Sequence analysis of proteins related to Dof
When Dof was initially characterized, the sequence of the protein did not resemble any other in the database. Two vertebrate proteins that function in B-cell signaling, BCAP and BANK, which share structural domains with Dof, have since been described (30
). These proteins possess a C-terminal coiled coil and have a high degree of similarity to Dof over the ankyrin repeats. Mouse BCAP shows 47% identity over 55 amino acids with this region of Dof, while BANK shows 43% identity. Interestingly, the similarity among Dof, BCAP, and BANK extends beyond the ankyrin repeats toward the N terminus. Mouse BCAP and human BANK show 19 and 17% identity, respectively, with amino acids 216 to 353 of Dof (Fig. ). We have designated this the Dof, BCAP, and BANK (DBB) region.
FIG.1. Sequence analysis of Dof and related proteins. (A) Aligment over the DBB motif and the ankyrin repeats of Drosophila Dof (accession number O96757), Anopheles Dof (accession number Q8T5J9), mouse BCAP (accession number Q9EQ32), chicken BCAP (accession (more ...)
The completion of the Anopheles gambiae
genome has revealed a homolog of Dof (accession number Q8T5J9
) that shows 37% identity and 50% similarity over 924 amino acids of the Drosophila
protein (Fig. ). A number of expressed sequence tags derived from this gene, such as AJ285928 and AJ281683, indicate that it is expressed. The degree of identity shared by the Drosophila
Dof homologs is considerably lower than 56%, which is the average degree of conservation between clear Drosophila
), so dof
may represent an example of a fast-evolving gene. However, the regions of Dof that are similar to the vertebrate proteins BCAP and BANK are also among the most highly conserved parts of the Anopheles
Dof homologs (Fig. ; see also Table S1 in the supplemental material) and have not been interrupted by amino acid insertions during evolution. Interestingly, the sequence between the DBB motif and ankyrin repeats diverges (Fig. ), suggesting that the DBB region may represent an independent domain of the protein. Within the C-terminal half, the coiled coil shows the highest degree of similarity and is the most extensive block of conservation. Notably, the coiled-coil motif of Dof also shows 48% identity and 84% similarity with the hypothetical open reading frame of a partial Bombyx
cDNA (accession number AU000659
), suggesting that the motif may be important functionally. Thus, the sequences and organizations of three distinct domains of Dof appear to have been conserved among the homologs in insects and the most closely related vertebrate proteins. Furthermore, these domains are the regions with the highest degree of conservation among the vertebrate BCAP proteins (see Table S1 in the supplemental material) and as such define the general structure and features of this family of proteins.
In vitro mutagenesis of Dof
To determine if specific domains of Dof mediate different cellular functions, we generated by in vitro mutagenesis a series of mutants of the protein encoded by transcript II of dof
. We chose to analyze the protein encoded by this transcript, which will be referred to as Dof below, since it has been shown that it can rescue the defects in the development of the tracheae and mesoderm of homozygous dof
mutant embryos (39
). To assay the in vivo functions of the mutant Dof proteins, we used the Gal4 upstream activation sequence system (7
) to express transgenes encoding the mutant protein in wild-type and homozygous dof
Characterization of the functions of the mutant Dof proteins in vivo
FGF receptor-dependent signal transduction regulates transcriptional activity and morphogenesis within the tracheae and the mesoderm. We tested Dof mutant constructs for FGF-dependent morphogenesis by examining the tracheae of homozygous dof mutant embryos expressing the mutant forms of Dof under the control of btl-Gal4. To determine their abilities to activate transcription of FGF-dependent genes, we monitored the expression of Even-skipped in the mesoderm. In this case, the constructs were expressed in dof mutant embryos under the control of twi-Gal4.
Differences in the behaviors of the mutant Dof proteins in these biological assays may not simply reflect differences in the functions of the proteins but could also reflect differences in the protein stabilities. We assessed whether the mutant Dof transgenes could produce stable proteins by expressing FLAG-tagged versions in Schneider S2 cells in tissue culture. Western blots probed with antibodies against the FLAG tag showed that in this system, all mutant proteins were expressed. They showed only slight variations in abundance, except for Dof[1-522], which was significantly more abundant than the full-length form, and Dof[168-1012], which appeared to be much less stable (see Fig. S1 in the supplemental data). When the proteins were expressed ectopically in the ectoderm of embryos by using prd-Gal4 and were visualized with an antiserum directed against Dof, we were able to detect expression of all mutant proteins (see below for more details), with the exception of the large C-terminal truncations (Dof[1-277] and Dof[1-446]). The fact that Dof[1-446] can be immunoprecipitated from Schneider S2 cells but is undetectable in formaldehyde-fixed embryos using the Dof antibody suggests that formaldehyde destroys the epitopes remaining in this part of the molecule. Furthermore, in yeast cells, the truncation Dof[1-446] fused to the Gal4 DNA binding domain must be stable, since it can interact with the kinase domain of Htl in the yeast two-hybrid assay (see below). Thus, we included these transgenes in further functional tests.
The DBB domain is necessary for the function of Dof
In homozygous dof
mutant embryos, differentiation of the lumen of the tracheae can be recognized using the antibody 2A12, as shown in Fig. . In comparison to the wild type (Fig. ), the tubular structures lack connections to one another; in particular, the dorsal and lateral trunks are absent and the prominent visceral branches do not grow out normally. The morphogenesis of the tracheae in dof
mutants can be completely rescued by expression of Dof (38
), and this is not compromised by the addition of a FLAG epitope tag to the amino terminus of the protein (Fig. ).
FIG. 2. Abilities of different mutant Dof proteins to rescue defects in the development of the mesoderm and tracheae. (A to K) Tubular network of the tracheae, revealed using the monoclonal antibody 2A12, is shown in brown. Even-skipped, visualized as a blue (more ...)
A truncated form of Dof that lacks the last 210 amino acids (Dof[1-802]) (Fig. ) is able to rescue morphogenesis of the tracheae completely. This was consistently observed with independently derived transgenic lines, suggesting that these C-terminal amino acids are not essential for this function in vivo. With transgenes encoding shorter proteins lacking the last 338 amino acids, or the last 481 amino acids, and which do not contain the coiled-coil motif, we still observed a partial rescue of the dof tracheal phenotype that was variable between embryos (Fig. and ). The formation of the dorsal trunk was least efficiently rescued by the transgenes, particularly towards the anterior of the embryo. We used this feature as a semiquantitative measure to compare different transgenic constructs (Fig. ). In a few cases, complete rescue was observed with these constructs, indicating that the last 481 amino acids, which include the coiled coil, are not absolutely essential for the transmission of the signal from the FGF receptor. In contrast, transgenes encoding proteins with more extensive C-terminal deletions (data summarized in Fig. ) had no activity in this assay.
FIG. 3. Functions of mutant Dof proteins in in vivo assays. The Dof constructs are shown schematically on the left. The amino acids present in each construct are noted. A flag epitope tag is represented by a red circle, while the membrane-targeting SH4 domain (more ...)
Dof constructs lacking the first 88 or 168 amino acids at the N terminus partially rescued development of the tracheae (Fig. ). With both of these N-terminal truncations, the degree of rescue was more variable than with the C-terminal truncations with biological activity. Surprisingly, rescue with Dof[89-1012] was worse than with Dof[168-1012]. However, the breakpoint at amino acid 89 appears to have a nonspecific deleterious effect on the protein, since we have also observed that the 89-to-1012 truncation has less activity than the 168-to-1012 truncation in yeast two-hybrid experiments irrespective of the interaction being tested (A. Battersby, unpublished results). Notably, the N-terminal amino acids deleted in these mutants are not absolutely essential for signal transmission. However, further deletions, extending into the DBB motif (up to amino acid 227 or 277), resulted in complete failure of the constructs to rescue tracheal morphogenesis (data summarized in Fig. ).
When we examined the activities of proteins with combined amino- and carboxy-terminal truncations, namely, Dof[89-802] and Dof[168-802], we found that they had no activity in the assays. Since neither of these deletions alone abolished the activity of the protein, this indicates that the amino and carboxy termini of the protein could have overlapping functions. None of the mutant Dof transgenes in these experiments had a dominant-negative effect on the function of the wild-type protein in the heterozygous sibling embryos.
The experiments described above suggested that the region of Dof containing the ankyrin repeats and the DBB motif, which has the most similarity to the vertebrate proteins BCAP and BANK, may be important for the function of the protein. To investigate the importance of these domains, we examined the activities of a number of transgenes with internal deletions. A protein that lacked the DBB motif and the ankyrin repeats, flag-Dof[Δ233-449], was severely compromised in its ability to support tracheal morphogenesis (Fig. ). Formation of a normal dorsal trunk was never observed in these embryos; however, occasional misdirected outgrowth of visceral branches and partial formation of the lateral trunk distinguished them from homozygous dof mutant embryos. A transgene in which just the ankyrin repeats were deleted could fully rescue tracheal morphogenesis (Fig. ). Thus, surprisingly, even though the ankyrin repeats are the most highly conserved part of the protein, they are not essential for transmission of the signal from the FGF receptor in this assay. By contrast, deletion of just the DBB region had the same phenotype as the larger deletion (Fig. ), suggesting that the DBB domain provides a critical function of Dof.
We also assayed the constructs for the ability to participate in FGF signaling in the mesoderm by expressing them in homozygous dof
mutant embryos and determining the number of Even-skipped-expressing cell clusters. In wild-type embryos, Even-skipped is expressed in the dorsal mesoderm in clusters comprised of two or three cells, which include the pericardial cells (2
). Mutations in the genes encoding the FGF receptor Htl (5
) and Dof (20
) result in the loss of these Even-skipped-positive cells. The activities of the Dof mutants in this assay were similar to those in the tracheae (summarized in Fig. ), although the degree of rescue of Even-skipped-positive cells with the N-terminally truncated forms of Dof was more variable than that observed in the tracheal assay, and the activity of flag-Dof[1-674] in this assay was poor in comparison with that in the tracheal assay. Thus, in summary, for any Dof mutant that was capable of rescuing tracheal morphogenesis, we could also detect activation of Even-skipped within the mesoderm. The mutants we have analyzed have allowed us to identify a domain within the N-terminal half of the protein that is critical for efficient transmission of the FGF signal in both situations.
Subcellular localization of the mutant Dof proteins
We examined whether the truncations and deletions within Dof altered the subcellular localization of the protein. The wild-type protein is observed in a punctate pattern within the cytoplasm of mesodermal, glial, and tracheal cells and is slightly enriched at the cell cortex (39
). An identical or similar subcellular distribution was also observed for full-length Dof with an N-terminal FLAG epitope tag upon expression of the protein in the ectoderm with prd
-Gal4 and most of the mutant Dof proteins (Fig. ). However, both flag-Dof[277-1012] and flag-Dof[Δ233-449] (Fig. ) were distributed in a punctate fashion throughout these cells, whereas flag-Dof[Δ361-449] (Fig. ) appeared to be more tightly associated with the cell cortex than the wild-type protein and comparable in distribution to full-length Dof protein fused to the C terminus of the SH4 domain of human Src (Fig. ). Surprisingly, flag-Dof[Δ233-364] appeared to be enriched within nuclei and only present at low levels in the cytoplasm (Fig. ; for double labeling with markers for the nuclear envelope and the plasma membrane, see Fig. S2 in the supplemental material).
FIG. 4. Subcellular localization of mutant Dof proteins and influence of Dof on localization of ectopically expressed Htl. (A to L) The stabilities and subcellular localization of mutant forms of Dof were assessed using a prd-Gal4 transgene to express each construct (more ...)
There are several molecules involved in signal transduction, such as Corkscrew, the Drosophila SHP-2 homolog, and Raf, which become active when recruited to the plasma membrane. To investigate whether high levels of Dof at the plasma membrane are sufficient to rescue the defects observed in homozygous dof mutant embryos, we expressed variants of Dof with an N-terminal SH4 epitope tag. When targeted to the plasma membrane in this way (Fig. ), both the full-length Dof protein and the C-terminal truncation lacking the last 210 amino acids gave a good rescue (Fig. and ). However, we found that targeting Dof to the membrane was not sufficient to induce signaling independent of an active receptor, since the SH4-Dof fusion proteins did not rescue homozygous btl-dof double-mutant embryos, which lack both the FGF receptor Btl and Dof. Similar results were also obtained with Torso4021-Dof chimeras (Fig. ).
Interaction of Dof with the FGF receptors
In a yeast two-hybrid screen, in which we used Dof[1-802] as bait (4
), we found a number of proteins that interact with Dof, including the FGF receptor Heartless (Fig. ). Dof[1-802] can interact with a fragment covering most of the intracellular domain of Heartless, as well as a fragment lacking most of the smaller lobe of the kinase domain. We confirmed the interaction by coimmunoprecipitation from lysates of Schneider S2 cells expressing Dof and the activated FGF receptor (Fig. ).
FIG. 5. Interaction of the FGF receptor Heartless with mutant forms of Dof. (A) Diagram of Heartless (accession number Q07407) showing the transmembrane domain and the ATP binding and autophosphorylation sites. The two blue bars represent the longest and shortest (more ...)
We used the yeast two-hybrid assay to investigate which regions of Dof are required for the interaction with Heartless. When we tested the Dof deletion constructs in this assay, we found that the first 446 or the last 844 amino acids of Dof were able to interact with both fragments of Htl (Fig. ). However, the constructs with more extensive N-terminal deletions or constructs lacking the DBB motif were unable to interact with the long form of Htl, and their interaction with the short form of Htl was significantly reduced. This resembled results from coimmunoprecipitation of the Dof deletion constructs with the activated receptor from Schneider S2 cell lysates (Fig. ). Deletions that did not affect the DBB region had minimal effects on the interaction (note that even Dof[168-1012], although expressed at very low levels, is able to coprecipitate the receptor, albeit at similarly low levels). By contrast, constructs lacking the DBB region alone or in combination with the ankyrin repeat showed strongly reduced or even abolished binding, with a certain degree of experimental variation.
The long fragment of Htl interacts with the C-terminally truncated DB-Dof[1-802] under both low- and high-stringency selection conditions. Surprisingly, the full-length form of Dof could interact only under the less stringent selection conditions (Fig. ), indicating that the C terminus of Dof can weaken the interaction of Dof with Heartless (4
Thus, in summary, the integrity of the region that lies between amino acids 168 and 364, which spans the DBB domain, is critical for the efficient interaction of Dof with the cytoplasmic domain of an FGF receptor in Drosophila. Whether the ability of the DBB deletion mutants to interact weakly with the activated receptor in Schneider S2 cells and the large lobe of the kinase domain in the yeast assay, but not with the whole kinase domain in yeast, is a reflection of a difference in affinity for the activated and nonactivated kinase is an interesting problem that remains to be elucidated.
We investigated whether Dof influences the distribution of Htl protein expressed ectopically in the ectodermal cells. In the absence of Dof, we observed Htl largely in a punctate pattern at the membranes of these cells (Fig. ), and coexpression of Dof did not change this distribution significantly (Fig. ), nor did the presence of the FGF receptor have any gross effect upon the distribution of Dof in these cells (compare Fig. ). Thus, the subcellular localizations of Htl and Dof are independent of each other and are not dependent upon an interaction between the proteins.
Dof is phosphorylated by the FGF receptors
To investigate whether Dof could be phosphorylated in the presence of activated FGF receptors, we expressed the protein in Drosophila S2 cells and examined the effect of adding an activated form of the FGF receptor Btl. A phosphorylated protein with an apparent molecular mass of roughly 130 kDa was present specifically in cells expressing both the activated FGF receptor and Dof (Fig. , left) (the predicted molecular mass for Dof with the Flag tag is 112.8 kDa). We confirmed that this protein was indeed Dof by immunoprecipitation with an anti-Dof antiserum followed by Western blotting with an anti-phosphotyrosine antibody (Fig. , right). Thus, these experiments indicate that Dof is phosphorylated in the presence of an active FGF receptor, although not whether the phosphorylation is mediated directly by the FGF receptors.
FIG. 6. Dof phosphorylation in response to FGF receptor activation and the effect of specific tyrosine mutations on the function of Dof. (A) Drosophila Schneider S2 cell lines were stably transformed as indicated above each lane with different combinations of (more ...)
The FGF receptors require Dof to generate high levels of diphosphorylated MAP kinase (20
). The analysis of the truncated forms of Dof described above indicates that the last 210 amino acids are not essential for the function of the molecule. Thus, we focused our attention upon the potential binding sites for Drk (Grb2), PI 3-kinase, and Corkscrew (SHP2) that are present at positions 97, 486, and 515 of Dof, respectively (Fig. ).We constructed a number of transgenes with different combinations of mutations that result in the production of proteins containing phenylalanine residues in place of these tyrosines (Fig. ). When expressed in Schneider S2 cells, these constructs are stably expressed at levels comparable to those of the full-length wild-type protein (see Fig. S3 in the supplemental material). The level of phosphorylation of these constructs is reduced compared to the full-length wild-type protein, but even in Dof1-802(FFF), phosphorylation is not completely abolished (see Fig. S3 in the supplemental material). This suggests that tyrosines other than those that lie within recognizable consensus binding sites for known adapter proteins can also be phosphorylated. Comparison of the phosphorylation states of the deletion mutants (see Fig. S3 in the supplemental material) indicates that these tyrosines lie in the region between amino acids 522 and 802, which contains seven tyrosines.
In the tracheal assay, the mutation of individual sites had little effect on the activity of Dof, irrespective of whether the full-length or the truncated form was used (data summarized in Fig. and ). However, the combination of these mutations gave a less reliable rescue (Fig. ), and the mutation of all three tyrosines in a transgene that lacked the C terminus reduced the activity in this assay further (Fig. ). In the mesoderm assay, the single mutations of the potential Drk and PI 3-kinase binding sites also had little effect (Fig. ). However, constructs in which the potential Corkscrew binding site was mutated were compromised in the ability to rescue the expression of Even-skipped, and a transgene lacking the C terminus and with mutations in all three tyrosines had much less activity. Taken together, these results indicate that the most important of the potential phosphorylation sites is Y515, suggesting that one function of Dof may be to recruit Corkscrew/SHP-2 upon the activation of an FGF receptor.