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.
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 mitogenactivated 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 from 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.
Diggle
T A
TA
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Moule
S K
SK
Avison
M B
MB
Flynn
A
A
Foulstone
E J
EJ
Proud
C G
CG
Denton
R M
RM
D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002352:Carrier Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004822:Epididymis; D039561:Eukaryotic Initiation Factor-4E; D007328:Insulin / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D010448:Peptide Initiation Factors / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011090:Polyenes / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D019893:Ribosomal Protein S6 Kinases; D020123:Sirolimus; D000077191:Wortmannin
D000730:Androstadienes; D002352:Carrier Proteins; C499526:Eif4ebp1 protein, rat; D004791:Enzyme Inhibitors; D039561:Eukaryotic Initiation Factor-4E; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D011090:Polyenes; D004815:Epidermal Growth Factor; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D007545:Isoproterenol; D020123:Sirolimus; D000077191:Wortmannin