Figure 1.
Rapamycin activates PP2A in an mTOR-dependent manner.
Serum-starved Rh30 cells were exposed to rapamycin (Rapa, 100 ng/ml) for the indicated time (A), or pretreated with or without rapamycin (Rapa, 100 ng/ml) or okadaic acid (OA, 100 nM) for 2 h, and then stimulated with or without IGF-1 (10 ng/ml) for 1 h (B), or the indicated serum-starved Rh30 cells were exposed to rapamycin (Rapa, 100 ng/ml) for 2 h (C, D). PP2A in cell lysates was immunoprecipitated with antibodies to PP2A catalytic subunit (PP2Ac) plus protein A/G agarose beads, followed by in vitro phosphatase assay using Ser/Thr Phosphatase Assay Kit 1 (Millipore). Data represent mean ± SE from 3–4 independent experiments. aP<0.05 vs. controls, bP<0.05 vs. Rapa group.
Figure 2.
Rapamycin suppresses IGF-1 stimulated phosphorylation of Erk1/2 in an mTOR-dependent manner.
(A) Serum starved Rh1 cells were pretreated with or without rapamycin (Rapa, 100 ng/ml) for 2 h, and then stimulated with IGF-1 (10 ng/ml) for the indicated time. (B) Serum-starved Rh1/pcDNA and Rh1/mTORrr cells were exposed to increasing concentrations of rapamycin (Rapa, 0–1000 ng/ml) for 2 h, and then to IGF-1 for 15 min. (C) Serum-starved Rh30/pcDNA and Rh30/mTORrr cells were pretreated with or without rapamycin (Rapa, 100 ng/ml) for 2 h, and then stimulated with or without IGF-1 (10 ng/ml) for 15 min. (D) Serum-starved Rh1 cells, infected with lentiviral shRNA to GFP (control) or mTOR, were stimulated with IGF-1 (10 ng/ml) for 1 h (Left panel), or exposed to increasing concentrations of rapamycin (Rapa, 0–1000 ng/ml) for 2 h, and then to IGF-1 for 15 min. For (A–D), cell lysates were analyzed by Western blotting using indicated antibodies.
Figure 3.
Inhibition of PP2A with okadaic acid confers resistance to rapamycin inhibition of IGF-1-induced phosphorylation of Erk1/2 and cell motility.
(A) Serum-starved Rh1 cells were pretreated treated with or without OA (100 nM) for 1 h, and then treated with or without rapamycin (Rapa, 100 ng/ml) for 2 h, followed by stimulation with or without IGF-1 (10 ng/ml) for 15 min. Cell lysates were analyzed by Western blotting using indicated antibodies. β-tubulin served as loading control. (B) Motility of Rh1 cells was determined by the wound healing assay, as described in Materials and Methods. Following wounding, serum-starved Rh1 cells were pretreated treated with or without OA (100 nM) for 1 h, and then treated with or without rapamycin (Rapa, 100 ng/ml) for 2 h, followed by stimulation with or without IGF-1 (10 ng/ml) for 22 h. Cells migrated over the denuded area were observed and photographed with an Olympus inverted phase-contrast microscope equipped with Quick Imaging system. The number of cells migrating per millimeter of scratch was counted. Results are means ± SE of 3–4 independent experiments. aP<0.05, difference vs. control group; bP<0.05, difference vs. IGF-1 group.
Figure 4.
Expression of dominant negative PP2A confers resistance to rapamycin inhibition of cell motility.
(A) Rh1 cells were stably transfected with vector alone (Rh1/pcDNA) or with a vector expressing HA-tagged dn-PP2Ac (L199P), serum-starved for 24 h, and then treated with or without rapamycin (Rapa, 100 ng/ml), followed by in vitro phosphatase assay using Ser/Thr Phosphatase Assay Kit 1 (Millipore). Data represent mean ± SE from 3–4 independent experiments. aP<0.05 vs. Rh1/pcDNA control, bP<0.05 vs. Rh1/pcDNA Rapa group. (B) Serum-starved Rh1/pcDNA and Rh1/dn-PP2A cells were stimulated with or without IGF-1 (10 ng/ml) for 15 min, followed by Western blotting using the indicated antibodies. (C) Motility of Rh1/pcDNA and Rh1/dn-PP2A cells was determined by the wound healing assay, as described in Materials and Methods. Serum-starved cells were pretreated with or without rapamycin (Rapa, 100 ng/ml) or PD98059 (PD, 10 µM) for 2 h, and then stimulated with or without IGF-1 (10 ng/ml) for 22 h. Results are means ± SE of 3–4 independent experiments. aP<0.05, difference vs. control group; bP<0.05, difference vs. IGF-1 group.
Figure 5.
Inhibition of Erk1/2 with PD98059 blocks IGF-1-stimulated cell motility.
(A) Serum-starved Rh1 cells were exposed to increasing concentrations of PD98059 (0–50 µM) for 2 h, and then to IGF-1 for 15 min, followed by Western blotting using the indicated antibodies. (B) Motility of Rh1 cells was determined by the wound healing assay. Serum-starved cells were pretreated for 2 h with rapamycin (Rapa) (100 ng/ml) or PD98059 (PD) (10 µM) alone, or in combination as indicated, followed by stimulation with or without IGF-1 (10 ng/ml) for 22 h. Results are means ± SE of 3–4 independent experiments. aP<0.05, difference vs. control group; bP<0.05, difference vs. IGF-1 group.
Figure 6.
Expression of constitutively active MKK1 confers resistance to rapamycin inhibition of cell motility.
(A) Rh30 cells, infected with Ad-MKK1-R4F, Ad-MKK1-K97M, and Ad-GFP (control), respectively, were serum-starved for 24 h, pretreated with or without rapamycin (Rapa, 100 ng/ml) for 2 h, and then stimulated with or without IGF-1 (10 ng/ml) for 15 min, followed by Western blotting with indicated antibodies. (B) Motility of Rh30 cells infected with Ad-MKK1-R4F, Ad-MKK1-K97M or Ad-GFP was determined by the wound healing assay. Results are means ± SE of 3–4 independent experiments. aP<0.05, difference vs. Ad-GFP control group; bP<0.05, difference vs. IGF-1 group.