160 Biochimica et Biophysica Acta, 1177 (1993) 160-166 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00 BBAMCR 13392 Epidermal growth factor stimulates phosphorylation of eukaryotic initiation factor 4B, independently of protein kinase C Rob M.F. Wolthuis, Alfons F.M. Cremers, Marcell6 A.M. Kasperaitis, Cor van der Mast, Harry O. Voorma and Johannes Boonstra Department of Molecular Cell Biology, University of Utrecht, Utrecht (The Netherlands) (Received 24 November 1992) Key words: Epidermal growth factor; Protein kinase C; Phosphorylation; Eukaryotic initiation factor; Epidermoid carcinoma; (A431 cell); (Human) We demonstrate that exposure of human epidermoid carcinoma A431 cells to epidermal growth factor (EGF) results in phosphorylation of elF-4B within minutes after addition of EGF. The EGF-induced phosphorylation of elF-4B is not caused by the EGF receptor tyrosine kinase itself, since no tyrosine-phosphorylated elF-4B could be detected upon immunoprecipitation using an anti-phosphotyrosine antibody. Enhanced phosphorylation of elF-4B was also detected upon exposure of the cells to phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), suggesting that elF-4B may be a substrate of PKC. However, down-regulation of PKC did not influence the EGF-induced elF-4B phosphorylation, which indicates that elF-4B is phosphorylated by an as yet unknown kinase, activated early in the EGF-induced signal transduction cascade. Introduction Polypeptide growth factors play an important role in the regulation of cell proliferation [1]. EGF, one of the best-characterized growth factors, exerts its effects in the target cells by binding to and activation of the EGF-receptor. Following tyrosine kinase activation, the receptor phosphorylates a number of proteins, includ- ing the receptor itself, which results in the activation of the signal transduction pathway. Ultimately these events lead to enhanced DNA synthesis and cell divi- sion in most cells [2-5]. Cell division requires DNA synthesis, enhanced rates of RNA- and protein synthesis, and therefore it is attractive to suggest that these cellular processes are under direct control of the receptor kinase in order to realize the specific adaptation of the cell. With respect to protein synthesis, the targets of the growth factor induced signal transduction cascade are most likely the eukaryotic initiation factors (elFs), since regulation of protein synthesis is realized at the level of these fac- Correspondence to: J. Boonstra, Department of Molecular Cell Biology, Padualaan 8, 3584 CH Utrecht, The Netherlands. Abbreviations: EGF, epidermal growth factor; elF, eukaryotic initia- tion factor; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; NBT, nitroblue tetrazolium salt; BCIP, 5-bromo-4-chloro- 3-indolyl phosphate; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DTT, dithiothreitol. tors. The initiation factors are involved in modulating rate limiting steps such as the recognition of the 5' cap structure, unwinding of the 5' untranslated region of mRNA and in promoting binding of the 40S ribosomal subunit to the mRNA [6]. Of particular interest are the observations that the eukaryotic initiation factors 3, 4B and 4F as well as the ribosomal protein $6 may be phosphorylated in vivo [7-11] and in vitro [12,13]. Fur- thermore it has been shown that processes leading to a stimulation of overall translational initiation rates cor- relate with enhanced phosphorylation of elF-3, elF-4B, elF-4F and $6, suggesting that the phosphorylation of these translation factors is involved in the regulation of protein synthesis [13,14]. In view of the above men- tioned possible regulation of protein synthesis by growth factor-induced signal tl'ansduction, it is of inter- est that one of the initiation factors, elF-4B, is phos- phorylated upon treatment of cells with the phorbol ester PMA, an activator of protein kinase C [10,15], or insulin [15,16]. Furthermore stimulation of protein syn- thesis during recovery of heat-shocked cells or by addi- tion of serum to serum-starved cells, is accompanied by increased phosphorylation of elF-4B [7-10]. elF-4B is an 80-kDa phosphoprotein containing at least 8 serine residues that may be modified by phosphorylation [14,15]. The protein can be phosphorylated in vitro by at least five different protein kinases [12]. Sequence analysis revealed a consensus RNA recognition motive unique for initiation factors, but which is found in several different RNA binding proteins [17]. elF-4B