Site-Selective Tyrosine Phosphorylation in the Activation of the p50 Subunit of NF-κB for DNA Binding and Transcription.
Site-Selective Tyrosine Phosphorylation in the Activation of the p50 Subunit of NF-κB for DNA Binding and Transcription.
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位点选择性酪氨酸磷酸化激活 NF-κB 的 p50 亚基以实现 DNA 结合和转录。
DOI：
10.1021/acschembio.2c00678
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发表时间：
2023
影响因子：
4
通讯作者：
Hecht,SidneyM
中科院分区：
文献类型：
--
作者：
Chen,Shengxi;Ji,Xun;Dedkova,LarisaM;Potuganti,GalReddy;Hecht,SidneyM
核因子-κB转录激活剂家族控制着许多基因的表达，包括那些与细胞生存和发育有关的基因。该家族由五个亚基组成的同源和异源二聚体组成。P50亚基包括13个酪氨酸残基，但特异性酪氨酸磷酸化与p50功能之间的关系尚不清楚。体外制备的p50和p65亚基形成异源二聚体，但该NF-κB不与白细胞介素2(IL-2)启动子结合。用三磷酸鸟苷(GTP)和Jurkat细胞裂解液处理p50，可使p50迅速磷酸化，在野生型p65存在的情况下，同时伴随着IL-2启动子DNA的结合。修饰后的p50含有七种化学计量的磷酸化酪氨酸中的一种，在NF-κB p50/p65异源二聚体中，包括三种，比野生型更容易与IL-2DNA启动子区域结合。这三个化学计量比的NF-κB的p50/p65异源二聚体中的一个，在p50亚基中包含pTyr60，用激活的Jurkat细胞的裂解物+GTP处理，并被证明在与野生型p50相同的时间尺度上被磷酸化。这种修饰的NF-κB还具有与野生型相同的时间尺度的IL-2启动子结合活性，但与IL-2DNA启动子的结合比野生型更强。使用代谢稳定的pTyr衍生物(位于p50的第60位)和细胞磷酸酶更详细地研究了这种增强结合的性质。我们认为，p50亚基中含有pTyr60的修饰的NF-κB的DNA结合增强可能反映了化学计量比的NF-κB的磷酸化，该位点通常不完全磷酸化，或者根本不磷酸化，并且相对抵抗Jurkat细胞酪氨酸磷酸酶活性的影响。这一结论得到了证实，用代谢稳定的亚甲基膦酸部分修饰p50的Tyr60进一步增加了形成的NF-κB p50/p65异源二聚体对抗激活的Jurkat细胞磷酸酶作用的稳定性。
The family of NF-κB transcriptional activators controls the expression of many genes, including those involved in cell survival and development. The family consists of homo- and heterodimers constituted by combinations of five subunits. Subunit p50 includes 13 tyrosine residues, but the relationship between specific tyrosine phosphorylations and p50 function is not well understood. Subunits of p50 and p65 prepared in vitro formed a heterodimer, but this NF-κB would not bind to the interleukin-2 (IL-2) promoter DNA. Treatment of p50 with guanosine triphosphate (GTP) and a lysate from activated Jurkat cells, effected rapid p50 phosphorylation, and, in the presence of wild-type subunit p65, was accompanied on the same time scale by IL-2 promoter DNA binding. Modified p50s containing one of seven stoichiometrically phosphorylated tyrosines in NF-κB p50/p65 heterodimers, included three that facilitated binding to the IL-2 DNA promoter region to a greater extent than the wild type. One of these three stoichiometrically phosphorylated p50/p65 heterodimers of NF-κB, containing pTyr60 in the p50 subunit, was treated with a lysate from activated Jurkat cells + GTP and shown to be phosphorylated on the same time scale as wild-type p50. This modified NF-κB also developed IL-2 promoter DNA binding activity on the same time scale as the wild type but exhibited greater binding to the IL-2 DNA promoters than the wild type. The nature of this enhanced binding was studied in greater detail using a metabolically stable pTyr derivative at position 60 of p50 and cellular phosphatases. We suggest that enhanced DNA binding of modified NF-κB containing pTyr60 in the p50 subunit may reflect stoichiometric NF-κB phosphorylation at a site that is not normally fully phosphorylated, or not phosphorylated at all, and is relatively resistant to the effects of Jurkat cell tyrosine phosphatase activity. This conclusion was reinforced by demonstrating that modification of Tyr60 of p50 with a metabolically stable methylenephosphonate moiety further increased the stability of the formed NF-κB p50/p65 heterodimer against the action of activated Jurkat cell phosphatases.
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