CONICET | Buscador de Institutos y Recursos Humanos

ZEB1 (Zn Finger E-box binding Homeobox) is a key transcription factor for Epithelial Mesenchymal Transition which not only induces an aberrant motility triggering dissemination and metastasis in cancer cells, but also confers stemness properties. ZEB1 is associated to metastasis initiation, aggressive behavior, treatment resistance and poor prognosis in lung, pancreas and breast cancers. ZEB1 is target of post translational modifications and the presence of many consensus sites for SUMOylation on its sequence suggests a possible role of this addition in the regulation of ZEB1. The SUMOylation is a covalent modification that adds the SUMO protein (Small Ubiquitin-like Modifier) to a Lysin residue (K) in a consensus motif ΨKX(D/E), where Ψ is an hydrophobic aminoacid. SUMOylation regulates a variety of activities such as protein subcellular localization and stability, transcriptional regulation and others. Our goal was to determine whether ZEB1 was SUMOylated (by in vivo assays) and what K residues were modified. An in silico comparison using SUMOplot identified 7 K residues with high score on ZEB1. The experimental approach chosen was to use small fragments of ZEB1 fused to GFP with all the potential SUMO sites: 1(K88, K175), 2 (K327, K473), 3 (K175), 4 (K473, K495, K635), 5 (K752) and 6 (K473,K495,K635,K752). HEK293T cells were cotransfected by lipofection with expression vectors (EV) of His6x-SUMO1 or His6x-SUMO2, Ubc-9 and the EV of full length ZEB1 or the GFP clones. His6x tagged proteins were purified from cell lysates in Ni2+-NTA-agarose affinity columns, immunoblotted and developed with anti ZEB1 and anti GFP antibodies. The results showed that ZEB1 can be SUMOylated in different K sites. We also verified the colocalization of ZEB1 and SUMO-1 by immunofluorescence of HEK293T cells transfected with EV of SUMO-HA, Ubc9, ZEB1 and mutant deletion clones. The results suggest that ZEB1 could be modified by SUMOylation which could affect its oncogenic role.