AKAP95 interacts with nucleoporin TPR in mitosis and is important for the spindle assembly checkpoint

Faithful chromosome segregation during mitosis relies on a proofreading mechanism that monitors proper kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) is based on the concerted action of numerous components that maintain a repressive signal inhibiting transition into anaphase until all chromosomes are attached. Here we show that A-Kinase Anchoring Protein 95 (AKAP95) is necessary for proper SAC function. AKAP95-depleted HeLa cells show micronuclei formed from lagging chromosomes at mitosis. Using a BioID proximity-based proteomic screen, we identify the nuclear pore complex protein TPR as a novel AKAP95 binding partner. We show interaction between AKAP95 and TPR in mitosis, and an AKAP95-dependent enrichment of TPR in the spindle microtubule area in metaphase, then later in the spindle midzone area. AKAP95-depleted cells display faster prometaphase to anaphase transition, escape from nocodazole-induced mitotic arrest and show a partial delocalization from kinetochores of the SAC component MAD1. Our results demonstrate an involvement of AKAP95 in proper SAC function likely through its interaction with TPR.

有丝分裂过程中忠实的染色体分离依赖于监测动粒-微管正确附着的校对机制。纺锤体组装检验点（spindle assembly checkpoint, SAC）依靠众多组分的协同作用维持抑制信号，以阻止细胞向有丝分裂后期转换，直至所有染色体均完成正确附着。本研究证实，A激酶锚定蛋白95（A-Kinase Anchoring Protein 95, AKAP95）对于纺锤体组装检验点的正常功能不可或缺。敲低AKAP95的海拉（HeLa）细胞在有丝分裂阶段会出现由滞后染色体形成的微核。通过基于BioID邻近标记的蛋白质组学筛选，我们鉴定出核孔复合物蛋白TPR为一种新型AKAP95结合伴侣。实验证实，AKAP95与TPR在有丝分裂过程中存在相互作用，且在有丝分裂中期时，AKAP95依赖的TPR会富集于纺锤体微管区域，后续则富集于纺锤体中区。敲低AKAP95的细胞表现出前中期向后期的转换速率加快，能够逃脱诺考达唑诱导的有丝分裂阻滞，且纺锤体组装检验点组分MAD1在动粒上的定位出现部分缺失。本研究结果表明，AKAP95可能通过与TPR的相互作用参与纺锤体组装检验点的正常功能调控。