Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 1998 Feb 15;12(4):502–513. doi: 10.1101/gad.12.4.502

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 1998, Cold Spring Harbor Laboratory Press

PMC Copyright notice

PI 3-kinase and Akt elicit phosphorylation of 4E-BP1. (A) Insulin-mediated phosphorylation of 4E-BP1 is both rapamycin and wortmannin sensitive. Human embryonic kidney (HEK) 293 cells were transfected transiently with a hemaglutinin (HA) epitope-tagged 4E-BP1 expression vector. After transfection, cells were deprived of serum for 36 hr, and either mock treated (lane 1) or stimulated with insulin (1 μg/ml) for 30 min (lanes 2–4) in the presence of either wortmannin, [200 nm (Wort.)] (lane 3) or rapamycin [20 ng/ml (Rap.)] (lane 4). Cell extracts were prepared as described in Materials and Methods and HA–4E-BP1 was detected by immunoblot analysis with an anti-HA antibody (12CA5). Molecular size markers (in kD) are indicated. Arrows indicate the different phosphorylated isoforms of HA–4E-BP1. (B) The catalytic subunit of PI 3-kinase p110α elicits phosphorylation of HA–4E-BP1. HEK 293 cells were cotransfected with HA–4E-BP1 expression vector along with one of the following: control vector (lane 1), p110α expression vector (lane 2), or p110αcaax (p110α*) expression vector (lane 3). After transfection, cells were deprived of serum for 36 hr. HA–4E-BP1 was detected as described in A. Small arrows indicate the different phosphorylation forms of 4E-BP1. p110α and p110αcaax were detected as described in Materials and Methods. (C) Akt elicits phosphorylation of HA–4E-BP1. HEK 293 cells were mock transfected (lane 1) or cotransfected with HA–4E-BP1 expression vector and one of the following: control vector (lane 2), HA–c-Akt expression vector (lane 3), or HA–MyrAkt expression vector (lane 4). Cells were deprived of serum for 36 hr. HA–4E-BP1 was detected as described above. Small arrows indicate the different phosphorylation forms of 4E-BP1. (D) A kinase-deficient mutant of Akt inhibits phosphorylation of 4E-BP1 by insulin. HEK 293 cells were cotransfected with a HA–4E-BPI expression vector (100 ng) and the following: control vector (lanes 1,2) or HA–AktK179M expression vector [Akt(kin−)] (lanes 3,4). After transfection, cells were serum-deprived for 36 hr and then stimulated with 100 ng/ml of insulin for 45 min (lanes 2,4). HA–4E-BP1 was detected as described above. HA–AktK179M was detected on the same immunoblot. Small arrows indicate the different phosphorylated forms of 4E-BP1. The results shown are representative of three independent experiments.

PI 3-kinase and Akt elicit phosphorylation of 4E-BP1. (A) Insulin-mediated phosphorylation of 4E-BP1 is both rapamycin and wortmannin sensitive. Human embryonic kidney (HEK) 293 cells were transfected transiently with a hemaglutinin (HA) epitope-tagged 4E-BP1 expression vector. After transfection, cells were deprived of serum for 36 hr, and either mock treated (lane 1) or stimulated with insulin (1 μg/ml) for 30 min (lanes 2–4) in the presence of either wortmannin, [200 nm (Wort.)] (lane 3) or rapamycin [20 ng/ml (Rap.)] (lane 4). Cell extracts were prepared as described in Materials and Methods and HA–4E-BP1 was detected by immunoblot analysis with an anti-HA antibody (12CA5). Molecular size markers (in kD) are indicated. Arrows indicate the different phosphorylated isoforms of HA–4E-BP1. (B) The catalytic subunit of PI 3-kinase p110α elicits phosphorylation of HA–4E-BP1. HEK 293 cells were cotransfected with HA–4E-BP1 expression vector along with one of the following: control vector (lane 1), p110α expression vector (lane 2), or p110αcaax (p110α*) expression vector (lane 3). After transfection, cells were deprived of serum for 36 hr. HA–4E-BP1 was detected as described in A. Small arrows indicate the different phosphorylation forms of 4E-BP1. p110α and p110αcaax were detected as described in Materials and Methods. (C) Akt elicits phosphorylation of HA–4E-BP1. HEK 293 cells were mock transfected (lane 1) or cotransfected with HA–4E-BP1 expression vector and one of the following: control vector (lane 2), HA–c-Akt expression vector (lane 3), or HA–MyrAkt expression vector (lane 4). Cells were deprived of serum for 36 hr. HA–4E-BP1 was detected as described above. Small arrows indicate the different phosphorylation forms of 4E-BP1. (D) A kinase-deficient mutant of Akt inhibits phosphorylation of 4E-BP1 by insulin. HEK 293 cells were cotransfected with a HA–4E-BPI expression vector (100 ng) and the following: control vector (lanes 1,2) or HA–AktK179M expression vector [Akt(kin−)] (lanes 3,4). After transfection, cells were serum-deprived for 36 hr and then stimulated with 100 ng/ml of insulin for 45 min (lanes 2,4). HA–4E-BP1 was detected as described above. HA–AktK179M was detected on the same immunoblot. Small arrows indicate the different phosphorylated forms of 4E-BP1. The results shown are representative of three independent experiments.