Phosphatidylinositol 3-kinase signaling does not activate the wnt cascade

Abstract

Mutational activation of the phosphatidylinositol 3-kinase (PI3K) pathway occurs in a wide variety of tumors, whereas activating Wnt pathway mutants are predominantly found in colon cancer. Because GSK3 is a key component of both pathways, it is widely assumed that active PI3K signaling feeds positively into the Wnt pathway by protein kinase B (PKB)-mediatefd inhibition of GSK3. In addition, PKB has been proposed to modulate the canonical Wnt signaling through direct stabilization and nuclear localization of beta-catenin. Here, we show that compartmentalization by Axin of GSK3 prohibits cross-talk between the PI3K and Wnt pathways and that Wnt-mediated transcriptional activity is not modulated by activation of the PI3K/PKB pathway.

FIGURE 1.

Wnt ligands, β-catenin, and LiCl induce TCF/β-catenin-dependent transcription in PC3 (A), BT549 (B), LNCaP (C), and MDA-MB-468, (D) cell lines in a PI3K/Akt-independent manner. Cells were plated at 50–60% confluency in 24-well plates and transfected with pTOPFlash reporters, with or without Wnt1, Wnt3A, β-catenin, or TCF4 plasmids as indicated. 20 mm LiCl, 100 nm (BT509) or 250 nm (PC3, LNCaP, and MDA-MB-468) PI3K-inhibitor wortmannin, and 50 nm Akt inhibitor IV were added as indicated 16 h following transfection, and measurements were done after 24 h. The TOPFlash data (top panel) are representative of at least two independent experiments. Error bars represent ± S.D. Effective inhibition of the PI3K/PKB pathway in response to wortmannin and Akt inhibitor IV treatment is shown by Western blotting as indicated in the panels below the pertinent bar graphs.

FIGURE 2.

PI3K/PKB pathway activity does not modulate Wnt/β-catenin signaling through GSK3β. A, mouse monoclonal A5 antibody is Axin1-specific, whereas A6 antibody recognizes both Axin1 and Conductin. 0.5 μg of DNA/well were transfected into HEK293T cells in a 6-well plate. All samples were lysed in 2× SDS sample buffer after 24 h, and Western blot analysis was performed using anti-FLAG, A6, and A5 antibodies, respectively. B–D, activation of PI3K/PKB pathway does not influence the phosphorylated or total amount of GSK3β protein that is bound to AXIN. HEK293T cells were cultured in 5% fetal calf serum/RPMI 1640 medium to 80% confluency and then starved in serum-free medium for at least 12 h before treatment with 10 mg/liter insulin for 10 and 30 min (B) and 1 and 4 h (C) or 200 nm wortmannin for 30 min (B) and 1 and 4 h (D). Cells were lysed and immunoprecipitated (IP) with either control mouse IgG or anti-Axin antibody. Resulting immunocomplexes were subjected to immunoblotting (IB) using the indicated antibodies.

FIGURE 3.

Tcf/β-catenin transcriptional activity is not modulated by activation of the PI3K/PKB pathway. HEK293T cells were plated at 30–40% confluency in 24-well plates and transfected with pTOPFlash reporters, Wnt3a and Dkk1 (Wnt inhibitor) plasmids as indicated. 16 h after transfection, half of the cells were starved with serum-free medium for at least 12 h before treatment with 20 mm LiCl, 10 mg/liter insulin, or 200 nm wortmannin as indicated. Measurement was done another 16–24 h later. Incubation periods of the nonstarved cells were the same for the starved cells. Data are from three independent duplicate experiments (except ★, n = 2). Error bars indicate mean ± S.D.