Phosphorylation Regulates Functions of ZEB1 Transcription Factor

Abstract

ZEB1 transcription factor is important in both development and disease, including many TGFβ-induced responses, and the epithelial-to-mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 is differentially phosphorylated in different cell types; however the role of phosphorylation in ZEB1 activity is unknown. Luciferase reporter studies and electrophoresis mobility shift assays (EMSA) show that a decrease in phosphorylation of ZEB1 increases both DNA-binding and transcriptional repression of ZEB1 target genes. Functional analysis of ZEB1 phosphorylation site mutants near the second zinc finger domain (termed ZD2) show that increased phosphorylation (due to either PMA plus ionomycin, or IGF-1) can inhibit transcriptional repression by either a ZEB1-ZD2 domain clone, or full-length ZEB1. This approach identifies phosphosites that have a substantial effect regulating the transcriptional and DNA-binding activity of ZEB1. Immunoprecipitation with anti-ZEB1 antibodies followed by western analysis with a phospho-Threonine-Proline-specific antibody indicates that the ERK consensus site at Thr-867 is phosphorylated in ZEB1. In addition to disrupting in vitro DNA-binding measured by EMSA, IGF-1-induced MEK/ERK phosphorylation is sufficient to disrupt nuclear localization of GFP-ZEB1 fusion clones. These data suggest that phosphorylation of ZEB1 integrates TGFβ signaling with other signaling pathways such as IGF-1. J. Cell. Physiol. 231: 2205-2217, 2016. © 2016 Wiley Periodicals, Inc.

FIGURE 1. The phosphorylation state of ZEB1 changes its DNA-binding activity

EMSAs were performed with Jurkat and CHO-K1 nuclear extracts (NE) and DNA oligonucleotides [³²P]ZEB1, [³²P]α4-integrin or [³²P]p73E5. NE were treated with calf intestinal phosphatase (CIP) or CIP plus phosphate (CIP + P) except in “NE Jurkat”, “NE CHO” and “PMA/IO” lanes. (A) Lane 1, untreated NE; Lane 2, CIP treated; Lane 3, PMA/IO: cells were preincubated for 30 minutes with PMA+Iono as in M&M; Lanes 4 + 5, CIP-treated without or with phosphate buffer; Lane 6, ZEB1 antibody (R17) was incubated with CIP-treated NE; Lane 7, [³²P]ZEB1 probe was competed with a 50-fold (50X) molar excess of unlabeled ZEB1 oligonucleotide. (B) Untreated (lanes 1, 7) or CIP-treated NE was incubated with [³²P]α4-integrin probe or [³²P]p73E5 probe. Binding was competed by 20X, 50X or 100X excess of cold oligonucleotides. N-terminus ZEB1 antibody (E20) was used to block ZEB1-binding. (C) [³²P]p73E5 was incubated with untreated (Lane 1) or CIP-treated CHO NE. Binding was completed by 20X excess of unrelated Actin oligonucleotide, 20-50X excess of p73E5 oligonucleotide, or 20-50X excess of mutated p73E5 oligonucleotide.

FIGURE 2. Signaling pathways regulate of ZEB1 binding to target genes

(A) EMSAs were performed with COS-7 NE incubated with [³²P]p73 E5 or [³²P]CD4. Cells were pre-incubated with DMSO (solvent), 10-50 μM PD98059 (PD) or 5–10 μM LY294002 (LY) prior to making NE as indicated in M&M. (B) EMSAs were performed with COS-7 NE incubated with [³²P]α4-integrin. Cells were incubated with DMSO, PD98059 (PD), SB203580 (SB), LY294002 (LY) or KT5720 (KT) prior to making NE. (C) EMSAs were performed with CHO-K1 NE incubated with [³²P]CD4. Cells were incubated with DMSO (solvent) (lane 1), and consecutively with Calphostin C (CalC) (lane 2), followed by PMA+Iono (lane 3), followed by CalC-PMA+Iono (lane 4). (A–C) Graphs represent quantification of the results shown in the adjacent EMSA, which are representative of three independent experiments. (D) Expression of endogenous ZEB1 protein is not altered by 20% Fetal Bovine Serum (FBS), IGF-1, PMA/Iono, LY, PD, SB or CalC. Immunoblots of ZEB1 in NE from cells incubated with indicated reagents as in M&M. Arrows indicate ZEB1 positions.

FIGURE 3. PMA/Iono treatment of cells inhibits ZEB1-mediated repression

COS-7 and CHO-K1 cells were co-transfected and treated with PMA/Iono for 30 minutes as indicated in M&M. (A) In the absence of cotransfected ZEB1, PMA/Iono has no effect. Results are expressed as mean ± S.E.M. of the fold luciferase activation normalized to the vector pGL3-basic. (B-D) The effect of kinase activation on ZEB1-mediated repression is tested with (B) the ZEB1 promoter, (C) the p73intr promoter, or (D) the CD4 promoter. Results are expressed as mean ± S.E.M. (n=4-6) of promoter activation. DMSO or PMA+Iono treatment without transfected ZEB1 is set as 100%. *P<0.05; ** P<0.01; ***P<0.001.

FIGURE 4. Phosphorylation within ZD2 inhibits binding to DNA and transcriptional activity

(A) Cartoons showing the ZEB1 structure representing its zinc finger domains (ZD1 and ZD2), the central homeodomain (HD) and an acidic domain (EE). The ZEB1 subclones used in different experiments are also illustrated. (B) EMSAs were performed with RRL programmed with ZD2 mRNA. ZD2-RRLs were incubated with [³²P]α4-integrin (lane 1), and ZEB1 R17 antibody (lane 2), or an anti-actin antibody (lane 3), or were competed by 20X excess of unlabelled α4-integrin oligonucleotide (lane 4). ZD2-programmed RRL was treated with buffer alone (lanes 5, 7) or CIP at 30°C (lane 6) or 37°C (lane 8). (C) Luciferase reporter assay in CHO-K1 cells showing the CD4 promoter-luciferase activity co-transfected with the expression vectors for full-length ZEB1 or wtZD2 after PMA/Iono incubation for 10 min. to 30 min. The results indicate mean ± S.E.M. (n=2).

FIGURE 5. Mutagenesis of phosphorylation sites for transcriptional regulation of ZD2

(A) Alignment by CLUSTALW of three mammalian sequences (human, rat and mouse) for a carboxy-terminal fragment of ZEB1 (788-994 in rat) showing the conserved putative phosphorylation sites in text boxes. Amino acids were numbered according to UniProtKB/Swiss-Prot Q62947 for rat ZEB1. (B) Schematic representation of the rat wtZD2 and mutant constructs. The wtZD2 clone is identical to ZEB1-ZD2 in Figure 4A. CtBP binding site is indicated. (C) Luciferase reporter assays in CHO-K1 cells testing the ZEB1 promoter-luciferase activity co-transfected with mutant ZD2-1 and mZD2-2 constructs (mZD2-1A/B, mZD2-2A/B/C). The activity of the promoter in the absence of ZD2 was set as 100%. The results represent mean ± S.E.M. of 3-4 experiments. (D) Effect of PMA/Iono treatment on the transcriptional role of mZD2-1 and mZD2-2 on the E-cadherin promoter. The results represent mean ± S.E.M. of 2 experiments. NS = not significant compared to DMSO or control. *P<0.05; ***P<0.001.

FIGURE 6. The phosphorylation sites in mZD2-1 are relevant for ZEB1 transcriptional regulation by MAPKs

Luciferase reporter assays in CHO-K1 cells showing the effect of transfected wtZD2 or the mutants, mZD2-1A, -B and mZD2-C, on the ZEB1 target promoter E-cadherin (A, B and, C) were performed (n=2). Treatments: (A) IGF-1, (B) LY294002 or, (C) PD98059. (D) Whole-cell lysates of CHO-K1 cells transfected with ΔZD1 or ZD2E expression vectors were immunoprecipitated with two anti-ZEB1 antibodies: anti-ZEB1-HD and ZEB1 R-17 or an IgG control antibody. The blot was revealed with phospho-TP MAPK antibody and R-17 as control. (E) Luciferase reporter assays in CHO-K1 cells showing the effect of IGF-1 on the transcriptional activity induced by wtZD2, mZD2-T867A and mZD2-T867E clones. The percentage of increase of ZEB1 promoter activity by IGF-1 effect is shown. (F) Luciferase reporter assays in CHO-K1 cells incubated with or without IGF-1 showing the effect of FLZEB1 and the mutants, mZEB1-1A, mZEB1-1B and mZEB1-T867A on the ZEB1 target promoter. The activities of the promoter in the absence of ZEB1 (Control) were set as 100% *P<0.05; ** P<0.01.

FIGURE 7. Phosphorylation of ZD2 alters nuclear localization

(A) Confocal microscopy of CHO-K1 cells transfected with the pEGFP-GKLF, GFP-ZD2 or GFP-ZD2-NLS expression vectors treated as indicated. (B) Confocal microscopy of CHO-K1 cells transfected with Full-Length ZEB1, mZEB1-T867A, mZEB1-1B, eGFP-ZD2 or eGFP-ZD2 T867A clones and treated with IGF-1 as indicated. The location of ZEB1 signal after IGF-1 incubation is indicated by Cyt (cytoplasmic) or Nuc (nuclear) inside the panels. A color reproduction with the counterstaining of DAPI is shown in Supplementary material S6B.

FIGURE 7. Phosphorylation of ZD2 alters nuclear localization

(A) Confocal microscopy of CHO-K1 cells transfected with the pEGFP-GKLF, GFP-ZD2 or GFP-ZD2-NLS expression vectors treated as indicated. (B) Confocal microscopy of CHO-K1 cells transfected with Full-Length ZEB1, mZEB1-T867A, mZEB1-1B, eGFP-ZD2 or eGFP-ZD2 T867A clones and treated with IGF-1 as indicated. The location of ZEB1 signal after IGF-1 incubation is indicated by Cyt (cytoplasmic) or Nuc (nuclear) inside the panels. A color reproduction with the counterstaining of DAPI is shown in Supplementary material S6B.

FIGURE 8. Kinase activity inhibits ZEB1 function through the ZD2 region

Specific phosphosites are critical for regulation of ZEB1 activities. ERK1/2 phosphorylation of T867 allows PKC activity at adjacent sites, blocking ZEB1-mediated transcriptional repression.