Huang Guocun, Chen She, Li Shaojie, Cha Joonseok, Long Chengzu, Li Lily, He Qiyang, Liu Yi
Department of Physiology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA;
Genes Dev. 2007 Dec 15;21(24):3283-95. doi: 10.1101/gad.1610207.
Regulation of circadian clock components by phosphorylation plays essential roles in clock functions and is conserved from fungi to mammals. In the Neurospora circadian negative feedback loop, FREQUENCY (FRQ) protein inhibits WHITE COLLAR (WC) complex activity by recruiting the casein kinases CKI and CKII to phosphorylate the WC proteins, resulting in the repression of frq transcription. On the other hand, CKI and CKII progressively phosphorylate FRQ to promote FRQ degradation, a process that is a major determinant of circadian period length. Here, by using whole-cell isotope labeling and quantitative mass spectrometry methods, we show that the WC-1 phosphorylation events critical for the negative feedback process occur sequentially-first by a priming kinase, then by the FRQ-recruited casein kinases. We further show that the cyclic AMP-dependent protein kinase A (PKA) is essential for clock function and inhibits WC activity by serving as a priming kinase for the casein kinases. In addition, PKA also regulates FRQ phosphorylation, but unlike CKI and CKII, PKA stabilizes FRQ, similar to the stabilization of human PERIOD2 (hPER2) due to the phosphorylation at the familial advanced sleep phase syndrome (FASPS) site. Thus, PKA is a key clock component that regulates several critical processes in the circadian negative feedback loop.
磷酸化对生物钟组件的调控在生物钟功能中起着至关重要的作用，并且从真菌到哺乳动物都保守存在。在粗糙脉孢菌的昼夜节律负反馈回路中，频率（FRQ）蛋白通过招募酪蛋白激酶CKI和CKII对白领（WC）复合物蛋白进行磷酸化，从而抑制WC复合物的活性，导致frq转录受到抑制。另一方面，CKI和CKII逐步磷酸化FRQ以促进FRQ降解，这一过程是昼夜节律周期长度的主要决定因素。在这里，通过使用全细胞同位素标记和定量质谱方法，我们表明对负反馈过程至关重要的WC-1磷酸化事件是依次发生的——首先由引发激酶作用，然后由FRQ招募的酪蛋白激酶作用。我们进一步表明，环磷酸腺苷依赖性蛋白激酶A（PKA）对生物钟功能至关重要，并且通过作为酪蛋白激酶的引发激酶来抑制WC活性。此外，PKA还调节FRQ磷酸化，但与CKI和CKII不同，PKA使FRQ稳定，类似于由于家族性早睡综合征（FASPS）位点的磷酸化导致的人类周期蛋白2（hPER2）的稳定。因此，PKA是调节昼夜节律负反馈回路中几个关键过程的关键生物钟组件。
