Figure 5
Rapamycin-induced MAPK activation is downstream of S6K/IRS-1/PI3K.
(A) ERK phosphorylation in MCF7 transfected for 24 hours with wild-type S6K1 (HAS6K1wt) or a rapamycin-insensitive constitutively active form of S6K1 (HAS6K1 E389 D3E) and treated with rapamycin (20 nM, 24 h; n = 3). (B) ERK and RpS6 phosphorylation status in starved (24 h, 0.1% FBS) T24 cells treated with vehicle or rapamycin (20 nM, 24 h) and stimulated with insulin (200 nM, 15 min) or IGF-1 (100 ng/ml, 15 min; n = 3). (C) ERK and RpS6 phosphorylation in Tsc2-null p53-null MEFs 24 hours after transfection with empty vector or 0.5 or 4 μg of Tsc2-expressing vector (n = 3). Asterisk indicates non-specific band. HSP90 was used as a loading control. (D) Effect of rapamycin (20 nM, 24 h) and/or PI3K inhibitor LY294002 (10 μM, 24 h) on AKT, ERK, and RpS6 phosphorylation in MCF7 cells (n = 3). (E) AKT, ERK, and RpS6 phosphorylation status in starved (24 h, 0.1% FBS) MCF7 cells preincubated with DMSO or wortmannin (500 nM, 45 min) and stimulated with insulin (100, 200, and 400 nM; n = 3). Numbers indicate the ratio of phosphorylated protein related to total protein levels.
