裂殖酵母中定位在SPB处的CK2激酶磷酸化Wee1激酶并促进其泛素化降解的分子机制研究

Wee1 kinase is a critical rate-limiting factor for G2/M transition in eukaryotes. It negatively regulates Cdk1 kinase activity during the G2 phase of the cell cycle to inhibit the mitotic entry. In human somatic cells, it has been demonstrated that Wee1 can be phosphorylated by Cdk1, CK2 and Polo kinases and its ubiquitylation and degradation is usually followed. Although the fission yeast (Schizosaccharomyces pombe) Wee1 was the first identified member of Wee1 protein kinase family, how its activity is regulated by phosphorylation is largely unclear. Our preliminary data revealed that fission yeast CK2 kinase affects the protein level and activity of Wee1, and Ser37 is one of the potential sites phosphorylated by CK2. Interestingly, the spindle pole body (SPB) (the counterpart of centrosome in higher eukaryotes) most likely functions as a “triggering center” for phosphorylation of Wee1 by CK2 kinase. In addition, we also found that Ckb1, the regulatory subunit of CK2 kinase, and nucleolar protein Dnt1 affect the localization of CK2 at SPBs. Our previously published data uncovered that Dnt1 downregulates Wee1 protein level during G2/M transition. Thus, based on these results, we propose that fission yeast Dnt1 might regulate the protein level and activity of Wee1 through promoting localization of CK2 at SPBs, namely the Dnt1-CK2-Wee1 cascade controls G2/M transition using SPB as a signaling center. This is a very novel mechanism for cell cycle regulation. In this project, we intend to dissect the molecular details of this SPB-based mechanism for regulation of G2/M transition.

Wee1激酶是真核细胞G2/M期过渡的关键限速因子，它负调控Cdk1激酶的活性来限制细胞进入M期。人类中Wee1被Cdk1, CK2和Polo等激酶磷酸化并促进其泛素化降解。尽管裂殖酵母Wee1是这一家族中最早被分离鉴定的成员，但一直缺乏针对其被磷酸化调控的细致研究。我们前期研究发现：裂殖酵母CK2激酶影响Wee1蛋白水平和活性，Ser37为其潜在磷酸化位点；相当于中心体的纺锤体极体(SPB)可能是CK2调控Wee1的“指令中心”；另外，CK2的调节亚基Ckb1及核仁蛋白Dnt1影响CK2激酶在SPB处的定位。我们已发表的研究表明Dnt1在G2/M期下调Wee1蛋白水平。因而，我们推测Dnt1通过促进CK2在SPB处定位来调控Wee1活性和水平，即Dnt1-CK2-Wee1三者以SPB作为信号中心调控G2/M期过渡。这是一种非常新颖的细胞周期调控机制，本项目拟研究这一调控机制的具体细节。

Wee1激酶是真核细胞G2/M期过渡的关键限速因子，它负调控Cdk1激酶的活性来限制细胞进入M期。人类中Wee1被Cdk1, CK2和Polo等激酶磷酸化并促进其泛素化降解。. 在本研究中，我们利用遗传学、细胞生物学以及分子生物学手段研究了裂殖酵母中Wee1被CK2激酶磷酸化调控的可能机制。我们发现，CK2激酶主要是通过促进Wee1蛋白的稳定性，而不是通过影响其转录水平来调控其活性的。CK2激酶对于Wee1的磷酸化有利于抑制其被泛素连接酶复合体SCF的泛素化修饰和蛋白酶体介导的降解。我们还确定了，裂殖酵母细胞中组装到SCF复合体中负责降解Wee1的F-box蛋白主要为Pof3和Pof1。. 通过定点突变Wee1中潜在的磷酸化位点，我们发现Wee1中有8个可能被CK2磷酸化的Ser/Thr位点。尽管我们并未检测到CK2和Wee1之间的相互作用，但是我们发现在ckb1∆细胞中，Wee1的泛素化水平升高。因而，我们推测，当Wee1被磷酸化后，极有可能会抑制其被泛素化修饰和蛋白酶体介导的降解，这与人类中的情形有很大不同。. 另外，我们还发现，dnt1∆细胞中CK2更多地入核，同时，Wee1的泛素化水平降低。暗示Dnt1主要是通过限制CK2对于Wee1的磷酸化，来去除CK2对于Wee1的保护作用，进而促进Wee1的泛素化修饰和降解。. 综上所述，我们的研究发现Dnt1-CK2-Wee1三者以SPB作为信号中心，通过调控Wee1的磷酸化和泛素化来调控G2/M期过渡。