Rapid and specific enhancement of phosphorylation of two normal rat kidney cell membrane polypeptides of Mr = 170,000 and 150,000 by epidermal growth factor in vitro.

Abstract

Incubation of membranes of normal rat kidney (NRK)cells in vitro with [y3'P]ATP demonstrated at least 10 components whose phosphorylation was enhanced severalfold by epidermal growth factor (EGF).One major component of M, = 170,000 and a minor band of M, = 150,000, which were either undetectable or very weakly prosphorylated in the basal state, were particularly affected by exposure to EGF.The phosphorylation of these two polypeptides required the presence of the divalent cations Mn2+ or Co2+; M 8 + , Cu2+, Ca2+, Fez+, and Zn'+ were ineffective.The enhanced phosphorylation of the M , = 170,000 and 150,000 polypeptides was specific for EGF since other growth factors and numerous other hormones were ineffective.The NRK membranes catalyzed the phosphorylation of certain exogenously added proteins such as partially dephosphorylated casein hydrolysates and histones and their phosphorylation was further enhanced by EGF.The M, = 170,000 and 150,000 proteins were effectively labeled by [y3'P]GTP in both the basal and EGF-stimulated reactions.Although the phosphorylation of various NRK membrane proteins was significantly enhanced by CAMP, cGMP, or dibutyryl-CAMP, none of these nucleotides appeared to be specific for phosphorylation of the M, = 170,000 and 150,000 proteins in the presence or absence of EGF.Dephosphorylation reactions of the M, = 170,000 and 150,000 bands occurred rapidly even at 0 "C; these reactions were not significantly affected by EGF.The M, = 170,000 and 150,000 membrane components phosphorylated in the presence or absence of EGF were digested by the proteolytic enzymes, trypsin or pronase.The phosphorylated components of M, = 170,000 and 150,000 co-migrated with corresponding ['*C]glucosamine-labeled and periodic acid-Schiff basestained bands indicating the possible glycoprotein nature of the phosphorylated components.Additional experiments demonstrated that membrane preparations solubilized with the nonionic detergent Triton X-100 retained the ability to phosphorylate the M, = 170,000 protein in the presence or absence of EGF.These findings suggest that one of the earliest biochemical steps in the mechanism of action of EGF in NRK cells is increased phosphorylation of an intrinsic membrane glycoprotein or M , = 170,000 by a membrane-associated cyclic nucleotide-independent protein kinase.Therefore, a specific M, = 170,000 protein phosphorylationmediated process may play a central role in the mech-