Synergistic activities of multiple phosphotyrosine residues mediate full signaling from the Drosophila Torso receptor tyrosine kinase.
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.
Gayko
U
U
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Cleghon
V
V
Copeland
T
T
Morrison
D K
DK
Perrimon
N
N
D000818:Animals; D030801:Animals, Genetically Modified; D004330:Drosophila / Q000196:embryology / Q000235:genetics*; D029721:Drosophila Proteins; D047108:Embryonic Development; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000235:genetics / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D013329:Structure-Activity Relationship
D029721:Drosophila Proteins; D019476:Insect Proteins; D010748:Phosphopeptides; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D020794:Receptor Protein-Tyrosine Kinases; C080148:tor protein, Drosophila