Hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites
Hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites
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DOI：
10.1152/ajplung.00203.2002
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发表时间：
2004-01-01
影响因子：
4.9
通讯作者：
Dashnamoorthy, R
中科院分区：
文献类型：
--
作者：
Das, KC;Dashnamoorthy, R
高氧已被证明会导致DNA损伤，导致细胞在p53依赖性和p53非依赖性途径中的生长停滞。虽然H2 O2和其他过氧化物已被证明诱导共济失调毛细血管扩张突变（ATM）依赖性p53磷酸化的DNA损伤，响应高氧的信号转导机制目前尚不清楚。在这里，我们表明，高氧磷酸化的Ser 15残基的p53独立的ATM。在DNA依赖性蛋白激酶空（DNA-PK-/-）细胞中，高氧使p53（Ser 15）磷酸化，表明p53（Ser 15）的磷酸化可能不依赖于DNA-PK。我们发现，Ser 37和Ser 392残基的p53也磷酸化的ATM独立的方式在高氧。相反，H2 O2在ATM（+/+）或ATM（-/-）细胞中不磷酸化Ser 37。此外，H_2O_2不能磷酸化ATM（-/-）细胞中的Ser 15。此外，与仅转染载体的细胞相比，HEK 293 T细胞中激酶失活的ATM和Rad 3相关（ATR）的过表达减少了Ser 15，Ser 37和Ser 392的磷酸化。与此相反，野生型ATR过表达并没有减少Ser 15，Ser 37，或Ser 392磷酸化。我们还表明，检查点激酶1（Chk 1）的丝氨酸345磷酸化反应高氧，这可以抑制咖啡因或渥曼青霉素，磷酸肌醇3-激酶相关激酶的有效抑制剂。高氧也使ATM（+/+）和ATM（-/-）细胞中的Chk 1磷酸化，证明Chk 1磷酸化的ATM非依赖性机制。总之，我们的数据表明，高氧激活ATR-Chk 1通路和磷酸化p53在多个位点在ATM独立的方式，这是不同于其他形式的氧化应激，如H2 O2或紫外线。
Hyperoxia has been shown to cause DNA damage resulting in growth arrest of cells in p53-dependent, as well as p53-independent, pathways. Although H2O2 and other peroxides have been shown to induce ataxia telangiectasia-mutated (ATM)-dependent p53 phosphorylation in response to DNA damage, the signal transduction mechanisms in response to hyperoxia are currently unknown. Here we demonstrate that hyperoxia phosphorylates the Ser15 residue of p53 independently of ATM. Hyperoxia phosphorylated p53 ( Ser15) in DNA-dependent protein kinase null (DNA-PK-/-) cells, indicating that it may not depend on DNA-PK for phosphorylation of p53 ( Ser15). We show that Ser37 and Ser392 residues of p53 are also phosphorylated in an ATM-independent manner in hyperoxia. In contrast, H2O2 did not phosphorylate Ser37 in either ATM(+/+) or ATM(-/-) cells. Furthermore, H2O2 failed to phosphorylate Ser15 in ATM(-/-) cells. Additionally, overexpression of kinase-inactive ATM-and-Rad3-related (ATR) in HEK293T cells diminished Ser15, Ser37, and Ser392 phosphorylation compared with vector-only transfected cells. In contrast, wild-type ATR overexpression did not diminish Ser15, Ser37, or Ser392 phosphorylation. We also show that checkpoint kinase 1 (Chk1) is phosphorylated on Ser345 in response to hyperoxia, which could be inhibited by caffeine or wortmannin, potent inhibitors of phosphoinositide 3-kinase-related kinases. Hyperoxia also phosphorylated Chk1 in ATM(+/+) as well as in ATM(-/-) cells, demonstrating an ATM-independent mechanism in Chk1 phosphorylation. Together, our data suggest that hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites in an ATM-independent manner, which is different from other forms of oxidative stress such as H2O2 or UV light.