Carboxy-terminal zinc finger domain (ZD2) of ZEB1 contains its main phosphorylation sites

LORENZATTI, GUADALUPE; CAVALLO, NATALIA; CABANILLAS, ANA MARIA

Carlos paz, Cordoba, Argentina.

Congreso; XLIV Reunión Anual SAIB; 2008

SAIB

The activity of a transcription factor (TF) can be controlled by phosphorylation.ZEB1 exists as two phosphorylated forms. ZEB1(Zn Finger E-box Binding Homeobox) is a TF involved in cell differentiation and metastasis of tumors. ZEB1contains two Zn finger domains (ZD1, ZD2) flanking a homeodomain (HD) and an acidic domain (E). The isoform ZEB1-b lacks ZD1.We showed that the hypophosphorylated ZEB1 binds to its target genes stronger than the hyperphosphorylated one. Our aim here is to delimit which of the ZEB1 domains are involved.  Rabbit reticulocyte lysates (RRL) were programmed with expression vectors that encode the ZEB1 protein fragments ZEB1-b, HD, ZD2E and ZD2. RRL were incubated with phosphatase (CIP) or CIP+phosphate. EMSAs were done with the RRLs and 32P-oligonucleotides harboring ZEB1 binding sites from á4integrin, CD4 and p73 promoters.  CIP treatment increased ZEB1 binding capacity to all the probes assayed with all the RRLs but HD. Binding was competed by either anti-ZEB1 antibodies or an excess of cold oligonucleotides. COS/CHO cells were transfected with same vectors and CD4/p73-luciferase promoters.Then, cells were incubated with TPA/ionomycin. As expected, luciferase activities (normalized by âGal) were lowered by ZEB1/ZD2E/ZD2. TPA/IO treatment reverted that repression to the target genes. The results suggest that ZD2 contains main phosphorylation sites of ZEB1.