p19INK4d function linked to DNA repair relies on sequential phosphorylation

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

Foz do Iguaçu, PR, Brazil

Congreso; XXXIX ANNUAL MEETING OF THE BRAZILIAN BIOCHEMISTRY AND MOLECULAR BIOLOGY SOCIETY (SBBQ); 2010

BRAZILIAN BIOCHEMISTRY AND MOLECULAR BIOLOGY SOCIETY (SBBQ)

p19INK4d (p19), a member of INK4 cell cycle inhibitors, plays a role in DNA repairand becomes phosphorylated upon DNA damage (UV - beta-amyloid peptide). Weaimed to evaluate first the involvement of predicted p19 phosphorylation sites in itsDNA repair activity, second to demonstrate the phosphorylation event in thosesites, and last to establish the subcellular localization of the phosphorylationprocess. p19 mutants were made replacing Ser or Thr by Ala (A) at S13, S66, S76,T89, T141. Wi-38 cells were transfected with the mutants and cell cycle arrestability by 3H-timidine incorporation, DNA repair capacity by UDS and apoptosis bycaspase-3 activity were assayed. Although none of the mutated sites are involvedin cell cycle arrest capacity, S76 and T141 are necessary for p19 DNA repairfunction. Mutation of S76 and T141 to glutamic acid (E) to mimic phosphorylationleads to the recovery of the function.To evaluate in vivo phosphorylation of thesemutants we performed metabolic labeling with 32P-orthophosphate. While S76Aentirely supressed phosphorylation, T141A didn´t completely abrogated. Themutant S76E retrieves the phosphorylation potential. p19 translocates into thenucleus after genotoxic treatment.We found that p19 phosphorylation begins in thecytoplasm. These results show p19 is sequentially phosphorylated at S76 and thenat T141 and this events results essential for exerting an effective DNA repairfunction.