Phosphorylation of specific residues of the carboxi-terminal Zinc finger domain (ZD2) regulates the activity of the transcriptional repressor ZEB1

LORENZATTI, GUADALUPE; CABANILLAS, ANA MARIA

Mar del Plata, Buenos Aires.

Congreso; LV Reunion Cientificia Annual Sociedad Argentina de Investigacion Clinica (SAIC); 2010

Sociedad Argentina de Investigacion Clinica (SAIC)

ZEB1 (Zn Finger E-box Binding Homeobox) is a master regulator of Epithelial-Mesenchymal Transition, a critical program for tumor metastasis. ZEB1 activity can be regulated by changes in its phosphorylation (PO4) status. We previously reported that the hypo-PO4 ZEB1 binds to target genes more strongly than the hyper-PO4 protein and that the C-term Zn finger domain (ZD2) of ZEB1 contains key phosphorylation sites. Our aim is to identify the specific sites in ZD2 that regulate ZEB1biological role. We performed site-directed mutagenesis of the ZD2 sites with highest phosphorylation scores generating the mutants ZD2-1A (T904A), ZD2-1B (T904A, S932A), ZD2-2A (S895A), ZD2-2B (S895A, S947A, S951A), ZD2-2C (S895A, S947A, S951A, S961A), ZD2-3A (T851A, S852A, S853A), ZD2-3B (T851A, S852A, S853A, S857A) and ZD2-3C (T851A, S852A, S853A, S857A, T867A, T873A). EMSA and gene reporter assays were used to test the binding capacity and the biological role of the mutants. The mutants or the control ZD2 wt and the E-cadherin/ZEB1-luciferase promoters were transfected into CHO-K1 cells. The mutants ZD2-1A, ZD2-1B, ZD2-3A and ZD2-3C repressed luciferase more than ZD2 wt, while the mutants ZD2-2 showed no change in promoters activity compared to ZD2 wt. The treatment with a PKC activator (PMA/ionomycin) reverted the repression induced by both ZD2 wt and ZD2-2 mutants, but not the effect obtained with the other mutants indicating that key phosphorylation sites contained in the mutants ZD2-1A, B and -3A, C were made unresponsive to PMA/iono by mutagenesis. As expected, these unresponsive mutants weren’t able to increase their binding capacity to ZEB1 target promoters after treatment with alkaline phosphatase (CIP) in EMSAs. ZD2 wt and ZD2-2 mutants induced a bigger binding band after CIP treatment. Our results point out to 8 S/T residues in ZD2 domain as responsible for the transcriptional activity of ZEB1. ZEB1 (Zn Finger E-box Binding Homeobox) is a master regulator of Epithelial-Mesenchymal Transition, a critical program for tumor metastasis. ZEB1 activity can be regulated by changes in its phosphorylation (PO4) status. We previously reported that the hypo-PO4 ZEB1 binds to target genes more strongly than the hyper-PO4 protein and that the C-term Zn finger domain (ZD2) of ZEB1 contains key phosphorylation sites. Our aim is to identify the specific sites in ZD2 that regulate ZEB1biological role. We performed site-directed mutagenesis of the ZD2 sites with highest phosphorylation scores generating the mutants ZD2-1A (T904A), ZD2-1B (T904A, S932A), ZD2-2A (S895A), ZD2-2B (S895A, S947A, S951A), ZD2-2C (S895A, S947A, S951A, S961A), ZD2-3A (T851A, S852A, S853A), ZD2-3B (T851A, S852A, S853A, S857A) and ZD2-3C (T851A, S852A, S853A, S857A, T867A, T873A). EMSA and gene reporter assays were used to test the binding capacity and the biological role of the mutants. The mutants or the control ZD2 wt and the E-cadherin/ZEB1-luciferase promoters were transfected into CHO-K1 cells. The mutants ZD2-1A, ZD2-1B, ZD2-3A and ZD2-3C repressed luciferase more than ZD2 wt, while the mutants ZD2-2 showed no change in promoters activity compared to ZD2 wt. The treatment with a PKC activator (PMA/ionomycin) reverted the repression induced by both ZD2 wt and ZD2-2 mutants, but not the effect obtained with the other mutants indicating that key phosphorylation sites contained in the mutants ZD2-1A, B and -3A, C were made unresponsive to PMA/iono by mutagenesis. As expected, these unresponsive mutants weren’t able to increase their binding capacity to ZEB1 target promoters after treatment with alkaline phosphatase (CIP) in EMSAs. ZD2 wt and ZD2-2 mutants induced a bigger binding band after CIP treatment. Our results point out to 8 S/T residues in ZD2 domain as responsible for the transcriptional activity of ZEB1. ZEB1 (Zn Finger E-box Binding Homeobox) is a master regulator of Epithelial-Mesenchymal Transition, a critical program for tumor metastasis. ZEB1 activity can be regulated by changes in its phosphorylation (PO4) status. We previously reported that the hypo-PO4 ZEB1 binds to target genes more strongly than the hyper-PO4 protein and that the C-term Zn finger domain (ZD2) of ZEB1 contains key phosphorylation sites. Our aim is to identify the specific sites in ZD2 that regulate ZEB1biological role. We performed site-directed mutagenesis of the ZD2 sites with highest phosphorylation scores generating the mutants ZD2-1A (T904A), ZD2-1B (T904A, S932A), ZD2-2A (S895A), ZD2-2B (S895A, S947A, S951A), ZD2-2C (S895A, S947A, S951A, S961A), ZD2-3A (T851A, S852A, S853A), ZD2-3B (T851A, S852A, S853A, S857A) and ZD2-3C (T851A, S852A, S853A, S857A, T867A, T873A). EMSA and gene reporter assays were used to test the binding capacity and the biological role of the mutants. The mutants or the control ZD2 wt and the E-cadherin/ZEB1-luciferase promoters were transfected into CHO-K1 cells. The mutants ZD2-1A, ZD2-1B, ZD2-3A and ZD2-3C repressed luciferase more than ZD2 wt, while the mutants ZD2-2 showed no change in promoters activity compared to ZD2 wt. The treatment with a PKC activator (PMA/ionomycin) reverted the repression induced by both ZD2 wt and ZD2-2 mutants, but not the effect obtained with the other mutants indicating that key phosphorylation sites contained in the mutants ZD2-1A, B and -3A, C were made unresponsive to PMA/iono by mutagenesis. As expected, these unresponsive mutants weren’t able to increase their binding capacity to ZEB1 target promoters after treatment with alkaline phosphatase (CIP) in EMSAs. ZD2 wt and ZD2-2 mutants induced a bigger binding band after CIP treatment. Our results point out to 8 S/T residues in ZD2 domain as responsible for the transcriptional activity of ZEB1.