Keap1 is deemed as a suppressor of Nrf2 in cytoplasm by sequestrating Nrf2 to proteolysis as an adapter of the Cul3-Rbx1 E3 ubiquitin ligase complex. In the study, it was proposed that post-translational modification might affect the interaction between Nrf2 and Keap1, and the profiles of the phosphorylation of amino acid residues of Keap1 and its effects on the binding of Keap1 to Nrf2 was investigated. A mass spectrometry analysis revealed that S53 and S293 were phosphorylated upon an oxidative stress. Using Keap1 proteins with amino acid residues mutated to glutamate to simulate the introduction of a negative charge by phosphorylation, it was found that a potential phosphorylation of S53 affected Keap1-Nrf2 binding in the pull-down assay, and induced nuclear translocation of Nrf2 in the electrophoretic mobility shift assay. Sequence homology analysis showed that S53 was highly conserved. Structural modeling around BTB domain of wild type and S53E-mutant Keap1 showed that the negative charge introduced by S53E mutation generates a salt bridge between E53 and ionized guanidine group of Arg50. Real-time qRT-PCR for transcription levels of antioxidant genes that are modulated by Nrf2 further proved the effects of the potential phosphorylation of S53 under an oxidative stress condition.

In summary, S53 is a potential phosphorylation site of Keap1, and the phosphorylation could enhance the antioxidative capacity of cells in response to an oxidative stress.

Keap1通过将Nrf2隔离为蛋白水解物（作为Cul3-Rbx1 E3泛素连接酶复合体的衔接子）而被认为是细胞质中Nrf2的抑制剂。在这项研究中，有人提出翻译后修饰可能会影响Nrf2与Keap1之间的相互作用，并研究了Keap1氨基酸残基的磷酸化概况及其对Keap1与Nrf2结合的影响。质谱分析表明，S53和S293在氧化应激时被磷酸化。使用具有突变为谷氨酸的氨基酸残基的Keap1蛋白模拟通过磷酸化引入负电荷的过程，发现S53的潜在磷酸化影响了下拉测定中的Keap1-Nrf2结合，并诱导了Nrf2的核易位。电泳迁移率变动分析。序列同源性分析表明S53是高度保守的。野生型BTB结构域和S53E突变的Keap1周围的结构模型表明，由S53E突变引入的负电荷在E53和Arg50的离子化胍基之间形成盐桥。实时定量RT-PCR检测Nrf2调节的抗氧化剂基因的转录水平，进一步证明了在氧化应激条件下S53潜在磷酸化的作用。

总之，S53是Keap1的潜在磷酸化位点，磷酸化可以增强细胞对氧化应激的抗氧化能力。