Serine-76 phosphorylation and the fourth ankyrin domain are involved in p19INK4d DNA repair activity

MARIELA C. MARAZITA; PABLO F. SIRKIN; OMAR O. PIGNATARO; EDUARDO T. CÁNEPA

Rosario, Provincia de Santa Fé

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

CB-P76 SERINE 76 PHOSPHORYLATION AND THE FOURTH ANKYRIN DOMAIN ARE INVOLVED IN p19INK4d DNA REPAIR ACTIVITY Marazita, Mariela C.; Sirkin, Pablo F.; Pignataro, Omar P.; Cánepa, Eduardo T. Laboratorio de Biología Molecular, Depto. Química Biológica, FCEN, UBA, Buenos Aires, Argentina E-mail: marazita@qb.fcen.uba.ar We have previously reported that p19INK4d (p19), a member of INK4 cell cycle inhibitors, enhances DNA repair ability and increases the capacity to survive after UV damage. However, the mechanism by which p19 confers this ability remains unknow. p19 has 4 putative serine phosphorylation sites, 2 of which, serine (ser) 66 and ser76, were found to be phosphorylated in vivo. Besides, p19 has 4 ankyrin domains, the fourth (ANK4) markedly differs in DNA sequece from the other 3 INK4 family members. This work aims to study if p19 phosphorylation at ser 66 and/or 76 are required to increase DNA repair activity upon DNA damage and if the ANK4 has a role in this process as well. First, we made a time course phosphorylation curve applying UV, cisplatin and beta amyloid protein as damaging agents. The three types of damages promotes p19 phosphorylation beginning within 2hs upon damage. Four p19 mutations were made, replacing ser for alanine at ser66 or ser76 or at both sites and truncating the last ankyrin domain. For all of them, cell cycle arrest ability, DNA repair capacity and caspase activity were assayed. We conclude that ser76 but not ser66 is necessary to increase DNA repair activity and diminish apoptosis although neither of them are involved in cell cycle arrest ability. As for ANK4, it´s found to be essential to enhance DNA repair but also to exert an efficient cell cycle arrest.