There is mounting evidence that in fat and other insulin-sensitive cells activation of protein synthesis may involve the dissociation of a protein (4E-BP1) from eukaryotic initiation factor (eIF)-4E thus allowing formation of the eIF-4F complex. This study compares the effects of insulin and epidermal growth factor (EGF) on the phosphorylation of 4E-BP1 in fat-cells (followed by gel-shift assays and incorporation of 32P) and on its association with eIF-4E. Several lines of evidence suggest that mitogen-activated protein kinase (MAP kinase) is not involved in these effects of insulin. Insulin causes much more extensive phosphorylation and dissociation of 4E-BP1 from eIF-4E than EGF, although EGF activates MAP kinase to a much greater extent than insulin. Moreover, MAP kinase does not phosphorylate 4E-BP1 when it is complexed with eIF-4E. In contrast, insulin activates the 40S ribosomal protein S6 kinase (p70S6K) 18-fold compared with a 2-fold activation by EGF, and the time course of this activation is similar to the phosphorylation and dissociation of 4E-BP1. Rapamycin, a specific inhibitor of the activation of this latter kinase, inhibits dissociation of 4E-BP1 from eIF-4E in cells incubated with insulin but reveals a phosphorylated form of 4E-BP1 which remains bound to eIF-4E. It is concluded that in rat epididymal fat-cells, the effects of insulin on 4E-BP1 involves multiple phosphorylation events. One phosphorylation event is rapamycin-insensitive, occurs only on bound 4E-BP1 and does not initiate dissociation. The second event does result in dissociation and is blocked by rapamycin, suggesting that the p70S6K signalling pathway is involved: p70S6K itself is probably not involved directly as this kinase does not phosphorylate 4E-BP1 in vitro.
Both rapamycin-sensitive and -insensitive pathways are involved in the phosphorylation of the initiation factor-4E-binding protein (4E-BP1) in response to insulin in rat epididymal fat-cells
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Tricia A. DIGGLE, S. Kelly MOULE, Matthew B. AVISON, Andrea FLYNN, Emily J. FOULSTONE, Christopher G. PROUD, Richard M. DENTON; Both rapamycin-sensitive and -insensitive pathways are involved in the phosphorylation of the initiation factor-4E-binding protein (4E-BP1) in response to insulin in rat epididymal fat-cells. Biochem J 1 June 1996; 316 (2): 447–453. doi: https://doi.org/10.1042/bj3160447
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