TGFbeta-stimulated Smad1/5 phosphorylation requires the ALK5 L45 loop and mediates the pro-migratory TGFbeta switch

Abstract

During the course of breast cancer progression, normally dormant tumour-promoting effects of transforming growth factor beta (TGFbeta), including migration, invasion, and metastasis are unmasked. In an effort to identify mechanisms that regulate the pro-migratory TGFbeta 'switch' in mammary epithelial cells in vitro, we found that TGFbeta stimulates the phosphorylation of Smad1 and Smad5, which are typically associated with bone morphogenetic protein signalling. Mechanistically, this phosphorylation event requires the kinase activity and, unexpectedly, the L45 loop motif of the type I TGFbeta receptor, ALK5, as evidenced by studies using short hairpin RNA-resistant ALK5 mutants in ALK5-depleted cells and in vitro kinase assays. Functionally, Smad1/5 co-depletion studies demonstrate that this phosphorylation event is essential to the initiation and promotion of TGFbeta-stimulated migration. Moreover, this phosphorylation event is preferentially detected in permissive environments such as those created by tumorigenic cells or oncogene activation. Taken together, our data provide evidence that TGFbeta-stimulated Smad1/5 phosphorylation, which occurs through a non-canonical mechanism that challenges the notion of selective Smad phosphorylation by ALK5, mediates the pro-migratory TGFbeta switch in mammary epithelial cells.

Figure 1

TGFβ stimulates Smad1/5 phosphorylation and migration in 4T1 cells. (A) Transwell migration of cells treated for 20 h with indicated ligands. Data shown are representative of at least two independent experiments. (B) Western blot analysis of cells treated with indicated ligands for 45 min. Arrow indicates band corresponding to phosphorylated Smad8. (C) Western blot analysis of Smad phosphorylation over time in cells treated with indicated ligands.

Figure 2

ALK5 kinase activity is required for TGFβ-stimulated Smad1/5 phosphorylation and migration in 4T1 cells. Control or ALK5-depleted cells expressing empty vector (MSCV) or indicated shRNA-resistant HA-tagged ALK5 isoforms were subjected to (A) western blot analysis after 45 min. TGFβ treatment or (B) transwell migration for 20 h in the presence or absence of TGFβ. Data shown are representative of at least two independent experiments. (C) Transwell migration of cells treated for 20 h with vehicle or TGFβ in the presence or absence of ALK5 kinase inhibitor, SB431542 (SB). Representative images of stained migratory cells on transwell membranes are shown. (D) Western blot analysis of cells pretreated with SB431542 for 30 min and then treated with vehicle or TGFβ for 45 min.

Figure 3

Depletion of Smad1/5 but not Smad2/3 inhibits TGFβ-stimulated migration in 4T1 cells. Puromycin-selected cells expressing empty vector (pSicoR-puro), Smad1 targeting shRNA (1A or 1B), or Smad5 targeting shRNA (5B or 5C) were subjected to (A) western blot analysis after 45 min. TGFβ treatment or (B) transwell migration for 20 h in the presence or absence of TGFβ. Asterisk indicates detection of Smad1 by Smad5 antibody. Puromycin-selected, GFP⁺ cells expressing empty vector (pSicoR-puro+GFP), 1A+5C shRNA, 5B+1B shRNA, and 1B+5C shRNA were subjected to (C) western blot analysis after 45 min. TGFβ treatment or (D) transwell migration for 20 h in the presence or absence of TGFβ. Two-tailed Student's t-test was performed (^**P=0.05, ^***P=0.01, ^****P=0.11). Cells expressing control or S3+S2 shRNA were subjected to (E) western blot analysis after 45 min. TGFβ treatment or (F) transwell migration for 20 h in the presence or absence of TGFβ. Migration assays represent the average of at least three independent experiments.

Figure 4

Expression of various ALK5 isoforms in ALK5-1 shRNA cells differentially affects TGFβ-stimulated Smad1/5 phosphorylation and migration. (A) Summary of wild-type and mutant ALK5 amino-acid sequences and previously reported Smad phosphorylation activities. Underlined residues indicate substitutions. ALK5-depleted 4T1 cells expressing the indicated shRNA-resistant HA-tagged ALK5 isoforms were subjected to (B) luciferase assay as described in Supplementary data, (C) western blot analysis after 45 min. TGFβ treatment or (D) transwell migration for 20 h in the presence or absence of TGFβ. Data shown are representative of at least two independent experiments. (E, F) In vitro kinase assay measuring GST–Smad phosphorylation by anti-HA immunoprecipitates derived from 293T cells co-expressing the indicated TGFβ receptors. The western blot analysis of assay samples using the indicated antibodies is shown. Coomassie-stained GST–Smad serves as a GST–Smad loading control.

Figure 5

TGFβ-stimulated Smad1/5 phosphorylation and migration are independent of BMP signalling and lateral type I receptor signalling in 4T1 cells. (A) Western blot analysis of cells co-treated for 45 min with indicated ligands and soluble antagonists. (B) Transwell migration of cells after 20-h co-treatment with indicated ligands and soluble antagonists. Asterisks denote untested conditions. (C, left) Thirty-five-cycle qualitative RT–PCR analysis with or without reverse transcriptase (RT) or (C, right) quantitative real-time RT–PCR analysis of relative mRNA levels in PY-4-1 (PY) and 4T1 cells. Control or ALK2-depleted cells were subjected to (D) western blot analysis after 45-min treatment with indicated ligands or (E) transwell migration for 20 h in the presence of vehicle or TGFβ. Arrow indicates band corresponding to phosphorylated Smad8. Control or ALK3-23 shRNA-expressing cells were subjected to (F) BRE-luciferase assay in the presence or absence of BMP4 as described in Supplementary data, (G) western blot analysis after 45-min TGFβ treatment, or (H) transwell migration for 20 h in the presence of vehicle or TGFβ.

Figure 6

Tumorigenic cells provide a permissive context for TGFβ-stimulated Smad1/5 phosphorylation. Western blot analysis of cells after 45-min TGFβ treatment.

Figure 7

TGFβ-stimulated Smad1/5 phosphorylation is essential for the initiation of sensitivity to TGFβ-stimulated migration. (A) Western blot analysis of cells co-treated as indicated for 45 min. (B) Western blot analysis of cells cultured in growth media and treated with TGFβ for 45 min. (C) Transwell migration of cells and co-treated as indicated for 20 h. (D) Western blot analysis of cells transfected with control, Smad3 (S3), or Smad1/5 (S1+S5) targeting duplex siRNA co-treated as indicated for 45 min. (E) TGFβ-stimulated growth inhibition in siRNA-transfected MCF10AN cells as described in Materials and methods. (F) Transwell migration of siRNA-transfected, AP1510-treated MCF10AN cells following 20 h vehicle or TGFβ treatment. TGFβ-stimulated migration is shown. Data from (E, F) represent average of at least three independent experiments.