Genetic Identification of Phosphotyrosine Specific Drosophila Effectors of Neu/Erb-B2 Signaling

Abstract

The Drosophila Epidermal Growth Factor Receptor (DEgfr) is the only fly orthologue of the vertebrate Neu/ErbB2 receptor tyrosine kinase (RTK) family. In Drosophila, DEgfr signaling is required in the developing wing discs, and for the determination and differentiation of wing veins. Expression of constitutively active rat Neu/ErbB2 transgenes, each having a single phosphotyrosine (pTyr) residue in the adaptor domain, generates cellular responses in Drosophila consistent with the activation of signaling cascades employed by intrinsic DEgfr. We have performed extensive genetic screening to identify adaptor and second messenger pathways that are activated by individually reconstituted pTyr, by determining which signaling gene mutants alter neu wing phenotypes. In addition, we have screened for genetic deletions on the second and third chromosome that enhance or suppress the wing phenotypes generated by the neu add-back alleles. This approach has enabled us to identify several signaling proteins that differentially affect the Neu signaling pathway via association with specific pTyr residues. We have also identified 41 genomic regions in Drosophila, which modify signals from either individual or multiple neu add-back alleles. As Neu signaling appears to function in a manner similar to the DEgfr, we sought to determine whether these receptors were capable of heterodimerizing. In order to examine this we co-expressed a kinase inactive version of the neu^YD allele with activated DEgfr. Co-expression of these receptors suggested that the DEgfr was unable to dimerize with, and transphosphorylate, the YD pTyr on Neu, as no potent anti-apoptotic phenotype was detected. Additionally, co-expression of activated neu and the kinase inactive version of neu^YD resulted in a decrease in glial cell numbers, in relation to mis-expression of the activated neu allele alone. These findings suggest that Neu does not interact with the DEgfr, but rather functions via homodimerization of it's receptor subunits.