Abstract
The proper orientation of centrosome and spindle is essential for genome stability; however, the mechanism that governs these processes remains elusive. Here, we demonstrated that polo-like kinase 1 (Plk1), a key mitotic kinase, phosphorylates residue Thr76 in VCP/p97 (an AAA-ATPase), at the centrosome from prophase to anaphase. This phosphorylation process recruits VCP to the centrosome and in this way, it regulates centrosome orientation. VCP exhibits strong co-localization with Eg5 (a mitotic kinesin motor), at the mitotic spindle, and the dephosphorylation of Thr76 in VCP is required for the enrichment of both VCP and Eg5 at the spindle, thus ensuring proper spindle architecture and chromosome segregation. We also showed that the phosphatase, PTEN, is responsible for the dephosphorylation of Thr76 in VCP; when PTEN was knocked down, the normal spread of VCP from the centrosome to the spindle was abolished. Cryo-EM structures of VCPT76A and VCPT76E, which represent dephosphorylated and phosphorylated states of VCP, respectively, revealed that the Thr76 phosphorylation modulates VCP by altering the inter-domain and inter-subunit interactions, and ultimately the nucleotide-binding pocket conformation. Interestingly, the tumor growth in nude mice implanted with VCPT76A-reconstituted cancer cells was significantly slower when compared with those implanted with VCPWT-reconstituted cancer cells. Collectively, our findings demonstrate that the phosphorylation and dephosphorylation switch of VCP regulates the architecture of centrosome and spindle for faithful chromosome segregation.
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Data supporting the present study are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank members of Yue laboratory for advice on the manuscript. We would like to thank the Cryo-EM center of Southern University of Science and Technology for Cryo-EM data collection and the HPC-Service Station in Cryo-EM center of Southern University of Science and Technology for data processing.
Funding
This work was supported by Hong Kong Research Grant Council grants (11101717 and 11103620 to JY), Ministry of Science and Technology of the People’s Republic of China (2019YFA0906000 to HZ), NSFC (21778045, 32070702, and 82161128014 to JY), ITF (MRP/064/21 and GHP/097/20GD to JY), Guangdong Science and Technology Program (2017B030301018 to HZ), Shenzhen Science and Technology Innovation Committee (SGDX20201103093201010, JSGG20200225150702770, and JCYJ20210324134007020 to JY, and ZDSYS20140509142721429 to HZ).
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KZ, SL, HZ, and JY designed the experiments; KZ, YC, XS, ZM, HZ, ZY, RW and JY performed the experiments; KZ, YC, ZY, YG, CL, LZ, SL, HZ, and JY analyzed the data; and KZ, YC, HZ and JY wrote the paper.
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All animal experiments were performed following the university laboratory animal guidelines and with approval from the Animal Committee of City University of Hong Kong. Pathological sections of lung cancer patients were obtained from the Pathology Department of the Second Affiliated Hospital of Chongqing Medical University, with informed consent and approval from the Clinical Research Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University. The patients were provided and signed the consent forms.
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Zhu, K., Cai, Y., Si, X. et al. The phosphorylation and dephosphorylation switch of VCP/p97 regulates the architecture of centrosome and spindle. Cell Death Differ 29, 2070–2088 (2022). https://doi.org/10.1038/s41418-022-01000-4
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DOI: https://doi.org/10.1038/s41418-022-01000-4
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