CNRS, UMR144, Institute Curie, PSL Research University, F-75005 Paris, France.
Laboratoire de Spectrométrie de Masse Protéomique, Institute Curie, PSL Research University, Paris, France.
J Biol Chem. 2019 Aug 30;294(35):12992-13005. doi: 10.1074/jbc.RA118.006937. Epub 2019 Jul 11.
Although Merlin's function as a tumor suppressor and regulator of mitogenic signaling networks such as the Ras/rac, Akt, and Hippo pathways is well-documented, in mammals as well as in insects, its role during cell cycle progression remains unclear. In this study, using a combination of approaches, including FACS analysis, time-lapse imaging, immunofluorescence microscopy, and co-immunoprecipitation, we show that Ser-518 of Merlin is a substrate of the Aurora protein kinase A during mitosis and that its phosphorylation facilitates the phosphorylation of a newly discovered site, Thr-581. We found that the expression in HeLa cells of a Merlin variant that is phosphorylation-defective on both sites leads to a defect in centrosomes and mitotic spindles positioning during metaphase and delays the transition from metaphase to anaphase. We also show that the dual mitotic phosphorylation not only reduces Merlin binding to microtubules but also timely modulates ezrin interaction with the cytoskeleton. Finally, we identify several point mutants of Merlin associated with neurofibromatosis type 2 that display an aberrant phosphorylation profile along with defective α-tubulin-binding properties. Altogether, our findings of an Aurora A-mediated interaction of Merlin with α-tubulin and ezrin suggest a potential role for Merlin in cell cycle progression.
尽管 Merlin 在肿瘤抑制和调节丝裂原信号通路（如 Ras/rac、Akt 和 Hippo 通路）方面的功能已得到充分证实，无论是在哺乳动物还是昆虫中，但其在细胞周期进程中的作用仍不清楚。在这项研究中，我们使用了包括流式细胞术分析、延时成像、免疫荧光显微镜和共免疫沉淀在内的多种方法，表明 Merlin 的丝氨酸 518 是 Aurora 蛋白激酶 A 在有丝分裂期间的底物，其磷酸化有助于新发现的 Thr-581 位点的磷酸化。我们发现，在 HeLa 细胞中表达同时在两个位点磷酸化缺陷的 Merlin 变体，会导致中期中心体和有丝分裂纺锤体定位缺陷，并延迟从中期到后期的过渡。我们还表明，双重有丝分裂磷酸化不仅减少了 Merlin 与微管的结合，而且还及时调节 ezrin 与细胞骨架的相互作用。最后，我们鉴定了几种与神经纤维瘤病 2 型相关的 Merlin 点突变，它们表现出异常的磷酸化谱和缺陷的α-微管结合特性。总之，我们发现 Aurora A 介导的 Merlin 与α-微管和 ezrin 的相互作用表明 Merlin 可能在细胞周期进程中发挥作用。
