Requirement of ATM for Rapid p53 Phosphorylation at Ser46 without Ser/Thr-Gln Sequences
Requirement of ATM for Rapid p53 Phosphorylation at Ser46 without Ser/Thr-Gln Sequences
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DOI：
10.1128/mcb.00810-09
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发表时间：
2010-04-01
影响因子：
5.3
通讯作者：
Taya, Yoichi
中科院分区：
文献类型：
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作者：
Kodama, Masami;Otsubo, Chihiro;Taya, Yoichi
DNA损伤后p53在Ser 46的磷酸化对于促凋亡基因的优先反式激活是重要的。在这里，我们报告，共济失调毛细血管扩张突变（ATM）激酶是负责Ser 46磷酸化的p53在早期阶段的DNA损伤的反应。为了阐明ATM对p53丝氨酸46位的直接磷酸化作用，设计了一种对ATP类似物敏感的ATM突变体（ATM-AS）。体外激酶测定显示，p53被ATM-AS在Ser 46处磷酸化，即使当ATP类似物被用作磷酸供体时也是如此，尽管该磷酸化位点不在SQ基序中，这是一个共识ATM位点。此外，Ser 46的ATM磷酸化依赖于N-和C-末端结构域的p53，不像Ser 15磷酸化。免疫荧光分析表明，Ser 46-磷酸化的p53被观察到作为响应于DNA损伤的病灶，并与γ-H2 AX或Ser 1981-磷酸化的ATM共定位。这些结果表明，ATM磷酸化p53上的一个非典型丝氨酸残基的机制不同于Ser 15的磷酸化。
p53 phosphorylation at Ser46 following DNA damage is important for preferential transactivation of proapoptotic genes. Here, we report that ataxia-telangiectasia mutated (ATM) kinase is responsible for Ser46 phosphorylation of p53 during early-phase response to DNA damage. To elucidate the direct phosphorylation of p53 at Ser46 by ATM, an ATM mutant (ATM-AS) sensitive to ATP analogues was engineered. In vitro kinase assays revealed that p53 was phosphorylated at Ser46 by ATM-AS, even when ATP analogues were used as phosphate donors, although this phosphorylation site is not in an SQ motif, a consensus ATM site. Furthermore, Ser46 phosphorylation by ATM was dependent on the N- and C-terminal domains of p53, unlike Ser15 phosphorylation. Immunofluorescence analyses showed that Ser46-phosphorylated p53 was observed as foci in response to DNA damage and colocalized with gamma-H2AX or Ser1981-phosphorylated ATM. These results suggest that ATM phosphorylates a noncanonical serine residue on p53 by mechanisms different from those for the phosphorylation of Ser15.