Handling Editor: David Glover
ABSTRACT
Proper orientation of the mitotic spindle is critical for accurate development and morphogenesis. In human cells, spindle orientation is regulated by the evolutionarily conserved protein NuMA, which interacts with dynein and enriches it at the cell cortex. Pulling forces generated by cortical dynein orient the mitotic spindle. Cdk1-mediated phosphorylation of NuMA at threonine 2055 (T2055) negatively regulates its cortical localization. Thus, only NuMA not phosphorylated at T2055 localizes at the cell cortex. However, the identity and the mechanism of action of the phosphatase complex involved in T2055 dephosphorylation remains elusive. Here, we characterized the PPP2CA-B55γ (PPP2R2C)–PPP2R1B complex that counteracts Cdk1 to orchestrate cortical NuMA for proper spindle orientation. In vitro reconstitution experiments revealed that this complex is sufficient for T2055 dephosphorylation. Importantly, we identified polybasic residues in NuMA that are critical for T2055 dephosphorylation, and for maintaining appropriate cortical NuMA levels for accurate spindle elongation. Furthermore, we found that Cdk1-mediated phosphorylation and PP2A-B55γ-mediated dephosphorylation at T2055 are reversible events. Altogether, this study uncovers a novel mechanism by which Cdk1 and its counteracting PP2A-B55γ complex orchestrate spatiotemporal levels of cortical force generators for flawless mitosis.
Footnotes
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: S.K.; Methodology: R.K., S.K.; Validation: R.K., A.R., S.K.; Formal analysis: R.K., A.R., S.K.; Investigation: R.K., S.K.; Resources: S.K.; Data curation: R.K., S.K.; Writing - original draft: S.K.; Writing - review & editing: R.K., A.R., S.K.; Visualization: S.K.; Supervision: S.K.; Project administration: S.K.; Funding acquisition: S.K.
Funding
This work is supported by the Department of Biotechnology, Ministry of Science and Technology, India – Indian Institute of Science Partnership Program and by grants from The Wellcome Trust DBT India Alliance Fellowship (IA/I/15/2/502077 to S.K.). S.K. is a The Wellcome Trust DBT India Alliance Intermediate Fellow. Deposited in PMC for release after 6 months.
Supplementary information
Supplementary information available online at https://jcs.biologists.org/lookup/doi/10.1242/jcs.243857.supplemental
- Received January 9, 2020.
- Accepted June 16, 2020.
- © 2020. Published by The Company of Biologists Ltd
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