Ludwig Institute for Cancer Research, La Jolla, CA 92093, USA; Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA.
Ludwig Institute for Cancer Research, La Jolla, CA 92093, USA.
Curr Biol. 2020 Aug 17;30(16):3101-3115.e11. doi: 10.1016/j.cub.2020.05.090. Epub 2020 Jul 2.
Cytokinesis partitions the cell contents to complete mitosis. During cytokinesis, polo-like kinase 1 (PLK1) activates the small GTPase RhoA to assemble a contractile actomyosin ring. PLK1 is proposed to pattern RhoA activation by creating a docking site on the central spindle that concentrates the RhoA guanine nucleotide exchange factor ECT2. However, ECT2 targeting to the central spindle is dispensable for cytokinesis, indicating that how PLK1 controls RhoA activation remains unresolved. To address this question, we employed an unbiased approach targeting ∼100 predicted PLK1 sites in two RhoA regulators: ECT2 and the centralspindlin complex, composed of CYK4 and kinesin-6. This comprehensive approach suggested that the only functionally critical PLK1 target sites are in a single cluster in the CYK4 N terminus. Phosphorylation of this cluster promoted direct interaction of CYK4 with the BRCT repeat module of ECT2. However, mutational analysis in vitro and in vivo led to the surprising finding that the interaction was independent of the conserved "canonical" residues in ECT2's BRCT repeat module that, based on structurally characterized BRCT-phosphopeptide interactions, were presumed critical for binding. Instead, we show that the ECT2 BRCT module binds phosphorylated CYK4 via a distinct conserved basic surface. Basic surface mutations mimic the effects on cytokinesis of loss of CYK4 cluster phosphorylation or inhibition of PLK1 activity. Together with evidence for ECT2 autoinhibition limiting interaction with CYK4 in the cytoplasm, these results suggest that a spatial gradient of phosphorylated CYK4 around the central spindle patterns RhoA activation by interacting with ECT2 on the adjacent plasma membrane.
胞质分裂将细胞内容物分隔以完成有丝分裂。在胞质分裂过程中，类 Polo 激酶 1（PLK1）激活小 GTP 酶 RhoA 以组装收缩性肌动球蛋白环。PLK1 通过在中心纺锤体上创建一个停泊位点来集中 RhoA 的鸟嘌呤核苷酸交换因子 ECT2，从而提出了对 RhoA 激活进行模式化的设想。然而，ECT2 靶向中心纺锤体对于胞质分裂是可有可无的，这表明 PLK1 如何控制 RhoA 激活仍未解决。为了解决这个问题，我们采用了一种无偏方法，针对两个 RhoA 调节剂中的约 100 个预测的 PLK1 位点：ECT2 和由 CYK4 和驱动蛋白-6 组成的中心纺锤体复合物。这种全面的方法表明，唯一具有功能重要性的 PLK1 靶位点位于 CYK4 N 端的单个簇中。该簇的磷酸化促进了 CYK4 与 ECT2 的 BRCT 重复模块的直接相互作用。然而，体外和体内的突变分析导致了一个令人惊讶的发现，即相互作用不依赖于 ECT2 的 BRCT 重复模块中的保守“经典”残基，这些残基基于结构表征的 BRCT-磷酸肽相互作用，被认为对结合至关重要。相反，我们表明，ECT2 BRCT 模块通过独特的保守碱性表面结合磷酸化的 CYK4。碱性表面突变模拟了 CYK4 簇磷酸化缺失或 PLK1 活性抑制对胞质分裂的影响。结合 ECT2 自动抑制限制细胞质中与 CYK4 相互作用的证据，这些结果表明，围绕中心纺锤体的磷酸化 CYK4 的空间梯度通过与相邻质膜上的 ECT2 相互作用来对 RhoA 激活进行模式化。
