ras GTPase-activating protein. No interactions were seen between Sprouty and several other proteins involved in the Ras pathway: Sos, Dos, Csw, Ras1, Raf, and Leo (14-3-3). The interactions with Drk and Gap1 did not require the presence of the C-terminal cysteine-rich domain, the region of Sprouty most conserved between flies and humans. Since the well-conserved cysteine-rich domain of Sprouty is not required for binding to Drk or Gap1, it might instead target the protein to the plasma membrane. To test this, two truncated forms of Sprouty were expressed in cultured cells. One form lacks the conserved cysteine-rich domain, whereas a second exclusively comprises the cysteine-rich domain. The form with the cysteine-rich domain is membrane associated and is indistinguishable from the wild-type protein. In sharp contrast, the form lacking the cysteine-rich domain is distributed uniformly throughout the cell, with no specific localization to membranes. Cell fractionation confirms these results. It is concluded that the 147-residue cysteine-rich domain in Sprouty, which corresponds to the most conserved region in the published human ESTs, is responsible for the specific localization of Sprouty to the plasma membrane (Casci, 1999).

Sprouty's function is, however, more widespread. It also interacts genetically with the receptor tyrosine kinases Torso and Sevenless, and it was first discovered through its effect on FGF receptor signaling. In contrast to an earlier proposal that Sprouty is extracellular, biochemical analysis suggests that Sprouty is an intracellular protein, associated with the inner surface of the plasma membrane. Sprouty binds to two intracellular components of the Ras pathway, Drk and Gap1. These indicate that Sprouty is a widespread inhibitor of Ras pathway signal transduction (Casci, 1999).

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