Fig. 7. Mechanism of EGFR inhibition by Kek1. (A) Inhibition of EGF binding and EGF-stimulated receptor tyrosine phosphorylation by Kek1. Sf9 insect cells were infected with baculoviruses encoding either Kek1-Myc or EGFR, or co-infected with both viruses. Cells were treated without or with 30 nM EGF as indicated. For the [¹²⁵I]-labeled EGF crosslinking experiment (lower panel), trace levels (0.1 nM) of iodinated growth factor and 1 mM BS³ crosslinker were added to all samples at the time of EGF addition. Lysates from cells were immunoprecipitated with antibodies to either Myc epitope or to EGFR. Precipitates were exposed to autoradiography (lower panel), or were blotted with antibodies to phosphotyrosine (upper panel) or EGFR (middle panel). (B) Co-localization of Kek1 and EGFR at the cell surface. Sf9 insect cells were infected at a low multiplicity of infection with baculoviruses encoding Kek1-Myc and human EGFR. Cells were fixed and stained with both rabbit anti-EGFR (left panel) and mouse anti-Myc epitope (middle panel). Images were merged to show co-localization (right panel). (C,D) Kek1 can inhibit DER in trans. (C) 6% of the eggs (n=112) laid by females UASp-kek1/nos-Gal4 are weakly ventralized (partial or total fusion of the dorsal appendages). (D) Among the 64% of the ventralized eggs (n=96) laid by top/+; UASp-kek1/nos-Gal4 females, 8% are strongly ventralized.