DNA double-strand break formation upon UV-induced replication stress activates ATM and DNA-PKcs kinases.
The phosphatidylinositol 3-kinase-like protein kinases, including ATM (ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3 related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit), are the main kinases activated following various assaults on DNA. Although ATM and DNA-PKcs kinases are activated upon DNA double-strand breaks, evidence suggests that these kinases are rapidly phosphorylated by ATR kinase upon UV irradiation; thus, these kinases may also participate in the response to replication stress. Using UV-induced replication stress, we further characterize whether ATM and DNA-PKcs kinase activities are also involved in the cellular response. Contrary to the rapid activation of the ATR-dependent pathway, ATM-dependent Chk2 and KAP-1 phosphorylations, as well as DNA-PKcs Ser2056 autophosphorylation, reach their peak level at 4 to 8 h after UV irradiation. The delayed kinetics of ATM- and DNA-PKcs-dependent phosphorylations also correlated with a surge in H2AX phosphorylation, suggesting that double-strand break formation resulting from collapse of replication forks is responsible for the activation of ATM and DNA-PKcs kinases. In addition, we observed that some phosphorylation events initiated by ATR kinase in the response to UV were mediated by ATM at a later phase of the response. Furthermore, the S-phase checkpoint after UV irradiation was defective in ATM-deficient cells. These results suggest that the late increase of ATM activity is needed to complement the decreasing ATR activity for maintaining a vigilant checkpoint regulation upon replication stress.
Yajima
Hirohiko
H
Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, 2201 Inwood Road, Room NC7.502, Dallas, TX 75390-9187, USA.
Lee
Kyung-Jong
KJ
Zhang
Shichuan
S
Kobayashi
Junya
J
Chen
Benjamin P C
BP
D064007:Ataxia Telangiectasia Mutated Proteins; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D004247:DNA; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases