The highly conserved Chk2 kinase has a key role in delaying the cell cycle progression in response to DNA damage in eukaryotic cells. Using human lymphoblastoid and fibroblast cell lines we have characterized the time-dependent Chk2 phosphorylation/dephosphorylation kinetics in response to different doses and types of genotoxic agents. In normal, AT (Ataxia Telangiectasia) and AT-heterozygous cells exposed to low doses of gamma-radiation (4Gy), we have detected within 60min of treatment, two phosphorylated forms of Chk2, bearing different kinase activities in vitro to phosphorylate Cdc25C substrate. Both of these modifications depend on ATM (Ataxia Telangiectasia Mutant), since no phosphorylation is detectable in AT-derived cells negative for ATM protein. Chk2 phosphorylation and activation occurs even in AT-heterozygotes, hence indicating that ATM haploinsufficiency may not affect Chk2 response. We have additionally found that in response to high doses of gamma-radiation (50Gy), Chk2 still undergoes a two-step phosphorylation kinetics, but these modifications are clearly detectable also in ATM negative cells, indicating that a protein kinase other than ATM is activated by extensive DNA double strand breaks (DSBs). Using different doses of genotoxic agents, like UV, hydroxyurea and hydrogen peroxide, which induce different types of DNA injuries, we demonstrate the selective response of Chk2 to DSBs and the likely involvement of this protein not only in G2/M, but also in G1/S cell cycle checkpoint.
|Titolo:||ATM-dependent and -independent Chk2 phosphorylation, activation and cell cycle regulation in response to DNA damage|
|Data di pubblicazione:||2000|
|Settore Scientifico Disciplinare:||Settore BIO/11 - Biologia Molecolare|
|Citazione:||ATM-dependent and -independent Chk2 phosphorylation, activation and cell cycle regulation in response to DNA damage / G. Buscemi, C. Savio, L. Zannini, F. Miccichè, D. Masnada, M. Nakanishi, S. Mizutani, L. Chessa, D. Delia. ((Intervento presentato al 65. convegno Cold Spring Harbor Laboratory Symposium : Biological Response to DNA Damage tenutosi a Cold Spring Harbor nel 2000.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|