Phosphorylation Regulates Functions of ZEB1 Transcription Factor

Abstract

SUMMARY 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. This article is protected by copyright. All rights reserved.

CLCA2 epigenetic regulation by CTBP1, HDACs, ZEB1, EP300 and miR‐196b‐5p impacts prostate cancer cell adhesion and EMT in metabolic syndrome disease

“…ZEB1 plasmids were reported previously, HDACs plasmids were kindly provided by Dr. Martin Monte (Universidad de Buenos Aires, Argentina) and EP300 plasmid was described previously …”

“…10 pGL4 plasmids with different lengths of the CLCA2 promoter, pGIPZ.shCLCA2 and pGIPZ control, pLEX-CLCA2 and pLEX were described previously. 16,18 ZEB1 plasmids were reported previously, 22 HDACs plasmids were kindly provided by Dr. Martin Monte (Universidad de Buenos Aires, Argentina) and EP300 plasmid was described previously. 23 The 3 0 UTR CLCA2 (1,031 bp) was amplified using specific primers (Supporting Information Table 1) and cloned in Spe I and Hind III restriction sites of pMIR-REPORT firefly luciferase vector plasmid (pMIR, Ambion, Grand Island, NY).…”

EMT Factors and Metabolic Pathways in Cancer

“…ZEB1/2 and the miR-200 family expression levels are anticorrelated, with a double-negative feedback loop between them, which is described in the section of microRNAs. Posttranslational modifications of both TFs can also modulate their expression levels, an example being the phosphorylation of ZEB1 (117) and the SUMOylation of ZEB2 [reviewed in (118)].…”

Oncogenic functions of the EMT-related transcription factor ZEB1 in breast cancer

“…After the formation of the ZEB1 protein, insulin-like growth factor 1 (IGF-1) induced ZEB1 phosphorylation at residues T851, S852, and S853 by protein kinase C (PKC), showing involvement in the EMT process and resulting in transcriptional activity [38]. Another posttranslational modification of ZEB1 is polycomb protein (Pc2)-induced sumoylation at residues K347 and K777 [39].…”