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

TA Diggle, SK Moule, MB Avison, A Flynn, EJ Foulstone, CG Proud, RM Denton

Research output: Contribution to journalArticle (Academic Journal)peer-review

83 Citations (Scopus)

Abstract

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 P-32) 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 (p70(S6K)) 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 p70(S6K) Signalling pathway is involved: p70(S6K) itself is probably not involved directly as this kinase does not phosphorylate 4E-BP1 in vitro.

Translated title of the contributionBoth rapamycin-sensitive and -insensitive pathways are involved in the phosphorlyation of the initiation factor-4E-binding protein (4E-BP1) in response to insulin in rat epididymal fat cells
Original languageEnglish
Pages (from-to)447-453
Number of pages7
JournalBiochemical Journal
Volume316
Publication statusPublished - 1 Jun 1996

Keywords

  • ADIPOSE-TISSUE
  • ADIPOCYTES
  • STIMULATE

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