Wang Yimeng, Cao Siyu, Tone Daisuke, Fujishima Hiroshi, Yamada Rikuhiro G, Ohno Rei-Ichiro, Shi Shoi, Matsuzawa Kyoko, Yada Saori, Kaneko Mari, Sakamoto Hirokazu, Onishi Taichi, Ukai-Tadenuma Maki, Ukai Hideki, Hanashima Carina, Hirose Kenzo, Kiyonari Hiroshi, Sumiyama Kenta, Ode Koji L, Ueda Hiroki R
Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, Suita, Osaka, Japan.
Nature. 2024 Dec;636(8042):412-421. doi: 10.1038/s41586-024-08132-2. Epub 2024 Nov 6.
The phosphorylation of synaptic proteins is a significant biochemical reaction that controls the sleep-wake cycle in mammals. Protein phosphorylation in vivo is reversibly regulated by kinases and phosphatases. In this study, we investigate a pair of kinases and phosphatases that reciprocally regulate sleep duration. First, we perform a comprehensive screen of protein kinase A (PKA) and phosphoprotein phosphatase (PPP) family genes by generating 40 gene knockout mouse lines using prenatal and postnatal CRISPR targeting. We identify a regulatory subunit of PKA (Prkar2b), a regulatory subunit of protein phosphatase 1 (PP1; Pppr1r9b) and catalytic and regulatory subunits of calcineurin (also known as PP2B) (Ppp3ca and Ppp3r1) as sleep control genes. Using adeno-associated virus (AAV)-mediated stimulation of PKA and PP1-calcineurin activities, we show that PKA is a wake-promoting kinase, whereas PP1 and calcineurin function as sleep-promoting phosphatases. The importance of these phosphatases in sleep regulation is supported by the marked changes in sleep duration associated with their increased and decreased activities, ranging from approximately 17.3 h per day (PP1 expression) to 4.3 h per day (postnatal CRISPR targeting of calcineurin). Localization signals to the excitatory post-synapse are necessary for these phosphatases to exert their sleep-promoting effects. Furthermore, the wake-promoting effect of PKA localized to the excitatory post-synapse negated the sleep-promoting effect of PP1-calcineurin. These findings indicate that PKA and PP1-calcineurin have competing functions in sleep regulation at excitatory post-synapses.
突触蛋白的磷酸化是一种重要的生化反应，它控制着哺乳动物的睡眠-觉醒周期。体内的蛋白质磷酸化受到激酶和磷酸酶的可逆调节。在本研究中，我们研究了一对相互调节睡眠时间的激酶和磷酸酶。首先，我们通过使用产前和产后CRISPR靶向技术生成40个基因敲除小鼠品系，对蛋白激酶A（PKA）和磷蛋白磷酸酶（PPP）家族基因进行了全面筛选。我们确定PKA的一个调节亚基（Prkar2b）、蛋白磷酸酶1（PP1；Pppr1r9b）的一个调节亚基以及钙调神经磷酸酶（也称为PP2B）的催化和调节亚基（Ppp3ca和Ppp3r1）为睡眠控制基因。使用腺相关病毒（AAV）介导的PKA和PP1-钙调神经磷酸酶活性刺激，我们表明PKA是一种促进觉醒的激酶，而PP1和钙调神经磷酸酶作为促进睡眠的磷酸酶发挥作用。这些磷酸酶在睡眠调节中的重要性得到了睡眠时间显著变化的支持，这些变化与它们活性的增加和降低有关，范围从每天约17.3小时（PP1表达）到每天4.3小时（产后CRISPR靶向钙调神经磷酸酶）。这些磷酸酶发挥其促进睡眠作用需要定位到兴奋性突触后。此外，定位到兴奋性突触后的PKA的促觉醒作用抵消了PP1-钙调神经磷酸酶的促睡眠作用。这些发现表明，PKA和PP1-钙调神经磷酸酶在兴奋性突触后睡眠调节中具有相互竞争的功能。
