School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Ireland.
Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Ireland.
Cell Cycle. 2023 Apr;22(8):951-966. doi: 10.1080/15384101.2023.2167430. Epub 2023 Jan 23.
The centrosome acts as a protein platform from which proteins are deployed to function throughout the cell cycle. Previously, we have shown that the prolyl isomerase Cyclophilin A (CypA) localizes to the centrosome in interphase and re-localizes to the midbody during mitosis where it functions in cytokinesis. In this study, investigation of CypA by SDS-PAGE during the cell cycle reveals that it undergoes a mobility shift during mitosis, indicative of a post-translational modification, which may correlate with its subcellular re-localization. Due to the lack of a phospho-specific antibody, we used site-directed mutagenesis to demonstrate that the previously identified serine 77 phosphorylation site within CypA is important for control of CypA centrosome localization. Furthermore, CypA is shown to interact with the mitotic NIMA-related kinase 2 (Nek2) during interphase and mitosis, while also interacting with the Nek2-antagonist PP1 during interphase but not during mitosis, suggesting a potential role for the Nek2-PP1 complex in CypA phospho-regulation. In support of this, Nek2 is capable of phosphorylating CypA . Overall, this work reveals that phosphorylation of CypA at serine 77 is important for its release from the centrosome during mitosis and may be regulated by the activity of Nek2 and PP1 during the cell cycle.
中心体作为一个蛋白质平台，从这里可以向整个细胞周期中的细胞中部署蛋白质。之前，我们已经表明，脯氨酰异构酶亲环素 A（CypA）在有丝分裂间期定位于中心体，并在有丝分裂中期重新定位于中间体，在那里它在胞质分裂中发挥作用。在这项研究中，通过 SDS-PAGE 在细胞周期中对 CypA 的研究表明，它在有丝分裂过程中发生了迁移变化，表明发生了翻译后修饰，这可能与其亚细胞重新定位有关。由于缺乏磷酸特异性抗体，我们使用定点突变来证明 CypA 中先前鉴定的丝氨酸 77 磷酸化位点对于控制 CypA 中心体定位很重要。此外，CypA 在有丝分裂间期和有丝分裂期间与有丝分裂 NIMA 相关激酶 2（Nek2）相互作用，而在有丝分裂间期与 Nek2 拮抗剂 PP1 相互作用，但在有丝分裂期间不相互作用，表明 Nek2-PP1 复合物在 CypA 磷酸化调节中可能发挥作用。支持这一点的是，Nek2 能够磷酸化 CypA。总的来说，这项工作表明，CypA 丝氨酸 77 的磷酸化对于其在有丝分裂期间从中心体释放很重要，并且可能受到细胞周期中 Nek2 和 PP1 活性的调节。
