Phosphorylation Regulates the Function of ZEB1 Transcriptional Repressor

LORENZATTI, GUADALUPE; CABANILLAS, ANA MARIA

Bariloche, Rio Negro

Congreso; South American Spring Symposium in Signal Transduction and Molecular Medicine (SISTAM); 2012

SISTAM

ZEB1 transcription factor is important in both development and disease, including the epithelial-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. It was our interest to characterize the significance of phosphorylation in the biological function of this transcription factor.  Previous studies of gene reporter and electrophoresis mobility shift assays (EMSA) showed that a decrease in ZEB1 phosphorylation increases both DNA binding and transcriptional repression of ZEB1 target genes. Functional analysis of phosphorylation site mutants around the second zinc finger domain (termed ZD2), in the presence of either inhibitors or activators of signaling pathways, show that PMA plus ionomycin, or IGF-1 can prevent the transcriptional repression by ZD2. In the present study, we identified critical phosphosites that have a substantial effect regulating the transcriptional and DNA-binding activity of ZEB1. ZD2 mutants demonstrate that MEK/ERK phosphorylation of T867 is sufficient to reduce the transcriptional activity of ZEB1.  Immunoprecipitation with ZEB1 antibodies followed by western analysis with a phospho-Threonine-Proline-specific antibody indicates that the ERK1 consensus site at T867 is phosphorylated in ZEB1.  GFP-ZEB1 fusion clones show that MEK/ERK phosphorylation is sufficient to disrupt nuclear localization of the transcription factor.  We propose that the activation of ZEB1 is modulated by kinase pathways, allowing ZEB1 to serve as an integrating factor of external signals.