Research ArticleBIOCHEMISTRY

BRK phosphorylates SMAD4 for proteasomal degradation and inhibits tumor suppressor FRK to control SNAIL, SLUG, and metastatic potential

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Science Advances  23 Oct 2019:
Vol. 5, no. 10, eaaw3113
DOI: 10.1126/sciadv.aaw3113
  • Fig. 1 BRK is overexpressed in several human tumors and regulate different signaling pathways in normal and cancer cells.

    (A) Differential expression of BRK in five major cancer types. Data obtained from The Cancer Genome Atlas database, median ± one quartile; ***P < 0.001. Tissue samples are denoted N for normal and C for cancer tissue. (B) Activity of BRK–wild-type (WT) and BRK-Y447F (BRK-YF) mutants in transfected human embryonic kidney (HEK) 293 cells. BRK-WT and BRK-YF were transfected in HEK293 cells, and cell lysates were subjected to immunoblot with antiphosphotyrosine antibody (PY20), and anti-BRK and anti–β-tubulin served as a loading control. (C) Flow diagram of peptide arrays for kinome analysis. (D) Signaling pathways significantly (P < 0.05) affected by activated BRK as identified by kinome analysis in HEK293.

  • Fig. 2 Ectopically expressed BRK and SMAD4 interact and colocalize in HEK293 cells.

    (A and B) SNAP-FLAG-BRK-YF (SF-BRK-YF) and HALO-SMAD2/3/4 were expressed into HEK293 cells, and cell lysates were subjected to affinity purification (AP) with Halo magnetic beads (A) or SNAP capture magnetic beads (B) antibodies, followed by immunoblotting using anti-Halo and anti-FLAG antibodies. Bottom: The ectopic expression of BRK and SMAD2/SMAD3/SMAD4 as detected by anti-Halo and anti-FLAG antibodies. During affinity purification, either the SNAP_Flag or Halo tags were clipped off using Precision or Tev proteases, respectively. Halo-SMAD4 is ~93 kDa; after Halo-Tag removal, SMAD4 is ~60 kDa. Similarly, SNAP-Flag-BRK is ~73 kDa, and after SNAP-Tag removal, BRK is ~50 kDa. In (B), the three different blots in the middle were probed with the SMAD2, SMAD3, and SMAD4 antibodies. (C) HEK293 cells were cotransfected with SF-BRK-YF, Halo-SMAD2, Halo-SMAD3, and Halo-SMAD4, and the cell lysates were subjected to affinity purification with Halo magnetic beads or SNAP capture magnetic beads, followed by immunoblotting using anti-SMAD2, anti-SMAD3, anti-SMAD4, and anti-Flag antibodies. Total cell lysates were also analyzed by immunoblotting using antibodies against Halo-SMAD2, Halo-SMAD3, Halo-SMAD4, and Flag. (D) GFP-SMAD4 was cotransfected with BRK-W44A, BRK-∆SH2, BRK-∆SH3, BRK-WT, BRK-Y342F, or BRK-YF. The corresponding protein extracts were immunoprecipitated with anti-GFP and mouse immunoglobulin G (IgG; lysates from SMAD4 and BRK-YF cotransfected cells as a representative) and immunoblotted with anti-BRK and anti-GFP antibodies and β-actin as a loading control. IP, immunoprecipitation. (E) SF-BRK-YF was expressed either alone or with Halo-SMAD4 [full length (FL)] or SMAD4 deletion mutants (A to E) in HEK293 cells. Total cell lysates were subjected to Halo affinity purification and analyzed by immunoblotting with FLAG and Halo antibodies. (F) Halo-SMAD4 or Halo plasmid alone with SF-BRK-WT or SF-BRK-YF was ectopically expressed in HEK293T cells. SNAP affinity purification followed by MudPIT mass spectrometry analysis showed that Halo-SMAD4 copurified with SNAP-Flag-BRK-WT or SNAP-Flag-BRK-YF but not with Halo alone. (G) Halo-SMAD4 or SF-BRK-YF alone or in combination was transfected into HEK293T cells. Halo-Tag TMRDirect fluorescent ligand (red) and SNAP-Cell 505-Star (green) were used to label Halo-Tag and SNAP-Tag proteins, respectively; DNA was stained with Hoechst dye (blue). DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 3 Targeted proteomics reveals BRK-mediated tyrosine phosphorylation of SMAD4.

    (A) Workflow of global phosphorylation analysis by MudPIT mass spectrometry and targeted proteomics. MS/MS, tandem mass spectrometry. (B) Phosphorylation sites identified in this study (S, serine; T, threonine; Y, tyrosine) tabulated with known phosphorylation sites (www.phosphosite.org/proteinAction.action?id=1845&showAllSites=true on 7 February 2018). The frequency of detection and total spectral counts for the phosphorylated peptides are reported for the Halo-SMAD4 affinity purifications with or without BRK-YF. (C) Validation of previously unidentified tyrosine phosphorylations on Halo-SMAD4 Y353 and Y412 in the presence of SF-BRK-YF by MRM. For each phosphopeptide, at least four fragment ions containing the modified residue were targeted for MRM including respective nonmodified resides. (D) Halo-SMAD4 or Halo-SMAD4 Y353F and Y412F, with or without SNAP-Flag-BRK-YF were cotransfected into HEK293T cells, and the cell lysates were subjected to affinity purification, followed by immunoblotting (IB) with anti-PY20 or anti-SMAD4 antibodies. The expression of Halo-SMAD4, Halo-SMAD4 Y353F and Y412F, and SNAP-Flag-BRK-YF were analyzed by immunoblotting using anti-Halo and anti-Flag specific antibodies. β-Tubulin was used as a loading control. (E) An in vitro kinase assay was performed using the active kinase, Flag-BRK, and the substrate SMAD4, in the presence or absence of adenosine 5′-triphosphate (ATP). Antiphosphotyrosine antibody PY20 was used to detect phosphotyrosine. The blots were reprobed with anti-Flag and anti-SMAD4 antibody (bottom).

  • Fig. 4 BRK and SMAD4 are expressed in most breast cancer cells and tissues.

    (A) BRK expression was detected by immunoblotting in the indicated normal mammary epithelial and breast cancer cell lines (TNBC, HER2, and ER), and β-actin was used as a loading control. (B) Differential expression of SMAD4 and BRK in breast cancer cell lines, as obtained from the Cancer Cell Line Encyclopedia. (C) Absolute expression of BRK and SMAD4 in patient-driven tumor tissues of three major breast cancer subtypes: ER/PR, HER2, and TNBC. Super-stable isotope labeling by amino acids–based absolute proteins expression data were obtained from Tyanova et al. (19). (D) Immunoblotting analysis of total cell lysates from HEK293, MDA231, and MCF10A cells with or without ectopically expressed GFP-BRK-YF. Stable cell lysates were analyzed by immunoblotting using SMAD2, SMAD3, SMAD4, antiphosphotyrosine (4G10), and GFP antibodies. β-Actin served as a loading control. (E) SF-BRK-YF was ectopically expressed in HEK293 cells and treated with MG132 for 8 hours, followed by immunoblotting analysis using Flag and SMAD4 antibodies. β-Tubulin served as a loading control. (F) Total cell lysates from HEK293 and MCF10A; GFP-BRK-YF–expressing HEK293 and MCF10A stable cell lines and GFP-SMAD4–transfected GFP-BRK-YF–expressing stable HEK-293 and MCF10A stable cell lines were analyzed by immunoblotting using p21, SMAD4, and BRK-specific antibodies. β-Actin was a loading control.

  • Fig. 5 Tyrosine phosphorylated SMAD4 interacts with enzymes of the ubiquitin pathway.

    (A) A workflow for discovery proteomics using MudPIT mass spectrometry for protein identifications. (B) SMAD4 polyubiquitination under denaturing condition: HEK293T cells were transiently transfected with Halo-SMAD4, SF-BRK-YF, or in combination with HA-ubiquitin plasmid. After 36 hours, the cells were treated with 10 μM MG132 for an additional 8 hours. The total cell lysates were subjected to immunoprecipitation, followed by immunoblotting with anti-HA and anti-SMAD4 antibodies. (C and D) SMAD4 stability: Tet-On–inducible Halo-SMAD4 plasmid alone or Tet-On–inducible Halo-SMAD4 plasmid with SNAP-Flag-BRK-YF was transfected into HEK293T cells for the indicated time points and analyzed by immunoblotting with anti-Halo, anti-Flag, and β-tubulin antibodies. The protein expression was quantified using ImageJ software and plotted as a bar chart. (E) Interaction with ubiquitin modifying enzymes: Relative QSPEC log2 fold changes measured for the specified proteins in the APMS analyses of Halo-SMAD4/Halo-SMAD4 Y353F and Y412F with or without BRK-YF were plotted as heat maps (Genesis software package developed by A. Sturn and R. Snajder: http://genome.tugraz.at/genesisclient/genesisclient_description.shtml). (F) SMAD4 Y353F and Y412F polyubiquitination under denaturing condition: HEK293T cells were transiently transfected with Halo-SMAD4 Y353F and Y412F or SF-BRK-YF or in combination with HA-ubiquitin plasmid and analyzed as described above. (G and H) SMAD4 stability: Tet-On–inducible Halo-SMAD4 Y353F and Y412F plasmid with or without SNAP-Flag-BRK-YF was transfected into HEK293T cells and analyzed as described above. a.u., arbitrary units.

  • Fig. 6 BRK regulates tumor suppressor, EMT markers, and metastatic potential in a SMAD4-dependent manner.

    (A) Luciferase reporter constructs were transfected in HEK293 cells with and without SMAD4 to measure the transcriptional activation of the FRK promoter. (B) ChIP-qPCR experiment shows the relative abundance of SMAD4 in FRK promoter. (C and D) The mRNA levels of FRK were quantified via quantitative RT-PCR and protein levels were analyzed by immunoblotting of the total proteins extracted from parental, stably expressing BRK-YF and SMAD4-transfected BRK-YF expressing MDA-MB-231 stable cell lines. (E and F) The mRNA levels of SNAIL and SLUG were quantified via quantitative RT-PCR in the parental cell line, BRK-YF stable expressing MDA-MB-231 cell line and ectopically expressed SMAD4 in BRK-YF expressing stable cell line. (G) Cell adhesion assay shows the cell adhesion properties of HEK293 and MCF10A, BRK-YF stably expressing HEK293 and MCF10A cell lines and SMAD4-transfected stable cell lines. *P ≤ 0.05; **P ≤ 0.001; ***P ≤ 0.0001. (H) Activated BRK regulates EMT markers (SNAIL and SLUG) and cell adhesion by modulating SMAD4-FRK.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaaw3113/DC1

    Fig. S1. The kinase activity of BRK and regulated signaling pathways.

    Fig. S2. Identification of Halo-SMAD4–associated proteins.

    Fig. S3. Activated BRK phosphorylates tyrosine-353 and tyrosine-412 on SMAD4.

    Fig. S4. BRK and SMAD4 mRNA and protein expression in different cells.

    Fig. S5. Halo-SMAD4/Halo-SMAD4 Y353F– and Halo-SMAD4 Y412F–associated proteins in the presence or absence of SF-BRK-YF and their ubiquitination.

    Fig. S6. Gene ontology analyses for cellular components represented in the proteins associated with Halo-SMAD4 and phosphorylated Halo-SMAD4.

    Fig. S7. FRK-dependent regulation of EMT markers.

    Table S1. Differential protein interaction of Halo-SMAD4 in the presence of SNAP-F-BRK-WT or SNAP-F-BRK-YF (QSPEC log2 fold change, ≥1; QSPEC false discovery rate, ≤0.05).

    Table S2. Phosphorylation sites on SMAD4 detected by MudPIT analyses of in presence or absence of BRK-YF.

    Table S3. Gene ontology analysis for cellular component of Halo-SMAD4 in the presence of SNAP-F-BRK-WT or SNAP-F-BRK-YF in ClueGO FDR_0.05; Zscore_3: Tab: 3. Primers: Tab: 3.

    References (41, 42)

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. The kinase activity of BRK and regulated signaling pathways.
    • Fig. S2. Identification of Halo-SMAD4–associated proteins.
    • Fig. S3. Activated BRK phosphorylates tyrosine-353 and tyrosine-412 on SMAD4.
    • Fig. S4. BRK and SMAD4 mRNA and protein expression in different cells.
    • Fig. S5. Halo-SMAD4/Halo-SMAD4 Y353F– and Halo-SMAD4 Y412F–associated proteins in the presence or absence of SF-BRK-YF and their ubiquitination.
    • Fig. S6. Gene ontology analyses for cellular components represented in the proteins associated with Halo-SMAD4 and phosphorylated Halo-SMAD4.
    • Fig. S7. FRK-dependent regulation of EMT markers.
    • Legends for tables S1 to S3
    • References (41, 42)

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Differential protein interaction of Halo-SMAD4 in the presence of SNAP-F-BRK-WT or SNAP-F-BRK-YF (QSPEC log2 fold change, ≥1; QSPEC false discovery rate, ≤0.05).
    • Table S2 (Microsoft Excel format). Phosphorylation sites on SMAD4 detected by MudPIT analyses of in presence or absence of BRK-YF.
    • Table S3 (Microsoft Excel format). Gene ontology analysis for cellular component of Halo-SMAD4 in the presence of SNAP-F-BRK-WT or SNAP-F-BRK-YF in ClueGO FDR_0.05; Zscore_3: Tab: 3. Primers: Tab: 3.

    Files in this Data Supplement:

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