IDENTIFICATION OF P19INK4D PHOSPHORYLATION SITES INVOLVED IN DNA REPAIR FUNCTION

MARAZITA MC; CASTILLO DS; PIGNATARO OP; CÁNEPA ET

San Miguel de Tucumán

Congreso; XLV Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2009

SAIB

We have reported that p19INK4d (p19), a member of INK4 cell cycle inhibitors, has a role in DNA repair and becomes phosphorylated upon DNA damage. We identified putative phosphorylation sites replacing serine or threonine by alanine in predicted sites. We found two mutations that abrogates p19 DNA repair capacity. In this work, we aimed to study first the phosphorylation ability of other three p19INK4d mutants and second to test if the replacement of serine or threonine by glutamic acid, in those mutants that lose the DNA repair function, can mimic the phosphorylation event and recall DNA repair ability. p19 mutants were made replacing serine or threonine by alanine in predicted sites. To evaluate in vivo phosphorylation we performed metabolic labeling with 32P. We found that none of the mutants containing alanine diminishes p19 phosphorylation level concluding that this sites aren´t phosphorylated upon DNA damage. As for the mutant bearing glutamic acid in both sites linked to DNA repair, the phosphorylation event was entirely supressed. However, this mutant didn´t lose its DNA repair ability as measured by UDS. We conclude that glutamic acid mimics the phosphorylation state of p19 after DNA damage, that these are the only two phosphorylation sites upon DNA damage and the phosphorylation event is absolutely necessary for exerting an effective DNA repair function.