Serine phosphorylation of mCRY1 and mCRY2 by mitogen-activated protein kinase
Serine phosphorylation of mCRY1 and mCRY2 by mitogen-activated protein kinase
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DOI：
10.1111/j.1356-9597.2004.00758.x
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发表时间：
2004-08-01
期刊：
影响因子：
2.1
通讯作者：
Fukada, Y
中科院分区：
文献类型：
--
作者：
Sanada, K;Harada, Y;Fukada, Y
昼夜节律振荡器由一个基于转录/翻译的自动调节反馈环组成，其中隐花色素和周期作为其自身基因表达的负调节因子。尽管这些调节子的磷酸化等翻译后修饰似乎对昼夜节律机制至关重要，但对负责任的蛋白激酶及其对调节器功能的贡献知之甚少。我们发现丝裂原活化蛋白激酶(MAPK)与小鼠隐花色素(mCRY1和mCRY2)结合并使其磷酸化。质谱分析表明，mCRY2的Ser265和Ser557为体外磷酸受体残基。Ser残基和Ala残基的突变完全取消了MAPK介导的mCRY2的磷酸化，表明这两个残基是mCRY2的主要磷酸化位点。类似地，MAPK在Ser247处磷酸化mCRY1，该位置对应于mCRY2的Ser265。通过将mCRY1的Ser247和mCRY2的Ser265突变为Asp来研究Ser磷酸化的影响，这导致每个mCRY抑制BMAL1：时钟介导的转录的能力减弱，而mCRY2的Ser557位类似的突变没有引起其活性的明显变化。这些结果说明了MAPK通过特异性Ser残基的磷酸化来负调控mCRY功能的模型。
The circadian oscillator is composed of a transcription/translation-based autoregulatory feedback loop in which Cryptochromes and Periods function as negative regulators for their own gene expression. Although post-translational modifications such as phosphorylation of these regulators appear crucial for circadian time-keeping mechanism, less is known about responsible protein kinases and their contribution to the function of the regulators. We found that mitogen-activated protein kinase (MAPK) associates with and phosphorylates mouse Cryptochromes (mCRY1 and mCRY2). Mass spectrometry analysis identified Ser265 and Ser557 of mCRY2 to be in vitro phospho-acceptor residues. Mutations of both the Ser residues to Ala completely abolished MAPK-mediated mCRY2 phosphorylation, suggesting that the two residues are the principal phosphorylation sites in mCRY2. Similarly, MAPK phosphorylates mCRY1 at Ser247, a site corresponding to Ser265 of mCRY2. An effect of the Ser phosphorylation was investigated by mutating Ser247 of mCRY1 and Ser265 of mCRY2 to Asp, which resulted in attenuation of each mCRYs' ability to inhibit BMAL1: CLOCK-mediated transcription, whereas a similar mutation at Ser557 of mCRY2 induced no measurable change in its activity. These results illustrate a model of MAPK-mediated negative regulation of mCRY function by phosphorylation at the specific Ser residue.