FGF receptor 1


Transcriptional Regulation

Embryos mutant for twist or snail fail to express FR1. Neural expression of FR1 is lacking in embryos lacking achaete-scute expression (Shishido, 1993).

Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. Wingless and Decapentaplegic confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects of three signal-activated transcription factors (dTCF, Mad, and Pointed that function in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted transcription factors (Twist and Tinman) on Even-skipped, an progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman determines inductive Ras signaling specificity in muscle and heart development (Halfon, 2000).

Cell fate specification in the somatic mesoderm of the Drosophila embryo has been examined as a model for dissecting the molecular basis of combinatorial signaling involving receptor tyrosine kinases (RTKs). The somatic musculature and the cells that compose the heart develop from specialized cells called progenitors. Each progenitor divides asymmetrically to produce two founder cells that possess information that specifies individual muscle fate and that seed the formation of multinucleate myofibers. The focus of this study has been a small subset of somatic mesodermal cells that express the transcription factor Even skipped. Eve is expressed in the progenitors and founders of both the dorsal muscle fiber DA1 and a pair of heart accessory cells, the Eve pericardial cells or EPCs. Since eve is the earliest known marker for these cells and is required for their formation, eve is referred to here as a progenitor identity gene (Halfon, 2000).

Previous genetic experiments have defined multiple intercellular signaling events that govern the progressive determination of the Eve progenitors. Signaling from both the Wnt family member Wingless (Wg) and the TGF family member Decapentaplegic (Dpp) prepatterns the mesoderm and renders cells competent to respond to Ras/MAPK activation. Localized Ras activation within the competence domain determined by the intersection of Wg and Dpp expression occurs through the action of two RTKs: the Drosophila epidermal growth factor receptor (Egfr) and the Heartless (Htl) fibroblast growth factor receptor. This RTK signaling induces two distinct equivalence groups, each of which expresses Eve. Lateral inhibition mediated by Notch then selects a single progenitor from each equivalence group (Halfon, 2000).

The present study explores how the prepattern genes wg and dpp establish competence for mesodermal cells both to activate and to respond to the Ras/MAPK cascade; how multiple intercellular signals are integrated to establish Eve progenitor fates, and how muscle- and cardiac-specific responses to Ras signaling are generated. Wg provides competence for the generation of the Ras/MAPK inductive signal by regulating the expression of key proximal components of the Egfr and Htl RTK pathways. Wg and Dpp then create competence for a specific response to the inductive signal both through their own respective downstream transcriptional effectors, dTCF and Mothers against dpp (Mad), and through their regulation of the mesoderm-specific transcription factors Tinman (Tin) and Twist (Twi). Specificity of the Ras/MAPK response is achieved though the integration of these signal-activated and tissue-restricted transcription factors, along with the Ras/MAPK-activated Ets domain transcription factor PointedP2 (Pnt), at a single transcriptional enhancer. These results provide a direct link between the initial axis patterning processes in the early embryo and the subsequent combinatorial signaling events that lead to the progressive determination of muscle and cardiac progenitors (Halfon, 2000).

The Eve progenitors in each mesodermal hemisegment arise during embryonic stage 11 in a dorsal region demarcated by the intersecting domains of Wg and Dpp expression. The cells exposed to both Wg and Dpp are competent to respond to localized Ras signaling, which induces the initial expression of Eve in two clusters of equipotent cells. In each of these equivalence groups, activity of the Notch pathway leads to the rapid refinement of Eve expression to a single muscle or cardiac progenitor. The two Eve equivalence groups arise sequentially. Cluster C2, from which progenitor P2 derives, is first to form. P2 divides asymmetrically, with one daughter maintaining Eve expression and becoming the founder of the two EPCs (F2EPC), and the other losing Eve expression and becoming the founder of muscle DO2. The second Eve-expressing cluster, C15, forms slightly later and produces the progenitor P15, which in turn divides to yield the founder of the Eve-expressing muscle, DA1, and an Eve-negative cell of as-yet-undetermined identity. Activation of the Ras/MAPK pathway in C15 depends on both the Egfr and Htl RTKs, but only Htl signaling is required for C2 formation (Halfon, 2000).

The progressive determination of Eve mesodermal progenitors requires that Wg prepattern the mesoderm, rendering cells competent to respond to inductive RTK/Ras signaling. To further investigate the basis of this competence, whether or not the Ras pathway is active in the absence of Wg signaling was examined by monitoring the expression of the activated, diphosphorylated form of MAPK in wg mutant embryos. Diphospho-MAPK is expressed in progenitor P2 in early stage 11 wild-type embryos. Not only is this progenitor missing from wg mutant embryos, but activation of MAPK in the C2 equivalence group, which is dependent on Htl, fails to occur. Similarly, Wg is essential both for P15 formation and for the Egfr- and Htl-dependent activation of MAPK in the equivalence group from which this progenitor is derived (Halfon, 2000).

Next to be determined was at what level in the RTK/Ras pathway Wg is required for MAPK activation. In wg mutant embryos, there is loss of (1) the P2-specific expression of Htl; (2) its specific downstream signaling component, Heartbroken (Hbr, also known as Dof and Stumps), and (3) Rhomboid (Rho), a protein involved in the presentation of the Egfr ligand Spitz. Conversely, constitutive Wg signaling, achieved by ectopic expression of Wg or an activated form of the downstream Wg pathway component Armadillo (Arm), induces Htl, Hbr, and Rho expression in more dorsal mesodermal cells than the single P2 progenitor found at a comparable developmental stage. This effect is less prominent for Rho than for Htl and Hbr, which may reflect different threshold responses to Wg. Alternatively, the effect on Htl and Hbr may be more pronounced because ectopic Wg signaling prolongs their earlier expression in the entire C2 cluster; Rho, in contrast, is normally expressed in P2 but not in C2, possibly making it more refractory to a prepattern factor such as Wg. Expanded expression of these RTK pathway components is associated with increased MAPK activation and Eve expression. However, these effects of Wg hyperactivation are transient, with a normal number of Eve progenitors eventually segregating. Moreover, activated Arm is able to fully rescue Htl, Hbr, Rho, diphospho-MAPK, and Eve expression in wg mutant embryos. Htl, Hbr, and Rho expression, as well as MAPK activation, are also Dpp dependent. In summary, Wg and Dpp regulate the production of several key proximal components of the Egfr and Htl signal transduction pathways (Halfon, 2000).

Given the involvement of Wg in the expression of Htl, Hbr, and Rho, it was reasoned that a constitutively activated form of Ras1 might bypass the requirement of Wg for MAPK activation. Constitutively activated Ras1, when targeted to the mesoderm of wild-type embryos, leads to an overproduction of Eve progenitors, as well as to the expected hyperactivation of MAPK in these cells. In the absence of Wg signaling, diphospho-MAPK expression is restored by activated Ras1. However, despite this recovery of MAPK activation, constitutive Ras1 does not rescue Eve progenitor formation in a wg mutant background. This is in marked contrast to the ability of activated Arm to fully rescue RTK signaling and Eve progenitor specification in a wg mutant. These results suggest that, in addition to enabling activation of Ras/MAPK signaling as a result of the induction of Htl, Hbr, and Rho expression, Wg signaling must contribute other factors that are essential for the specification of mesodermal Eve progenitors (Halfon, 2000).

It is concluded that Wg and Dpp coordinate a series of signal-activated (dTCF and Mad) and mesoderm-specific (Twi and Tin) transcription factors in a temporal and spatial pattern that facilitates cooperation with the Ras transcriptional effector Pnt. The synergistic integration of these five transcription factors by a single enhancer generates a specific developmental response to Ras/MAPK signaling. Moreover, Wg and Dpp exert proximal effects in this signaling network by enabling Ras/MAPK activation through the regulated localized expression of upstream components of the RTK signal transduction machinery. A model governing the acquisition of developmental competence, signal integration and response specificity in this system is presented. Wg and Dpp provide competence through the regulation of tissue-specific transcription factors (Tin and Twi), signal-responsive transcription factors (Mad and dTCF), and proximal components of the RTK/Ras pathways (Htl, Hbr, and Rho). The Ras signaling cascade leads to activation of the inductive transcription factor, Pnt, and inactivation of the Yan repressor. While a direct role for Mad in regulating Tin expression has been demonstrated, Wg regulation of Tin, Twi, Htl, Hbr, and Rho may be either direct or indirect. Dpp has additional effects on the proximal RTK factors. The five transcriptional activators assemble at and are integrated by the MHE, where they function synergistically to promote eve expression. Specificity of the response to inductive RTK/Ras signaling derives from the combinatorial effects of the tissue-restricted and signal-activated transcription factors that converge at the MHE. In the absence of inductive signaling, Yan would repress eve by binding to the Ets sites. Since eve is a muscle and heart identity gene, the regulatory mechanisms are inferred to have a more general function in determining progenitor fates. Additional complexity attendant upon the control of RTK activity in this system derives from positive feedback regulation of the Ras/MAPK cascade and from reciprocal regulatory interactions between the Ras and Notch pathways (Halfon, 2000).

Convergent intercellular signals must be precisely integrated in order to elicit specific biological responses. During specification of muscle and cardiac progenitors from clusters of equivalent cells in the Drosophila embryonic mesoderm, the Ras/MAPK pathway -- activated by both epidermal and fibroblast growth factor receptors -- functions as an inductive cellular determination signal, while lateral inhibition mediated by Notch antagonizes this activity. A critical balance between these signals must be achieved to enable one cell of an equivalence group to segregate as a progenitor while its neighbors assume a nonprogenitor identity. Whether these opposing signals directly interact with each other has been investigated, and how they are integrated by the responding cells to specify their unique fates was been examined. Two distinct modes of lateral inhibition, one Notch based and a second based on the epidermal growth factor receptor antagonist Argos, are described that have complementary and reinforcing functions. Argos/Ras and Notch do not function independently; rather, several modes of cross-talk between these pathways have been uncovered. Ras induces Notch, its ligand Delta, and Argos. Delta and Argos then synergize to nonautonomously block a positive autoregulatory feedback loop that amplifies a fate-inducing Ras signal. This feedback loop is characterized by Ras-mediated upregulation of proximal components of both the epidermal and fibroblast growth factor receptor pathways. In turn, Notch activation in nonprogenitors induces its own expression and simultaneously suppresses both Delta and Argos levels, thereby reinforcing a unidirectional inhibitory response. These reciprocal interactions combine to generate the signal thresholds that are essential for proper specification of progenitors and nonprogenitors from groups of initially equivalent cells (Carmena, 2002).

This study involves the origin of two progenitors from a single cell cluster. The two progenitors are characterized by expression of the segmentation gene eve and are specified in a distinct temporal order in the Drosophila embryonic mesoderm. Progenitor 2 (P2) develops first; it originates from the preC2 cluster which develops into the C2 cluster and subsequently gives rise to a single P2 cell. P2 divides asymmetrically to give rise to two founder cells, one specific for a pair of persistently Eve-positive heart-associated or pericardial cells (EPCs) in every hemisegment and a second of previously undetermined identity. This second founder coexpresses Eve along with the gap gene Runt, with Eve levels rapidly fading but Runt persisting as development proceeds. By the time that Eve is evident in the EPCs, Runt labels a single somatic muscle, dorsal oblique muscle 2 (DO2). Runt is also detected in the muscle DO2 precursor during germband retraction (Carmena, 2002).

The second Eve progenitor, P15, which also has its origin in the preC2 cluster (which gives rise to a C15 cluster) forms later than P2 and divides asymmetrically to yield the founders of dorsal acute muscle 1 (DA1) and another cell whose fate cannot be followed since a specific, stably expressed marker for it is unavailable (Carmena, 2002).

To further substantiate the lineage relationships among these progenitors and founders, observations related to RTK signaling dependence of P2 and P15 specification were used: whereas P15 requires the activities of both Egfr and Htl, only Htl is involved in P2 formation. In this way, targeted mesodermal expression of a dominant negative form of Egfr strongly blocks formation of DA1 but not the EPCs. Also, consistent with DO2 and EPC founders being the progeny of P2, DO2 development, like that of the EPCs, is not affected by dominant negative Egfr. Additional support for the sibling relationship between the DO2 and EPC founders derived from the analysis of targeted expression of a dominant negative form of Htl. Under conditions in which early mesoderm migration is not perturbed, dominant negative Htl generates an incompletely penetrant phenotype in which different hemisegments lose derivatives of P2, P15, or both progenitors. With such partial inhibition of Htl activity, muscle DO2 and the EPCs are consistently either both present or both absent from any given hemisegment; in no cases did one of these cell types develop without the other, as expected for cells derived from a common progenitor. In contrast, muscle DA1 frequently forms in the absence of muscle DO2 and the EPCs, consistent with its derivation from an independent progenitor. Taken together, these data establish that the EPC and DO2 founders are sibling cells of the P2 division, whereas the other Eve-expressing muscle founder arises from a different progenitor (Carmena, 2002).

This model differs from one derived on the basis of clonal analysis in which it was proposed that the two Eve-positive mesodermal cell types originate from the same progenitor. This discrepancy may relate to the fact that muscles form by sequential cell fusions involving both founders and fusion-competent cells of potentially different parental cell origins, thereby confounding the interpretation of clonal analysis in which the cytoplasm of a single myotube is labeled by the lineage tracing marker (Carmena, 2002).

Autoregulation of a signal transduction cascade can cause either enhancement or attenuation of the transduced signal, depending on whether the feedback loop acts positively or negatively. Both types of feedback control occur during the Ras- and N-mediated specification of Eve mesodermal progenitors. Ras activation leads to increased expression of several proximal components of both the Fgfr and Egfr pathways that serve to amplify and/or prolong both fate-inducing RTK/Ras signals in the emerging Eve progenitors. A similar amplification of Egfr signaling occurs via induction of Rho during Drosophila oogenesis and mesothoracic bristle formation, and via upregulation of Egfr expression during C. elegans vulva development. The present analysis also uncovers a positive feedback mechanism for inductive Fgfr signaling, in this case via increased expression of not only the Htl receptor but also its specific signal transducer, Heartbroken (Hbr). Interestingly, the data suggest that the downstream components may respond to different thresholds of Ras activity since Rho exhibits a less robust response than either Htl or Hbr to Ras activation (Carmena, 2002).

Competitive cross-talk between Ras and N is manifest by the ability of the latter to block the expression of proximal components of the two RTK pathways—namely Htl/Hbr and Rho -- as well as to prevent the associated activation of MAPK. An antagonistic relationship between the RTK and N pathways is also revealed by the strong genetic interaction between Dl and Egfr, in agreement with previously reported genetic studies. Collectively, these results establish that the RTK and N pathways are not simply acting in parallel to exert opposing influences on progenitor specification; rather, N must be interfering with the generation and/or transmission of the inductive RTK signal. This effect could occur at multiple levels. The ability of activated N to at least partially block MAPK activation induced by constitutive Ras argues that N functions downstream of Ras. An additional direct effect of N on expression of Ras-responsive target genes cannot be excluded, particularly since Enhancer of split repressors are involved in the specification of progenitor cell fates. Such targets could include eve itself, or, given positive autoregulation of RTK signaling, one or more RTK pathway components (Carmena, 2002).

Targets of activity

Fr1 is required for the specification of even-skipped expressing pericardial and somatic muscle cells, but not for eve expressing CNS cells (Gisselbrecht, 1996).

FR1 is expressed in a segmentally reiterated pattern within mesoderm and prefigures the ventral, lateral and dorsal somatic muscle cell clusters. FR1 is expressed in large clusters of mesodermal cells in a pattern similar to the wild type distribution of nautilus expressing cells. A disruption of the DRF1 locus results in severly reduced nautilus expression (Shishido, 1993).

adrift is expressed in the leading cells of growing tracheal branches, near clusters of branchless FGF-expressing cells and in a pattern very similar to that of several known branchless-induced genes including pointed, DSRF/pruned and sprouty. This suggested that adrift expression might also be induced by the bnl signaling pathway. Expression of an adrift lacZ reporter was examined in embryos mutant for four components of the branchless pathway: bnl, breathless, pnt and pruned. Initial expression of the adrift reporter in stage 11 tracheal cells is normal in all four mutants, but subsequent expression in the leading cells of the branches is absent in bnl, btl and pnt mutants. Expression in pruned mutants is unaffected. In a complementary experiment in which bnl was misexpressed under the control of the hsp70 promoter, expression of the adrift reporter expands to include additional cells in each branch. Thus, the Branchless FGF pathway induces adrift expression in the leading cells of tracheal branches, and this induction requires the bnl FGF, the btl FGF receptor and the pointed ETS domain transcription factor (Englund, 1999).

Protein Interactions

The active state of receptor tyrosine kinases (RTKs) and the RTK signaling cascade pathways were followed in situ. This was achieved by monitoring, with a specific monoclonal antibody, the distribution of the active, dual phosphorylated form of MAP kinase (ERK). A dynamic pattern is observed during embryonic and larval phases of Drosophila development, which can be attributed, to a large extent, to the known RTKs. Torso-dependent, Egfr-dependent, Breathless-dependent, and Heartless dependent activation profiles have all been identified. This specific detection has enabled the determination of the time of receptor activation, the visualization of gradients and boundaries of activation, and has allowed the postulation of the distribution of active ligands. A novel pattern is observed in the visceral mesoderm at stage 11 that is not Heartless dependent, as patches of cells display activated ERK at normal intensity in heartless mutants. Since the antibody was raised against the phosphorylated form of a conserved ERK peptide containing the TEY motif, this approach is applicable to a wide spectrum of multicellular organisms (Gabay, 1997).

Receptor tyrosine kinases (RTKs) transmit signals to the cell nucleus via the MAP kinase (MAPK) cascade, using specific molecules to link the activated receptors to the MAPK cascade activator, Ras. A component of the FGF receptor (FGFR) signal transduction pathway Downstream of FGFR (Dof), also termed heartborken, has been identified. Dof is an intracellular protein that is essential for signal transmission by the FGFR and acts downstream of the receptor and upstream of Ras. Unlike other signaling molecules that act downstream of RTKs, Dof is not expressed ubiquitously but is present exclusively in cells that express FGFRs. DOF mRNA is first expressed on the ventral side of the embryo at the late syncytial blastoderm stage, in a region slightly narrower than the mesoderm primordium. It disappears from the mesoderm during germ band extension, and is seen in the tracheal placodes by stage 9/10. As the tracheae branch and start to differentiate, the transcript disappears from the primary branches and is seen mainly in the extended secondary branches. These expression patterns resemble those of the Drosophila FGF receptors htl and btl. The dof gene is also expressed transiently in the anterior and parts of the posterior midgut primordium (as is btl) and, like htl in a subset of heart cells and a group of migrating visceral mesodermal cells. Expression is also seen in glia cells. Dof is needed in these cells for activation of the MAPK cascade via FGF signaling, but not for activation via other RTK ligands (Vincent, 1998).

The open reading frame of transcript II of DOF encodes a protein of 1009 amino acids. Database searches fail to identify any other proteins with significant overall homology to Dof. However the protein has two ankyrin repeats and another region (predicted to fold into a coiled coil) with some similarity to the myosins. The protein is likely to be phosphorylated and phosphorylated residues may serve as binding sites for the SH2 domain of the Drosophila Grb2 homolog. The context of another tyrosine suggests that it might represent a binding site for the SH2 domain of the regulatory subunit of phosphatidyl 3-kinase, while yet another tyrosine could represent a binding site for RasGAP. There is also a potential binding site for the protein tyrosine phosphatase SH-PTP2 (coded for by the corkscrew gene). Dual phosphorylate MAP kinase is absent in dof mutant embryos. Dof therefore appears to be committed exclusively to FGFR-mediated signal transduction. Dof is unlikely to act as an adaptor protein sensing the activated (phosphorylated) state of the receptor, because it does not possess SH2 or SH3 domains (Vincent, 1998).

A functional domain of Dof that is required for fibroblast growth factor signaling

Signal transduction by fibroblast growth factor (FGF) receptors in Drosophila depends upon the intracellular protein Dof, which has been proposed to act downstream of the receptors and upstream of Ras. Dof is the product of a fast-evolving gene whose vertebrate homologs, BCAP and BANK, are involved in signaling downstream of the B-cell receptor. Mapping functional domains within Dof revealed that neither of its potential interaction motifs, the ankyrin repeats and the coiled coil, is essential for the function of Dof. However, a region has been identified within the N terminus of the protein with similarity to BCAP and BANK, that is referred to as the Dof, BCAP, and BANK (DBB) motif; the DBB motif is required for FGF-dependent signal transduction and is necessary for efficient interaction of Dof with the FGF receptor Heartless. In addition, Dof is phosphorylated in the presence of an activated FGF receptor and tyrosine residues can contribute to the function of the molecule (Wilson, 2004).

Three different ways are envisioned by which Dof could function, all of which are consistent with the physical interaction of Dof with the FGF receptors. (1) Dof could be required for the transport of the FGF receptors to the cell surface. However, the detection of Heartless at the peripheries of cells in the absence of Dof argues against such a function. (2) Dof may have a role in the activation of the receptors. It could, for example, facilitate or stabilize conformational changes or autophosphorylation of the receptors. No data is available that would specifically support this model, but it is not ruled out by any of the results. (3) Dof is phosphorylated in the presence of an activated FGF receptor and could be involved in transmission of the signal from the receptors (Wilson, 2004).

Although Dof is a large protein, the only motifs that can be identified in the primary sequence are two ankyrin repeats and a coiled coil. Comparison of the Drosophila protein with its Anopheles homolog and the most closely related vertebrate proteins, BCAP and BANK, suggests that dof may be an example of a fast-evolving gene used for FGF signaling in Drosophila but which has acquired a novel function with the development of the immune system in higher vertebrates. Dof shares a number of distinct parts with these proteins, namely, the ankyrin repeats, the coiled coil, and the region adjacent to the ankyrin repeats, which has been termed the DBB motif. Surprisingly, despite the conservation of these domains, only the DBB domain appears to be indispensable for FGF signaling in Drosophila. In this respect, it is interesting that the association of BANK with the IP-3 receptor, which stimulates the release of calcium from intracellular stores upon activation of the B-cell receptor, also depends upon the N-terminal part of the protein but not upon the ankyrin repeats that are present within this region. However, two caveats apply to the in vivo assays that were used to test the function of Dof: (1) there are aspects of FGF signaling besides those examined in the assays, such as feedback regulation and the response to oxygen deprivation, which could be affected by the Dof mutations; (2) the consequence of a particular deletion was determined based on the overexpression of the mutant protein, and this may have masked certain physiological requirements for particular domains of the protein. Thus, all of the functional domains of Dof might not yet been determined; nevertheless, this approach has revealed a critical part of the protein (Wilson, 2004).

The most important region for the function of Dof corresponded to the DBB motif, which is critical for FGF signaling and for the efficient interaction of Dof with the receptor. Dof mutants with deletions that disrupted the DBB motif have only miminal biological activity and are no longer capable of interacting efficiently with the FGF receptor. These observations suggest that the DBB domain interacts directly with the FGF receptors. This is unlikely to be the only function of the DBB motif, since this domain is conserved in BCAP and BANK, which are expressed in B cells and macrophages and are required for the function of the B-cell receptor and thus are unlikely to interact with FGF receptors. Indeed, it was found that the DBB domain in both Dof and BCAP is required to mediate self-association in yeast cells, indicating that this domain may have a more general role in mediating protein-protein interactions (Wilson, 2004).

Constructs lacking this domain still provide above-background biological activity, suggesting that perhaps other parts of the molecule participate in receptor binding, allowing residual signal transmission in the absence of the DBB. Conversely, the DBB region in itself is clearly not sufficient for transmission of the signal, showing that other essential functions reside elsewhere in the molecule. The smallest N-terminal fragment of Dof with biological activity was Dof[1-522]. A mutant with a more extensive C-terminal truncation, Dof[1-446], had no biological activity but was still able to interact with the cytoplasmic domain of the FGF receptor Htl. Together, these findings imply that in addition to the DBB domain there are essential properties of Dof that are located within the 76 amino acids between residues 446 and 522. Intriguingly, there are two tyrosine residues in this region that are potential binding sites for PI 3-kinase and Corkscrew. The mutation of the potential Corkscrew binding site has a clear effect upon the activation of Even-skipped within the mesoderm, suggesting that this binding site contributes to the function of the molecule (Wilson, 2004).

In summary, two parts of Dof are important for its function. The DBB motif is necessary for the efficient interaction of Dof with the receptor, and tyrosines between the ankyrin repeat and the coiled-coil region also contribute to the function of Dof, possibly by recruiting Csw. Thus, similar to FRS2 in vertebrate FGF signal transmission, Dof uses a protein-protein interaction domain to interact with the receptor and acts as a substrate for phosphorylation, and it can therefore recruit other signaling molecules. It is intriguing that although the FGF signaling pathway must have existed in the common precursor of insects and vertebrates, different molecules have taken on this role in the two lineages, while their respective homologs in the other lineage have diverged in sequence and function (Wilson, 2004).

The RhoGEF Pebble is required for cell shape changes during cell migration triggered by the Drosophila FGF receptor Heartless

The FGF receptor Heartless (HTL) is required for mesodermal cell migration in the Drosophila gastrula. Mesoderm cells undergo different phases of specific cell shape changes during mesoderm migration. During the migratory phase, the cells adhere to the basal surface of the ectoderm and exhibit extensive protrusive activity. HTL is required for the protrusive activity of the mesoderm cells. Moreover, the early phenotype of htl mutants suggests that HTL is required for the adhesion of mesoderm cells to the ectoderm. In a genetic screen pebble was identfied as a novel gene required for mesoderm migration. pbl encodes a guanyl nucleotide exchange factor (GEF) for RHO1 and is known as an essential regulator of cytokinesis. The function of Pbl in cell migration is shown to be independent of the function of Pbl in cytokinesis. Although the small GTPase Rho1 acts as a substrate for Pbl in cytokinesis, compromising Rho1 function in the mesoderm does not block cell migration. These data suggest that the function of Pbl in cell migration might be mediated through a pathway distinct from Rho1. This idea is supported by allele-specific differences in the expressivity of the cytokinesis and cell migration phenotypes of different pbl mutants. Pbl is shown to be autonomously required in the mesoderm for cell migration. Like Htl, Pbl is required for early cell shape changes during mesoderm migration. Expression of a constitutively active form of Htl is unable to rescue the early cellular defects in pbl mutants, suggesting that Pbl is required for the ability of Htl to trigger these cell shape changes. These results provide evidence for a novel function of the Rho-GEF Pbl in Htl-dependent mesodermal cell migration (Schumacher, 2004).

In Drosophila the mesoderm originates from a ventral population of cells in the monolayered blastoderm epithelium. At the onset of gastrulation these cells are first internalized through an invagination of the epithelium. After internalization, the cells undergo mitosis, lose their epithelial characteristics, and start to spread as an aggregate between the central yolk sac and the basal cell surfaces of the ectoderm (Schumacher, 2004).

To follow cell shape changes of mesoderm cells, a transgene was used driving expression of the transmembrane protein CD2 from rat under the control of the twist (twi) promotor (twi::CD2). twi::CD2 is already expressed during invagination and represents a cell-surface marker specific for the mesoderm. Mesoderm migration can be divided into three phases with characteristic cell shape changes. After invagination, the mesoderm initially forms an epithelial tube. At phase 1 of migration, the surface of the mesoderm cells appears relatively smooth. After disassembly of the epithelial tube and mitosis, phase 2 begins, in which the mesodermal aggregate migrates out in dorsolateral direction. Cells at the leading edge of the aggregate are stretched along the dorsoventral axis and extend multiple cellular protrusions. The longest cellular protrusions often measure half to two-thirds the size of a cell diameter (to a length of 10-15 µm in fixed samples). Cross-sections reveal that not only the leading edge cells exhibit this polarized morphology, but that the cells immediately following the leading edge cells frequently also extend in dorsolateral direction. The term 'protrusive activity' is used to describe the formation and/or the dynamics of the filoform and lamelliform protrusions that are observed in fixed preparations. The protrusive activity is specific for the migratory phase, because when the cells have reached their final positions (phase 3) and form a coherent monolayer, large extensions are absent and only few filoform protrusions are observed (Schumacher, 2004).

Examination of embryos homozygously mutant for htl reveal that Htl is required for cell shape changes during phase 1 and 2 of mesoderm migration. In phase 1, the mesodermal epithelial tube extends further into the interior of the embryo when compared with wild type. During phase 2, the leading edge cells do not extend dorsolaterally. This phenotype is not simply explained by the possibility that htl mutant mesoderm cells were not able to contact the ectoderm, because cells directly apposed to the ectoderm also fail to extend. In phase 3, mesoderm cells of htl mutant embryos do not establish a monolayer configuration. Interestingly, during and after phase 3, htl mutant mesoderm cells exhibit directional protrusions suggesting that some migration might occur at these stages. This result indicates that Htl is not generally required for protrusive activity of the mesoderm cells. This late migration in htl mutants is never able to rescue the defects in mesoderm differentiation, most probably because of a second requirement of Htl for mesoderm differentiation (Schumacher, 2004).

These observations suggest that Htl is required for the early interaction of the mesoderm with the ectoderm. By analyzing sections, wild-type cells at the base of the mesodermal tube are observed to be attached to the basal surfaces of the ectoderm. By contrast, htl mutant mesoderm cells at the respective stage and position fail to establish contact to the ectoderm. This phenotype correlates well with a misalignment of the mesodermal tube in htl mutants. It is concluded that Htl is required for the effective attachment of mesoderm cells to the ectoderm, which might promote the protrusive activity of mesoderm cells during migration (Schumacher, 2004).

htl and dof represent the only zygotically expressed genes that have thus far been described to be essential for mesoderm migration. To obtain a better insight into the genetic control of mesoderm migration, a genetic screen was performed to identify zygotically expressed genes involved in mesoderm migration. Three loci mapped to the third chromosome and were characterized using chromosomal deletions and available point mutations. Two loci corresponded to the genes htl and dof, respectively. Embryos lacking the chromosomal interval 61A to 68 (based on breakpoints of the chromosomal translocation T(2;3)C309) displayed defects in mesoderm migration. Genetic mapping revealed that small overlapping chromosomal deletions, which exhibited the phenotype, all removed the gene pbl. Analysis of a strong loss-of-function point mutation in pbl, pbl3, indicated that pbl is required for mesoderm morphogenesis. Embryos homo- or hemizygously mutant for pbl3 show a dramatic reduction in the number of Eve-positive mesoderm cells at the extended germband stage. These results demonstrate a thus far unrecognized function for pbl in mesoderm differentiation (Schumacher, 2004).

pbl encodes a RHO1-GEF most similar to the vertebrate ect2 proto-oncogene. Both pbl and ect2 are required for the assembly of the contractile actin ring during cytokinesis. Interfering with the function of PBL or ECT2 results in a failure of cytokinesis and the generation of multinucleate cells. Because mutations in pbl affect cell shape changes before mitoses in the mesoderm occur, it was suspected that the requirement of pbl for mesoderm migration might be independent from its cytokinesis function. To determine, whether the defects in mesoderm migration in pbl mutants are direct rather than a secondary consequence of the failure in cytokinesis, the pbl phenotype was examined in division-defective embryos (Schumacher, 2004).

Postblastoderm mitotic divisions are controlled by zygotic expression of the cell cycle regulator String. Since mesoderm migration and specification of Eve-positive mesoderm cells occur normally in stg mutant embryos, this mutation provides a genetic condition to assay cytokinesis-independent functions of pbl. The cytokinesis defect of pbl is completely blocked by stg. In pbl stg double mutant embryos, migration of the mesoderm and specification of dorsal mesodermal derivatives are impaired similar to pbl single mutants. Moreover, cell shape changes in phase 2 occur normally in stg mutant embryos, but protrusive activity of the mesoderm cells is blocked in pbl stg double mutants. These results indicate that the activity of pbl is required for mesoderm migration even in the absence of mitosis and thus in the absence of cytokinesis defects. It is therefore concluded that Pbl has independent functions in cytokinesis and cell migration (Schumacher, 2004).

Thus Htl is required for protrusive activity only during phase 1 and 2 of mesoderm migration. However, Htl activity is not essential for the protrusive activity of the cells per se, because cells do extend dorsolaterally during phase 3 in htl mutant embryos. These data demonstrate that Htl activation is unlikely to provide the only directional cue in mesoderm migration. The results presented in this paper suggest that Htl signaling provides temporal information for protrusion formation during phases 1 and 2, and might be therefore acting as a permissive factor during mesoderm migration (Schumacher, 2004).

Pbl is required for protrusive activity of mesoderm cells also in phase 3 and later. It is therefore possible that Pbl function might be required in a more general way for the cell to extend protrusions. The specificity of Pbl for protrusive activity is also supported by the fact that loss of epithelial characteristics is unaffected in pbl mutant embryos. Although the specific mechanism of Pbl function in cell migration is currently unknown, it is important to note that not all morphogenetic movements are compromised in pbl mutants. For example, cephalic furrow formation, invagination of the ventral furrow and germband extension movements, which all depend on a functional cytoskeleton are normal in pbl mutant embryos. It is therefore proposed that Pbl might constitute an important component for cytoskeletal changes, which are triggered by FGFR signaling events (Schumacher, 2004).

Of the multiple responses generated downstream of FGFR activation, only little is known of the molecular pathways by which FGFRs trigger cell shape changes in vivo. The Rho GEF Pbl represents a good candidate for mediating cell shape changes triggered by Htl signaling. Importantly, the early phenotypes of htl and pbl mutants are almost identical, indicating that both gene products are required in a narrow time window for early cell shape changes after invagination of the mesoderm. Furthermore, in both mutants this phenotype is completely penetrant, indicating that the gene products do not act in a redundant fashion (Schumacher, 2004).

The function of Pbl for mesoderm migration is specific for mesoderm cells. Because htl is expressed only in the mesoderm at this stage of development, Pbl might be involved in the presentation of the receptor or its ligand and thus acting upstream, or Pbl might be involved in downstream events triggered by the Htl signaling cascade. If Pbl was acting upstream of Htl, signaling events downstream of Htl should be blocked in such mutants. By contrast, Pbl is shown to be dispensable for activation of MAP kinase in the early mesoderm cells. These results suggest that Pbl does not act upstream of Htl, and a model is favored in which Pbl acts downstream of the Htl signaling cascade (Schumacher, 2004).

The present results render it unlikely that Pbl is directly involved in a signaling pathway downstream of Htl FGFR. In contrast to htl mutants, no cell shape changes and no protrusive activity was observed in pbl mutant mesoderm cells in phase 3. In addition, the pbl null mutant phenotype still allows a few cells to undergo eve expression, probably owing to the larger cells and abnormal cytoarchitecture in the division defective embryos. This is in contrast to htl loss of function mutants where EVE-positive mesoderm cells are never observed. If pbl is essential for signaling downstream of the Htl receptor, the phenotype of htl and pbl mutants should be more similar with respect to mesoderm differentiation: in fact, the phenotypes of htl and dof mutant embryos are identical. It is therefore proposed that Pbl might represent a regulator of the cytoskeleton or adhesive mechanisms of the cell, which provide targets of the Htl signaling cascade to trigger cell shape changes (Schumacher, 2004).

Although the activation of MAP kinase in the mesoderm depends on Htl, it is not known whether this is a direct response or whether it is indirect, i.e. MAP kinase may not be directly activated by Htl itself, but through interactions of the mesoderm with the ectoderm. In this case, activation of MAP kinase would be a response rather than a cause of the cell shape changes. The phenotype of pbl mutants, however, argues against the latter possibility, because it shows that in the absence of cell shape changes, MAP kinase can still be activated. This result also suggests that activation of MAP kinase alone cannot account for the cell shape changes that occur. This idea is supported by the fact that activated forms of RAS1 are unable to completely rescue the defects in mesoderm migration of htl or dof mutant embryos, including the defects in cell shape changes in phase 1 and 2. These results suggest the presence of a signaling pathway acting in parallel to the Ras/Raf MAP kinase pathway to be involved in mesoderm migration (Schumacher, 2004).

The pbl gene was originally identified and characterized as an essential factor for cytokinesis. Two lines of evidence indicate that the function of Pbl in cell migration is mediated through a pathway different from the cytokinesis pathway. (1) Expression of a dominant-negative form of the small GTPase Rho1, which blocks cytokinesis in the mesoderm, has no effect on mesoderm migration, cell shape changes associated with it or expression of differentiation markers specific for dorsal mesoderm derivatives. (2) A mutation in pbl, pbl11D exhibits significantly weaker defects in mesoderm differentiation compared to the strong loss of function mutation pbl3. These allele-specific differences indicate distinct requirements of the Pbl protein for its two functions, because both alleles exhibit identical cytokinesis defects and only differ in mesoderm differentiation defects significantly. It is therefore proposed that the function of Pbl for cell migration might not involve Rho1 and might therefore be using another mechanism (Schumacher, 2004).

How does Pbl act in cell migration and which GTPase represents its substrate? Both Pbl and its mammalian orthologs belong to the Dbl family of Rho-GEFs, which promote activation of Rho GTPases through a conserved Dbl-homology (DH) domain. The DH domain is required for both functions of Pbl, because a missense mutation in pbl, called pbl5, in which an amino acid exchange renders the DH domain inactive, exhibits equally strong defects in cell migration and cytokinesis. Data from yeast two-hybrid assays, as well as genetic interactions indicate that Pbl binds to and interacts with RHO1. During cytokinesis, Pbl is proposed to locally activate RHO1, which then interacts with its effector Diaphanous, a Drosophila homolog of the Formin family of actin regulators. Although Pbl appears to interact with RHO1, but not with CDC42 or RAC1, mammalian homologs of Pbl promote GTP/GDP exchange of the GTPases RHO1, RAC1 and CDC42. Because these discrepancies might reflect differences in the sensitivity of the assays applied, it remains to be determined which substrate Pbl uses for its function in cell migration (Schumacher, 2004).

Although a role of Pbl in FGFR triggered cell migration has been detected, it is currently unclear how general the requirement of Pbl is for the protrusive activity of migrating cells. Interestingly, mutations in pbl have been discovered in a screen for genes required for the development of the peripheral nervous system. These mutants affect the correct migration of the axons in the PNS without obvious defects in cytokinesis. It will therefore be interesting to assess the function of Pbl in a variety of migrating cells to further characterize its potential role as a mediator of cell shape changes triggered by extracellular signals (Schumacher, 2004).

The epithelial-mesenchymal transition of the Drosophila mesoderm requires the Rho GTP exchange factor Pebble: Pbl function is not required for Htl-dependent activation of the MAP kinase signalling pathway

Drosophila pebble encodes a Rho-family GTP exchange factor (GEF) required for cytokinesis. The accumulation of high levels of Pbl protein during interphase and the developmentally regulated expression of pbl in mesodermal tissues suggests that the primary cytokinetic mutant phenotype might be masking other roles. Using various muscle differentiation markers, it was found that Even skipped (Eve) expression in the dorsal mesoderm is greatly reduced in pbl mutant embryos. Eve expression in the dorsalmost mesodermal cells is induced in response to Dpp secreted by the dorsal epidermal cells. Further analysis has revealed that this phenotype is likely to be a consequence of an earlier defect. pbl mutant mesodermal cells fail to undergo the normal epithelial-mesenchymal transition (EMT) and dorsal migration that follows ventral furrow formation. This phenotype is not a secondary consequence of failed cytokinesis, since it is rescued by a mutant form of pbl that does not rescue the cytokinetic defect. In wild-type embryos, newly invaginated cells at the lateral edges of the mesoderm extend numerous protrusions. In pbl mutant embryos, however, cells appear more tightly adhered to their neighbours and extend very few protrusions. Consistent with the dependence of the mesoderm EMT and cytokinesis on actin organisation, the GTP exchange function of the PBL RhoGEF is required for both processes. By contrast, the N-terminal BRCT domains of Pbl are required only for the cytokinetic function of Pbl. These studies reveal that a novel Pbl-mediated intracellular signalling pathway operates in mesodermal cells during the transition from an epithelial to migratory mesenchymal morphology during gastrulation (Smallhorn, 2004).

Heartless (HTL), a receptor tyrosine kinsase (RTK) of the fibroblast growth factor receptor (FGFR) subfamily is required for the mesoderm EMT, where it is known to activate the conserved Ras/MAP kinase pathway. In htl mutant embryos, mesodermal cells fail to dissociate from each other following invagination and fail to migrate dorsally. Mesoderm migration also fails in embryos mutant for three other genes: Downstream-of-FGFR (Dof), Sugarless and Sulphateless. In each case, the failure in mesoderm migration is accompanied by a failure in the activation of the Ras1/MAPK pathway (Smallhorn, 2004).

To investigate whether the pbl mutant phenotype is also due to a failure in the activation of the HTL/MAPK pathway, pbl mutant embryos were stained with an antibody directed towards the dual phosphorylated form of MAP kinase (dp-ERK). In wild-type embryos following gastrulation, dp-ERK is expressed in the dorsalmost mesodermal cell rows on each lateral surface of the embryo, a staining pattern that is Htl dependent. In pbl mutant embryos, dp-ERK staining is seen in the dorsalmost mesodermal cell rows similar to wild type. This result shows that Pbl function is not required for Htl-dependent activation of the MAP kinase signalling pathway, and that the mesoderm migration defect in pbl mutants is not due to a failure in the activation of the MAPK pathway (Smallhorn, 2004).

Downstream-of-FGFR is a fibroblast growth factor-specific scaffolding protein and recruits Corkscrew upon receptor activation

Fibroblast growth factor (FGF) receptor (FGFR) signaling controls the migration of glial, mesodermal, and tracheal cells in Drosophila melanogaster. Little is known about the molecular events linking receptor activation to cytoskeletal rearrangements during cell migration. A functional characterization has been performed of Downstream-of-FGFR (Dof), a putative adapter protein that acts specifically in FGFR signal transduction in Drosophila. By combining reverse genetic, cell culture, and biochemical approaches, it was demonstrated that Dof is a specific substrate for the two Drosophila FGFRs. After defining a minimal Dof rescue protein, two regions were identified that are important for Dof function in mesodermal and tracheal cell migration. The N-terminal 484 amino acids are strictly required for the interaction of Dof with the FGFRs. Upon receptor activation, tyrosine residue 515 becomes phosphorylated and recruits the phosphatase Corkscrew (Csw). Csw recruitment represents an essential step in FGF-induced cell migration and in the activation of the Ras/MAPK pathway. However, the results also indicate that the activation of Ras is not sufficient to activate the migration machinery in tracheal and mesodermal cells. Additional proteins binding either to the FGFRs, to Dof, or to Csw appear to be crucial for a chemotactic response (Petit, 2004).

Genetic epistasis experiments have shown that Dof functions downstream of the activated FGFRs and upstream or in parallel to Ras. However, the biochemical function of Dof in the interpretation of the chemotactic response to FGFR signaling has not been addressed so far. Using in vivo rescue assays, a minimal Dof protein containing the first 600 amino acids of Dof was identified that allows rescue of both mesodermal and tracheal cell migration. Although the rescue in the tracheal system is not as efficient as the rescue observed with the wild-type construct, all six branches can migrate out, demonstrating that the first 600 amino acids of Dof retain the capacity to read out the local activation state of the FGFRs and to relay the signal to the migration machinery, albeit with somewhat reduced efficiency. Deletion from the C terminus of this dof minigene, as well as internal deletions, results in loss of rescue capacity, thus identifying regions of functional importance (Petit, 2004).

All of the constructs were examined in a Drosophila S2 cell culture assay, in which either the FGFR or the Torso signaling system was activated. Both full-length Dof and Dof600 are phosphorylated on tyrosine residues upon FGF signaling, but Torso cannot use Dof as a substrate. These results are consistent with in vivo data showing that Dof is exclusively needed for FGF-mediated signal transduction and that Torso is able to activate the MAPK cascade in the absence of Dof in dof mutant embryos. Using coimmunoprecipitation experiments, it was shown that Dof forms a complex with both FGFRs and that the first 484 amino acids, although not phosphorylated upon FGF signaling, are required and sufficient for the association with the FGFRs, demonstrating that phosphorylation of Dof is not necessary for complex formation. Cell culture analysis is in line with studies showing that the N-terminal part of Dof directly interacts with the kinase domains of Btl and Htl in yeast two hybrid assays. In addition, it was observed that both the juxtamembrane and the C terminus of Btl can be deleted without affecting considerably the quality of the rescue capacity of the receptor. Thus, it appears that Dof directly docks onto the kinase domain of the FGF receptor, in contrast to the vertebrate FGFR adapter SNT/FRS2, which interacts with a sequence motif in the juxtamembrane region of the receptor (Petit, 2004).

Since Dof becomes phosphorylated upon FGFR signaling in S2 cells, it was asked whether it was possible to identify functionally important phosphorylation sites, the proteins recognizing these sites in the phosphorylated state, and confirm the results in vivo by making use of the rescue assay and genetic analysis. Two potential phosphorylation target sites were identified by sequence analysis in the essential region comprising amino acids 485 to 600. While mutation of each individual site results in reduced phosphorylation of Dof600 in S2 cells upon FGFR signaling, mutation of only one of these sites, tyrosine 515, abolished the migration rescue capacity in vivo. Since the functionally required tyrosine residue was part of a putative consensus binding site for the SH2 domain of the nonreceptor tyrosine phosphatase Csw/SHP-2, the interaction of Csw with Dof was tested using coimmunoprecipitation experiments; Csw is indeed recruited to the activated signaling complex via Dof. It was found in rescue assays that both the region 485 to 600 as well as the region from 600 to the C terminus (construct dofDelta485-600) are able to confer function to the signaling-deficient N terminus (residues 1 to 484). It is known that the C-terminal sequences also recruit the Csw phosphatase in the absence of tyrosine 515, but it is not known know whether they do so directly or indirectly. Further deletion analyses and biochemical studies will be required to address this question (Petit, 2004).

Genetic evidence supporting an interaction between Dof and Csw was provided some time ago by the finding that mutations in csw produce a phenotype identical to bnl, btl, and dof; i.e., tracheal and mesodermal cells fail to migrate. The sum of these results clearly assign a crucial role for both Dof and Csw downstream of the FGFRs in the migratory response, indicating that the ligand-dependent phosphorylation of Dof leads to the recruitment of Csw to the signaling complex, ultimately triggering cell locomotion. SHP-2, the vertebrate homologue of Csw, has been shown to be required at the initial steps of gastrulation, as mesodermal cells migrate away from the primitive streak in response to chemotactic signals initiated by fibroblast growth factors. In addition, SHP-2 has also been found to be crucial for tubulogenesis and for the sustained stimulation of the ERK/MAPK pathway upon induction of another chemotactic factor, the hepatocyte growth factor/scatter factor, thus placing SHP-2/Csw as a key player in branching morphogenesis induced by diverse chemotactic factors. Therefore, it appears that both in invertebrates and vertebrates, SHP-2/Csw plays a major role in RTK signaling in the control of cell migration. The similarity of the Drosophila FGF signal transduction pathway to the vertebrate FGF pathway make the fly system accessible to address future issues not resolved in vertebrates, such as the targets of SHP-2/Csw involved in Ras activation and/or cell migration (Petit, 2004).

Using the dpERK antibody as a readout for the activation of the Ras/MAPK pathway in vivo, it was found that abolishing the interaction between the Dof600 minimal protein and Csw abolishes the activation of the MAPK cascade upon FGFR signaling. The strong correlation found between migration and MAPK activation when analyzing all mutant dof constructs in this assay might indicate that local activation of the Ras/MAPK pathway in tracheal tip cells is sufficient to trigger the migratory response upon Btl signaling. However, two lines of evidence suggest that this might not be the case (Petit, 2004).

In one case, it has been observed that under conditions in which all tracheal cells sustain high levels of Ras/MAPK activity (upon RasV12 overexpression), tracheal cells migrate normally in wild-type embryos. In sharp contrast, ectopic expression of the Bnl ligand leads to a complete disruption of directed migration. Therefore, high levels of Ras/MAPK activity do not appear to produce the same migratory response as ligand-activated FGFR signaling. Indeed, and again in contrast to ectopic Bnl, overexpression of RasV12 in wild-type embryos does not produce significant filopodial activity in DT tracheal cells, confirming that the activation of Ras is not sufficient to produce cytoskeletal rearrangements by itself (Petit, 2004).

In the other case, it was also observed that while the Dof600 protein lacking the ankyrin repeats did allow FGFR-dependent activation of the Ras/MAPK pathway and downstream nuclear response genes, this protein failed to induce migration. Thus, even local Ras activation under the control of the endogenous ligand Bnl, Btl, and Dof600DeltaAR is unable to activate the migratory machinery. Interestingly, it has also been reported that Ras activation is insufficient to guide RTK-mediated border cell migration during Drosophila oogenesis (Petit, 2004).

Is Ras activation then required at all for cells to produce a cytoskeletal response and migrate directionally? Unfortunately, genetic analysis cannot be used to directly address this question in the embryo since maternal and zygotic loss of Ras activity results in embryos that do not develop far enough to analyze the tracheal system. However, when activated Ras (RasV12) is expressed in the tracheal system or in the mesoderm of dof mutant embryos, a certain rescue of migration can be obtained. This suggests that Ras signaling is essential but not sufficient for efficient FGFR-dependent cell migration; additional proteins binding to the receptor, to Dof or to Csw appear to be crucial for a chemotactic response. To analyze the role of Ras experimentally and in detail, mitotic clones lacking Ras activity should be analyzed with regard to their migration properties. Recent reports concerning the role of FGF signaling in the migration of mesodermal and tracheal cells during late larval development might provide the basis for such analyses (Petit, 2004).

p120 Ras GTPase-activating protein associates with fibroblast growth factor receptors in Drosophila

Btl (breathless) and Htl (heartless), the two FGFRs (fibroblast growth factor receptors) in Drosophila melanogaster, control cell migration and differentiation in the developing embryo. These receptors signal through the conserved Ras/mitogen-activated protein kinase pathway, but how they regulate Ras activity is not known. The present study shows that there is a direct interaction between p120 RasGAP (Ras GTPase-activating protein), a negative regulator of Ras, and activated FGFRs in Drosophila. The interaction is dependent on the SH2 (Src homology 2) domains of RasGAP, that have been shown to interact with a phosphotyrosine residue within the consensus sequence (phospho)YXXPXD. A potential binding site that matches this consensus is found in both Btl and Htl, located between the transmembrane and kinase domains of each receptor. A peptide corresponding to this region is capable of binding RasGAP only when the tyrosine residue is phosphorylated. This tyrosine residue appears to be conserved in human FGFR-1 and mediates the association with the adapter protein CrkII, but no association between dCrk (Drosophila homologue of CrkII) and the activated FGFRs was detected. RasGAP is a substrate of the activated FGFR kinase domain, and mutation of the tyrosine residue within the potential binding site on the receptor prevents tyrosine phosphorylation of RasGAP. RasGAP attenuates FGFR signalling in vivo and this ability is dependent on both its SH2 domains and its GAP activity. On the basis of these results, it is proposed that RasGAP is directly recruited into activated FGFRs in Drosophila and plays a role in regulating the strength of signalling through Ras and the mitogen-activated protein kinase pathway (Woodcock, 2004).

Alignments of the mammalian FGFR-1 to the Drosophila FGFRs revealed a potential binding site for the RasGAP SH2 domains, conserved between species, in the form of a tyrosine residue within the consensus sequence, (phospho)YXXPXD, lying between the transmembrane domain and the cytoplasmic tyrosine kinase domain of the receptors. The fact that the corresponding tyrosine residue was shown to be both an autophosphorylation site and a docking site for SH2 domains in mammalian FGFR-1 suggested that this juxtamembrane tyrosine is a good candidate for a RasGAP-binding site on Btl and Htl. This was shown to be the case in three separate experiments. (1) A peptide corresponding to this region of Btl and Htl can precipitate RasGAP expressed in Drosophila only when the peptide is phosphorylated on the tyrosine. (2) This phosphorylated peptide readily competes with Tor-Htl for binding to immobilized RasGAP SH2 domains, whereas the non-phosphorylated peptide can not compete with this binding. (3) If the juxtamembrane Tyr402 of Htl is mutated, RasGAP is no longer tyrosine-phosphorylated when expressed in S. pombe. This suggests that RasGAP can not be recruited to the mutated receptor and therefore is not in a position to be phosphorylated by the active receptor kinase domain. The evidence provided indicates that the juxtamembrane tyrosine residue of Btl and Htl is both an autophosphorylation site and a binding site for the SH2 domains of RasGAP (Woodcock, 2004).

Why does RasGAP have tandem SH2 domains? Two possible reasons for the existence of tandem SH2 domains in the adapter region of RasGAP are (1) they may be required to bind two separate docking sites simultaneously and (2) they may stabilize the interaction with one docking site by increasing the avidity. The individual requirement of the SH2 domains in the interaction with Btl and Htl was tested in two experiments. One of them showed that the immobilized pTyr peptide of Btl/Htl was unable to precipitate RasGAP with single N- and C-terminal mutant SH2 domains to the same extent as wild-type RasGAP. This shows that the two SH2 domains in RasGAP work together to increase the strength of the interaction with the juxtamembrane phosphotyrosine of FGFRs. The second experiment showed that both SH2 domains of RasGAP were required for its maximal tyrosine phosphorylation in response to FGFR signalling in Drosophila. This suggests that when RasGAP has only one intact SH2 domain, it cannot form a stable enough complex with an activated tyrosine kinase, probably the FGFR in this case, to be phosphorylated efficiently. These results indicate that the tandem SH2 domains of the RasGAP adapter region are essential for both the initial interaction with FGFRs and its subsequent phosphorylation on tyrosine. The fact that the activated FGFRs will be dimerized means that there will be two juxtamembrane phosphotyrosine-binding sites in the receptor complex, both of which may be bound to the tandem SH2 domains of RasGAP, adding to the avidity of the association and stabilizing the interaction. The requirement for both SH2 domains in the binding of RasGAP to FGFRs is consistent with the requirement of both SH2 domains for the attenuation of ectopic FGFR signalling by RasGAP in the wing (Woodcock, 2004).

There is a corresponding juxtamembrane tyrosine residue equivalent to that of Btl and Htl in mammalian FGFR-1 that is autophosphorylated and binds the SH2 domain of mammalian CrkII. However, when dCrk, the Drosophila homologue of CrkII, was tested for its ability to bind the FGFR homologues in Drosophila, no association could be detected. This suggests that the interaction between Crk and FGFRs is not conserved between mammals and Drosophila. Therefore it is unlikely that in Drosophila, RasGAP and dCrk compete in vivo for binding to the juxtamembrane tyrosine residues of Btl and Htl. In mammals, it is possible that CrkII and p120 RasGAP may both compete with each other for binding to the juxtamembrane tyrosine residue of FGFR-1 (Woodcock, 2004).

Drk, the Drosophila homologue of mammalian Grb2, has been shown to act upstream of Ras in RTK signalling pathways and acts by recruiting the Ras activator, Sos, to the site of receptor activation. Drk binds directly to activated RTKs or to substrates of the activated RTKs. Since activation of Ras has been shown to partially rescue both btl and htl mutant phenotypes, the ability of Drk to interact directly with these FGFRs was tested. However, a GST fusion protein of wild-type Drk was not capable of associating with activated Btl or Htl expressed in adult Drosophila. This result suggests that the recruitment of Drk to the site of FGFR activation in Drosophila is not directly to the receptor, but requires a receptor substrate, as in the case of mammals that utilize the adapter-like protein FRS2. The lack of an FRS2 homologue in Drosophila suggests the involvement of a novel adapter in FGFR signalling; one candidate is the cytoplasmic protein Dof, which is essential for FGFR signalling in Drosophila. It acts between FGFRs and Ras, and it contains a number of tyrosine residues that lie within a consensus binding sequence for the Drk SH2 domain (Woodcock, 2004).

In conclusion, a model is proposed in which, after ligand binding, the FGFRs Btl and Htl undergo autophosphorylation on the juxtamembrane tyrosine residue, thereby providing a docking site for RasGAP. This association is stabilized by the fact that RasGAP possesses two SH2 domains, both of which are required for maximal binding to the FGFRs. Once recruited, RasGAP becomes a substrate for the active kinase domain of the receptor, potentially providing further docking sites at the location of receptor activation. Recruitment of RasGAP to the activated FGFRs would allow it to act on its substrate, namely RasGTP, thus negatively regulating the downstream signal through Ras effector pathways, such as the MAPK pathway. This model is consistent with the observations that the ability of RasGAP to attenuate FGFR signalling in vivo requires its GAP activity and both its SH2 domains, but not its SH3 domain, which is dispensable for FGFR binding. However, the physiological relevance of the association of RasGAP with FGFRs remains to be established. The recent identification of mutants defective in the gene encoding RasGAP, vacuolar peduncle (vap), will make it possible to examine the effects of loss of RasGAP activity on FGFR signalling pathways regulating morphogenesis and differentiation in the Drosophila embryo (Woodcock, 2004).

FGF receptor 1: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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