Department of Microbiology and Cell Biology, Indian Institute of Science, 560012 Bangalore, India.
Department of Microbiology and Cell Biology, Indian Institute of Science, 560012 Bangalore, India
J Cell Sci. 2020 Jul 31;133(14):jcs243857. doi: 10.1242/jcs.243857.
Proper orientation of the mitotic spindle is critical for accurate development and morphogenesis. In human cells, spindle orientation is regulated by the evolutionarily conserved protein NuMA, which interacts with dynein and enriches it at the cell cortex. Pulling forces generated by cortical dynein orient the mitotic spindle. Cdk1-mediated phosphorylation of NuMA at threonine 2055 (T2055) negatively regulates its cortical localization. Thus, only NuMA not phosphorylated at T2055 localizes at the cell cortex. However, the identity and the mechanism of action of the phosphatase complex involved in T2055 dephosphorylation remains elusive. Here, we characterized the PPP2CA-B55γ (PPP2R2C)-PPP2R1B complex that counteracts Cdk1 to orchestrate cortical NuMA for proper spindle orientation. reconstitution experiments revealed that this complex is sufficient for T2055 dephosphorylation. Importantly, we identified polybasic residues in NuMA that are critical for T2055 dephosphorylation, and for maintaining appropriate cortical NuMA levels for accurate spindle elongation. Furthermore, we found that Cdk1-mediated phosphorylation and PP2A-B55γ-mediated dephosphorylation at T2055 are reversible events. Altogether, this study uncovers a novel mechanism by which Cdk1 and its counteracting PP2A-B55γ complex orchestrate spatiotemporal levels of cortical force generators for flawless mitosis.
正确的有丝分裂纺锤体取向对于准确的发育和形态发生至关重要。在人类细胞中，纺锤体的取向受进化上保守的蛋白 NuMA 调控，NuMA 与动力蛋白相互作用，并使其在细胞皮层富集。皮层动力蛋白产生的拉力使有丝分裂纺锤体定向。Cdk1 介导的 NuMA 丝氨酸 2055 位（T2055）磷酸化负调控其皮层定位。因此，只有未磷酸化 T2055 的 NuMA 才能定位在细胞皮层。然而，参与 T2055 去磷酸化的磷酸酶复合物的身份和作用机制仍不清楚。在这里，我们描述了 PPP2CA-B55γ（PPP2R2C）-PPP2R1B 复合物，该复合物与 Cdk1 拮抗，协调皮层 NuMA 以实现有丝分裂纺锤体的正确取向。重建实验表明，该复合物足以进行 T2055 去磷酸化。重要的是，我们确定了 NuMA 中的多碱性残基，这些残基对于 T2055 去磷酸化以及维持适当的皮层 NuMA 水平以实现准确的纺锤体伸长至关重要。此外，我们发现 Cdk1 介导的磷酸化和 PP2A-B55γ 介导的 T2055 去磷酸化是可逆事件。总之，这项研究揭示了 Cdk1 及其拮抗的 PP2A-B55γ 复合物协调皮层力发生器时空水平以实现完美有丝分裂的新机制。
