A Novel p53 Phosphorylation Site within the MDM2 Ubiquitination Signal II. A MODEL IN WHICH PHOSPHORYLATION AT SER269 INDUCES A MUTANT CONFORMATION TO p53
A Novel p53 Phosphorylation Site within the MDM2 Ubiquitination Signal II. A MODEL IN WHICH PHOSPHORYLATION AT SER269 INDUCES A MUTANT CONFORMATION TO p53
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DOI：
10.1074/jbc.m110.143107
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发表时间：
2010-11-26
影响因子：
4.8
通讯作者：
Hupp, Ted R.
中科院分区：
文献类型：
--
作者：
Fraser, Jennifer A.;Madhumalar, Arumugam;Hupp, Ted R.
p53 DNA结合域具有构象灵活的多蛋白结合位点，调节p53泛素化。一个新的磷酸化位点存在于该区域内的Ser（269），其磷酸化模拟突变使p53失活。拟磷酸化p53（S269 D）表现出突变型p53的特征：体内稳定结合HSP 70，体内泛素化水平升高，DNA结合和转录无活性，使用热位移测定增加的热不稳定性，以及在403 nm而不是346 nm处的内源性色氨酸荧光λ（max），这是野生型p53的特征。这些数据表明，p53构象稳定性是由一个磷酸受体网站内暴露的灵活的表面环，这可以通过磷酸化不稳定。为了测试p53内的其他基序是否也类似地进化，我们分析了Ser（215）突变对p53功能的影响，因为Ser（215）是构象柔性PAb 240表位中的另一个失活磷酸化位点。p53（S215 D）蛋白与p53（S269 D）一样无活性，而p53（S215 A）与p53（S269 A）一样有活性。然而，双突变体p53（S215 A/S269 A）是转录失活和更热不稳定比任何一个单独的Ser-Ala环突变。分子动力学模拟表明（i）磷酸-Ser（215）和磷酸-Ser（269）被带正电荷的残基或溶剂水溶剂化导致局部解折叠，这伴随着N-末端环的局部不稳定和p53的全局不稳定，和（ii）双丙氨酸215/269突变破坏通常由Ser（215）和Ser（269）两者稳定的氢键。这些数据表明，p53在构象灵活的表位内进化出两个丝氨酸磷酸受体残基，这些表位通常稳定p53 DNA结合结构域，但其磷酸化
The p53 DNA-binding domain harbors a conformationally flexible multiprotein binding site that regulates p53 ubiquitination. A novel phosphorylation site exists within this region at Ser(269), whose phosphomimetic mutation inactivates p53. The phosphomimetic p53 (S269D) exhibits characteristics of mutant p53: stable binding to Hsp70 in vivo, elevated ubiquitination in vivo, inactivity in DNA binding and transcription, increased thermoinstability using thermal shift assays, and lambda(max) of intrinsic tryptophan fluorescence at 403 nm rather than 346 nm, characteristic of wild type p53. These data indicate that p53 conformational stability is regulated by a phosphoacceptor site within an exposed flexible surface loop and that this can be destabilized by phosphorylation. To test whether other motifs within p53 have similarly evolved, we analyzed the effect of Ser(215) mutation on p53 function because Ser(215) is another inactivating phosphorylation site in the conformationally flexible PAb240 epitope. The p53(S215D) protein is inactive like p53(S269D), whereas p53(S215A) is as active as p53(S269A). However, the double mutant p53(S215A/S269A) was transcriptionally inactive and more thermally unstable than either individual Ser-Ala loop mutant. Molecular dynamics simulations suggest that (i) solvation of phospho-Ser(215) and phospho-Ser(269) by positive charged residues or solvent water leads to local unfolding, which is accompanied by local destabilization of the N-terminal loop and global destabilization of p53, and (ii) the double alanine 215/269 mutation disrupts hydrogen bonding normally stabilized by both Ser(215) and Ser(269). These data indicate that p53 has evolved two serine phosphoacceptor residues within conformationally flexible epitopes that normally stabilize the p53 DNA-binding domain but whose phosphorylation