Insulin treatment of S2 cells and potential phosphorylation sites of d4E-BP. (A) Time course of d4E-BP phosphorylation in response to insulin. S2 cells were starved for 36 h and stimulated with bovine insulin (1 μg/ml) for the indicated times. (B) Phosphatase treatment. Extracts from starved or insulin-treated S2 cells were treated with CIP for 30 min at the indicated temperatures. The effect of insulin treatment on d4E-BP in panels A and B was assessed by immunoblotting with anti-phospho-4E-BP1(Thr37/46) (α4E-BP1) (top) and by gel shift of the α phosphoisoform to β as visualized by anti-d4E-BP (αd4E-BP) (bottom) using extracts from S2 cells. P, phosphorylated. (C) Sequence alignment of the putative phosphorylation sites of d4E-BP and human and murine 4E-BP1. Identical (solid boxes) and conserved (shaded boxes) amino acids are highlighted. Conserved phosphorylation sites are colored red and marked by asterisks. The arrows indicate differences in (for Thr37, Ser65, and Thr70) or identity of (for Thr46) the amino acids surrounding the putative phosphorylation sites of d4E-BP.

Analysis by IEF-SDS-PAGE of d4E-BP phosphorylation. (A) Selected S2 cell extracts for IEF-SDS-PAGE (arrows). Rapamycin-treated cell extract (not shown) is similar to unstimulated cell extract. (B to F) IEF-SDS-PAGE of extracts from serum-starved (B), insulin-treated (15 min) (C), insulin-treated (30 min) (D and E), and rapamycin- and insulin-treated (15 min) (F) cells were probed with the indicated antibodies. d4E-BP (B to F) was assessed by blotting it with anti-d4E-BP (αd4E-BP) or anti-phospho-4E-BP1(Thr37/46) (α4E-BP1 P-T37/46) as indicated. The individual isoforms are represented by the letters a to e. P, phosphorylated.

Effects of RNAi of dAkt, dTSC1, and dTOR on d4E-BP phosphorylation in S2 cells. (A) A reduction in the amount of dAkt blocks signaling to d4E-BP and reduces insulin-induced phosphorylation. The phosphorylation of dS6K at Thr398 is also reduced. +, treated; −, untreated; P, phosphorylated. (B) A reduction in the amount of dTsc1 augments signaling to d4E-BP, causing an increase in the basal levels of phosphorylated d4E-BP. The phosphorylation of dS6K at Thr398 is also increased. (C) A reduction in the amount of dTOR blocks signaling to d4E-BP and dS6K and abolishes the insulin-induced phosphorylation of both proteins. RNAis of dAkt, dTSC1, and dTOR were performed at least three times. The effects on d4E-BP and dS6K were assessed as for Fig. 4.

Analysis of mutations at d4E-BP phosphorylation sites. (A) Mutation of Thr37 and/or Thr46 to Ala (T37A and T46A, respectively) prevents the insulin-induced phosphorylation of d4E-BP. As expected, the double mutant (T37A/T46A) also showed no phosphorylation. The last lane (-) is an untransfected control. WT, wild type; +, treated; −, untreated; P, phosphorylated. (B) Mutation of Ser65 and/or Thr70 to Ala (S65A and T70A, respectively) had no effect on phosphorylation. As expected, the double mutant (S65A/T70A) also had no effect. S2 cells transfected with 3HA-tagged d4E-BP mutant cDNAs were starved for 24 h before insulin treatment. The effect of insulin was assessed with anti-phospho-4E-BP1(Thr37/46) (α4E-BP1 P-T37/46) (middle), and protein expression levels were compared using anti-HA-11 (αHA) (bottom). Total protein loading levels were compared using anti-deIF4A (αdeIF4A).

Effects of RNAi of dPI3K, dPTEN, and dPDK1 on d4E-BP phosphorylation in S2 cells. (A) Northern analysis confirms that the dPI3K (Dp110) and dPTEN transcript amounts are reduced compared with control cells. (B) RNAis of dPI3K and dPTEN have different effects on d4E-BP phosphorylation. Whereas dPI3K RNAi reduces the insulin-stimulated phosphorylation of d4E-BP, dPTEN RNAi increases the phosphorylation of d4E-BP in unstimulated cells. The phosphorylation of dS6K Thr398 is increased by RNAi of dPI3K but not by RNAi of dPTEN. RNAi of Dp110 and dPTEN was performed at least three times. +, treated; −, untreated. (C) Northern analysis confirms that the dPDK1 transcript is reduced compared with control cells. (D) A reduction in dPDK1 amounts reduces the endogenous levels of d4E-BP but does not affect its insulin-induced phosphorylation. dPDK1 RNAi does, however, block phosphorylation of dS6K at Thr398. RNAi of dPDK1 was performed four times. The effect on d4E-BP was assessed by blotting with anti-phospho-4E-BP1(Thr37/46) (α4E-BP1 P-T37/46) and by gel shift of the α phosphoisoform to β with anti-d4E-BP (αd4E-BP). The phosphorylation of dS6K was detected with anti-phospho-S6K Thr389 (αS6K P-T389). Antibodies against translation factors deIF4E (αdeIF4E) and deIF4A (αdeIF4A) were used to confirm equal loading of the gels in panels B and D, respectively. RpS15a is the probe for ribosomal protein S15a, which was used to confirm equal loading in panels A and C. P, phosphorylated.

Models of the insulin-signaling pathway in Drosophila and regulation of d4E-BP by phosphorylation. (A) A common pathway regulates 4E-BP and S6K. The regulation of 4E-BP is dependent on signaling from Inr-PI3K-Akt-TSC and TOR. The regulation of S6K is also effected by PDK1 and the same upstream elements. The different size of the Akt typeface is to illustrate that the intensity of signaling toward TSC affects 4E-BP and S6K differentially. (B) The pattern of phosphorylation of d4E-BP as observed by IEF-SDS-PAGE may result from one of three possible models. (i) In the primed model, d4E-BP is already phosphorylated at Thr37 and is subsequently phosphorylated on one additional site, Thr46, after insulin stimulation. (ii) In the sequential model, d4E-BP is phosphorylated on Thr37 and then Thr46 (or vice versa). (iii) In the coordinated model, d4E-BP is phosphorylated coordinately on Thr37 and Thr46.