餘建軍^1,2,*,劉慧潔^1,2,*,高瑞^1,*,王濤^1,2,*,李成鋼^1,2,*,劉玉祥^1,2,楊璐^1,2,徐穎³,崔雲鳳²,賈辰熙³,黃娟¹,陳鵬¹,饒毅^1,2,4,# 

¹Laboratory of Neurochemical Biology, Peking-Tsinghua Center for Life Sciences,Peking-Tsinghua-NIBS (PTN) Graduate Program, School of Life Sciences, Peking University, Beijing, China; Chinese Institute for Brain Research, Beijing (CIBR); Department of Chemical Biology, College of Chemistry and Chemical Engineering; School of Pharmaceutical Sciences, PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing, China.

²Chinese Institutes for Medical Research, Beijing (CIMR), Capital Medical University, Beijing, China.

³National Center for Protein Sciences Phoenix, Beijing, China.

⁴Lead contact.

傳統上，研究睡眠的機理依賴於電生理學和遺傳學。由於睡眠只能透過行為觀察和物理學手段在整體動物上測量，因此沒有透過生化和化學生物學方法來找到調控睡眠分子的先例。此前發現影響睡眠的蛋白激酶SIK3，其磷酸化位點對於功能很重要，我們以此為靶點，用生物化學和化學生物學的方法作為這項研究的起點。我們發現在小鼠的SIK3進行點突變後（將469位的蘇氨酸變為丙氨酸，T469A），突變小鼠的睡眠增加。我們使用了生化純化和光交聯兩個方法，卻殊途同歸地發現，鈣調磷酸酶（簡稱CaN）無論在體外和體內還是體內，都能去除SIK3的T469（和S551位）的磷酸基團，但卻不影響SIK3在T221位點的磷酸化。降低腦內CaN調節亞基的的基因表達量後，小鼠每晝夜睡眠時間減少5個小時以上，變化幅度超過所有已知的小鼠遺傳突變體。我們發現了CaN在小鼠睡眠中的關鍵生理作用，並開創了用生化純化和化學生物學揭示睡眠分子機理的有效途徑。

睡眠的重要性不言而喻，但其分子和細胞機制仍然是個謎。包括我們在內的科學家已用遺傳學方法在果蠅和小鼠中尋找參與睡眠調控的基因。但是在本研究中，我們率先應用生化和化學生物學方法來揭示睡眠調節的機理。我們發現鈣磷酶（CaN）控制SIK3 去磷酸化有位點特異性：可以作用於兩個調節位點，但不能作用於酶活性所需的位點。用分子生物學方法降低CaN 會降低睡眠5 個多小時，這是在遺傳突變小鼠身上觀察到的最顯著睡眠表型。這項研究揭示了睡眠調節中的蛋白磷酸酶–蛋白激酶途徑，彰顯了生化純化和化學生物學方法作為研究大腦功能有效技術的價值。

本研究利用培養細胞進行蛋白質分離純化，腦組織光交聯，質譜分析腦內磷酸化蛋白組，發現磷酸酶對同一個蛋白質上不同位點的高度選擇性去磷酸化，在體外用細菌表達的蛋白質重組磷酸酶活性，同時用了4種基因修飾小鼠、兩種病毒介導的基因敲低的小鼠，密切結合分子生物學、生物化學、遺傳學和腦電圖記錄而闡明基因與睡眠的關係。

睡眠是動物的重要生理過程¹，受晝夜節律和穩態過程的調節^2,3。事實證明，在果蠅、小鼠、狗和人類身上採用遺傳學方法能有效發現調節睡眠的基因^4-10。例如，發現了食慾素及其受體在維持覺醒方面的作用^11-13，以及鹽誘導激酶3（SIK3）在調節睡眠方面的作用¹⁴。

在評估了這些策略的優勢和侷限之後，我們決定嘗試同時將經典的生物化學純化方法和現代的化學生物學途徑用於研究睡眠調控機制。我們的第一個目標底物是 SIK3¹⁴，Yanagisawa組^14,20,21和我們自己^18,19發現其特異位點磷酸化對睡眠調控非常重要。此前我們發現SIK3的特異磷酸化位點可指示睡眠需求並參與睡眠調節¹⁹，因此我們使用SIK3蛋白作為底物，並對其調節因子進行生化鑑定，然後再在體內測試這些體外發現的磷酸化調節因子是否也能在體內調控睡眠。

蛋白質磷酸化水平在不同的睡眠–覺醒相關狀態中有所不同^22-25。哺乳動物的睡眠被認為涉及蛋白激酶 A（PKA）^26-30、細胞外訊號調節激酶（ERK）^31-33、單磷酸腺苷（AMP）啟用蛋白激酶（AMPK）^34-36、鈣（Ca²⁺）/鈣調蛋白（CaM）激酶 II（CaMKII）a和b^37-39、c-Jun N 端激酶（JNK）40、SIK3、1 和 2^14、19-21以及肝臟激酶 B1（LKB1）^18、41-43。CaMK2b基因敲低的小鼠被報道每24小時的睡眠時間減少了120分鐘以上³⁸，超過了其他已知的基因突變小鼠^{9,12-14,18-21,27,30,33,39,44-53}。

蛋白磷酸酶（PPases）是否參與哺乳動物睡眠知之甚少。1970 年代發現一種蛋白質^54-56被命名為鈣調磷酸酶（也稱鈣神經蛋白，calcineurin, CaN、PP2或PPP3）⁵⁷ ，到1980 年代確定其作為磷酸酶的功能^58,59。CaN是唯一由鈣離子（Ca²⁺）和鈣調素（CaM）啟用的磷酸酶，由二聚體組成，具有催化亞基A三種之一（PPP3CA、PPP3CB或PPP3CC）和調節亞基B兩種之一（PPP3R1或PPP3R2）^56,60。PPP3CA、PPP3CB 和PPP3R1 表達的組織器官較為廣泛，而PPP3CC和PPP3R2則在睪丸中特異表達^61-65。在腦中，PPP3CA 是含量最高的催化亞基，PPP3R1 是含量最高的調節亞基。

本項工作中，我們在確定了SIK3中469位點蘇氨酸（T469）磷酸化的重要性後，再開始尋找 SIK3的磷酸酶。雖然小鼠SIK1和SIK2中與SIK3絲氨酸（S）551 同等位點的缺失會導致與SIK3 S551A相同的功能增益（gain of function, GOF）表型²¹，但我們發現Sik1或Sik2基因的缺失不影響小鼠的睡眠¹⁹。因此，我們重點研究Sik3。PKA磷酸化SIK3 T469和S551位點後促進SIK3與14-3-3蛋白的相互作用⁶⁶。生化上，14-3-3蛋白抑制SIK3活性，而T469或S551 的缺失增加了 SIK3 的訊號轉導⁶⁶。T469磷酸化的體內生理學功能意義尚不清楚。在此，我們將小鼠SIK3的T469突變為丙氨酸（A）（T469A），發現它導致小鼠睡眠增加。

我們採用了兩種途徑尋找去磷酸化SIK3的T469和S551的磷酸酶：從人胚胎腎臟（HEK）293T細胞中用經典生物化學純化T469和S551的磷酸酶，以及在小鼠腦中用化學生物學光交聯尋找與SIK3相互作用的蛋白。都找到了CaN的催化亞基PPP3CA。進一步體外生物化學重組實驗表明，在 Ca²⁺、CaM和PPP3R1的存在下，PPP3CA可去除T469和S551的磷酸基團。PPP3CA 不能去除T221的磷酸基團，後者的磷酸化增強SIK3的激酶活性，並促進睡眠，且其水平可以反應睡眠需求¹⁹。在體外培養的HEK293T細胞中，敲除PPP3CA、PPP3CB 或 PPP3R1，可以抑制鈣離子誘導的 T469和S551（而非 T221）去磷酸化。在鼠腦透過分子生物學敲除PPP3CA或PPP3R1時，鼠腦中 T469 和 S551 的磷酸化水平增加，T221的磷酸化不受影響。敲除PPP3CA 基因在 24 小時內減少睡眠約 3小時（187.6±13.0分鐘）。敲除PPP3R1基因減少5小時以上（349.3±21.5分鐘），超過了所有已知小鼠遺傳突變體的睡眠變化。從睡眠變化的程度來看，CaN是迄今為止發現的最重要的睡眠調節因子。我們成功地利用生化純化和化學生物學發現了重要的睡眠調控分子，顯示了遺傳學外，還有生物化學和化學生物學對睡眠研究有巨大潛力。

我們首先構建了一株攜帶 T469A 點突變的小鼠品系（圖 1、S1-S4、表 S1 和 S2）。睡眠通常是在雄性小鼠中測量。由於基因型為Sik3^T469A/T469A的點突變純合體雄性小鼠胚胎致死，我們比較了兩種基因型（Sik3^+/+野生型純合體和Sik3^T469A/+點突變雜合體）的雄性小鼠睡眠表型。我們還研究了所有三種基因型（Sik3^+/+和Sik3^T469A/+和Sik3^T469A/T469A點突變純合體）雌性小鼠的睡眠表型（圖S3和S4，表S1和S2）。雄、雌小鼠的睡眠表型在趨勢上相似，雌性Sik3^T469A/T469A點突變純合體的表型強於雄性 Sik3^T469A/+點突變雜合體，在總睡眠和非快速眼動睡眠（NREMS）方面分別相差25.2分鐘和 37.1分鐘（表S2，雌性Sik3^T469A/T469A和Sik3^+/+之間的睡眠增量與雄性 Sik3^T469A/+和Sik3^+/+之間的睡眠增量）。

代表性腦電圖（EEG）和肌電圖（EMG）見圖 1L，典型的腦電熱圖見圖 S2E。與 Sik3^+/+相比，Sik3^T469A/+雄性24 小時內總睡眠時長以及非快動眼睡眠（NREMS ）持續時間增加，尤其是在暗期（圖1A、1B、1C，表S2）。NREMS發生次數和單次持續時間均無明顯變化（圖1D和1E）。快速眼動睡眠（REMS）在Sik3^T469A/+和Sik3^+/+雄性之間無顯著差異（圖S1 E-H，表S2）。腦電圖的功率譜在δ波段增加、α波段減少（圖1F和1G）。透過NREMS δ波功率密度測量的睡眠需求在Sik3^T469A/+雄性顯著增加^2、67-70（圖1H）。

Sik3^T469A/T469A雌鼠在暗期的NREMS時長顯著增加（圖S3A和 S3B，表S1和S2），原因是NREMS發生次數增加（圖S3C），但單次持續時間無變化（圖S3D）。REMS時長在暗期沒有差異，但在亮期有所減少（圖S3E和S3F，表S1和S2），原因是REMS 發生次數減少（圖S3G）、單次持續時間無變化（圖S3H）。從NREMS到REMS的轉換機率減少，而其他狀態間轉換機率沒有變化（圖S3 M-P）。與Sik3^+/+雌性相比，Sik3^T469A/T469A和Sik3^T469A/+雌性的 NREMS δ波功率密度都有所增加（圖S4E）。

Sik3^T469A/+雄性小鼠的睡眠表型與另一實驗室報道的Sik3^S551A/+不同²⁰。我們隨後構建了Sik3^S551A/+突變小鼠。我們的Sik3^S551A/+雄性小鼠表現出與 Sik3^T469A/+雄性小鼠高度相似的睡眠表型，即在暗期NREMS 持續時間和δ功率密度增加（圖S5A-B、S6E、表S3和S4），但清醒（Wake）至 NREMS的過渡機率和睡眠剝奪（sleep deprivation, SD）後REMS 反彈降低（圖S5M、圖S6G）。在生化上，我們發現T469A 或 S551A點突變會降低另一個位點的磷酸化水平（圖S6M和S6N）。

我們也構建了Sik3^T469E/+點突變小鼠，但沒有觀察到任何睡眠表型（圖S7和S8）。

雖然 SIK3 功能喪失性突變（loss of function, LOF）和GOF小鼠突變體只表現有限的睡眠表型，但 SIK3 的上游或下游可能存在更重要的調節因子。為研究這種可能性，我們從 HEK293T 細胞中純化了其上游磷酸酶，使用SIK3 在 T469 和 S551 位點的去磷酸化程度作為磷酸酶的活性指標。我們從 HEK293T 細胞中純化能使 SIK3 在T469 和 S551 位點去磷酸化的磷酸酶（圖2）。

我們先用大腸桿菌（E. coli）合成並純化的重組 SIK3 蛋白證實了抗SIK3 T469 和 S551磷酸化形式抗體的特異性（圖S9A）。

隨後，我們裂解了 HEK293T 細胞，並將含500 mg總蛋白的細胞裂解液（濃度為 10 mg/ml）依次在 Q HP、Blue HP、SP HP、heparin HP、HAP HP和Superdex 200 色譜柱上分餾（圖2A）。每一步都從每個餾分中抽取相等組分檢測對T469 和 S551的磷酸酶活性。將一個步驟中的活性餾分合並，並在下一個色譜柱上進一步分餾。因此，Q HP 柱的10至 13號餾分、Blue HP 柱的流過（FL）餾分、SP HP 柱的FL餾分、heparin HP柱的FL餾分以及 HAP HP 柱的 5 至 7 號餾分被分別裝載到下一個色譜柱上（圖2B-G）。

在純化過程中，我們觀察到針對T469和S551的磷酸酶活性高度相關（圖2B-F），而且在每一步純化之後，整體磷酸酶活性都有所提高（圖S9B）。

將來自Superdex 200柱的10至18號餾分透過銀染檢測蛋白含量（圖2H）。其中餾分15含有最強的磷酸酶活性，從中切取一條箭頭所示的條帶進行質譜（MS）分析，最後我們檢測到四種磷酸酶：PPP3CA、PPP3CB、PPP3CC 和 PPP5C（圖2I）。

為研究小鼠大腦中的內源蛋白相互作用，我們（陳鵬和高瑞）發明了光交聯方法來尋找與SIK3相互作用的內源蛋白。與神經元培養物或細胞裂解液相比，腦切片能更好地儲存原位蛋白質互作網路。在目前研究蛋白質–蛋白質相互作用（protein-protein interactions, PPIs）的方法中，透過光照共價捕獲相互作用蛋白的光交聯策略因其良好的時間解析度和低於化學交聯的細胞毒性而被認為是在活體系統中更受歡迎的方法。然而，將光交聯試劑加入感興趣的蛋白質通常依賴於遺傳密碼擴增策略，即設計相應的tRNA合成酶以插入可光啟用的非天然氨基酸⁷¹，或透過代謝過程插入含有氨基酸類似物的可光啟用分子^72-74，而這兩種方法都很難在組織中實現。此外，用於光交聯的傳統分子（如重氮啶和芳基疊氮化物）通常對紫外線照射敏感⁷⁵，而紫外線具有高能光毒性，組織穿透力弱⁷⁶。因此，在組織樣本中原位捕獲 PPI 的光交聯策略還鮮有報道。

基於之前的工作⁷⁷，我們（陳鵬和高瑞）開發了一種光催化化學交聯（photocatalytic chemical crosslinking，PhotoCAX）策略，用於捕獲小鼠腦片中的 PPIs（圖 3A）。我們選擇eosin-Y作為光催化劑，1,6-diaminohexane作為連線劑。每種化合物都具有良好的溶解性，尤其是eosin-Y，被廣泛用於組織染色。eosin-Y 的最大吸收波長為517 nm，在綠光照射下會產生單線態氧（¹O₂）⁷⁸，單線態氧會啟用某些氨基酸殘基（如酪氨酸）的側鏈，形成親電中間體，從而與胺彈頭形成共價連線⁷⁹。我們的方法不需要轉染或基因修飾就能將光催化劑或交聯劑加入樣品中進行 PPI 捕獲。

我們隨後構建了SIK3-3xHA小鼠，其中 SIK3 蛋白的羧基末端標記了血凝素 (HA) 抗原決定簇⁸⁰，以便我們能在光交聯後檢測與 SIK3-HA 相關的蛋白質。

我們在新鮮製備的 SIK3-HA 小鼠腦片中應用了 PhotoCAX 策略。樣品中加入eosin-Y 和 1,6-diaminohexane，然後用綠色 LED（GL）照射。透過免疫印跡分析驗證了SIK3相互作用組，其中+GL組出現了明顯的交聯條帶，表明探針被有效啟用（圖3B）。

在交聯組和非交聯組中，SIK3 蛋白都能被歐聯抗-HA抗體的磁珠所富集，交聯產生的相互作用組也能同時被拉下。為進一步分析蛋白質，我們在製備 MS 樣品時切除了分子量大於 SIK3 的條帶。在胰蛋白酶消化後，採用二甲基標記法對兩組蛋白質進行定量研究。如Venn圖（圖3C）所示，81 個蛋白質在三個獨立實驗中重複出現，且在 +GL 組中富集程度較高（富集倍數 +GL/-GL > 4, log2(+GL/-GL) > 2）。透過火山圖進一步分析富集結果（圖3D），其中只有 p < 0.05 且3個重複中平均富集倍數超過4的蛋白質才被認為是 SIK3 的重要互作候選蛋白。作為陽性對照，我們檢測到了一些已知的 SIK3 相互蛋白，如 14-3-3蛋白。PPP3CA 及其相應的調控亞基 PPP3R1也被富集（圖3D）。

為證實SIK3和PPP3CA在鼠腦中的相互作用，我們在免疫沉澱實驗中使用抗體富集SIK3，發現沉澱物中既有SIK3也有PPP3CA（圖 3E）。同樣，使用抗PPP3CA抗體處理鼠腦裂解液時，沉澱物中也發現了PPP3CA 和SIK3（圖3F）。將FLAG標記的SIK3和HA標記的PPP3CA匯入HEK293T 細胞過表達時，SIK3和PPP3CA的相互作用可在HEK293T細胞中再現（圖 3G, 3H）。

我們透過免疫沉澱從HEK293T細胞中富集過表達的被FLAG 標記的 SIK3，這種SIK3在T221、T469和S551位點都已經被磷酸化。我們在HEK293T細胞表達和富集每個候選磷酸酶（PPP3CA、PPP3CB、PPP3CC、PPP5C和PPP3R1）。在PPP3R1存在時，SIK3的T469和S551可被PPP3CA、PPP3CB和PPP3CC去磷酸化。而PPP3CA、PPP3CB和PPP3CC不能去磷酸化T221。在相同條件下，PPP5C不能去磷酸化SIK3的T469、S551 或T221（圖4A），但它能使Tau蛋白的S396位點去磷酸化（圖 S9C），表明PPP5C具有催化活性。

由於PPP3CA和PPP3R1在腦中含量豐富，而PPP3CC和PPP3R2在睪丸中含量豐富或特異表達^61-65,81，因此我們重點研究PPP3CA和PPP3R1。

在PPP3R1存在時，從HEK293T細胞富集純化的PPP3CA可去磷酸化MEK1的S217，但不能去磷酸化AKT1的T308、PDK1的S241、MARK1的T215、GSK3β的S9、HDAC4的S246和S632、JNK1的T183/Y185以及 ERK2的T185/Y187（圖4B-E）。因此，廣為流傳的所謂磷酸酶具有 “非特異性”對PPP3CA來說並不成立。

為排除從HEK293T細胞中富集純化的酶含有與磷酸酶相關其他蛋白質的可能性，我們在大腸桿菌中表達了CaM、PPP3CA和PPP3R1。我們發現，體外重組SIK3的T469和S551去磷酸化活性需要CaM、PPP3CA、PPP3R1和Ca²⁺同時存在（圖4F）。

Ionomycin是一種鈣離子載體，可使Ca²⁺流入細胞。當對HEK293T細胞施用ionomycin時，ionomycin以劑量（圖4G-H）和時間（圖4I-J）依賴的方式使SIK3 T469和S551（而不是 T221）去磷酸化。

PPP3CA/PPP3R1的過表達可以增強ionomycin誘導的T469和S551的去磷酸化（圖4K-L）。過表達無活性的PPP3CA形式PPP3CA-H151A（組氨酸或 H 突變為 A）不影響T469或S551的磷酸化水平（圖4M），但降低ionomycin的去磷酸化活性，表明該 LOF 突變體具有顯性負效應⁸²。

為研究ionomycin誘導的去磷酸化對CaN的依賴性，我們用sgRNAs 構建了 PPP3CA、PPP3CB 和 PPP3R1 的單基因或雙基因敲除HEK293T細胞系。HEK293T細胞中PPP3CA或PPP3CB 單基因敲除的表型不穩定。然而，在HEK293T細胞中，PPP3CA 和 PPP3CB 雙基因敲除或 PPP3R1 單基因敲除的表型穩定。PPP3R1 基因敲除或PPP3CA 和 PPP3CB 雙基因敲除抑制ionomycin誘導的T469和S551（但不是 T221）的去磷酸化（圖4N-P）。

為研究它們在體內的作用，我們使用 CRISPR-Cas9 策略在小鼠腦中靶向敲除PPP3CA或PPP3R1（圖S10）。對照組有兩種：WT^Ctrl是野生型（WT）小鼠，注射了靶向PPP3CA或PPP3R1的sgRNA表達病毒；eGFP^Ctr是表達Cas9的小鼠（Rosa^Cas9/+），注射了靶向增強型綠色熒光蛋白（eGFP）的sgRNA表達病毒；PPP3CA基因敲除（PPP3CA^KD）小鼠為Rosa^Cas9/+小鼠注射AAV2/PHP.eB-CMV-mScarlet-PPP3CA-sgRNA-WPRE病毒而產生；PPP3R1^KD小鼠為Rosa^Cas9/+小鼠注射AAV2/PHP.eB-CMV-mScarlet-PPP3R1-sgRNA-WPRE病毒而產生。

免疫印跡分析表明，靶向PPP3CA或PPP3R1的sgRNA降低了其蛋白水平（圖5）。PPP3CA被靶向時，PPP3R1蛋白也會減少（圖5A）。在 PPP3R1^KD小鼠中，PPP3R1、PPP3CA和PPP3CB均減少（圖5B）。在鼠腦中敲除PPP3CA或PPP3R1後，SIK3 T469和S551的磷酸化均增加，但T221無變化（圖5A-D）。

敲除PPP3CA或PPP3R1後，ERK1/2 T202/Y204、MEK1 S217、JNK T183/Y185、AKT1 S473、AMPKα T172、CaMK2a/b T286、PDK1 S241 或 GSK3β S9 等位點的磷酸化均無明顯變化（圖5A-B）。

為進一步研究CaN的底物特異性，我們進行了磷酸化蛋白質組學分析。將 PPP3R1^KD與WT^Ctrl或eGFP^Ctrl對照組進行比較後發現，在8117和8121個已確定的磷酸化位點中，分別只有148個（1.82%）和221個（2.72%）位點的磷酸化水平出現了顯著上調（圖S11A-B）。在兩組比較中，96個相同位點的磷酸化水平上調，包括MP2K2和IP3KA等激酶、CAC1C和SCN2A等離子通道（圖S11C）。在合計81個蛋白質中發現了這些位點，其中 69 個至少含有兩個典型CaN底物結合基序之一（圖S11D）。

為了研究內源性PPP3CA在小鼠中的生理作用，我們分析了PPP3CA^KD小鼠的睡眠表型，並將其與對照組（WT^Ctrl和eGFP^Ctrl）的表型進行了比較。在EEG或EMG的一般模式上，三種基因型無顯著差異(圖6L和 S13E)。

在24小時內，PPP3CA^KD小鼠的總睡眠時長比eGFP^Ctrl或WT^Ctrl對照小鼠減少了大約3小時(圖 6A和S12 A-D, 表 S5和 S6)。在PPP3CA^KD小鼠中，NREMS時長降低約204分鐘(圖6B-C，表S5和S6)。在亮期，NREMS次數無變化（圖6D），但NREMS單次持續時間減少(圖 6E)。在暗期，NREMS次數減少（圖6D），而單次持續時間無變化（圖6E）。

在亮期，REMS的時長變化較小且方向相反，與對照組相比，PPP3CA^KD小鼠的REMS增加約13分鐘 (圖S12E-F)。在亮期，REMS時長增加主要是因REMS次數增加（圖S12G），而REMS單次持續時間無變化 (圖S12H). 在暗期，REMS減少約6分鐘（圖S12E-F），主要因REMS次數（圖S12G）和單次持續時間（圖S12H）減少。

EEG功率譜分析僅在ZT15和ZT23時段顯示NREMS δ波功率密度減少，而其他時間點未變(圖6F-H)。REMS和NREMS轉換機率增加，但Wake和NREMS轉換機率減少 (圖6I-J和S12K-L)。

經過6小時的睡眠剝奪後，WTC^trl和eGFP^Ctrl小鼠的NREMS（圖6K）或總睡眠/覺醒時間（圖S13A）逐漸恢復，而PPP3CA^KD小鼠在睡眠剝奪後的相應恢復顯著減少。 SD後REMS的恢復在WT^Ctrl、eGFP^Ctrl和PPP3CA^KD小鼠之間沒有顯著差異 (圖 S13B)。

小鼠全腦的免疫印跡分析顯示，PPP3R1^KD小鼠的PPP3R1蛋白表達顯著降低 (圖5B)。代表性的EEG和EMG見圖7K，代表性的睡眠圖見圖S15J。

在每個ZT時段，除了光暗轉換前後的ZT 5，PPP3R1^KD小鼠的睡眠時長顯著減少，24小時內減少至少5小時(圖7A 和S14A-D, 表S7和S8)。相比eGFP^Ctrl或WT^Ctrl小鼠，PPP3R1^KD小鼠的平均NREMS時長在整個24小時內減少344分鐘（圖7B-C和表S8），這因NREMS單次持續時間減少（圖7D），而次數未變 (圖7E)。. 

在亮期，PPP3R1^KD小鼠與eGFP^Ctrl或WT^Ctrl小鼠的REMS無顯著差異，而在暗期，PPP3R1^KD小鼠的REMS時長平均減少10.4分鐘，主要因REMS次數減少(圖S14E-H，表S7和S8)。

不同睡眠和清醒狀態之間的轉換機率（圖7J）來自圖7I和圖S14K及S14L的資料。從Wake到NREMS和從NREMS到NREMS的轉換機率降低，而從NREMS到Wake和從Wake到Wake的轉換機率增加。

PPP3R1^KD小鼠在ZT0到ZT6的6小時睡眠剝奪後缺乏NREMS和REMS的恢復。從第8到第24小時的每個時間點均有顯著差異(圖7K和圖S15A-B)。我們注意到，PPP3R1^KD小鼠在ZT0到ZT6期間的睡眠時間顯著少於ZT6到ZT12期間。為確保在SD期間失去足夠的睡眠時長，我們在ZT6到ZT12重複了睡眠剝奪（SD）。與eGFP^Ctrl和WT^Ctrl小鼠相比，PPP3R1^KD小鼠的NREMS在SD後的回覆時長顯著減少（圖S15F），而REMS未受影響 (圖S15G)。

PPP3R1^KD小鼠的NREMS δ波功率降低，而α波功率增加 (圖7F-H和S14I-J)。SD後，PPP3R1^KD小鼠的NREMS δ波功率恢復也少於對照小鼠 (圖 S15C-D 和H)。PPP3R1^KD小鼠在ZT6-12期間SD後的NREMS δ功率密度反彈與兩個對照組無顯著差異 (圖S15I)。

在確認CaN在小鼠睡眠調節中的作用後，我們進一步分析了CRISPR-Cas9介導的PPP3CA或PPP3R1 KD在小鼠大腦中的效率。免疫組化分析顯示，與eGFP^Ctrl和WT^Ctrl小鼠相比，PPP3CA^KD或PPP3R1^KD小鼠的大腦皮層、丘腦、中腦、後腦和下丘腦中PPP3CA或PPP3R1蛋白水平顯著降低 (圖S16A-H)。為確定丟失CaN的細胞型別，我們進行了病毒標記mScarlet和神經元標記Neurotrace（圖S17A-C）或星形膠質細胞標記膠質纖維酸性蛋白（GFAP）（圖S17E-G）的免疫組化共染色。結果顯示，病毒主要侵染了神經元 (圖S17D和 S17H)，這表明CaN的丟失主要發生在敲除組的神經元中。

CaN LOF的表型比SIK3 LOF的表型顯著得多。為研究SIK3 T469磷酸化是否位於CaN調控睡眠的下游，我們在Sik3^T469A/+背景下敲低了PPP3R1。

在Rosa26^Cas9/+::Sik3^+/+背景下，與注射了AAV-sgRNA^EGFP的小鼠相比，PPP3R1^KD導致NREMS時長、NREMS δ波功率密度和SD後的NREMS反彈（從ZT6-12）減少(圖8A-H和S19B)。在Rosa26^Cas9/+::Sik3^T469A/+背景，NREMS的α波功率（圖8G）、SD前後的NREMS δ波功率密度（圖8H和圖8I），以及REMS到NREMS的轉換機率（圖8J-K）有部分回覆，而總睡眠時長和NREMS時長以及NREMS在SD後的反彈表型未回覆 (圖8A-E和S19B)。在Rosa26^Cas9/+::Sik3^+/+小鼠及其同窩Rosa26^Cas9/+::Sik3^T469A/+小鼠中，PPP3R1^KD不影響夜間REMS時長（圖S18E-H），而在後者中，它增加了ZT6-12期間SD導致的REMS反彈 (圖S19C)。

一些PPP3R1^KD表型，如NREMS時長和NREMS在SD後的反彈，無法透過SIK3-T469A來回復，這可以透過PPP3R1能夠去磷酸化鼠腦中的其他蛋白質（如我們圖S11中的那些）來解釋。換句話說，SIK3不可能是CaN介導其調節睡眠的唯一底物。

我們的研究結果表明，CaN在調節睡眠—特別是NREMS–起重要作用，部分透過SIK3 T469去磷酸化，並揭示了一個涉及蛋白激酶和磷酸酶的訊號通路在睡眠調節中的作用。我們的研究還揭示了CaN在體外和體內去磷酸化SIK3中特定S和T殘基的特異性，顯示磷酸酶有之前未觀察到的特異性。因SIK3敲除小鼠¹⁹和SIK3 T469A點突變小鼠的睡眠表型都比CaN突變的弱得多，很明顯SIK3及其T469和S551位點並非CaN的唯一下游靶點。

我們的Sik3^S551A/+小鼠睡眠表型遠弱於之前其他人報道的結果¹⁴。事實上，野生型對照小鼠的基礎NREMS時長也有所不同（我們研究中的約520分鐘/天 vs 他們研究中的約650分鐘/天）。這種差異可能因不同的遺傳背景、飼養和測量條件所造成。 Sik3^T469E/+小鼠無睡眠表型，這與之前的生化結果一致，即 SIK3 T469E未能模擬持續磷酸化，且無法結合14-3-3蛋白⁶⁶。Sik3^S551D/+小鼠同樣表現出與Sik3^S551A/+ 小鼠相似、而非相反的睡眠表型¹⁴。

雖然我們和其他研究人員已經研究了許多與果蠅睡眠相關的基因，但尚未發現它們的鼠類同源基因對小鼠睡眠都是必需的。目前尚無法預測在任何一種物種中發現的基因是否會在另一種物種中調節睡眠。因此，瞭解到CaN參與調節果蠅睡眠是令人欣慰的^83,84。SIK3在果蠅和小鼠中都參與調節睡眠的事實^14,18,19進一步支援了SIK3及其調節因子如LKB1和CaN在睡眠控制中的重要性。

CaN的三個催化亞基在體外都能在生化上去磷酸化SIK3的T469和S551，但不能去磷酸化T221。在體內，我們已證明PPP3CA和PPP3CB在HEK293T細胞中去磷酸化T469和S551，以及PPP3CA在小鼠腦中的作用。我們已發現PPP3CA和PPP3R1在調節小鼠睡眠方面具有生理作用。 PPP3CB的作用仍需進一步研究。

Sik3^T469A/+的部分回覆效果表明CaN調節睡眠可能涉及其他下游靶點。基於CaN的兩個經典底物結合基序（LxVP和PxIxIT），Wigington及同事發現了691種獨特的蛋白質，這些蛋白質至少包含這兩個基序中的一個，可能作為CaN的潛在底物⁸²。我們的磷酸化蛋白質組學資料還揭示了部分在CaN KD組中磷酸化狀態發生變化的候選蛋白，其中離子通道蛋白（Cacna1c、Cacna1h、Kcnb1、Scn2a等）尤為引人注目。

有研究表明，SIK3在穀氨酸能神經元調節小鼠的睡眠⁸⁵。穀氨酸能神經元在小鼠的許多腦區中廣泛分佈⁸⁶。很可能CaN也在穀氨酸能神經元中發揮作用，從而影響睡眠。CaN 還在位於丘腦皮層和丘腦網狀核的 GABA 能神經元中發揮作用⁸⁷。我們的免疫組化資料表明CaN的丟失主要發生在神經元中，但需在不同的腦區和神經元型別中系統性地操縱CaN，以找到CaN調節睡眠的主要區域。

我們發現CaN在睡眠中具有特定表型，這將激發對其他磷酸酶在睡眠中作用的進一步研究。例如，雖然CaN在NREMS中似乎比在REMS中更重要，但是否有其他磷酸酶在調節REMS方面更為重要？我們發現CaN在去磷酸化特定位點和調節睡眠特定成分方面的特異性，既帶來了問題，也激發我們進一步理解哺乳動物睡眠分子機理的熱情。應進一步研究磷酸酶在重要生理過程中的具體作用。

不同腦區的Ca²⁺成像研究已顯示出在不同的睡眠/覺醒狀態下細胞內和細胞外的Ca²⁺濃度是不同的^88-91。利用藥理學和遺傳學方法操縱影響Ca²⁺濃度的離子通道可以改變小鼠的睡眠模式^38,92-95。

我們對CaN作用的發現提供了Ca²⁺的一個可能下游組分，但仍需進一步研究Ca²⁺、激酶、磷酸酶、離子通道和轉錄因子如何相互作用從而調節睡眠。

第一個發現的與睡眠調節相關的激酶是PKA。一種能夠抑制磷酸二酯酶並增加環腺苷酸（cAMP）的抗抑鬱藥可以增加大鼠的清醒度²⁶。在小鼠中，過表達一種顯性負性的PKA突變體促進了REMS和NREM的碎片化，同時減少了SD後的睡眠反彈³⁰。對小鼠神經元中的ERK1或ERK2進行藥理抑制或基因敲除顯著減少睡眠時長³³。當發現AMPK的抑制劑會減少小鼠的睡眠，而其啟用劑會增加小鼠的睡眠時，AMPK被認為與睡眠調節有關³⁴。在特定腦區透過藥理學抑制CaMKII可以增加睡眠³⁷，而胚胎敲低CaMK2a和CaMK2b基因導致睡眠時間分別減少了大約50分鐘和120分鐘³⁸。透過小鼠的前向遺傳篩選發現，SIK3的GOF突變可以調節睡眠¹⁴。在Sik3^sleepy突變體中刪除了一個小片段，導致缺少PKA靶位點S551²⁰。SIK1和SIK2中的SIK3 S551同源位點的突變導致了GOF表型²¹，這就不清楚哪種SIK激酶或磷酸化位點在生理上為睡眠調節所必需。我們對每個SIK基因缺失的小鼠的研究表明，只有SIK3、而不是SIK1或SIK2，為小鼠睡眠所必需¹⁹。Lkb1是一個腫瘤抑制基因，其產物被發現能夠磷酸化AMPK的α亞基的T172位點^97-103，以及包括SIK3在內的AMPK相關激酶（ARKs）的相應位點¹⁰⁴。我們最近的體內功能研究表明，LKB1為果蠅和小鼠睡眠所必需¹⁸。除了LKB1，我們體外生化研究還發現了二十多種STE20亞家族的激酶可以作為ARKs上游^105,106，這使LKB1在睡眠調節中位於SIK3上游的簡單情景變得不確定。我們仍在研究這些透過生化方法鑑定的任何STE20激酶是否參與睡眠調節。

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(A-B)Profiles showing sleep time each hr in minutes/hr (min/hr) (A) or profiles of NREMS (B). The X axis shows zeitgeber time (ZT) with the white box indicating light phase (or daytime) and black box dark phase (or nighttime). The black line shows data from SIK3^+/+ mice (n = 11), the blue line data from SIK3^T469A/+ mice (n = 10). (C-E) NREMS duration (C), NREMS episode number per 24 hr or 12 hr in light/dark phase (D), and NREMS episode duration (E). (F) EEG power spectrum during NREMS. X-axis indicates frequency distribution of EEG power. (G) Normalized EEG power of δ, θ, α, σ and β waves during NREMS. (H) Diurnal NREMS delta power density. (I) Transition probabilities. W: Wake, NR: NREMS, R: REMS. (J) A diagrammatic illustration of transition probabilities of different sleep and wake states, summarized from data in Fig. 1H and Fig. S1K and S1L. (K) Recovery of NREMS after 6 hrs of sleep deprivation (SD, ZT0-6). (L) 1 hr representative EEG and EMG traces at different vigilance states (Wake, NREMS, REMS). ns, not significant; *p < 0.05; **p <0.01; ***p <0.001 and ****p <0.0001; mean ± standard error of the mean (mean ± SEM). Two-way ANOVA (A, B, I, J); One-way ANOVA (C); Kruskal-Wallis test (D, E); Two-way ANOVA with Sidak`s post-hoc test (G); Mixed-effects model (H); Two-way repeated measurement ANOVA (Two-way RM ANOVA) (F, K). 

(A)A schematic illustration of phosphatase purification with HEK cell lysates passing through Q HP, Blue HP, SP HP, heparin HP, HAP HP and Superdex 200 columns before silver staining and MS analysis. (B)SIK3 T469 and S551 dephosphorylation of fractions separated by Q-HP anionic chromatography column. 500 mg (at a concentration of 10 mg/ml) HEK cell lysates flowed through 0.45 mm filter was fractionated on a Q-HP anionic chromatography column, eluted with a linear gradient of NaCl (0-600 mM) into 20 column volumes (CVs). We used one CV per fraction as the standard fraction in our purification. A small aliquot was analyzed for of phosphatase activities with SIK3. (C)SIK3 T469 and S551 dephosphorylation of fractions separated by Blue-HP column. Fractions 10 to 13 from (B) were combined, dialyzed with buffer A and loaded onto a Blue-HP column. (D)Dephosphorylation activity of fractions separated by SP-HP column. The FL fraction from (C) was further fractionated on a SP-HP column. (E) Dephosphorylation activity of fractions separated by heparin column. The FL fraction from (D) was further fractionated on a heparin column. (F) Dephosphorylation activity of fractions separated by HAP-HP column. The FL fraction from (E) was further fractionated on a HAP-HP column. The rest of the fractionation was similar to (B) except that the final wash was with 5 CVs of 500 mM K₂PO₄, giving rise to samples 21 to 25. (G) Active fractions from the HAP-HP were fractionated on a Superdex 200 column 10/300 GL, eluted into 20 fractions. 1 ml from each fraction was collected and labeled as samples 1 to 20. Protein contents were monitored with UV at 280 nm. (H) Silver-stain result and corresponding dephosphorylation activity of Fractions 10 to 18 separated by Superdex 200 column on SIK3 T469 and S551. (I) Phosphatases detected by MS.

(A)A schematic diagram of photo crosslinking method. Brain slices were prepared from SIK3-3xHA mice and photocatalytic crosslinking was carried out in darkness with eosin Y (50 mM) and 1, 6 dihexamine (1 mM). The anti-HA antibody was used to pull down proteins crosslinked to SIK3. (B)Validation of photocatalytic crosslinking efficacy in mouse brain slices. 15 min green light was given 1 hr after photocatalytic reagents treatment and slices were collected and homogenized. The arrowhead indicates SIK3-interaction protein complexes. (C–D) Veen (C) and volcano (D)plot of putative SIK3 interaction proteins enriched in photo-linkage groups. The significant threshold in (D) was set at P value < 0.05 and Fold change > 4. Blue dots represented protein candidates reaching significant threshold. PPP3CA and PPP3R1 were highlighted in red. Members of 14-3-3 family and one PKA catalytic subunit PRKACB were highlighted in black. (E–F) Co-immunoprecipitation between PPP3CA and SIK3 from WT mouse brain homogenates using anti-SIK3 and anti-PPP3CA antibodies. (G–H) Co-immunoprecipitation between the HA tagged PPP3CA and FLAG tagged SIK3 in HEK293T cells using anti-FLAG and anti-HA antibodies. HEK293T cells were co-overexpressed with 1 μg HA-PPP3CA and 1 μg FLAG-SIK3 expressing plasmids for 24 hrs before cell collection and lysis.

(A) Dephosphorylation activity of PPP3CA, PPP3CB, PPP3CC and PPP5C. Each PPases, PPP3R1 and full length SIK3 were separately immunoprecipitated from HEK cells. (B-E)PPP3CA dephosphorylation of MEK1 at S217, AKT1 at T308, PDK1 at S241, MARK1 at T215, GSK3β at S9, HDAC4 at S246 and S632, JNK1 at T183/Y185, ERK2 at T185/Y187. Proteins were separately immunoprecipitated from HEK cells. (F) Subunits and cation dependence of calcineurin dephosphorylation of SIK3 T221, T469 and S551 in vitro. Recombinant PPP3CA, PPP3R1, CaM were purified from E. coli. Full-length SIK3 was immunoprecipitated from HEK 293T cells. (G-H) Dose-dependent dephosphorylation effect of ionomycin on SIK3 T469 and S551 in HEK cells. (I-J) Time-dependent dephosphorylation effect of ionomycin on SIK3 T469 and S551 in HEK cells. (K-L) Alteration of ionomycin dephosphorylation effect on SIK3 T469 and S551 by PPP3CA/PPP3R1 co-transfection. Numbers represent plasmid gram weight. After transfection for 24 hrs, cells were treated with 10 μg/ml ionomycin for 10 min. (M) Alteration of ionomycin dephosphorylation effect on SIK3 T469 and S551 by PPP3CA-H151A/PPP3R1 co-transfection. (N-P) Alteration of ionomycin dephosphorylation effect on SIK3 T469 and S551 by PPP3CA/PPP3CB double KO or PPP3R1 KO. ns, not significant; *p < 0.05; **p <0.01; ***p <0.001; mean ± standard error of the mean (mean ± SEM). One-way ANOVA (H, J, L); Student`s t-test (O, P).

(A) PPP3CA knockdown caused serine/threonine phosphorylation alteration in the brain. Each lane shows results from one mouse. From the left are: two wt mice injected with sgRNAs targeting GFP, five Cas9 expressing mice injected with sgRNAs targeting GFP, and five Cas9 expressing mice injected with sgRNAs targeting PPP3CA. (B) PPP3R1 knockdown caused serine/threonine phosphorylation alteration in the brain. From the left are: three WT mice injected with sgRNAs targeting GFP, three Cas9 expressing mice injected with sgRNAs targeting GFP, and three Cas9 expressing mice injected with sgRNAs targeting PPP3R1. (C-D) Statistical analysis for (A) and (B). ns, not significant; *p < 0.05; **p <0.01; mean ± standard error of the mean (mean ± SEM). One-way ANOVA with Tukey's multiple comparisons test.

(A–B)Profiles of total sleep (A) or NREMS (B) in PPP3CA^KD, eGFP^Ctrl and WT^Ctrl mice. (C)NREMS duration over 24 hrs. (D–E) NREMS episode number per 24 hr or 12 hr in light/dark phase (D) and episode duration (E). (F) EEG power spectrum during NREMS. (G) Normalized EEG power of δ, θ, α, σ and β waves during NREMS. (H) Diurnal NREMS delta power density. The jump points at ZT12-15 and ZT23 were due to lack of data because most mice in PPP3CA^KDgroup were awake at these time points. (I) NREMS related transition probabilities. (J) A diagrammatic illustration of transition probabilities of different sleep and wake states. (K) Recovery of NREMS after 6 hrs of SD (ZT0-6). (L) 1 hrrepresentative EEG and EMG traces at different vigilance states. ns, not significant; *p < 0.05; **p <0.01; ***p <0.001 and ****p <0.0001; mean ± standard error of the mean (mean ± SEM). Two-way ANOVA (A, B, I, J); One-way ANOVA (C); Kruskal-Wallis test (D, E); Two-way ANOVA with Sidak`s post-hoc test (G); Mixed-effects model (H); Two-way repeated measurement ANOVA (Two-way RM ANOVA) (F, K).

(A–B) Profiles of total sleep (A) or NREMS (B) in eGFP^Ctrl, WT^Ctrl and PPP3R1^KD mice. (C) Data and statistics of NREMS duration over 24 hrs. (D-E) NREMS episode number per 24 hr or 12 hr in light/dark phase (D) and episode duration (E). (F) NREMS EEG power spectrum analysis. (G) Normalized EEG power of δ, θ, α, σ and β waves during NREMS. (H) NREMS delta power density over 24 hrs. (I) NREMS related transition probabilities. (J) Transition probabilities of different sleep and wake states. (K) Recovery of NREMS after 6 hrs of SD (ZT0-6). (L) 1 hrrepresentative EEG and EMG traces at different vigilance states. ns, not significant; *p < 0.05; **p <0.01; ***p <0.001 and ****p <0.0001; mean ± standard error of the mean (mean ± SEM). Two-way ANOVA (A, B, I, J); One-way ANOVA (C); Kruskal-Wallis test (D, E); Two-way ANOVA with Sidak`s post-hoc test (G); Mixed-effects model (H); Two-way repeated measurement ANOVA (Two-way RM ANOVA) (F, K).

(A–B) Profiles of total sleep (A) or NREMS (B) in eGFP^Ctrl: Rosa26^Cas9/+; SIK3^+/+ mice injected with AAV-eGFP-sgRNA virus, PPP3R1^KD: Rosa26^Cas9/+; SIK3^+/+mice injected with AAV-ppp3r1-sgRNA virus and SIK3^T469A/+; PPP3R1^KD: Rosa26^Cas9/+; SIK3^T469A/+mice injected with AAV-ppp3r1-sgRNA virus. (C) NREMS duration over 24 hrs.(D-E) NREMS episode duration (D) and episode number per 24 hr or 12 hr in light/dark phase (E). (F) NREMS EEG power spectrum analysis. (G) Normalized EEG power of δ, θ, α, σ and β waves during NREMS. (H) NREMS delta power density over 24 hrs. (I) NREMS delta power density during the 24 hrs recovery after SD (ZT6-12). (J) REMS related transition probabilities. (K) Transition probabilities of different sleep and wake states. (L) 1hr representative EEG and EMG traces at different vigilance states. ns, not significant; *p < 0.05; **p <0.01; ***p <0.001 and ****p <0.0001; mean ± standard error of the mean (mean ± SEM). Two-way ANOVA (A, B, J, K); One-way ANOVA (C); Kruskal-Wallis test (D, E); Two-way ANOVA with Sidak`s post-hoc test (G); Mixed-effects model (H, I); Two-way repeated measurement ANOVA (Two-way RM ANOVA) (F).

(A-D) Profiles of wake over 24 hrs (A), total wake duration over 24 hrs (B), wake episode number per 24 hr or 12 hr in light/dark phase (C), wake episode duration (D) of Sik3^T469A/+(blue, n=10) and Sik3^+/+ mice (black, n = 11).

(E–H) Profiles of REMS over 24 hrs (E), total REM duration over 24 hrs (F), REM episode number per 24 hr or 12 hr in light/dark phase (G), REM episode duration (H).

(I–J) EEG power spectrum during Wake (I) or REMS (J).

(K–L) Self- (K) and REMS related (L) transition probabilities between different sleep and wake states. W: Wake, NR: NREMS, R: REMS.

(A–B)Recovery of Wake (A) or REMS (B) after 6 hrs of SD. ns, not significant; mean ± SEM (Two-way ANOVA with Tukey's multiple comparisons test). 

(C) NREMS delta power density during the 24 hrs recovery after SD. ns, not significant; *p< 0.05; **p <0.01; ***p<0.001; ****p <0.0001; mean ± SEM (Mixed-effects model). 

(D) Changes of NREMS delta power density after SD. ns, not significant; mean ± SEM (Two-way repeated measurement ANOVA with Tukey's multiple comparisons test). 

(E) Representative hypnograms of littermates.

(A-D) Profiles of NREMS over 24 hrs (A), total NREMS duration over 24 hrs (B), NREMS episode number per 24 hr or 12 hr in light/dark phase (C), NREMS episode duration (D).

(E–H) Profiles of REMS over 24 hrs (E), total REMS duration over 24 hrs (F), REMS episode number per 24 hr or 12 hr in light/dark phase (G), REMS episode duration (H).

(I-L) Profiles of wake over 24 hrs (I), total wake duration over 24 hrs (J), wake episode number per 24 hr or 12 hr in light/dark phase (K), wake episode duration (L).

(M–P) Probabilities of transition between different sleep and wake states.

(A–C) EEG power spectrum during NREMS (A), REMS (B) or wake (C).

(D) Normalized EEG power of δ, θ, α, σ and β waves during NREMS.

(F-H)Recovery of NREMS (F), REMS (G) and Wake (H) after 6 hrs of SD (ZT0-6).

(I) NREMS delta power density during the 24 hrs recovery time.

(J) Changes of NREMS delta power density after 6 hrs of SD.

(K) 1 hr representative EEG and EMG traces of littermates at each vigilance state.

(L) Representative hypnograms of littermates.

ns, not significant; *p < 0.05; **p <0.01; ***p <0.001 and ****p <0.0001; mean ± SEM. Two-way ANOVA (F, G, H); Mixed-effects model (E, I); Two-way repeated measurement ANOVA (Two-way RM ANOVA) (A, B, C, J). Two-way ANOVA with Sidak`s post-hoc test (D). 

(A-D) Profiles of NREMS over 24 hrs (A), total NREMS duration over 24 hrs (B), NREMS episode number per 24 hr or 12 hr in light/dark phase (C), NREMS episode duration (D). 

(E-H) Profiles of wake over 24 hrs (E), total wake duration over 24 hrs (F), wake episode number per 24 hr or 12 hr in light/dark phase (G), wake episode duration (H).

(I-L) Profiles of REMS over 24 hrs (I), total REMS duration over 24 hrs (J), REMS episode number per 24 hr or 12 hr in light/dark phase (K), REMS episode duration (L).

(M–P) Probabilities of transition between different sleep and wake states.

(A–C) EEG power spectrum during NREMS (A), REMS (B) or Wake (C). 

(D) Normalized EEG power of δ, θ, α, σ and β waves during NREMS.

(E) Diurnal NREMS delta power density.

(F-H)Recovery of NREMS (F), REMS (G) and Wake (H) after 6 hrs of SD (ZT0-6).

(I) NREMS delta power density during the 24 hrs recovery time.

(J) Changes of NREMS delta power density after 6 hrs of SD.

(K) 1 hr representative EEG and EMG traces of littermates at each vigilance state.

(L) Representative hypnograms of littermates.

(M-N) Western-blot showing phosphorylation change in SIK3 T221, T469 and S551 sites in SIK3^T469A/+ (L) and SIK3^S551A/+ (M) mouse brain.

(A-D) Profiles of NREMS over 24 hrs (A), total NREMS duration over 24 hrs (B), NREMS episode number per 24 hr or 12 hr in light/dark phase (C), NREMS episode duration (D).

(E-H) Profiles of wake over 24 hrs (E), total wake duration over 24 hrs (F), wake episode number per 24 hr or 12 hr in light/dark phase (G), wake episode duration (H).

(I-L) Profiles of REMS over 24 hrs (I), total REMS duration over 24 hrs (J), REMS episode number per 24 hr or 12 hr in light/dark phase (K), REMS episode duration (L).

(M–P) Probabilities of transition between different sleep and wake states.

ns, not significant; *p < 0.05; **p <0.01; ***p <0.001 and ****p <0.0001; mean ± SEM. Two-way ANOVA (A, E, I, M-P); One-way ANOVA (B, F, J); Kruskal-Wallis test (C, D, G, H, K, L).

(A–C) EEG power spectrum during NREMS (A), REMS (B) or Wake (C).

(D) Normalized EEG power of δ, θ, α, σ and β waves during NREMS.

(E) Diurnal NREMS delta power density.

(F-H)Recovery of NREMS (F), REMS (G) and Wake (H) after 6 hrs of SD (ZT0-6).

(I) NREMS delta power density during the 24 hrs recovery time.

(J) Changes of NREMS delta power density after 6 hrs of SD.

(K) 1 hr representative EEG and EMG traces of littermates at each vigilance state.

(L) Representative hypnograms of littermates.

(A)Specificity test of antibodies targeting SIK3 pT469 and pS551. Recombinant SIK3(1-558) was incubated with recombinant PKA^T197E in the presence or absence of ATP. SIK3 T469 and S551 phosphorylation were then detected using indicated antibodies.

(B) Purification table showing the enzymatic activity after each step.

(C) Verification of PPP5C dephosphorylation activity. PPP5C purified from HEK293T cells was incubated with either Tau IPed from HEK293T. Phosphorylation level of indicated sites were examined.

Host mice were either WT or had Cas9 inserted at its Rosa26 site (Rosa26^Cas9/+). Each host mouse was injected with an AAV virus two weeks before an EEG recorder was placed on its head.

(A-B) Volcano plots showing changes of phosphopeptides in PPP3R1^KD/WT^Ctrl (A) and PPP3R1^KD/eGFP^Ctrl (B) groups. The significant threshold was set as p value < 0.05 and fold change > 2 or < -2. Numbers in red color represented the amounts of phosphopeptides upregulated and numbers in brackets meant the amounts of total phosphopeptide identified.

(C) Venn diagram of significantly upregulated phosphopeptides among two groups.

(D) Venn diagram of calcineurin binding motif distribution among 81 proteins to which common upregulated phosphopeptides among two groups belonged.

(A–D)Profiles of wake over 24 hrs (A), total wake duration over 24 hrs (B), wake episode number per 24 hr or 12 hr in light/dark phase (C), wake episode duration (D).

(E–H)Profiles of REMS over 24 hrs (E), total REMS duration over 24 hrs (F), REMS episode number per 24 hr or 12 hr in light/dark phase (G), REMS episode duration (H).

(I–J)EEG power spectrum during Wake and REMS.

(K–L) Self- (K) and REMS related (L) transition probabilities between different sleep and wake states.

(A–B) Recovery of wake (A) and REMS (B) after 6 hrs of SD.

(C) NREMS delta power density during the 24 hrs recovery time.

(D) Changes of NREMS delta power density after 6 hrs of SD.

(E) Representative hypnograms of littermates.

ns, not significant; *p < 0.05; **p <0.01; ***p <0.001 and ****p <0.0001; mean ± SEM. Two-way ANOVA (A, B); Mixed-effects model (C); Two-way repeated measurement ANOVA (Two-way RM ANOVA) (D).

(A-D) Profiles of wake over 24 hrs (A), total wake duration over 24 hrs (B), wake episode number per 24 hr or 12 hr in light/dark phase (C), wake episode duration (D).

(E-H) Profiles of REMS over 24 hrs (E), total REMS duration over 24 hrs (F), REMS episode number per 24 hr or 12 hr in light/dark phase (G), REMS episode duration (H).

(I–J) EEG power spectrum analysis of Wake (I) and REMS (J).

(K–L) Self- (K) and REMS related (L) transition probabilities of different sleep and wake states.

ns, not significant; *p < 0.05; **p <0.01; ***p <0.001 and ****p <0.0001; mean ± SEM. Two-way ANOVA (A, E); One-way ANOVA (B, F); Kruskal-Wallis test (C, D, G, H); Two-way repeated measurement ANOVA (Two-way RM ANOVA) (I-L).

(A-B) Recovery of Wake (A) and REMS (B) after 6 hrs of SD.

(C) NREMS delta power density during the 24 hrs recovery period after 6 hrs of SD.

(D) Changes of NREMS delta power density after 6 hrs of SD.

(E-G) Recovery of wake (E), NREMS (F) and REMS (G) after 6 hrs of SD from ZT6-12.

(H) NREMS delta power density during the 24 hrs recovery period after 6 hrs of SD from ZT6-12.

(I) Changes of NREMS delta power density after 6 hrs of SD from ZT6-12.

(J) Representative hypnograms of littermates.

ns, not significant; *p < 0.05; **p <0.01; ***p <0.001 and ****p <0.0001; mean ± SEM. Two-way ANOVA (A, B, E-G); Mixed-effects model (C, H); Two-way repeated measurement ANOVA (Two-way RM ANOVA) (D, I).

Figure S17. Cell types of AAV infection within mouse brain. (A-C) Co-staining of mScarlet and Neurotrace in the brain slices from eGFPCtrl (A) and PPP3CAKD (B) and PPP3R1KD (C) groups. Scalebars, 2000 μm. N=3. The inset graphs below represent the overlap between mScarlet and Neurotrace signals. Scalebars, 100 μm. N=3. (D) The percentage of neurons in mScarlet positive cells. (E-G) Co-staining of mScarlet and GFAP in the brain slices from eGFPCtrl (E) and PPP3CAKD (F) and PPP3R1KD (G) groups. Scalebars, 2000 μm. N=3.The inset graphs on below represent the overlap between mScarlet and GFAP signals. Scalebars, 100 μm. N=3. (H) The percentage of GFAP in mScarlet positive cells.

(A-D) Profiles of wake over 24 hrs (A), total wake duration over 24 hrs (B), wake episode duration (C), wake episode number per 24 hr or 12 hr in light/dark phase (D).

(E-H) Profiles of REMS over 24 hrs (E), total REMS duration over 24 hrs (F), REMS episode duration (G), REMS episode number per 24 hr or 12 hr in light/dark phase (H).

(I–J) EEG power spectrum analysis of Wake (I) and REMS (J).

(K–L) Self- (K) and NREMS related (L) transition probabilities of different sleep and wake states.

ns, not significant; *p < 0.05; **p <0.01; ***p <0.001 and ****p <0.0001; mean ± SEM. Two-way ANOVA (A, E); One-way ANOVA (B, F); Kruskal-Wallis test (C, D, G, H); Two-way repeated measurement ANOVA (Two-way RM ANOVA) (I-L).

(A-C) Recovery of Wake (A), NREMS (B) and REMS (C) after 6 hrs of SD from ZT6-12.

(D) Changes of NREMS delta power density after 6 hrs of SD from ZT6-12.

(E) Representative hypnograms of littermates.

ns, not significant; *p < 0.05; **p <0.01; ***p <0.001 and ****p <0.0001; mean ± SEM. Two-way ANOVA (A-C);Two-way repeated measurement ANOVA (Two-way RM ANOVA) (D).

Supplemental information includes 19 figures and 8 tables.

WT C57 BL/6J mice (8 to 10 weeks old) were purchased from Beijing Vital River Laboratories Technology Co., Ltd. or Laboratory animal resource center in Chinese Institute for Brain Research. Rosa26-Cas9 knock-in (Rosa26^Cas9/+, RRID: IMSR_JAX:024858) mice were obtained from Jackson laboratory (Platt et al., 2014). SIK3-3xHA mice were constructed in BIOCYTOGEN by infusing a 3xHA-T2A-iCre cassette into the C terminus right before the SIK3 stop codon. SIK3 point mutant mice were constructed with CRISPR-Cas9 mediated homologous recombination. For SIK3^T469A/+, the gRNA sequence was 5’- TTTGTCAATGAGGAGGCACA-3’ and a single strand homologous arm was designed to introduce nucleotide mutation from ACG to GCG (T469A) as well as a restriction enzyme site BstUI for future genotyping, sequence of which was 5’-CCTTCTCCAGAAGCCTTGGTTCGCTATTTGTCAATGAGGAGGCACGCGGTGGGAGTGGCTGACCCACGGTAAGTACCTGGTCAGCATCCT-3’. For SIK3^T469E/+, the gRNA sequence was 5’-TTTGTCAATGAGGAGGCACA-3’, the single strand homologous arm with BstUI was 5’-CCTTCTCCAGAAGCCTTGGTTCGCTATTTGTCAATGAGGAGGCATGAAGTGGGAGTGGCTGACCCACGGTAAGTACCTGGTCAGCATCCT-3’. For SIK3^S551A/+, the gRNA sequence was 5’-GGCCGGAGAGCCTCAGATGG-3’, the single strand homologous arm with BstUI was 5’-ACACTACAGCTACTGAACGGAATGGGGCCCCTTGGCCGGAGAGCCGCGGACGGAGGCGCCAACATCCAACTGCATGCCCAGCAGCTGCTCAAG-3’. A mixture of Cas9-expressing mRNA, single strand homologous arm and sgRNA was injected into fertilized eggs through electroporation and the eggs were then transplanted into the womb of foster mothers. F0 and F1 mice were genotyped through PCR and BstUI digestion to make sure the presence of recombination. Mutant lines were back-crossed to C57BL/6J for at least 5 generations to exclude possible off-targeting.

All experimental procedures were performed in accordance with the guidelines and were approved by the Animal Care and Use Committee of Chinese Institute for Brain Research, Beijing. Mutant mice and wt littermates were maintained on a C57 BL/6J background. Mice were housed under a 12 hr:12 hr light/dark cycle and controlled temperature and humidity conditions. Food and water were delivered ad libitum. Mice used in all experiments were 10-14 weeks old.

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, C11995500BT) medium containing 10% fetal bovine serum (Transgen, FS101-02) and 1% Penicillin/Streptomycin (Gibco, 15070-063). cDNAs were transfected into HEK293T cells with Lipofectamine 3000 reagent (Thermo Fisher, L3000015) according to the manufacturer’s instructions and harvested 24 to 28 hrs after transfection.

HEK293T cells were treated with ionomycin (MCE, SQ23377) at indicated concentrations and time durations at 37℃. Cells were then harvested and lysed with 1 ml lysis buffer (0.3% Chaps, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, pH 7.4, 1x protease inhibitor cocktail (Roche, cOmplete™), 1x phosphatase inhibitor II (Sigma, P5726) and 1x phosphatase inhibitor III (Sigma, P0044)) before centrifugation of cell lysates at 13000 rpm for 10 mins at 4 ℃. Protein concentrations of cell lysates were determined with the bicinchoninic acid assay (Thermo Fisher, 23225) and normalized to 2 mg/ml. Samples were analyzed by immunoblotting with the indicated antibodies.

Whole brains of mice were quickly dissected, rinsed withP BS and homogenized by homogenizer (Wiggens, D-500 Pro) in ice-cold lysis buffer (150 mM NaCl, 1% Triton-X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-base, freshly supplemented with a protease and phosphatase inhibitor cocktail). Brain homogenates were centrifuged at 15000 rpm for 25 mins at 4℃. Supernatants were carefully transferred into a new microtube. Protein concentrations of brain lysates were determined with the bicinchoninic acid assay and normalized to 2 mg/ml. Before immunoblotting, samples were kept in liquid nitrogen, if necessary. 

Mice were anesthetized with 3% Tribromoethanol and perfused with 1× PBS followed by chilled 4% paraformaldehyde (PFA). Then the mouse brain was dissected. After 24h post-fixation at 4℃ and gradient dehydration from 20% sucrose to 30%, 40-μm sagittal brain slices were collected sequentially using a cryostat (CM3050 S, Leica). Free-floating sections were washed with PBS and then blocked in 5% albumin bovine serum (BSA, Sigma) in PBST (0.5% Triton X-100 in PBS) at room temperature for 2 h. After blocking, the sections were incubated with primary antibodies diluted in 3% albumin bovine serum (BSA, Sigma) in PBST (0.3% Triton X-100 in PBS) at 4℃ overnight. The primary used were as follows, rabbit anti-Calcineurin antibody (1:500, Abcam), rabbit anti-CNB antibody (1:500, Abcam), Chicken anti-GFAP antibody (1:500, Abcam), rabbit anti-RFP antibody (1:1000, Rockland). Then the sections were washed for 3 times with PBS before incubated in 3% albumin bovine serum with secondary antibody for 2h. Alexa 546 goat anti-rabbit IgG (H + L) (1:500, Invitrogen), alexa 488 goat anti-rabbit IgG (H + L) (1:500, Invitrogen), alexa 488 goat anti-chicken IgG (H + L) (1:500, Invitrogen) was used as the secondary antibody. Neurotrace 505/525 (1:500) was used. After 3 times wash, the brain slices were attached to adhesive slides. Images were collected using LSM880 confocal microscope (Zeiss) with 10×/0.45NA air objective and a 20×/0.8NA air objective. Mean fluorescence intensity was calculated for semi-quantitative fluorescence analysis using the software imageJ. Neuron positive cells were also counted using imageJ.

Lysates from HEK293T cells were prepared and filtered through 0.45 mm filters (Millipore, SLHV033R). 500 mg cell lysates at a concentration of 10 mg/ml were fractionated on a Q HP (Cytiva, 17115401) anionic chromatography column, eluted with a linear gradient of NaCl (0-600 mM) into 20 column volumes (CVs), with each fraction collected as one CV as samples 1 to 20, and the final wash with 1 M NaCl buffer A (20mM, HEPES, 10mM KCl, 1.5mM MgCl₂, 3mM DTT, pH 7.4) with 5 CVs gave rise to samples 21 to 25. Each fraction was dialyzed into buffer A, removing NaCl. 10 ml of each sample was used for analysis of activities removing phosphate from SIK3 T469 and S551. T469 and S551 of bacterially expressed recombinant SIK3 were phosphorylated by PKA in vitro before being used to test phosphatase activities of the fractions of HEK lysates. Fractions 10 to 13 contained significant activities removing phosphate from SIK3 T469 and S551.Fractions 10 to 13 from QHP were combined and dialyzed with buffer A before being loaded onto a Blue HP column (Cytiva, 17041301). It was eluted with a linear gradient of NaCl (0-600 mM) into 20 column volumes (CVs), with each fraction collected as one CV as samples 1 to 20, and the final wash with 1 M NaCl buffer A with 5 CVs gave rise to samples 21 to 25. The FL fraction from the Blue HP contained significant activities removing phosphate from SIK3 T469 and S551.The FL fraction from the Blue HP column was dialyzed with buffer A and loaded onto a SP HP column. The FL fraction from the SP-HP column (Cytiva, 17115201) contained significant activities removing phosphate from SIK3 T469 and S551.The FL fraction from the SP-HP column was dialyzed with buffer A and loaded onto a heparin HP column (Cytiva, 17040703). The FL fraction and Fraction 1 contained significant activities removing phosphate from SIK3 T469 and S551.The FL fraction from the heparin HP column was dialyzed with buffer A and loaded onto a HAP HP column (BioRad, 7510025). The rest of the fractionation was similar to the first column except that the final wash was with 5 CVs of 500 mM K₂PO₄, giving rise to samples 21 to 25. Fractions 5 to 7 contained significant activities removing phosphate from SIK3 T469 and S551.Active fractions from the HAP HP column were condensed into 0.5 ml, fractionated on a Superdex 200 molecular sieve column (Cytiva, 28990944), eluted with 200 mM NaCl into 20 ml. 1 ml from each fraction was collected and labeled as samples 1 to 20. Protein contents were monitored with UV at 280 nm.

Because green LED irradiation significantly initiates the crosslinking reaction which leads to covalent capture of PPIs, this strategy allows us to avoid the loss of interacting proteins caused by sample processing steps such as washing during subsequent enrichment and MS analysis. It thus reduces false-negative results caused by traditional PPI study methods such as affinity purification-mass spectrometry (AP-MS). Therefore, in our experiments, we prepared samples treated with and without green light irradiation treatment simultaneously, of which the latter served as background results of normal immunoprecipitation as no photo-crosslinking reaction occurs. By quantitatively comparing the enriched interacting proteins between the +GL and -GL groups with MS analysis, proteins significantly enriched in the light-irradiated group become primary candidates.

SIK3-3xHA mice were sacrificed and brain slices were kept in PBS. Neurobasal medium (Gibco, 21103049) supplied with B-27 (Gibco, 17504044) for photocatalytic crosslinking was prepared in darkness by adding eosin Y (Sigma-Aldrich, E4009) and 1,6-dihexamine (Sigma-Aldrich, H11696) to a working concentration of 50 μM and 1 mM, respectively. Cell culture chambers (Millipore, PICM0RR50) were placed in 6-well plates and rinsed with PBS. Brain slices were transferred to chambers carefully with a sterile dropper and each chamber finally contained four slices to ensure thorough stretch of each slice. PBS was discarded by pipetting from the outer side of chambers and 0.5 ml of neurobasal medium containing photocatalytic crosslinkers was added into each well to ensure that each slice was totally infiltrated with the probes. Samples were incubated at 37°C with 5% CO₂ for 1 hr before photo-irradiation. For photocatalytic crosslinking, the plates were placed on green LED (520 nm, 20 mW/cm²) equipment while an ice bag and a fan were used to reduce light irradiation-induced heat. After 15 mins of green light irradiation, the color of the medium was bleached, indicating effective activation of the eosin probe. The medium was discarded and after one round of PBS wash, the slices was collected into 1.5 ml Eppendorf tubes, which were placed into liquid N₂ to freeze the slices.

To each tube with frozen brain slice samples was added 1 ml of ice-cold lysis buffer containing 1% of protease cocktail and a steel ball. Samples were lysed through ultrasonication. After centrifugation (12,000 g, 10 mins, 4°C) to discard the residue, crosslinked proteins were enriched by anti-HA magnetic beads (Pierce, 88837) according to the manufacture protocol. Importantly, the beads should be washed with lysis buffer (with 0, 0.25, and 0.5 M NaCl) for three times to diminish non-specific binding proteins. Crosslinked proteins were eluted with 2x SDS-loading buffer and heated to 95 °C for 10 mins. Eluted proteins were subjected to further Western analysis and LC-MS/MS.

Proteins enriched with HA beads were loaded on an 8% SDS-PAGE gel and run at 150 V for 30 mins. After silver staining (Sigma-Aldrich, PROTSIL2), the desired bands of protein mixture were excised and cut into 1 mm³ pieces. The gel pieces were discolored in discoloring buffer until they all turned transparent. A dehydration process was carried out by adding pure acetonitrile into gel pieces until they were totally dehydrated to appear non-transparently white. Samples were then incubated in the reduction buffer (10 mM DTT (Sigma-Aldrich, D9779), 50 mM ammonium bicarbonate (Sigma-Aldrich, A6141)) at 56 °C for 30 mins and further incubated in alkylation buffer (55 mM iodoacetamide (Sigma-Aldrich, I6125), 50 mM ammonium bicarbonate) at 37 °C for 30 mins in the dark. After washed by 50 mM ammonium bicarbonate buffer twice, gel pieces were dehydrated through the same protocol. 20 ng/μltrypsin buffer in 50 mM ammonium bicarbonate was added and samples were incubated at 4 °C for one hr. The remaining buffer was discarded and 50 mM ammonium bicarbonate buffer was added for another 16 hrs of digestion at 37 °C. The resulting peptides were extracted with extraction buffer (50% acetonitrile, 45% water and 5% formic acid (Honeywell, 09676)), before being centrifuged to dryness under vacuum.

The collected peptides were reconstituted in 100 mM TEAB buffer (Sigma-Aldrich, T7408). 39.688 mg/mL of NaBH₃CN (Sigma-Aldrich, I56159) followed by 4% (v/v) CH2O or CD2O were added for light and heavy dimethyl labeling, respectively, following the addition of 39.688 mg/ml NaBH₃CN. After incubation in a fume hood for 30 mins at room temperature, enough 1% (v/v) ammonia solution and formic acid were added immediately to quench the labeling reaction. The light and heavy samples were combined together, and then desalted and dried under vacuum.

Trypsin digested peptides were analyzed on a Exploris 480 Hybrid Quadrupole Orbitrap Mass Spectrometer as well as Thermo Scientific Q Exactive Orbitrap Mass Spectrometer in conjunction with an Easy-nLC II HPLC (Thermo Fisher Scientific). The mobile phases were A: 0.1% formic acid in H₂O; B: 0.1% formic acid in 80% ACN–20% H₂O. MS/MS analysis was performed under the cationic mode with a full-scan m/z range from 350 to 1,800 and a mass resolution of 70,000.

For quantitative SIK3 interactome analysis, the quantification of light/heavy ratios was calculated with a precursor mass tolerance of 20 ppm. Alkylation of cysteine (+57.0215 Da) was set as the static modification, and oxidation of methionine (+15.9949 Da) and acetylation of N-terminal Lys (+42.0106 Da) was assigned as the variable modification. The isotopic modifications (28.0313 and 32.0557 Da for light and heavy labeling, respectively) were set as fixed modifications on the peptide N-terminus and lysine residues. Half-tryptic terminus and up to two missing cleavages were set within tolerance.

For HEK293T cells, plasmids expressing FLAG-tagged SIK3 and HA-tagged PPP3CA were transfected for 24 hrs. Cells were then collected and lysed with 1 ml lysis buffer (25 mM Tris-base pH 7.4, 150 mM NaCl, 1% NP40, 1mM EDTA, 5% glycerol,1x protease inhibitor cocktail, 1x phosphatase inhibitor II and 1x phosphatase inhibitor III) before centrifugation at 13000 rpm for 10 mins at 4℃. 40 μl supernatants were transferred into new microtubes as input samples and the rest was incubated either with 20 μl anti-FLAG (Millipore, M8823) or anti-HA antibody coated magnetic beads balanced by lysis buffer for 1 hr at 4 ℃. Beads were then washed with 1 ml lysis buffer three times at 4 ℃ and 40 μl PBS was added to transfer the beads into new microtubes as enriched samples. For mouse, anti-PPP3CA or anti-SIK3 antibody and corresponding IgG were pre-incubated with protein A/G beads (YEASEN, 36401ES08) for 2 hrs at 4℃ in lysis buffer. Protein samples from mouse brain were at first pre-absorbed using IgG-protein A/G beads for 30 mins at RT and 40 μl supernatants were transferred into a new microtube as input sample before anti-PPP3CA/SIK3 protein A/G beads was added and rotated overnight at 4℃. Beads were then washed with 1 ml lysis buffer three times at 4℃ and 40μl PBS was added to transfer the beads into a new tube as enriched samples.

cDNAs for specific proteins were subcloned into the pET-28a vector, with appropriate tags such as MBP, GFP or FLAG. Plasmids were transfected into E. coli BL21 and incubated at 37 ℃ until the OD reached 0.6, when 0.5 mM of IPTG (Sigma, I6758) was added at 18 ℃ to induce protein expression for 16 hrs. Cells were collected and treated with Ni column binding buffer (300 mM NaCl, 20 mM Tris-HCl, pH 7.5) containing protease inhibitors and thoroughly suspended. Cells were lysed with ultrasonication before centrifugation (14,000 rpm, 30 mins, 4℃). Supernatants were filtered through 0.45 mm filters and purified by Ni beads to 90% purity. Eluted proteins were measured with Coomasie blue (Thermo Scientific, 20278) and the rest of the proteins were stored at -80 ℃.

Plasmids expressing FLAG-tagged SIK3 were transfected into HEK293T cells for immunoprecipitation. After 24 hrs, cells were collected and lysed with 1 ml lysis buffer (25 mM Tris-base pH 7.4, 150 mM NaCl, 1% NP40, 1mM EDTA, 5% glycerol,1x protease inhibitor cocktail, 1x phosphatase inhibitor II and 1x phosphatase inhibitor III) before centrifugation at 13000 rpm for 10 min at 4 ℃. Cell lysates were incubated with 20μl anti-FLAG antibody coated magnetic beads balanced by lysis buffer for 1 hr at 4 ℃. Beads were washed with 1 ml lysis buffer three times at 4 ℃, before final elution with 30 μl buffer A containing 2 mg/ml 3xFLAG peptide. Phosphatase reactions were performed for 2 hrs at 37℃ by adding 4 μl FLAG-SIK3, 3 μg rPPP3CA, 3 μg rPPP3R1, 4 μg rCaM with a final concentration of 10 mM CaCl₂ and 20 mM MgCl₂. Samples were analyzed by immunoblotting with the indicated antibodies.

Viruses used in this study: AAV2/PHP.eB-CMV-mScarlet-PPP3CA-sgRNA-WPRE, AAV2/PHP.eB-CMV-mScarlet-PPP3R1-sgRNA-WPRE and AAV2/PHP.eB-CMV-mScarlet-eGFP-sgRNA-WPRE. PPP3CA^KD and PPP3R1^KD mice were generated with triple-targeted CRISPR-Cas9 technology (Sunagawa et al., 2016) by virus injection. Plasmids were generated before virus package. Three sgRNA sequences targeting PPP3CA or PPP3R1 were designed through VBC Score (vbc-score.org) and cloned into the PM04 plasmid, and sequentially inserted into pAAV-CMV-mScarlet-WPRE using Gibson assembly (NEB, E2611S). sgRNA sequences were shown as follows: 

PPP3CA-gRNA1: GACCATAGGATGTCACACAT;

PPP3CA-gRNA2: GCAGTCGAAGGCATCCATAC;

PPP3CA-gRNA3: GAGGCTGTTCGTACTTCTAC;

PPP3R1-gRNA1: GCTGATGAAATTAAAAGGCT;

PPP3R1-gRNA2: GCGATAAGGAACAGAAGTTG;

PPP3R1-gRNA3: GCAGAACCCTTTAGTACAGC.

Mice at 8 weeks old were anaesthetized using 4-5% isoflurane (Sigma, 792632, maintained at 1-2% for surgery), and 100uL virus (5×10¹² gc/ml) was injected through retro-orbital sinus (Yardeni et al., 2011). After two weeks to allow for expression, electroencephalogram (EEG) implantation surgery was performed according to the protocol published previously (Qu et al., 2010). Mice were fixed in stereotaxic (RWD Life Science, 68405) and skull was exposed. Two holes were drilled at the frontal and the parietal cortex over the right cerebral hemisphere (Frontal: lateral to middle 1.5mm, 1.0mm anterior to bregma; parietal: lateral to middle 1.5mm, 1.0mm anterior to lambda). Two stainless steel screws (RWD Life Science), each soldered to a short copper wire, were inserted into the holes. Two EMG wires were implanted bilaterally into the neck muscle. All the copper wires were attached onto a micro-connector and was fixed to the skull. After surgery, mice were single housed for five days of recovery in new cages and then were placed into the special recording cage for three days to habituate to the recording cables.

Extract protein from tissue:Remove the whole mouse brain tissue from liquid nitrogen, place it in a glass tissue grinder, and use 700 μl of pre-cooled lysis buffer (8 mol/L urea, protease inhibitor, phosphatase inhibitor) to its grinding. Ultrasonicate for 8 mins, centrifuge at 14,000g for 40 mins at 4°C, and take the supernatant.  Protein in the supernatant was quantified using BCA.

Mass spectrometry sample preparation: A protein lysate sample containing 1 mg of protein was reduced with 10 mM dithiothreitol (DTT) at 56°C for 1 hr, and then alkylated with 30 mM iodoacetamide (IAA) in the dark at room temperature for 30 mins.  The reacted liquid was added to the activated 10 kD ultrafiltration tube, centrifuged at 14,000g for 40 mins, and washed three times with 50mM ammonium bicarbonate after centrifugation.  Add trypsin to the ultrafiltration tube and digest overnight at 37 °C (1:50, enzyme: protein).  Centrifuge at 14,000g for 20 mins, collect the proteolytic solution, add 50 mM ammonium bicarbonate, centrifuge at 14,000g for 20 mins, repeat twice, combine the three obtained solutions, and centrifuge in vacuum until dry.

Enrichment of phosphopeptides:Resuspend the peptides in phosphopeptide binding buffer (70% acetonitrile (ACN), 5% TFA (trifluoroacetic acid), 8.3% LA (lactic acid)) for later use, and divide it into three equal portions of titanium dioxide (protein).  Amount: titanium dioxide = mass ratio 1:10), wash with phosphopeptide binding buffer 3 times, add the peptide mixture, gently rotate at room temperature for 30 mins, centrifuge at 3000g for 1 min, take out the supernatant, spin and combine each portion for 30 mins, and finally discard Remove the peptide mixture.  Add binding buffer and wash 2 times, wash buffer 1 (30% ACN, 0.5% TFA) for 2 times, and finally wash with wash buffer 2 (80% ACN, 0.5% TFA) 2 times, centrifuge at 3000g for 1 min each time. Then discard the supernatant. Elute twice with 200 μl of elution buffer (40% ACN, 15% ammonia), centrifuge at 3000 g for 2 mins, combine the two eluates and centrifuge in vacuum until dry.

EEG and EMG data recording and analysis were performed as our previous study (Liu et al., 2022). EEG and EMG data at basal sleep conditions were recorded for 2 consecutive days, with a sample frequency of 200 Hz and epoch length of 4 seconds. EEG and EMG data were initially processed using AccuSleep (Barger et al., 2019) and then were manual correction in SleepSign. EEG and EMG signals were classified into Wake (fast and low amplitude EEG, high amplitude and variable EMG), NREM (high amplitude and 1-4 Hz dominant frequency EEG, low EMG tonus) and REM (low amplitude and 6-9 Hz frequency EEG, complete silent of EMG). The state episode was defined as at least three continuous and unitary state epochs. Epoch contained movement artifacts were included in sleep duration analysis but excluded from the subsequent power spectrum analysis. For EEG power spectrum analysis, EEG signals were subjected to fast Fourier transform analysis (FFT) from 0-25 Hz with a 0.25 Hz bin resolution. Normalized power spectrum represents the mean ratio of each 0.25 Hz-bin which was normalized to total EEG power (0-25 Hz) during 24 hours baseline condition. The hourly NREMS EEG delta density represents the average ratio of delta (1-4 Hz) power to total EEG power (0-25 Hz) of NREM epochs in each hour. Cumulative rebound represented cumulative changes of time in post-SD compared with the same ZT under the baseline condition. For short, hourly time spent of each state in post-SD subtracts that of the corresponding ZT during baseline, the difference for each hour were then summed to generate cumulative rebound curve. Sleep/wake transition probabilities was analyzed as described in a previous study (Tone et al., 2022). For instance, P_{W to NR}= N_{W to NR} / (N_{W to W} + N_{W to R} + N_{W to NR}), N_{W to NR} denotes the number of transitions that transit from wakefulness epoch to NREMS epoch. W: wakefulness epoch, NR：NREM epoch, R: REM epoch. The changes of NRMES delta density denotes the difference before and after sleep deprivation, which are calculated as the value of the NRMES delta density after sleep deprivation minus that of the baseline at the equivalent ZT point.

After 2 consecutive days of EEG and EMG signals recording, mice were introduced into new cages at ZT0 or ZT6. Mice were gently handled or touched to keep them awake for 6 hrs of sleep deprivation, before being returned to the recording cage for another 24 hrs of recording.

All statistical analyses were performed using GraphPad Prism 9.0. One-way ANOVA was used to compare differences among more than three groups, followed by Tukey’s multiple comparisons tests. Kruskal-Wallis tests were used for non-parameters tests. Two-way ANOVA was used to compare the differences between different groups with different treatments, followed by Tukey’s multiple comparisons tests. Two-way ANOVA with repeated measurements (Two-way RM ANOVA) was used when the same individuals were measured on the same outcome variable more than once, followed by Tukey’s multiple comparisons test. Data are presented as mean±SEM. In all cases, p values more than 0.05 were considered not significant.

Data reported in this paper will be shared by the lead contact upon request. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

We are grateful to Dr. Juan Huang at CIBR for generating SIK3 mutant mice, to Dr. Lei Zhang at CIBR instrument core for help with customizing experimental devices for EEG recording, to Dr. Yuan Li for help with mouse brain slice preparation, to Linghao Kong and Ruixiang Wang from the CLS and Dr. Gongzheng Zhao from the Multi-Omics Mass Spectrometry Core of Shenzhen Bay Laboratory for assistance with mouse brain MS sample processing, to the National Center for Protein Sciences at Peking University for access to instrumentation, and to the Chinese Academy of Medical Sciences (2019RU003) for support. Research in the Rao Laboratory has never been contaminated by the Chinese Brain Initiative（饒毅實驗室的研究從未被中國腦計劃所汙染）。‍‍