Figure 3. Acidic Substitution of Y510 Promotes and Phenylalanine Substitution Impairs Activation of Full-Length Draf In Vitro and In Vivo.

(A) Draf^Y510E (lane 4) exhibited dramatically higher activities by in vitro kinase assay using bacterially expressed full-length Draf variants and GST-Dsor1. Dsor1 phosphorylation was detected by anti-pMEK. WT, wild type.

(B) Quantification of results represented in (A).

(C) V5-tagged full-length Draf variants were transfected into S2 cells and were immunoprecipitated with anti-V5 and subjected to kinase assay using bacterially expressed Dsor1 as substrate. Note that only Draf^Y510E exhibited prominent kinase activity toward purified Dsor1, as detected by anti-pMEK (lane 4).

(D) V5-tagged full-length Draf^WT or Draf^Y510E were transfected into S2 cells and were immunoprecipitated with anti-V5 and then subjected to kinase assay using bacterially expressed Dsor1 as substrate with or without purified GST-Src64B^act. Note that GST-Src64B^act stimulated the activity of Draf^WT to that of Draf^Y510E (cf. lanes 3 and 4).

(E–H) Effects of Y510 substitutions on Draf activity in vivo. (E and F) Injection of mRNA into early stage embryos. (i) Cuticles of a wild-type embryo exhibiting normal head skeleton (arrow), eight ventral denticle belts (numbered), and the Filzkörper (arrowhead). (ii) Cuticles from a buffer-injected (control) Draf ^−/− embryo exhibit characteristic Draf null phenotypes: collapsed head skeletons (arrow) and loss of all posterior structures (eighth denticle belt and the Filzkörper). (iii) A Draf null embryo rescued by injecting Draf^Y510E mRNA. Note the restored eighth denticle belt and the Filzkörper (arrowhead). Due to the limited diffusion of injected mRNA or different threshold requirement, the anterior (head skeleton; arrow) defects were not rescued, which serves as an internal control. The phenotypes of rescued torso null embryos are identical to Draf null embryos (unpublished data).

(F) Draf^Y510E expressed from injected mRNA exhibited higher basal and inducible activity than Draf^WT (p < 0.001) in vivo. Draf^Y510F had impaired inducible activity (p < 0.001). Basal Draf activity was measured by the percentage of rescue of posterior structures of torso null embryos by mRNA microinjection (shown in blue). Draf^Y510E rescued 9.5% of injected torso embryos, whereas no rescue was found for Draf^Y510F and Draf^WT. Inducible Draf activity was measured by the percentage of rescue of posterior structures of Draf null embryos by mRNA microinjection (shown in red). Draf^WT, Draf^Y510F, or Draf^Y510E rescued 52%, 9.2%, or 81% of Draf null embryos, respectively. The number of injected embryos scored is indicated. Chi-square tests were used to compare the differences, p-values are indicated.

(G and H) In vivo activities of Draf transgenes with different Y510 substitutions. (G) Virgin Draf^C110/FM7 females were mated with males with a recombinant chromosome carrying hsp70-Gal4 and an indicated UAS-Draf transgene. Surviving F1 Draf^C110/Y males were scored and normalized as percent viability relative to control crosses. Note that Draf^C110/Y males were not viable in the absence of UAS-Draf transgenes, but were rescued to different degrees by different Draf transgenes. (H) Females of Nanos-Gal4; torso^XR1; UAS-Draf variants were mated to wild-type males, and the F1 progeny were examined for viability and cuticle patterns. Note that expressing Draf^WT or Draf^Y510F did not rescue torso^XR1 phenotypes, whereas expressing Draf^Y510E fully rescued torso^XR1 female sterility (unpublished data) or restored the posterior cuticle structures.