Phosphorylation or Mutation of the ERK2 Activation Loop Alters Oligonucleotide Binding
Phosphorylation or Mutation of the ERK2 Activation Loop Alters Oligonucleotide Binding
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DOI：
10.1021/acs.biochem.6b00096
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发表时间：
2016-03-29
期刊：
影响因子：
2.9
通讯作者：
Cobb, Melanie H.
中科院分区：
文献类型：
--
作者：
McReznolds, Andrea C.;Karra, Aroon S.;Cobb, Melanie H.
丝裂原激活的蛋白激酶ERK2能够对不同的刺激引起广泛的上下文特异性反应，但这种多功能性背后的机制仍然存在疑问。ERK2的一些细胞功能是通过调节基因表达来实现的。除了使许多转录调节因子磷酸化外，ERK2已知还与染色质有关，并已被证明直接与寡核苷酸结合。ERK2被上游激酶MEK1/2激活，MEK1/2磷酸化酪氨酸185和苏氨酸183：ERK2需要这两个位点的磷酸化才能完全激活。一些额外的ERK2磷酸化位点也被报道，包括苏氨酸188。有人认为这种磷酸盐形式具有不同的性质。我们通过质谱仪检测到ERK2的细菌制剂中T188上的一些ERK2被磷酸化，进一步证明了该ERK2残基的拟磷取代会削弱其对明确底物的激酶活性，并影响其与DNA的结合。我们使用来自胰岛素基因启动子和其他区域的寡核苷酸的凝胶迁移率改变分析来检测磷酸化和突变对ERK2与DNA结合的影响。我们发现ERK2可以直接与寡核苷酸结合。磷酸化和突变改变了DNA的结合，并支持信号功能可能通过另一个磷酸化位点受到影响的观点。
The mitogen-activated protein kinase ERK2 is able to elicit a wide range of context-specific responses to distinct stimuli, but the mechanisms underlying this versatility remain in question. Some cellular functions of ERK2 are mediated through regulation of gene expression. In addition to phosphorylating numerous transcriptional regulators, ERK2 is known to associate with chromatin and has been shown to bind oligonucleotides directly. ERK2 is activated by the upstream kinases MEK1/2, which phosphorylate both tyrosine 185 and threonine 183: ERK2 requires phosphorylation on both sites to be fully active. Some additional ERK2 phosphorylation sites have also been reported, including threonine 188. It has been suggested that this phospho form has distinct properties. We detected some ERK2 phosphorylated on T188 in bacterial preparations of ERK2 by mass spectrometry and further demonstrate that phosphomimetic substitution of this ERK2 residue impairs its kinase activity toward well-defined substrates and also affects its DNA binding. We used electrophoretic mobility shift assays with oligOnucleotides derived from the insulin gene promoter and other regions to examine effects of phosphorylation and mutations on the binding of ERK2 to DNA. We show that ERK2 can bind oligonucleotides directly. Phosphorylation and mutations alter DNA binding and support the idea that signaling functions may be influenced through an alternate phosphorylation site.