p19INK4d function linked to DNA repair relies on sequential phosphorylation

MARAZITA M.C.; OGARA M.F.; SONZOGNI S. V.; PIGNATARO O.; CÁNEPA, E.T.

Anaheim

Congreso; ANNUAL MEETING OF THE AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY (ASBMB); 2010

THE AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY (ASBMB)

p19INK4d(p19), a member of INK4 cell cycle inhibitors, plays a role in DNA repair and becomes phosphorylated upon DNA damage. We aimed to evaluate first the involvement of predicted p19 phosphorylation sites in its DNA repair activity, second to demonstrate the phosphorylation event in those sites, and last to establish the subcellular localization of the phosphorylation process. p19 mutants were made replacing Ser or Thr by Ala at S13, S66, S76, T89, T141. Wi-38 cells were transfected with the mutants and cell cycle arrest ability by 3H-timidine incorporation, DNA repair capacity by UDS and apoptosis by caspase-3 activity were assayed. Although none of the mutated sites are involved in cell cycle arrest capacity, S76 and T141 are necessary for p19 DNA repair function. Mutation of S76 and T141 to glutamic acid to mimic phosphorylation leads to the recovery of the function. To evaluate in vivo phosphorylation of these mutants we performed metabolic labeling with 32P-orthophosphate. While S76A entirely supressed phosphorylation, T141A didn’t completely abrogated. The mutant S76E retrieves the phosphorylation potential. p19 translocates into the nucleus after genotoxic treatment. We found that p19 phosphorylation begins in the citoplasm. These results show p19 is sequentially phosphorylated starting in the cytoplasm and being necessary for exerting an effective DNA repair function.