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1 Department of Molecular Biology, University of Texas Health Center at Tyler, Tyler, TX, USA
* To whom correspondence should be addressed. E-mail: kumuda.das{at}uthct.edu.
Hyperoxia has been shown to cause DNA damage resulting in growth arrest of cells in p53-dependent, as well as p53-independent pathway. Although H2O2 and other peroxides have been shown to induce ATM-dependent p53 phosphorylation in response to DNA damage, the signal transduction mechanisms in response to hyperoxia are currently unknown. In this report we demonstrate that hyperoxia phosphorylates ser-15 residue of p53 independent of ATM. ATM-/- cells induced p53 expression and ser-15 phosphorylation suggesting that ATM may not be required for hyperoxia-dependent p53 ser-15 phosphorylation. Hyperoxia phosphorylated p53(ser-15) in DNA-PK-/- cells indicating that it may not depend on DNA-PK for phosphorylation of p53 (ser-15). Additionally, we have shown that ser-37 and ser-392 residues of p53 are also phosphorylated in an ATM-independent manner in hyperoxia. In contrast, H2O2 did not phosphorylate ser-37 in either ATM+/+ or ATM-/- cells. Furthermore, H2O2 failed to phosphorylate ser-15 in ATM-/- cells. Hyperoxia-mediated p53 phosphorylation was inhibited by wortmannin, a potent inhibitor of PIKKs. Additionally, overexpression of kinase-inactive ATR in HEK293T cells diminished ser-15, ser-37 and ser-392 phosphorylation compared to vector only transfected cells. In contrast, wildtype ATR overexpression did not diminish ser-15, ser-37 or ser-392 phosphorylation. We have also shown that Checkpoint kinase 1 (Chk1) is phosphorylated on ser-345 in response to hyperoxia, which could be inhibited by caffeine or wortmannin, potent inhibitors of PIKKs. In addition, hyperoxia also phosphorylated Chk1 in ATM+/+ as well as in ATM-/- cells demonstrating an ATM-independent mechanism in Chk1 phosphorylation. In contrast, UV light or H2O2 did not phosphorylate Chk1 in ATM-/- cells demonstrating that ATM is required for UV light or H2O2 -mediated Chk1 phosphorylation. Taken together, our data suggest that hyperoxia activates ATR-Chk1 pathway, and phosphorylates p53 at multiple sites in an ATM-independent manner, which is different than other forms of oxidative stress such as H2O2 or UV light.
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