“ZFHEP-1 AND -2 BINDING IS REGULATED BY PKC-INDUCED PHOSPHORYLATION”.

ARROYO D, KNUBEL C, MALDONADO N, CABANILLAS AM.

Pinamar

Congreso; XLI Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB).; 2005

Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB).

ZFHEP-1 AND -2 BINDING IS REGULATED BY PKC-INDUCED PHOSPHORYLATION Arroyo, Daniela, Maldonado, Noelia, Knubel, Carolina & Cabanillas, Ana María. CIBICI-CONICET, Facultad de CC QQ, UN Córdoba. daniarroyo@tutopia.com The activity of a transcription factor (TF) can be controlled, for instance by post-translational modification such as phosphorylation. Zfhep (Zinc Finger Homeodomain Enhancer-binding Protein) is a TF involved in lymphopoiesis, neurogenesis and myogenesis and it is expressed as two isoforms, Zfhep -1 and -2 which lacks the N-terminal DNA-binding domain of the larger Zfhep-1. Zfhep exists as two phosphorylated forms. Our goal was to examine the effect of phosphorylation on Zfhep binding to its promoters. Zfhep expressing cell lines were treated with 10 ng/ml phorbol esters (PMA) and 500 ng/ml ionomycin (IO) for 30 min. Nuclear extracts (NE) from untreated cells were incubated with calf intestinal phosphatase (CIP) or with CIP + phosphate. Band shift assays were performed with Jurkat/CHO-K1/COS-7 NE (CIP, CIP+phosphate or PMA/IO treated) and Zfhep-2 programmed rabbit reticulocyte lysates in the presence of [P]-labeled oligonucleotides harboring Zfhep binding sites from Zfhep, alfa4integrin, CD4 and p73 promoters. Probes and NE were incubated for 1 h at 20ºC. CIP- treated samples increased their binding capacity to all the probes assayed. Zfhep -1 and Zfhep -2 showed similar results. Retardation complexes were competed by either anti-Zfhep antibodies or an excess of cold probe. PMA/IO- treated cells shown no band of retardation. The results show that phosphorylation induced by PKC changes the affinity of Zfhep for its physiologically important target genes.