Netrin stimulates tyrosine phosphorylation of the UNC-5 family of netrin receptors and induces Shp2 binding to the RCM cytodomain

Caenorhabditis elegans UNC-5 and its mammalian homologues such as RCM are r eceptors for the secreted axon guidance cue UNC-6/netrin and are required t o mediate the repulsive effects of UNC-6/netrin on growth cones. We find th at C. elegans UNC-5 and mouse RCM are phosphorylated on tyrosine in vivo. C . elegans UNC-5 tyrosine phosphorylation is reduced in unc-6 null mutants, and RCM tyrosine phosphorylation is induced by netrin-1 in transfected HEK- 293 cells, demonstrating that phosphorylation of UNC-5 proteins is enhanced by UNC-6/netrin stimulation in both worms and mammalian cells. An activate d Src tyrosine kinase induces phosphorylation of RCM at multiple cytoplasmi c tyrosine residues creating potential binding sites for cytoplasmic signal ing proteins. Indeed, the NH2-terminal SH2 domain of the Shp2 tyrosine phos phatase bound specifically to a Tyr(568) RCM phosphopeptide. Furthermore, S hp2 associated with RCM in a netrin-dependent manner in transfected cells, and co-immunoprecipitated with RCM from an embryonic mouse brain lysate. A Y568F mutant RCM receptor failed to bind Shp2 and was more highly phosphory lated on tyrosine than the wild type receptor. These results suggest that n etrin-stimulated phosphorylation of RCM Tyr(568) recruits Shp2 to the cell membrane where it can potentially modify RCM phosphorylation and function.