Affinity labeling of the protein kinase associated with the epidermal growth factor receptor in membrane vesicles...

Epidermal growth factor (EGF), a mitogenic polypeptide hormone, stimulates the phosphorylation of certain endogenous proteins in membrane preparations derived from A431 cells, a human tumor cell line. Membrane vesicles prepared from A431 cells were reacted with 5'-p-fluorosulfonylbenzoyl adenosine (5'-p-FSO2BzAdo). Reaction of the vesicles with 5'-p-FSO2BzAdo results in a time-dependent inhibition of EGF-stimulable protein kinase activity which parallels an increase in incorporation into the vesicles of the 5'-p-sulfonylbenzoyl-[8-14C]adenosine moiety from 5'-p-FSO2Bz[14C]Ado. The primary bands labeled have Mr = 170,000 and 150,000. Labeling of these bands by 5'-p-FSO2Bz[14C]Ado is inhibited by incubation of the membrane vesicles with adenyl-5'-yl imidodiphosphate, an ATP analog. Inactivation of the kinase with N-ethylmaleimide or by heating results in a sharply decreased labeling of the proteins with Mr = 170,000 and 150,000. Proteins of these molecular weights have previously been identified in these cells as the EGF receptor and a degradation product of the receptor. These experiments provide chemical evidence that the EGF receptor and the EGF-stimulable kinase are the same protein.