Dof is a large molecule essential for signal transduction by the two FGF
receptors in Drosophila. It contains two ankyrin repeats and a coiled-coil
region, but has no other recognisable structural motif. Dof shares these
features with its closest vertebrate relatives, the B-cell signalling molecules
BCAP and BANK. In addition, this family of proteins shares a region of homology
upstream of the ankyrin repeats, which is called the Dof/BCAP/BANK (DBB) motif. Forty-four
proteins have been identified that interact with Dof in a yeast two-hybrid screen.
These include the Drosophila FGF-receptor Heartless and Dof itself.
The integrity of the DBB motif is required both for Dof and for BCAP to form
dimers. Analysis of the interactions between a set of deletion constructs of Dof
and the panel of interactors suggests that Dof may adopt different
conformations, with a folded conformation stabilized by interactions between the
DBB motif and the C-terminal part of the protein (Battersby, 2003).
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).
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).
A novel conserved phosphotyrosine motif in the Drosophila fibroblast growth factor signaling adaptor Dof with a redundant role in signal transmission
Csiszar, A., Vogelsang, E., Beug, H. and Leptin, M. (2010). A novel conserved phosphotyrosine motif in the Drosophila fibroblast growth factor signaling adaptor Dof with a redundant role in signal transmission. Mol. Cell. Biol. 30(8): 2017-27. PubMed Citation: 20154139
The fibroblast growth factor receptor (FGFR) signals through adaptors constitutively associated with the receptor. In Drosophila melanogaster, the FGFR-specific adaptor protein Downstream-of-FGFR (Dof) becomes phosphorylated upon receptor activation at several tyrosine residues, one of which recruits Corkscrew (Csw), the Drosophila homolog of SHP2, which provides a molecular link to mitogen-activated protein kinase (MAPK) activation. However, the Csw pathway is not the only link from Dof to MAPK. This study identified a novel phosphotyrosine motif present in four copies in Dof and also found in other insect and vertebrate signaling molecules. These motifs are phosphorylated and contribute to FGF signal transduction. They constitute one of three sets of phosphotyrosines that act redundantly in signal transmission: (1) a Csw binding site, (2) four consensus Grb2 recognition sites, and (3) four novel tyrosine motifs. Src64B binds to Dof and Src kinases contribute to FGFR-dependent MAPK activation. Phosphorylation of the novel tyrosine motifs is required for the interaction of Dof with Src64B. Thus, Src64B recruitment to Dof through the novel phosphosites can provide a new link to MAPK activation and other cellular responses. This may give a molecular explanation for the involvement of Src kinases in FGF-dependent developmental events (Csiszar, 2010).
Mutational analysis of Dof, which was used as an indirect approach to map tyrosines that are phosphorylated in the presence of an activated FGFR, showed that consensus tyrosine motifs for PI3K and Csw binding at amino acid positions 486 and 515 were substrates of phosphorylation. In addition, three tyrosine residues at positions 592, 613, and 629 were identified as phosphorylation targets, but these do not conform to known conserved tyrosine motifs. Finally, the last 200 amino acids of Dof also contain several phosphorylation target sites. Mutational analyses of this type do not prove that the same residues are phosphorylated in the wild-type situation, but the tyrosine at position 515 is required for the binding of Csw upon FGFR activation, and this study has shown that mutation of most of the identified sites resulted in impaired activity of the molecule in vivo, supporting the notion that these tyrosines are physiologically relevant phosphorylation targets in Dof (Csiszar, 2010).
The three tyrosine-containing motifs at positions 592, 613, and 629 do not resemble known conserved tyrosine motifs, but their positions and their sequences are conserved in Anopheles Dof. In Drosophila the sites are very close together, so that they could act as tandem interaction surfaces, but the fact that they are separated by longer insertions in Anopheles makes this unlikely. The motifs at 613 and 629 resemble each other, and the same sequence motif is present two more times in the C termini of both Drosophila and Anopheles Dof. Protein database searches with the consensus sequence motif Y-X3-P-X3-P, generated from these eight related sites, showed that this motif is present in several signaling molecules, in many cases as known phosphorylation target sites (e.g., in Shc and the mammalian FGFR-1). Mammalian Shc contains two consensus Grb2 binding sites. One, conserved in vertebrate Shc proteins, has been shown to bind Grb2 and activate the Ras-MAPK pathway. The other, part of a double-phosphorylation site of two adjacent tyrosines and conserved in all members of the Shc family from insects to vertebrates, does not interact with Grb2, and its function in mitogenic activation and apoptosis protection does not depend on Ras. This site is part of the novel tyrosine motif. In vitro kinase assays showed that this pair of adjacent tyrosines can be phosphorylation targets for EGFR and Src as well, but no proteins have been identified as binding partners. The highly conserved sequence patch around these two adjacent phosphorylated tyrosines in Shc goes beyond the Grb2 consensus site and outlines exactly the novel Y-X3-P-X3-P motif, suggesting that the whole motif is important for an evolutionarily conserved biological function (Csiszar, 2010).
In the mammalian FGFR-1, the novel tyrosine motif surrounds the one conserved autophosphorylation site at position Y583 and thus is located in the loop separating the small and large lobes of the kinase domain (28). This is the most variable intracellular sequence part within the FGFR family, and no other mammalian FGFRs share this motif. In spite of the fact that most autophosphorylation sites in the FGFR are conserved, many of these tyrosines are dispensable for signal propagation and only a few proteins that associate with these sites have been identified to date. Phosphorylated Y583 has no known binding partners, and no signaling function has been linked to it so far (Csiszar, 2010).
These findings lead to the speculation that the mammalian FGF-R1 has the ability to recruit a molecule directly that in the case of the Drosophila FGFR is recruited indirectly via Dof and, in the other mammalian FGF receptors, perhaps via other interactors, such as Shc (Csiszar, 2010).
The rescue experiments used to assay the functional relevance of the tyrosine mutations that influenced the phosphorylation levels of Dof in vitro yielded two important findings: first, three independent functional units in Dof were identified that contribute to signal propagation, and second, these units act redundantly, in that any one of them is sufficient to provide significant biological activity. However, it cannot be ruled out that the overexpression system used might have masked potential minor qualitative differences and therefore exaggerated the redundancy. Similarly, two of the phosphorylated tyrosines showed no functional relevance in this or previous studies. One is the tyrosine of a PI3K consensus site at position 486 for which there was no requirement in any of the assays, employing either full-length Dof or mutant forms retaining only the first 600 amino acids. The other identified phosphotyrosine site without an identified function is located at position 592. Though it is conserved in Anopheles Dof, including several surrounding amino acids, the mutation of this tyrosine alone or in combination with other tyrosines did not affect the biological function of Dof. Again, perhaps subtle effects of the loss of these sites might have been missed. Nevertheless, the presence of several copies of docking sites for downstream signaling molecules and the availability of alternative routes to activate the same signaling cascade may provide Dof with options for fine-tuning of signaling strength and duration (Csiszar, 2010).
Csw has previously been shown to interact with Dof. The phosphotyrosine of the Csw consensus site was required for efficient interaction and for MAPK activation in the context of a Dof construct that lacked any of the other phosphorylation sites. This study shows that the Csw site is indeed important only if other parts of Dof with MAPK activating capacity are deleted or mutated. The same is true for the phosphorylation sites in the C-terminal domain, which this study shows to be the consensus Grb2 binding sites (Csiszar, 2010).
The four novel phosphotyrosine motifs contributed to the activation of the MAPK cascade, although they were essential only if other parts of the molecule with MAPK activation capability were deleted or mutated. Phosphorylation of these tyrosines was essential for the efficient interaction of Dof with the protein kinase Src64B, and Src activity was in turn required for Dof-dependent MAPK activation in S2 cells. Src kinases can activate mitogenic signaling in many different ways. Recently, Drosophila Src64B has been shown to be essential in the regulation of Raf activity by phosphorylating a regulatory tyrosine residue in Raf, which is also conserved in mammalian B-Raf. Thus, it is reasonable to postulate that upon FGFR-dependent phosphorylation the novel tyrosine motif in Dof is utilized to recruit Src64B, which can then contribute to MAPK activation via Raf activation (Csiszar, 2010).
In addition to Src64B, a tyrosine phosphorylated protein of 29 kDa (p29) was found that coprecipitated with Dof802 and required the phosphorylated tyrosines of the novel motif for this interaction. This raises the possibility that other proteins might use these sites as docking surfaces as well, although no evidence was found that this protein directly binds these phosphosites (Csiszar, 2010).
Since the residues surrounding the tyrosines in the novel motifs are conserved, it was expected that they would be found to be important for function. However, while mutating the tyrosines had measurable effects on the function of Dof in vivo, replacing the prolines had only moderate or no effects in the same assays. Similarly, Src64B binding to Dof802 in S2 cells was strongly reduced when the tyrosines of the novel motifs were mutated (in the background of mutated Csw and PI3K sites) but not when the prolines were mutated (Csiszar, 2010).
The finding that the impact of tyrosine mutations in the novel motifs on Dof function was greater than that of the proline mutations agrees with the known general characteristics of the interaction of phosphotyrosine motifs with phosphotyrosine binding domains. The driving force of these interactions is the phosphorylated tyrosine itself, with additional lower-affinity interactions of surrounding residues contributing to specificity for the different phosphotyrosine binding domains, as has also been found for dissociation constants when measuring interactions of SH2 domains in phosphopeptide library interaction screens. Indeed, it has been proposed that the modest selectivity of SH2 domains to phosphotyrosine containing linear peptides (5- to 20-fold) is not sufficient to explain selectivity of signaling pathways in living cells. Recent work has identified additional components of these type of interactions and shows that the selectivity of phospholipase Cγ binding and signaling via activated FGFR-1 are determined by interactions between a secondary binding site on an SH2 domain and a region in the FGFR-1 kinase domain in a phosphorylation-independent manner. These data suggest that the mutation of two amino acids in a tyrosine motif might have only mild consequences compared to the loss of the phosphotyrosine site in the context of whole protein-protein interactions, based on the complexity of different binding interfaces and their different affinities of this interaction (Csiszar, 2010).
Since SH2 domains preferentially interact with residues C-terminal to the tyrosine, and these are the conserved residues in the novel motif, the motif is expected to interact with SH2-type domains. Why has the motif described in this study not been found in the extensive searches for SH2 target motifs? The answer may lie in the fact that no motifs with important conserved amino acids at positions +4 and +8 are known at all, and this may be primarily because of the way they have been screened for. The degenerate phosphotyrosine peptide libraries that have been used to determine SH2 domain specificities screened only positions +1 to +3, and the furthest that other studies have gone was up to position +5 (Csiszar, 2010).
It is not known if the SH2 domain of Src64B is involved in the interaction with the novel phosphomotif of Dof, but if so, it is not clear whether this motif could be accommodated by the same interaction surface as the one that binds to the consensus Src SH2 domain recognition sequence pYEEI, which has been defined by phosphopeptide library screening and structural studies on peptide-bound Src SH2 domains (Csiszar, 2010).
Finally, little is known about the interaction of Src family kinases with other vertebrate signaling molecules bearing the novel tyrosine motif. For example, the motif in Shc is a phosphorylation target of Src, but no interaction studies have been performed, and there are conflicting reports about direct interaction between FGFR-1 and Src. The results of this study suggest that Src might be a good candidate for interacting with mammalian FGFR-1 and other vertebrate signaling molecules via the novel motif. It should be worth probing the general validity of Src binding to this novel phosphotyrosine motif in the future (Csiszar, 2010).
Protein O-GlcNAcylation is required for fibroblast growth factor signaling in Drosophila
Glycosylation is essential for growth factor signaling through N-glycosylation of ligands and receptors and the biosynthesis of proteoglycans as co-receptors. This study shows that protein O-GlcNAcylation is crucial for fibroblast growth factor (FGF) signaling in Drosophila. nesthocker (nst) encodes a phosphoacetylglucosamine mutase; nst mutant embryos exhibited low amounts of intracellular uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc), which disrupted protein O-GlcNAcylation. Nst is required for mitogen-activated protein kinase (MAPK) signaling downstream of FGF but not MAPK signaling activated by epidermal growth factor. nst was dispensable for the function of the FGF ligands and the FGF receptor's extracellular domain but was essential in the signal-receiving cells downstream of the FGF receptor. The adaptor protein Downstream of FGF receptor (Dof), which interacts with the FGF receptor, was identified as the relevant target for O-GlcNAcylation in the FGF pathway, suggesting that protein O-GlcNAcylation of the activated receptor complex is essential for FGF signal transduction (Mariappa, 2011).