Medley Jeffrey C, Yim Nahyun, DiPanni Joseph, Sebou Brandon, Shaffou Blake, Cramer Evan, Wu Colin, Kabara Megan, Song Mi Hye
Department of Biological Sciences, Oakland University, MI, USA.
Department of Chemistry, Oakland University, MI, USA.
bioRxiv. 2023 May 30:2023.05.07.539463. doi: 10.1101/2023.05.07.539463.
Spindle bipolarity is critical for genomic integrity. Given that centrosome number often dictates mitotic bipolarity, tight control of centrosome assembly is vital for the fidelity of cell division. The kinase ZYG-1/Plk4 is a master centrosome factor that is integral for controlling centrosome number and is modulated by protein phosphorylation. While autophosphorylation of Plk4 has been extensively studied in other systems, the mechanism of ZYG-1 phosphorylation in remains largely unexplored. In , Casein Kinase II (CK2) negatively regulates centrosome duplication by controlling centrosome-associated ZYG-1 levels. In this study, we investigated ZYG-1 as a potential substrate of CK2 and the functional impact of ZYG-1 phosphorylation on centrosome assembly. First, we show that CK2 directly phosphorylates ZYG-1 and physically interacts with ZYG-1 Intriguingly, depleting CK2 or blocking ZYG-1 phosphorylation at putative CK2 target sites leads to centrosome amplification. In the non-phosphorylatable (NP)-ZYG-1 mutant embryo, the overall levels of ZYG-1 are elevated, leading to an increase in centrosomal ZYG-1 and downstream factors, providing a possible mechanism of the NP-ZYG-1 mutation to drive centrosome amplification. Moreover, inhibiting the 26S proteasome blocks degradation of the phospho-mimetic (PM)-ZYG-1, while the NP-ZYG-1 mutant shows partial resistance to proteasomal degradation. Our findings suggest that site-specific phosphorylation of ZYG-1, partly mediated by CK2, controls ZYG-1 levels via proteasomal degradation, limiting centrosome number. We provide a mechanism linking CK2 kinase activity to centrosome duplication through direct phosphorylation of ZYG-1, which is critical for the integrity of centrosome number.
纺锤体双极性对于基因组完整性至关重要。鉴于中心体数量通常决定有丝分裂双极性，对中心体组装的严格控制对于细胞分裂的保真度至关重要。激酶ZYG-1/Plk4是一种主要的中心体因子，对于控制中心体数量不可或缺，并受蛋白质磷酸化调节。虽然Plk4的自磷酸化在其他系统中已得到广泛研究，但ZYG-1磷酸化的机制在很大程度上仍未被探索。在秀丽隐杆线虫中，酪蛋白激酶II（CK2）通过控制与中心体相关的ZYG-1水平来负向调节中心体复制。在本研究中，我们研究了ZYG-1作为CK2的潜在底物以及ZYG-1磷酸化对中心体组装的功能影响。首先，我们表明CK2直接磷酸化ZYG-1并与ZYG-1发生物理相互作用。有趣的是，耗尽CK2或在假定的CK2靶位点阻断ZYG-1磷酸化会导致中心体扩增。在非磷酸化（NP）-ZYG-1突变体胚胎中，ZYG-1的总体水平升高，导致中心体ZYG-1及其下游因子增加，这为NP-ZYG-1突变驱动中心体扩增提供了一种可能的机制。此外，抑制26S蛋白酶体可阻断磷酸模拟（PM）-ZYG-1的降解，而NP-ZYG-1突变体对蛋白酶体降解表现出部分抗性。我们的研究结果表明，部分由CK2介导的ZYG-1位点特异性磷酸化通过蛋白酶体降解控制ZYG-1水平，从而限制中心体数量。我们提供了一种机制，通过ZYG-1的直接磷酸化将CK2激酶活性与中心体复制联系起来，这对于中心体数量的完整性至关重要。
