FIG. 2.
S101 is constitutively phosphorylated in vivo, and this affects phosphorylation at S65. (A) HEK293 cells were transfected with a vector encoding 4E-BP1(S65A) and 48 h later were treated (for 30 min) with either insulin (100 nM) (Ins), fetal calf serum (10% [vol/vol]) (FCS), LY294002 (30 μM) (LY), rapamycin (100 nM) (Rap), SB202190 (10 μM) (SB), or PD184352 (10 μM) (PD). The signal for the lower band, which corresponds to the endogenous 4E-BP1, is mainly due to phosphorylation of this protein at S65 and is accordingly increased by insulin and serum treatment. (B) HEK293 cells were transfected with wt 4E-BP1 or mutants in which S101 was mutated to A or to the acidic residue D or E. Where indicated, cells were stimulated with insulin (100 nM; 30 min). Extracts were analyzed by SDS-PAGE and Western blotting with the anti-S65[P] antibody (upper panel) or anti-Myc (lower panel). (C) Conditions were as described for panel B, but only the wild-type (wt) and S101A vectors were used. Blots were developed with anti-Myc or the indicated phosphospecific antisera for 4E-BP1. The signals for the endogenous 4E-BP1 in these extracts are too faint to be seen at this exposure of the immunoblots. (D) Conditions were as described for panel B, but extracts were subjected to affinity chromatography on m7GTP-Sepharose and SDS-PAGE followed by immunoblotting with the indicated antisera.