Cell-based inhibitor screening identifies multiple protein kinases important for circadian clock oscillations.
Cell-based inhibitor screening identifies multiple protein kinases important for circadian clock oscillations.
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DOI：
10.4161/19420889.2014.982405
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发表时间：
2015-07
影响因子：
--
通讯作者：
Fukada Y
中科院分区：
文献类型：
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作者：
Kon N;Sugiyama Y;Yoshitane H;Kameshita I;Fukada Y
生物钟的分子振荡是基于E-box介导的生物钟基因及其编码产物生物钟蛋白形成的转录反馈环。生物钟蛋白受翻译后修饰如磷酸化的调节。我们研究了一系列激酶抑制剂对Rat-1成纤维细胞基因表达节律的影响。用SB 203580（p38 MAPK抑制剂）、SP 600125（JNK抑制剂）、IC 261（CKI抑制剂）和Roscovitine（CDK抑制剂）处理培养的细胞昼夜节律的时间延长。另一方面，通过SB 216763（GSK-3抑制剂）或KN 93（CaMKII抑制剂）处理，该时间缩短。应用20 μM KN 93完全消除了节律基因的表达。CaMKII的活性在接近E盒介导的转录节律的阶段中表现出昼夜变化。体外激酶活性测定显示CaMK Ⅱ直接磷酸化CLOCK的N-末端和富含Ser/Pro的结构域，CLOCK是E-box介导的转录激活因子。这些结果表明，磷酸化依赖的调谐周期长度的调节网络的多种激酶，并揭示了一个重要的作用，CaMKII在细胞振荡机制。
Molecular oscillation of the circadian clock is based on E-box-mediated transcriptional feedback loop formed with clock genes and their encoding products, clock proteins. The clock proteins are regulated by post-translational modifications such as phosphorylation. We investigated the effects of a series of kinase inhibitors on gene expression rhythms in Rat-1 fibroblasts. The period of the cellular circadian rhythm in culture was lengthened by treatment with SB203580 (p38 MAPK inhibitor), SP600125 (JNK inhibitor), IC261 (CKI inhibitor) and Roscovitine (CDK inhibitor). On the other hand, the period was shortened by SB216763 (GSK-3 inhibitor) or KN93 (CaMKII inhibitor) treatment. Application of 20 μM KN93 completely abolished the rhythmic gene expression. The activity of CaMKII exhibited circadian variation in a phase close to the E-box-mediated transcriptional rhythms. In vitro kinase assay revealed that CaMKII directly phosphorylates N-terminal and Ser/Pro-rich domains of CLOCK, an activator of E-box-mediated transcription. These results indicate a phosphorylation-dependent tuning of the period length by a regulatory network of multiple kinases and reveal an essential role of CaMKII in the cellular oscillation mechanism.