p19INK4d function linked to DNA repair requires ATM/ATR activation, serine 76 phosphorylation and the fourth ankyrin domain

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

Austral, Pilar, Buenos Aires, República Argentina

Congreso; 1st ANNUAL IBEROAMERICAN PROTEOMICS CONGRESS - LAHUPO; 2007

Latinoamerican Human Proteomics Organization (LAHUPO)

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. We also found out that p19 is subjected to phosphorylation upon genotoxic insult. However, if this phosphorylation step is implicated in the mechanism by which p19 participates in DNA repair remained unkown. p19 has 4 putative serine phosphorylation sites, two of which, serine66 and 76, were found to be phosphorylated in vivo. Besides, p19 has four ankyrin domains, the fourth(ANK4) markedly differs in DNA sequece from the others three family members. This work had two aims, the first one sought to characterize the signal transduction pathways leading to p19 phosphorylation and the second one, to study if  ANK4 or p19 phosphorylation at serine66 and/or 76 are related to its DNA repair activity. To begin with, a time-course phosphorylation curve applying UV, cisplatin or beta-amyloid protein as damaging agents was made. The three types of damages promoted p19 phosphorylation at early times. Immunoprecipitation assays following metabolic labeling with [32P]orthophosphate revealed that inhibition of ATM/ATR by caffeine (5mM) blocks p19 phosphorylation. Moreover, ATR or ATM specific inhibition by suitable concentrations of wortmanin and the use of specific inhibitors for CHK1 and CHK2, pointed out p19 phosphorylation after cisplatin damage depends on  ATR and CHK1 activity while beta-amyloid insult  phosphorylates p19 through ATM and CHK2 pathway. Concluding this part we found that PKA and CDKs enzymes which have consensus phosphoprylation sequences in p19, also  participate in this modification following the three agents assayed. To address the second goal, four p19 mutations were made, replacing serine for alanine at serine66 or 76 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 concluded that serine76 but not serine66 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Ls found to be essential to enhance DNA repair but also to exert an efficient cell cycle arrest.