Phosphorylation of the C-terminal sites of human p53 reduces non-sequence-specific DNA binding as modeled with synthetic peptides.
Phosphorylation of the C-terminal sites of human p53 reduces non-sequence-specific DNA binding as modeled with synthetic peptides.
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人 p53 C 末端位点的磷酸化减少了非序列特异性 DNA 结合，如合成肽建模。
DOI：
10.1021/bi980760a
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
OtvosJr,L
中科院分区：
文献类型：
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作者：
Hoffmann,R;Craik,DJ;Pierens,G;Bolger,RE;OtvosJr,L
肿瘤抑制因子P53的磷酸化通常被认为改变了该蛋白五个独立结构域中的四个的性质。我们使用合成肽直接研究了磷酸化对非序列特异性DNA结合和C末端碱性结构域构象的影响。这些多肽对应于3 6 1个−393氨基酸，在蛋白激酶C(PKC)的Ser378位或酪蛋白激酶II(CKII)的Ser392位处被非磷酸化或磷酸化，或在PKC和CKII上都是双磷酸化的。荧光偏振分析表明，重组P53蛋白或合成肽与两个不相关的靶DNA片段结合。多肽在PKC或CKII位点的磷酸化明显减少了DNA结合，而第二个磷酸基团的加入几乎完全取消了结合。圆二色谱表明，这些多肽在水溶液中具有相同的无序结构。与Ser378磷酸化肽不同，未修饰的多肽在DNA存在下改变了构象。通过圆二色谱和核磁共振波谱可以检测到这些多肽在三氟乙醇α水混合物中形成−-螺旋的固有能力。单磷酸化或双磷酸化使磷酸化的Ser378残基周围的螺旋不稳定，但稳定了序列下游的螺旋。
Phosphorylation of the tumor suppressor p53 is generally thought to modify the properties of the protein in four of its five independent domains. We used synthetic peptides to directly study the effects of phosphorylation on the non-sequence-specific DNA binding and conformation of the C-terminal, basic domain. The peptides corresponded to amino acids 361−393 and were either nonphosphorylated or phosphorylated at the protein kinase C (PKC) site, Ser378, or the casein kinase II (CKII) site, Ser392, or bis-phosphorylated on both the PKC and the CKII sites. A fluorescence polarization analysis revealed that either the recombinant p53 protein or the synthetic peptides bound to two unrelated target DNA fragments. Phosphorylation of the peptide at the PKC or the CKII sites clearly decreased DNA binding, and addition of a second phosphate group almost completely abolished binding. Circular dichroism spectroscopy showed that the peptides assumed identical unordered structures in aqueous solutions. The unmodified peptide, unlike the Ser378 phosphorylated peptide, changed conformation in the presence of DNA. The inherent ability of the peptides to form an α-helix could be detected when circular dichroism and nuclear magnetic resonance spectra were taken in trifluoroethanol−water mixtures. A single or double phosphorylation destabilized the helix around the phosphorylated Ser378 residue but stabilized the helix downstream in the sequence.