Conserved motifs surrounding phosphotyrosines in Dof and other signaling molecules. (A) Protein sequence alignment of the C-terminal half of Drosophila and Anopheles Dof (DmDof and AgDof; UniProtKB accession numbers O96757 and Q8T5J9). Identified tyrosine phosphorylation sites in Drosophila Dof are labeled with a yellow P. Known consensus tyrosine motifs are underlined and shaded gray. Positions of predicted novel tyrosine motifs are shaded yellow and numbered 1 to 4. Note that one consensus tyrosine motif is embedded within one of the novel tyrosine motifs. (B) Alignment of the four novel tyrosine motifs from the two insect Dof sequences. Numbering of the motifs is as in panel A. Residues conserved in all motifs are shaded in green. Four or more identical or similar residues shared by the sequences are shaded black or gray, respectively. The consensus sequence is listed below the alignment. (C) Alignment of the novel tyrosine motifs from Dof, hBANK, and other conserved signaling molecules. Conserved residues are shaded green. Known consensus tyrosine motifs are underlined, and tyrosines known as phosphorylation target sites are marked in bold.

Requirement of phosphotyrosines for MAPK activation. The capacity of mutant Dof constructs to activate the MAPK pathway was tested by expressing them in the epidermis under the control of the pannier Gal4 driver together with activated Btl. The expression was monitored using anti-Dof antibody (right panels, red signal). Activation of the MAPK pathway was visualized using anti-dpErk antibody (green signal). Confocal projections are shown for embryos expressing the indicated transgenes (B and B′, full length [FL]; C and C′, to F and F′, various Y-to-F mutations as indicated by the cartoons below each panel) together with UAS-λBtl. Panel A shows a control embryo expressing UAS-λBtl alone. The green staining in this embryo shows the endogenous activation of MAPK induced by Heartless in the pericardial precursor cells and by DER in several muscle and ectodermal muscle-attachment cells. The weak red staining indicates the endogenous expression of Dof in the tracheal system.

Phosphotyrosine-dependent interaction of Dof with Src64B. (A) The relevance of Src kinase activity in FGFR-dependent MAPK activation was tested in S2 cells. FLAG epitope-tagged Dof was expressed together with an activated form of HA-tagged Htl (λHtl-2xHA) in the presence or absence of the Src kinase inhibitor PP2. MAPK activation was detected by Western blotting using an antibody against the diphosphorylated form of Erk. (B) The involvement of Src kinases in Dof phosphorylation was monitored in S2 cells. FLAG-Dof was expressed alone or together with λHtl-2xHA in the presence or absence of tyrosine phosphatase and Src kinase inhibitors pervanadate and PP2. The phosphorylation state of the Dof protein was determined by a phosphotyrosine antibody after immunoprecipitation (IP) from whole-cell lysates using a polyclonal Dof antibody. The asterisk marks the tyrosine-phosphorylated λHtl-2xHA coprecipitating with FLAG-Dof. Note that the apparent molecular weights for the Dof constructs appear not to be identical, although the constructs are the same in all lanes. We assume this is due to phosphorylation, as the migration behavior in the gel correlates with the phosphorylation state. W, West- ern blot analysis. (C) The ability of Src64B to interact with Dof and the requirement of phosphorylated tyrosines and novel tyrosine motifs in Dof for such an interaction were tested in S2 cells. Dof mutants 802, 802^[PI3K,csw], 802^{[PI3K,csw,613F,629F]}, 802^{[PI3K,csw,613AA,629AA]}, and 802^{[PI3K,csw,597F]} were expressed together with λHtl-2xHA and a V5 epitope-tagged form of Src64B (Src64B-V5). Dof protein complexes were immunoprecipitated from whole-cell lysates using an antibody against Dof and the precipitated proteins were detected by Western blotting using antibodies directed against FLAG or V5. Expression levels of Src64B-V5 and λHtl-2xHA were controlled by Western analysis of whole-cell lysates using antibodies against the V5 and HA epitope tags.

Mapping of phosphorylated tyrosines in Dof. (A) Schematic representation of the Dof protein. Two gray boxes and one oval represent the Dof-BCAP-BANK (DBB) domain, the two ankyrin repeats (Ank), and the coiled coil region. All tyrosines are marked in color; red highlights the tyrosines located within consensus motifs for binding sites for the indicated signaling molecules. The amino acid positions of these tyrosines are indicated on top of the scheme. Tyrosines that do not form part of a known consensus motif are marked in blue. Breakpoints of the Dof deletion constructs used or cited in this study—after amino acid positions 446, 522, and 802—are marked with dashed lines. (B to D) FLAG epitope-tagged mutant Dof constructs were expressed together with an activated form of HA-tagged Heartless (λHtl-2xHA) in S2 cells. FL, full-length Dof; 802 and 522, Dof constructs truncated after amino acid positions 802 and 522, respectively. Protein complexes were immunoprecipitated (IP) from whole-cell lysates using an antibody against the FLAG epitope tag of the Dof constructs. The precipitated FLAG-Dof and the coprecipitating λHtl-2xHA proteins were detected on Western blots (W) using antibodies directed against FLAG and HA. In addition to a protein of approximately the size predicted for each of the mutant FLAG-Dof constructs, for the FL and 802 Dof proteins the FLAG antibody also detects a smaller fragment of about 75 kDa representing an N-terminal cleavage product of Dof (see also reference 41). This additional protein band is absent in 522 constructs, because the cleavage site lies C-terminal to the position of truncation. The phosphorylation state of mutant Dof proteins was determined by a phosphotyrosine antibody. This antibody also recognizes the tyrosine-phosphorylated λHtl-2xHA construct, which interacts with Dof and therefore coprecipitates; these bands are marked with a black arrowhead. The asterisk marks the IgG heavy chain of the FLAG antibody. (B) FLAG-Dof constructs containing the first 522 amino acids of Dof with combinations of the Y to F point mutations at positions 97, 486, and 515 were coexpressed with λHtl-2xHA. (C) FLAG-Dof constructs containing the first 802 amino acids with combinations of Y to F point mutations at positions 486, 515, 592, 613, and 629 were coexpressed with λHtl-2xHA. As a positive control for phosphorylation, a Dof construct containing amino acids 1 to 802 without further mutations was used. (D) Full-length FLAG-Dof constructs with combinations of Y to F point mutations at positions 486, 515, 597, 613, and 629 (FL^Y5F, all of these five tyrosines mutated) were coexpressed with λHtl-2xHA.

Functional redundancy of the novel and Csw-binding phosphotyrosines in Dof. Mutant Dof constructs were tested for their ability to rescue defects in the development of the mesoderm and the tracheae in dof mutant embryos. Anti-Even-skipped antibody (blue signal) was used to visualize the FGF-dependent pericardial precursor cells (PPCs) in the mesoderm of stage 11 embryos (B to G). Anti-2A12 antibody (brown signal) was used to detect the tubular network of the tracheae (H to M). The UAS-Dof transgenes were expressed under the control of twistGal4 (B to G) or breathlessGal4 (H to M). Homozygous mutant embryos were identified by the lack of lacZ-marked Balancer chromosomes. Quantification was performed by counting the number of PPCs and dorsal trunk fusions on both sides of 10 to 15 mutant embryos for each experiment. (A) Summary of the rescue experiments. The left panel shows a schematic representation of mutant Dof constructs used in the assay. Bars in the right panels represent the average number of Even-skipped positive (Eve-positive) clusters in the mesoderm (middle panel) and of dorsal trunk fusions (right panel) ± standard errors of the means. Two independent transgenic fly lines were analyzed for most UAS-Dof constructs. (B to M) Representative examples of antibody-stained embryos from the above rescue experiments.

Additional level of redundancy in the C-terminal tail: functional relevance of Grb2 binding and tyrosines of the novel motifs in Dof. Mutant full-length (FL) Dof constructs were tested for their ability to rescue defects in the development of the mesoderm and the tracheae in dof mutant embryos. Grb2F, amino acid exchange of the tyrosines of all four consensus Grb2 recognition sites at positions Y914, Y930, Y988, and Y1009 to phenylalanines. 844AA and 891AA, an amino acid exchange of the two conserved prolines to alanines in the two novel motifs with tyrosine positions 844 and 891. #, reasonable rescue of dorsal and lateral tracheal branches despite a weak rescue of dorsal trunk fusions. For further details, see Fig. 3.