Synergistic activities of multiple phosphotyrosine residues mediate full signaling from the Drosophila Torso receptor tyrosine kinase

Abstract

Here, we identify four tyrosine residues (Y644, Y698, Y767, and Y772) that become phosphorylated after activation of the Torso (Tor) receptor tyrosine kinase. Previously, we characterized phosphotyrosine sites (P-Y630 and P-Y918). Of the six P-Y sites identified, three (Y630, Y644, and Y698) are located in the kinase domain insert region, one (Y918) is located in the C-terminal tail region, and two (Y767 and Y772) are located in the activation loop of the kinase domain. To investigate the function of each P-Y residue in Tor signaling, we have generated transgenic Drosophila embryos expressing mutant Tor receptors containing either single or multiple tyrosine to phenylalanine substitutions. Single P-Y mutations were found to have either positive, negative, or no effect on the signaling activity of the receptor. Elimination of all P-Y sites within the kinase insert region resulted in the complete loss of receptor function, indicating that some combination of these sites is necessary for Tor signaling. Mutation of the C-terminal P-Y918 site revealed that this site is responsible for negative signaling or down-regulation of receptor activity. Mutation of the P-Y sites in the kinase domain activation loop demonstrated that these sites are essential for enzymatic activity. Our analysis provides a detailed in vivo example of the extent of cooperativity between P-Y residues in transducing the signal received by a receptor tyrosine kinase and in vivo data demonstrating the function of P-Y residues in the activation loop of the kinase domain.

Figure 1

Identification of Tor autophosphorylation sites. (A and B) In vitro phosphorylated Tor^WT and Tor^Delta-Insert were separated by reverse phase HPLC, and fractions were collected. The relative amount of ³²P radioactivity in each fraction is shown. Peaks 1 + 1a (C), peak 3 (D), and peak 5 (E) were subjected to sequencing. A summary of the phosphotyrosine analysis is shown in F.

Figure 2

Effect of Tor tyrosine mutations on embryonic development. The effect of the tor P-Y mutations on the expression of tll (A1, B1, C1, D1, and E1) and hkb (A2, B2, C2, D2, and E2) and cuticle phenotypes (A3, B3, C3, D3, and E3) are shown. The mutations shown are: Tor^Y630F (A), Tor^Y644F (B), Tor^Y698F (C), Tor^Quadruple (D), and Tor^Y767+772F (E). The effect of these mutations was analyzed in the tor^XR1 (Tor protein null) mutant background. The domain of tll and hkb expression are indicated as percent egg length, with 0% corresponding to the posterior pole. Tor^Y630F shows a partial lof phenotype; Tor^Y644F, Tor^Y656F, and Tor^Y698F show a WT phenotype; and the Tor^Quadruple and Tor^Y767+772F show a lof phenotype.

Figure 3

Expression and tyrosine phosphorylation of mutant Tor proteins. Lysates were prepared from embryos expressing various Tor mutant proteins. Tor proteins were immunoprecipitated, and the precipitates were examined by immunoblot analysis using antibodies recognizing Tor (αTor). Blots then were stripped and reprobed with antibodies recognizing phosphotyrosine (αP-Y). The position of Tor and P-Y Tor is indicated on the right by an arrow.

Figure 4

Tor tyrosine phosphorylation and its function in mediating the developmental signal to downstream molecules. See text for details.