Research ArticleBiochemistry

TGF-β promotes PI3K-AKT signaling and prostate cancer cell migration through the TRAF6-mediated ubiquitylation of p85α

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Science Signaling  04 Jul 2017:
Vol. 10, Issue 486, eaal4186
DOI: 10.1126/scisignal.aal4186
  • Fig. 1 The interaction between AKT and TβRI depends on TRAF6.

    (A) Immunoblotting (IB) for the indicated proteins after immunoprecipitation (IP) for the HA tag from whole-cell lysates (WCL) of PC-3U cells transfected with HA–TβRI TD and treated with TGF-β. S473, Ser473; T308, Thr308; p, phosphorylated. (B) PC-3U cells were transfected with Flag-AKT1 and HA–TβRI TD or the HA–TβRI TD–E161A mutant. After treatment with TGF-β, cell lysates were subjected to immunoprecipitation with a polyclonal rabbit HA antibody, followed by immunoblotting using Flag antibody. (C) Cell lysates prepared from wild-type (WT) and TRAF6−/− MEFs treated or not with TGF-β were subjected to immunoprecipitation with a polyclonal rabbit AKT antibody or control (ctrl) immunoglobulin G (IgG) and then immunoblotted with a polyclonal goat TβRI antibody (V-22). (D) Immunofluorescence staining for the indicated proteins in HA–TβRI TD–transfected PC-3U cells, either untreated or after stimulation with TGF-β. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Scale bars, 20 μm. (E) PC-3U cells transfected with Flag-AKT1 and HA–TβRI TD were treated with dimethyl sulfoxide (DMSO) or 5 μM TβRI kinase inhibitor SB431542 (added 1 hour before TGF-β stimulation). Immunoprecipitation of cell lysates was performed, as described in the legend for (B). Blots and microscopy images are representative of three independent experiments. Graphs are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 within one set of samples (TGF-β–stimulated versus unstimulated cells); §P < 0.05 between different sets of samples [mutant complementary DNA (cDNA)–transfected versus wt cDNA–transfected or deficient versus wt cells]. a.u., arbitrary units.

  • Fig. 2 TGF-β–induced polyubiquitylation and activation of AKT require p85.

    (A) PC-3U cells treated with control or p85α siRNA were stimulated or not with TGF-β for different time periods and then lysed. Lysates were subjected to immunoprecipitation with rabbit AKT antibody, followed by immunoblotting using mouse monoclonal P4D1 to detect ubiquitylated AKT [AKT-(Ub)n]. The immunoprecipitation filter was reblotted with antibodies against AKT. The corresponding whole-cell lysates were subjected to immunoblotting for p85. (B) PC-3U cells treated with control or p85α siRNA were stimulated or not with TGF-β and then lysed after different time periods. Cell lysates were immunoblotted for pAKT Ser473 and pAKT Thr308, AKT, p85, and β-actin. (C) Ubiquitylation and activation of AKT were examined in WT and p85−/− MEFs treated or not with TGF-β for 30 min. Blots are representative of three independent experiments. Graphs are means ± SEM from three independent experiments. *P < 0.05, ***P < 0.001 within one set of samples (TGF-β–stimulated versus unstimulated); §P < 0.05, §§P < 0.01, §§§P < 0.001 between different sets of samples (specific siRNA–transfected versus control siRNA–transfected cells, deficient versus wt cells).

  • Fig. 3 Polyubiquitylation of p85α requires TRAF6.

    (A) PC-3U cells treated with control or TRAF6 siRNA were stimulated or not with TGF-β, and the ubiquitylation of p85α was examined. (B) Cell lysates prepared from WT and TRAF6−/− MEFs treated or not with TGF-β were examined for p85α ubiquitylation. (C) Lys63-linked (K63 clone Apu3) polyubiquitylation of p85 was examined in PC-3U cells, transfected with different amounts of Flag-TRAF6 cDNA, and treated or not with TGF-β for 30 min. (D) PC-3U cells transfected with Flag-tagged WT or C70A mutant TRAF6 cDNA were treated or not with TGF-β, and cell lysates were prepared. Ubiquitylation of p85α was monitored by immunoblotting with ubiquitin (P4D1) antibody after immunoprecipitation of p85α. (E) RAW264.7 cells were transfected with Flag-tagged WT or C70A mutant TRAF6 cDNA, and ubiquitylation of p85α and activation of AKT were examined. (F) Recombinant His-p85α proteins were incubated in the presence or absence of GST-TRAF6 protein (E3) in a reaction mixture containing E1, Ubc13-Uev1A (E2), ubiquitin, and adenosine triphosphate (ATP). After incubation at 30°C for 1 hour, reaction products were analyzed by immunoblotting with antibodies against ubiquitin and p85 to visualize synthesized polyubiquitin chains. Blots are representative of three independent experiments. Graphs are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 within one set of samples (TGF-β–stimulated versus unstimulated); §P < 0.05, §§P < 0.01, §§§P < 0.001 between different sets of samples (specific siRNA–transfected versus control siRNA–transfected cells, deficient versus wt cells, or mutant cDNA–transfected versus wt cDNA–transfected cells).

  • Fig. 4 TGF-β–induced polyubiquitylation of p85α is Lys63-linked and does not depend on TβRI kinase activity.

    (A) PC-3U cells were stimulated or not with TGF-β, and cell lysates were prepared and immunoprecipitated with rabbit p85 antibody. Beads were boiled in 1% SDS, and the supernatant was diluted in 0.5% NP-40 in phosphate-buffered saline (PBS) and thereafter subjected to immunoprecipitation with rabbit p85 antibody (double IP), followed by immunoblotting using rabbit monoclonal Apu3 against Lys63 (K63)–linked ubiquitin. (B) PC-3U cells transiently transfected with HA-tagged WT or mutated ubiquitin were treated or not with TGF-β, lysed, and subjected to immunoprecipitation using p85α antiserum and immunoblotting using an antiserum against HA. (C and D) Lysates of the cells treated or not with 10 μM SB505124 (C) or 5 μM LY2109761 (D) before TGF-β stimulation for 1 hour were exposed to immunoprecipitation with rabbit p85α antibody and immunoblotting with mouse ubiquitin antibody (P4D1). The corresponding whole-cell lysates were subjected to immunoblotting for pSMAD2, pAKT Ser473, pAKT Thr308, and p85. Blots are representative of three independent experiments. Graphs are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01 within one set of samples (TGF-β–stimulated versus unstimulated); §P < 0.05 between different sets of samples (mutant cDNA–transfected versus wt cDNA–transfected cells).

  • Fig. 5 TGF-β induces the interaction of p85α with TβRI in a TRAF6-dependent manner, which is mediated by binding of p85α to ubiquitin chains.

    (A) Lysates from PC-3U cells were subjected to immunoprecipitation with a TRAF6 antibody and immunoblotted for endogenous p85α. (B) HEK293T cells transfected with Flag-tagged full-length (FL) p85α, nSH2-iSH2-cSH2 (3SH2), or SH3-BH plasmids were subjected to immunoprecipitation with monoclonal mouse TRAF6 antibody and immunoblotting for Flag. (C) PC-3U cell lysates were subjected to immunoprecipitation with a TβRI antibody (goat V-22) and immunoblotting for endogenous p85. (D) HEK293T cells transfected with HA–TβRI TD and Flag-tagged full-length p85α, 3SH2, or SH3-BH plasmids were subjected to immunoprecipitation with polyclonal rabbit HA antibody or control IgG, followed by immunoblotting for Flag. (E) HEK293T cells transfected with Flag-p85α and HA–TβRI TD or HA–TβRI kinase-deficient (KR) plasmids were treated or not with TGF-β and subjected to immunoprecipitation with a polyclonal rabbit HA antibody or control IgG, followed by immunoblotting for Flag. (F) Cell lysates prepared from WT and TRAF6−/− MEFs were subjected to immunoprecipitation with a rabbit antibody against TβRI (V-22) and immunoblotting for mouse p85α. (G) Lysates of HEK293T cells transfected with Flag-tagged full-length p85α, del iSH2, del cSH2, SH3-BH, or iSH2-cSH2 plasmids were incubated with biotinylated Lys63-linked polyubiquitin chains and incubated with streptavidin-agarose beads, which were boiled in a sample buffer, and supernatants were subjected to immunoblotting with a Flag antibody. Blots are representative of three independent experiments. Graphs are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 within one set of samples (TGF-β–stimulated versus unstimulated); §P < 0.05, §§§P < 0.001 between different sets of samples (mutant cDNA–transfected versus wt cDNA–transfected cells or deficient versus wt cells).

  • Fig. 6 Ubiquitylation of p85α occurs on Lys513 and/or Lys519 in the p85α iSH2 domain.

    (A) Lysates of PC-3U cells transfected with HA-tagged WT ubiquitin and Flag-tagged full-length p85α or del iSH2, del cSH2, SH3-BH, or iSH2-cSH2 plasmids were subjected to immunoprecipitation with Flag or mouse IgG antiserum and immunoblotted with HA antibody. (B) Lysates of HEK293T cells transfected with HA-Ub and full-length Flag-p85α were subjected to SDS-PAGE. The protein bands used for mass spectrometry are marked by an arrowhead (nonubiquitylated) and arrows (polyubiquitylated). (C) Two detected branched peptide sequences in iSH2 domain of p85α. The red letters show the putative lysine residues. (D) Lysates of HEK293T cells transfected with HA-Ub and full-length WT Flag-p85α, K567/575R, or K513/519R Flag-p85α plasmids were examined for p85α polyubiquitylation. (E) Lysates of HEK293T cells transfected with HA-Ub and full-length WT Flag-p85α or K513R, K519R, or K513/519R mutant p85α plasmids were subjected to ubiquitylation assay by blotting with a rabbit antibody against Lys63-linked ubiquitin (K63-linked Ub). Blots are representative of three independent experiments. Graphs are means ± SEM from three independent experiments. *P < 0.05 within one set of samples (TGF-β–stimulated versus unstimulated and in panel), §P < 0.05 between different sets of samples (mutant cDNA–transfected versus wt cDNA–transfected cells).

  • Fig. 7 TGF-β–induced cell migration requires the PI3K pathway and TRAF6 and is not dependent on the kinase activity of TβRI.

    (A) RAW264.7 cells treated with the PI3K inhibitor wortmannin, TβRI kinase inhibitor SB431542, or 0.1% DMSO for 1 hour were subjected to Transwell migration assay. Scale bar, 100 μM. The corresponding whole-cell lysates were subjected to immunoblotting using antibodies against pAKT Ser473, pAKT Thr308, and pSMAD2. (B) PC-3U cells treated with the PI3K inhibitors NVP-BKM120, LY294002, and wortmannin, TβRI kinase inhibitor SB431542, or 0.1% DMSO as control were stimulated or not with TGF-β in a cell culture wound healing assay, as described in Materials and Methods. Scale bar, 100 μM. (C) WT and p85−/− MEF cells were exposed to Transwell migration assay as described for (A). Scale bar, 100 μM. (D) WT and TRAF6−/− MEFs treated or not with TGF-β for 24 hours were exposed to wound healing assay as described for (B). Scale bar, 100 μM. Blots and microscopy images are representative of three independent experiments. Graphs are means ± SEM from three independent experiments. **P < 0.01, ***P < 0.001 within one set of samples (TGF-β–stimulated versus unstimulated); §P < 0.05, §§P < 0.01, §§§P < 0.001 between different sets of samples (inhibitor-treated versus control cells or deficient versus wt cells).

  • Fig. 8 TGF-β–induced polyubiquitylation of p85α on Lys513 and Lys519 is important for PI3K and AKT activation and for cell migration.

    (A) PC-3U cells transfected with WT, K513R, K519R, or K513/519R plasmids of p85α were treated or not with TGF-β for 30 min. Cell lysates were prepared and subjected to a PI3K assay, followed by measurement of PIP3 concentration, as described in Materials and Methods. Data are means ± SEM from three independent experiments; *P < 0.05 within one set of samples (TGFβ-stimulated versus unstimulated), §P < 0.05 between different sets of samples (mutant cDNA-transfected versus wt cDNA-transfected cells). (B) Whole-cell lysates corresponding to (A) were subjected to immunoblotting with antiserum against pAKT Ser473, pAKT Thr308, pp85, p85, Flag, and pS6. (C) PC-3U cells silenced or not for endogenous p85α [by siRNA targeting 3′ untranslated region (3′-UTR)] and transfected with WT, K513R, K519R, or K513/519R plasmids of p85α were subjected to Transwell assay. Scale bar, 100 μM. Bar graphs show the means ± SEM from three independent experiments; *P < 0.05. (D) Cell lysates corresponding to the samples in (C) were subjected to immunoblotting for p85, Flag, and β-tubulin to check the knockdown and transfection efficiency. The corresponding whole-cell lysates were immunoblotted for p85 and Flag. The filters were stripped and reblotted for β-actin. Blots and microscopy images are representative of three independent experiments.

  • Fig. 9 Lys63-linked ubiquitylation of p85α and activation of SMAD2 and AKT in prostate cancer tissues are correlated with aggressiveness.

    (A and B) Prostate tumor tissue samples were stained with pSMAD2 (A) and pAKT Ser473 (B) antibodies. Quantification shows the means ± SEM of pAKT Ser473 of 10 patients in each group. (C) The association between p85α and Lys63 (K63) ubiquitin in prostate cancer patients (brown dots) was determined by PLA (300 cells were analyzed in each group). Data are means ± SEM of five patients in each group. **P < 0.01, ***P < 0.001. Scale bars, 50 μm. (D) Proposed model for TGF-β–induced polyubiquitylation of p85α and activation of PI3K and AKT. TGF-β–induced activation of PI3K is initiated by ligand-induced oligomerization of TGF-β receptors, which juxtaposes TRAF6 molecules, constitutively bound to TβRI, that undergo autoubiquitylation and ubiquitylate TβRI. Lys63-linked ubiquitin chains on TRAF6 or TβRI mediate the recruitment of PI3K to the complex by binding the SH3 domain of p85α. Activated TRAF6 then causes Lys63-linked ubiquitylation of p85α and activation of PI3K, which in turn leads to PIP3 production. Whether TRAF6, PI3K, AKT, and SMAD7 simultaneously form a complex or whether interactions occur sequentially remains to be elucidated. AKT is recruited to the complex whereby it is ubiquitylated and activated. The recruitment of p85α and AKT may be facilitated by SMAD7, which has been shown to have an adaptor function in the non-Smad TRAF6 pathway. Attachment of Lys63-linked polyubiquitin chains on p85α possibly favors a conformational change of p85α, thereby removing the inhibitory contacts of the SH2 domains of p85α from p110, which enhances the kinase activity of PI3K. AKT activation promotes cell migration, which has been shown to be dependent on TRAF6-mediated c-Jun activation as well (43). For simplicity, the PI3K-AKT-SMAD7 complex is shown only on one side of the symmetric TGF-β receptor complex.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/486/eaal4186/DC1

    Fig. S1. AKT interacts with TRAF6 in a TGF-β–dependent manner.

    Fig. S2. TGF-β induces Lys63-linked polyubiquitylation and activation of AKT.

    Fig. S3. TGF-β–induced polyubiquitylation and activation of AKT require the E3 ligase activity of TRAF6.

    Fig. S4. TGF-β–induced activation of AKT depends on the PI3K pathway and is not dependent on the kinase activities of TβRI or TβRII.

    Fig. S5. TGF-β–induced activation of AKT is not dependent on the kinase activity of TAK1 or the PDGFR.

    Fig. S6. SMAD7 is required for Lys63-linked polyubiquitylation of p85α and AKT.

    Fig. S7. TRAF6, pAKT Ser473, TβRI, and p85α colocalize at the cell membrane.

    Fig. S8. Mutation of Lys513 and Lys519 to arginine does not affect binding of p85α to p110α.

    Fig. S9. PI3K activity is increased by TGF-β stimulation and requires the E3 ligase activity of TRAF6 but not the kinase activity of TβRI.

    Fig. S10. TGF-β–induced polyubiquitylation of p85α on Lys513 and Lys519 is important for AKT activation and migration of p85−/− MEFs.

    Fig. S11. Negative control for immunohistochemistry and in situ PLA.

    Fig. S12. Mass spectrometry analysis.

  • Supplementary Materials for:

    TGF-β promotes PI3K-AKT signaling and prostate cancer cell migration through the TRAF6-mediated ubiquitylation of p85α

    Anahita Hamidi, Jie Song, Noopur Thakur, Susumu Itoh, Anders Marcusson, Anders Bergh, Carl-Henrik Heldin,* Maréne Landström*

    *Corresponding author. Email: marene.landstrom{at}medbio.umu.se (M.L.); c-h.heldin{at}licr.uu.se (C.-H.H.)

    This PDF file includes:

    • Fig. S1. AKT interacts with TRAF6 in a TGF-β–dependent manner.
    • Fig. S2. TGF-β induces Lys63-linked polyubiquitylation and activation of AKT.
    • Fig. S3. TGF-β–induced polyubiquitylation and activation of AKT require the E3 ligase activity of TRAF6.
    • Fig. S4. TGF-β–induced activation of AKT depends on the PI3K pathway and is not dependent on the kinase activities of TβRI or TβRII.
    • Fig. S5. TGF-β–induced activation of AKT is not dependent on the kinase activity of TAK1 or the PDGFR.
    • Fig. S6. SMAD7 is required for Lys63-linked polyubiquitylation of p85α and AKT.
    • Fig. S7. TRAF6, pAKT Ser473, TβRI, and p85α colocalize at the cell membrane.
    • Fig. S8. Mutation of Lys513 and Lys519 to arginine does not affect binding of p85α to p110α.
    • Fig. S9. PI3K activity is increased by TGF-β stimulation and requires the E3 ligase activity of TRAF6 but not the kinase activity of TβRI.
    • Fig. S10. TGF-β–induced polyubiquitylation of p85α on Lys513 and Lys519 is important for AKT activation and migration of p85−/− MEFs.
    • Fig. S11. Negative control for immunohistochemistry and in situ PLA.
    • Fig. S12. Mass spectrometry analysis.

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    Citation: A. Hamidi, J. Song, N. Thakur, S. Itoh, A. Marcusson, A. Bergh, C.-H. Heldin, M. Landström, TGF-β promotes PI3K-AKT signaling and prostate cancer cell migration through the TRAF6-mediated ubiquitylation of p85α. Sci. Signal.10, eaal4186 (2017).

    © 2017 American Association for the Advancement of Science

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