Biology 4 E - BP 1 phosphorylation is mediated by the FRAP - p 70 s 6 k pathway and is independent of mitogen - activated protein kinase ( insulin / signal transduction / translational control ) SABINE

It has previously been argued that the repressor of protein synthesis initiation factor 4E, 4E-BP1, is a direct in vivo target of p42mapk. However, the immunosuppressant rapamycin blocks serum-induced 4E-BP1 phosphorylation and, in parallel, p70s6k activation, with no apparent effect on p42mapk activation. Consistent with this finding, the kinetics of serum-induced 4E-BP1 phosphorylation closely follow those of p70s6k activation rather than those of p42mapk. More striking, insulin, which does not induce p42mapk activation in human 293 cells or Swiss mouse 3T3 cells, induces 4E-BP1 phosphorylation and p70s6k activation in both cell types. Anisomycin, which, like insulin, does not activate p42mapk, promotes a small parallel increase in 4E-BP1 phosphorylation and p70s6k activation. The insulin effect on 4E-BP1 phosphorylation and p70s6k activation in both cell types is blocked by SQ20006, wortmannin, and rapamycin. These three inhibitors have no effect on p42mapk activation induced by phorbol 12-tetradecanoate 13-acetate, though wortmannin partially suppresses both the p70s6k response and the 4E-BP1 response. Finally, in porcine aortic endothelial cells stably transfected with either the wild-type platelet-derived growth factor receptor or a mutant receptor bearing the double point mutation 740F/751F, p42mapk activation in response to platelet-derived growth factor is unimpaired, but increased 4E-BP1 phosphorylation is ablated, as previously reported for p70s6k. The data presented here demonstrate that 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase. Previous studies have suggested that a major target of p42maPk/ p44mapk activation following insulin stimulation of fat cells is a small molecular weight protein termed 4E-BP1 or PHAS-I (1, 2). Phosphorylation of 4E-BP1 disrupts its interaction with protein synthesis initiation factor eIF-4E, liberating eIF-4E to interact with the p220 subunit of the mRNA cap binding protein complex (2). Recent studies have shown that the interaction of eIF-4E with p220 is through a conserved hydrophobic region in p220 (3). A similar sequence in 4E-BP1 is also involved in eIF-4E binding (3) and competes with p220 for eIF-4E binding (4). The phosphorylation of 4E-BP1 and liberation of eIF-4E lead to upregulation of general translation, as free eIF-4E is thought to be the limiting component in this process (5). However, recent studies have shown that the immunosuppressant rapamycin blocks 4E-BP1 phosphorylation in NIH 3T3 cells (6). Rapamycin in a complex with FKBP12 is known to have no effect on p42maPk/p44maPk activation (7), but instead selectively prevents the activation of the p70s6k/p85s6k (7). Similar selective inhibition of the p70s6k pathway has also been obtained by using the phosphatidylinositol-30H kinase inhibitor wortmannin (8) or the methylxanThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. thine analogue SQ20006 (9). Consistent with these findings, dominant-negative mutants of p21ras and p74raf (10) as well as specific point mutants of the platelet-derived growth factor (PDGF) receptor demonstrate that these two signaling pathways bifurcate at the level of the receptor (8, 10). The inhibitory target of rapamycin action has been identified as a large molecular weight protein termed FRAP (11), mRAFT (12), or mTOR (13) that has structural homology to phosphatidylinositol-30H kinase. Because FRAP is an upstream element of the p70s6k signaling pathway (14), the studies with rapamycin would imply that in addition to the p42maPk/p44maPk pathway, the FRAP-p70s6k pathway is also implicated in the phosphorylation of 4E-BP1. The involvement of the FRAP-p70s6k signaling pathway in propagating the effects of insulin is strengthened by parallel studies in human 293 epithelial cells and Swiss 3T3 mouse fibroblasts. In these two cell types, insulin fails to activate p42mapk/p44mapk, whereas treatment with epidermal growth factor strongly activates both isoforms of the kinase (10, 15). Despite the difference between insulinand epidermal growth factor-induced p42maPk/p44maPk activation, insulin is at least as potent as epidermal growth factor as an activator of global translation in either cell type (ref. 16; S.R.v.M. and G.T., unpublished data). These findings imply that the p42maPk/ p44mapk pathway is not necessary to induce 4E-BP1 phosphorylation, or that 4E-BP1 itself may not play as central a role as was previously thought in global translation (2). Here we have used different mitogenic agents and specific inhibitors, including rapamycin, wortmannin, and SQ20006, to determine the relative contributions of the FRAP-p70s6k and p42mapk/p44mapk pathways to 4E-BP1 phosphorylation. We have also analyzed the phosphorylation of 4E-BP1 in cells that fail to activate p42maPk/p44maPk in response to insulin. Finally, receptor mutants were used to determine whether the signaling pathways leading to 4E-BP1 are redundant. EXPERIMENTAL PROCEDURES Cell Culture and Preparation of Cell Extracts. Swiss mouse 3T3 cells were seeded, maintained, and arrested on 15-cm tissue culture plates as described (15). Human embryonic kidney 293 cells (ATCC CRL 1573) were seeded at 106 cells per 10-cm dish in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). After 3 days, when cells had reached confluency, the medium was removed and replaced with DMEM for an additional 36 hr to bring the culture to quiescence. Porcine aortic endothelial (PAE) cells were grown and starved as described (17). Cultures were then stimulated with specific agonists or inhibitors as described in the text. FCS was used at 10%, insulin at 1 ,iM (stock 10 mg/ml Abbreviations: FCS, fetal calf serum; PAE, porcine aortic endothelial; PDGF, platelet-derived growth factor; TPA, phorbol 12-tetradecanoate 13-acetate; MBP, myelin basic protein.