Fig. 5.
Nemo phosphorylates Eya at consensus MAPK sites.(A–C). In vitro kinase assays using Flag-Nmo immunopurified from transfected HEK293T cells (see Methods for details). The upper panels show Coommassie staining for total protein, and the bottom panels show the phosphorimager exposure. (A) Full-length Eya protein is directly phosphorylated when incubated with wild type, but not the kinase inactive form of Nmo (K69M). Nmo autocatalytic activity is evident with the wild type, but not kinase dead form. (B) Schematic of the Eya protein, with the two MAPK phosphorylation consensus sites within the N-terminal P/S/T region of Eya represented by asterisks. In vitro kinase assays show that Nmo phosphorylates the N-terminal region of Eya, but not the ED. Immunoblot (IB) shows inputs for Nmo, Eya and GST. (C) In vitro kinase assays using a subfragment of the P/S/T-rich region of Eya (GST-EYA320–424). Mutating the phosphoacceptor serines in the MAPK sites significantly reduces Nmo-mediated phosphorylation. (D) Immunoblots (IB) showing HA-Nmo and Flag-Eya can be co-immunoprecipitated (IP) from transfected S2 cells. The lysates (bottom panel) were run together on the same gel, but out of order with respect to the IP gels, necessitating some cutting and splicing to align the lanes.