Dillac Louis, Porębska Klaudia, Kloc Malgorzata, Piprek Rafal P, Tassan Jean-Pierre, Kubiak Jacek Z
Dynamics and Mechanics of Epithelia Group, Institute of Genetics and Development of Rennes (IGDR), National Centre for Scientific Research (CNRS), Faculty of Medicine, University of Rennes, UMR 6290, 35043 Rennes, France.
Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA.
Int J Mol Sci. 2025 May 1;26(9):4309. doi: 10.3390/ijms26094309.
The control of cyclin-dependent kinase 1 (CDK1) kinase activity is crucial for cell cycle progression. Cell division cycle 6 (CDC6) inhibits this activity in embryonic mitoses, and thus regulates the timing of cell division progression. The meiotic cell cycle differs greatly from the mitotic one. Metaphase II (MII)-arrested oocytes remain in prolonged M-phase state due to the high activity of CDK1 in the presence of CytoStatic Factor (CSF). The role of CDC6 in the control of CDK1 during MII and oocyte activation remains unknown. Here, we studied the role of CDC6/CDK1 interactions in cell-free extracts arrested in MII (CSF extract) and upon calcium activation leading to meiotic-to-mitotic transition. The CSF extract allows analysis of biochemical processes based on immunodepletion of selected proteins and facilitates manipulations using addition of recombinant proteins. We show by glutathione S-transferase (GST)-CDC6 pull-down that CDC6 associates with CDK1 in CSF extract and by histone H1 kinase assay that it downregulates CDK1 activity. Thus, CDC6-dependent inhibition of CDK1 is involved in the homeostasis of the MII-arrest. Upon CSF extract activation with calcium exogenous GST-CDC6 provokes accelerated transition from MII to interphase, while the depletion of endogenous CDC6 results in a slower transition to interphase. We demonstrate this by following both the phosphorylation state of CDK1 substrate cell division cycle 27 (CDC27) and histone H1 kinase assay. Importantly, increasing doses of GST-CDC6 proportionally accelerate CDK1 inactivation showing that CDC6 controls the dynamics of MII to interphase transition in a dose-dependent manner. Thus, CDC6 is a CDK1 silencer acting upon both the MII arrest and CSF extract activation by assuring the physiological activity of CDK1 during this meiotic arrest and correct timely inactivation of this kinase during the second process. Thus, we show that CDC6 controls CDK1 not only during mitotic divisions, but also in MII-arrest and the meiotic-to-mitotic transition in cell-free extracts. This study aims to bridge that gap by investigating CDC6 function using a biochemically controlled system.
细胞周期蛋白依赖性激酶1（CDK1）激酶活性的控制对于细胞周期进程至关重要。细胞分裂周期6（CDC6）在胚胎有丝分裂中抑制这种活性，从而调节细胞分裂进程的时间。减数分裂细胞周期与有丝分裂细胞周期有很大不同。由于在细胞静止因子（CSF）存在下CDK1的高活性，中期II（MII）阻滞的卵母细胞保持延长的M期状态。CDC6在MII期间和卵母细胞激活过程中对CDK1的控制作用仍然未知。在这里，我们研究了CDC6/CDK1相互作用在停滞于MII期的无细胞提取物（CSF提取物）以及钙激活导致减数分裂向有丝分裂转变过程中的作用。CSF提取物允许基于所选蛋白质的免疫耗竭分析生化过程，并便于通过添加重组蛋白进行操作。我们通过谷胱甘肽S-转移酶（GST）-CDC6下拉实验表明CDC6在CSF提取物中与CDK1结合，并且通过组蛋白H1激酶测定表明它下调CDK1活性。因此，CDC6依赖性对CDK1的抑制参与了MII阻滞的稳态。在用钙激活CSF提取物后，外源性GST-CDC6会促使从MII期到间期的加速转变，而内源性CDC6的耗竭会导致向间期的转变更慢。我们通过跟踪CDK1底物细胞分裂周期27（CDC27）的磷酸化状态和组蛋白H1激酶测定来证明这一点。重要的是，增加剂量的GST-CDC6会按比例加速CDK1失活，表明CDC6以剂量依赖性方式控制MII期到间期转变的动力学。因此，CDC6是一种CDK1沉默因子，通过确保该激酶在减数分裂停滞期间的生理活性以及在第二个过程中正确及时失活，作用于MII阻滞和CSF提取物激活。因此，我们表明CDC6不仅在有丝分裂期间控制CDK1，而且在无细胞提取物的MII阻滞和减数分裂向有丝分裂转变过程中也起作用。本研究旨在通过使用生化控制系统研究CDC6的功能来填补这一空白。
