Sprouty proteins are targeted to membrane ruffles upon growth factor receptor tyrosine kinase activation - Identification of a novel translocation domain

Sprouty (Spry) was first identified in a genetic screen in Drosophila to be an antagonist of fibroblast growth factor and epidermal growth factor (EGF ) signaling, seemingly by inhibiting the Ras/MAP kinase pathway. Data base searches lead to the identification and cloning of, to date, four mammalian sprouty genes. The primary sequences of the mammalian sprouty gene product s share a well conserved cysteine-rich C-terminal domain with the Drosophil a protein, The N-terminal regions, however, do not exhibit significant homo logy, This study aimed at determining the disposition of Spry proteins in i ntact cells before and after stimulation of the EGF receptor tyrosine kinas e, Full-length or deletion mutants of Spry, tagged at the N termini with th e FLAG-epitope, were expressed in COS-1 cells by transient transfection and analyzed by immunofluorescence microscopy before and after EGF stimulation of the cells. In unstimulated cells, the Spry proteins were distributed th roughout the cytosol except for human Sprouty2 (hSpry2), which, although ge nerally located in the cytosol, co-localized with microtubules. In all case s, the Spry proteins underwent rapid translocation to membrane ruffles foll owing EGF stimulation. The optimal translocation domain was identified by d eletion and immunofluorescence analysis to be a highly conserved 105-amino acid domain in the C-terminal half of the hSpry2 protein. The translocation of this conserved domain, based on hSpry2 data, was independent of the act ivation of phosphatidylinositol-3 kinase.