ZEB1 is modified post-translationally by SUMOylation

VAGLIENTI, MARIA V.; LLORENS, M. CANDELARIA; CABANILLAS, ANA M.

Mar del Plata

Congreso; LXI Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica (SAIC), LXIV Reunión Científica Anual de la Sociedad Argentina de Inmunología (SAI), XLVIII Reunión Anual SAFE y VII Reunión Anual NANOMEDAR; 2016

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 andbreast 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 hydrophobicaminoacid. 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 allthe 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 expressionvectors (EV) of His6x-SUMO1 or His6x-SUMO2, Ubc-9 and theEV of full length ZEB1 or the GFP clones. His6x tagged proteinswere purified from cell lysates in Ni2+-NTA-agarose affinity columns,immunoblotted and developed with anti ZEB1 and anti GFPantibodies. The results showed that ZEB1 can be SUMOylated indifferent K sites. We also verified the colocalization of ZEB1 andSUMO-1 by immunofluorescence of HEK293T cells transfectedwith EV of SUMO-HA, Ubc9, ZEB1 and mutant deletion clones.The results suggest that ZEB1 could be modified by SUMOylationwhich could affect its oncogenic role.