Insulin-stimulated kinase from rat fat cells that phosphorylates initiation factor 4E-binding protein 1 on the rapamycin-insensitive site (serine-111)

The effects of insulin and rapamycin on the phosphorylation of the translat ion regulator, initiation factor 4E-binding protein 1 (4E-BP1) have been st udied in rat fat cells by following changes in the incorporation of P-32 fr om [P-32]Pi under steady-state conditions. Both unbound 4E-BP 1 and 4E-BP1 bound to eukaryotic initiation factor 4E (eIF4E) were isolated from the cel ls and then digested with trypsin and other proteases; the radiolabelled ph osphopeptides were then separated by two-dimensional thin-layer analysis an d HPLC. The results provide confirmation of the conclusion of Fadden, Hayst ead and Lawrence [J. Biol. Chem. (1997) 272, 10240-10247] that insulin incr eases the phosphorylation of four sites that fit a Ser/Thr-Pro motif (Thr-3 6, Thr-45, Ser-64 and Thr-69) and that taken together these phosphorylation s result in the dissociation of 4E-BP1 from eIF4E. The effects of insulin o n the phosphorylation of these sites, and hence dissociation from eIF4E, ar e blocked by rapamycin. However, the present study also provides evidence t hat insulin increases the phosphorylation of 4E-BP1 bound to eIF4E on a fur ther site (Ser-lll) and that this is by a rapamycin-insensitive mechanism. Extraction of rat epididymal fat cells followed by chromatography on Mono-S and Superose 12 columns resulted in the separation of both an insulin-stim ulated eIF4E kinase and an apparently novel kinase that is highly specific for Ser-1 11 of 4E-BP1. The 4E-BP1 kinase was activated more than 10-fold b y incubation of the cells with insulin and was markedly more active towards 4E-BP1 bound to eIF4E than towards unbound 4E-BP1. The effects of insulin were blocked by wortmannin, but not by rapamycin. A 14-mer peptide based on the sequence surrounding Ser-111 of 4E-BP1 was also a substrate for the ki nase, but peptide substrates for other known protein kinases were not. The kinase is quite distinct from casein kinase 2, which also phosphorylates Se r-lll of 4E-BP1. The possible importance of these kinases in the phosphoryl ation of 4E-BP1 in fat cells is discussed. It is suggested that the phospho rylation of Ser-lll might be a priming event that facilitates the subsequen t phosphorylation of Thr-36, Thr-45, Ser-64 and Thr69 by a rapamycin-sensit ive process that initiates the dissociation of 4E-BP1 from eIF4E and hence the formation of the eIF4F complex.