Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY, USA.
Cell Cycle. 2009 Dec 15;8(24):4138-46. doi: 10.4161/cc.8.24.10273. Epub 2009 Dec 8.
The circadian clock regulates biological processes from gene expression to organism behavior in a precise, sustained rhythm that is generated at the unicellular level by coordinated function of interlocked transcriptional feedback loops and post-translational modifications of core clock proteins. CLOCK phosphorylation regulates transcriptional activity, cellular localization and stability; however little is known about the specific residues and enzymes involved. We have identified a conserved cluster of serines that include, Ser431, which is a prerequisite phosphorylation site for the generation of BMAL dependent phospho-primed CLOCK and for the potential GSK-3 phosphorylation at Ser427. Mutational analysis and protein stability assays indicate that this serine cluster functions as a phospho-degron. Through the use of GSK-3 activators/inhibitors and kinase assays, we demonstrate that GSK-3beta regulates the degron site by increasing CLOCK phosphorylation/degradation, which correlates with an increase in the expression of CLOCK responsive promoters. Stabilization of phospho-deficient CLOCK delays the phase of oscillation in synchronized fibroblasts. This investigation begins the characterization of a complex phospho-regulatory site that controls the activity and degradation of CLOCK, a core transcription factor that is essential for circadian behavior.
生物钟以精确、持续的节奏调节从基因表达到生物体行为的生物过程，这种节奏是在单细胞水平上通过协调的转录反馈环和核心生物钟蛋白的翻译后修饰的功能产生的。CLOCK 磷酸化调节转录活性、细胞定位和稳定性；然而，关于涉及的特定残基和酶知之甚少。我们已经确定了一个保守的丝氨酸簇，包括 Ser431，它是产生 BMAL 依赖性磷酸化启动的 CLOCK 和潜在 GSK-3 在 Ser427 磷酸化的必要磷酸化位点。突变分析和蛋白质稳定性测定表明，这个丝氨酸簇作为一个磷酸化降解物发挥作用。通过使用 GSK-3 激活剂/抑制剂和激酶测定，我们证明 GSK-3β 通过增加 CLOCK 磷酸化/降解来调节降解物位点，这与 CLOCK 反应启动子表达的增加相关。磷酸化缺陷型 CLOCK 的稳定化延迟了同步成纤维细胞中振荡的相位。这项研究开始了对一个复杂的磷酸化调节位点的表征，该位点控制着 CLOCK 的活性和降解，CLOCK 是生物钟行为所必需的核心转录因子。
