Mechanism Of CRM1Regulating Mps1Subcellular Distribution

In eukaryotes, faithful segregation of chromosomes during mitotic cell division is essential for cell survival. To achieve this, cells employs a set of regulatory networks to monitor the key steps of mitosis, including mitotic entry, anaphase initiation and mitotic exit. SAC is an evolutionarily conserved mechanism that operates in prometaphase to prevent the onset of anaphase until every chromosome has successfully attached to the spindles. Defects in spindle assembly checkpoint are implicated in tumorigenesis.Mpsl is a dual specificity protein kinase, it can phosphorylate the serine、 threonine and tyrosine, and it also has auto-phosphorylation activity. It is a core component of spindle assembly checkpoint and apparently acts upstream in the SAC signaling cascade. The activity of Mpsl is required for activation of SAC in response to unoccupied kinetochores and the lack of tensions between the sister chromatids. The subcellular localization of Mpsl is both spatially and temporally regulated during cell cycle progression. In interphase cells, Mpsl primarily resides within the cytosol, centrosomes and nuclear pore. Upon mitotic entry Mpsl relocates to kinetochores, from which it is abruptly disassociated and diffusely redistributed throughout the cytosol as the metaphase plate is formed.The export of proteins from nucleus into cytoplasm is a dynamic process, which requires other transport related proteins including CRM1, Ran-GTP and Ran-GAP1. CRM1, a member of importinβ, exports proteins or RNAs via binding to the nuclear export sequence of cargo proteins. This process can be blocked by Leptomycin B (LMB), an inhibitor of Crml. According to the previous study and some literature reports, given that Mpsl is a cellcycle protein, we analyze Mpsl maybe regulated by CRM1. However, the mechanism of CRM1regulation of Mpsl thereby affecting the mitosis remains unclear.To reveal whether the subcellular distribution of Mpsl was regulated by CRM1, we first observed a physical interaction between Mps1and CRM1revealed by GST-pull down assay. Because CRM1usually bind the NES sequence of the protein, these evidences indicated that Mpsl should bear nuclear export sequence. We analyzed Mpsl protein sequence via using two online NES prediction sofwares NetNES and ELM and identified a putative nuclear export sequence, which bears the leusines enriched pattern similar to the classic NES sequence. Consistently, EGFP fused with pNES1(EGFP-pNES1) was transported into cytoplasm compared to the wild type EGFP which distributes through cell. Further, EGFP-pNES1relocated into cytoplasm upon LMB treatment. These findings strongly suggested that pNESl is a classic NES. To confirm the function of pNES1in Mpsl, we generated a Mpsl,which has some amino acid mutations, Unexpectedly, we found that the Mps1exclusively resided in nucleus even under LMB treatment. These studies suggested that pNES1could be essential for Mpsl export and import between both cytoplasm and nucleus. Next, the colon cancer cell line SW480stably expressing YFP fused Mpsl was employed as a cell model. We detected the dynamic distribution of YFPMps1upon LMB treatment through using immunofluorescence staining and live cell image. The result showed that the shuttling of Mpsl between cytoplasm and nucleus was dramatically affected by LMB in a time dependent way; the effect of LMB on Mpsl subcellular distribution is not limited to interphase as we also found that Mpsl accumulated on kinetochores through mitosis, including metaphase and anaphase; Interestingly, the mislocation of Mpsl in mitosis was also connected with a prolonged mitotic progression and extended approximately20minutes. Through the above study, we initially reveal the CRM1is needed in the metaphase kinetochore dissociation of Mps1, destroy the interation of CRM1and Mpsl will affect the mitosis process, and our study add a new layer to the functions of Mps1.