Abstract
Mitotic progression is regulated largely by reversible phosphorylation events that are mediated by mitotic kinases and phosphatases. Protein phosphatase 1 (PP1) has been shown to play a crucial role in regulation of mitotic entry, progression, and exit. We previously observed, in Xenopus egg extracts, that phosphatase 1 nuclear targeting subunit (PPP1R10/PNUTS) acts as a mitotic regulator by negatively modulating PP1. This study investigates the role of PNUTS in mitotic progression in mammalian cells, and demonstrates that PNUTS expression is elevated in mitosis and depletion partially blocks mitotic entry. Cells that enter mitosis after PNUTS knockdown exhibit frequent chromosome mis-segregation. Aurora A/B kinase complexes and several kinetochore components are identified as PNUTS-associated proteins. PNUTS depletion suppresses the activation of Aurora A/B kinases, and disrupts the spatiotemporal regulation of the chromosomal passenger complex (CPC). PNUTS dynamically localizes to kinetochores, and is required for the activation of the spindle assembly checkpoint. Finally, PNUTS depletion sensitizes the tumor cell response to Aurora inhibition, suggesting that PNUTS is a potential drug target in combination anticancer therapy. IMPLICATIONS: Delineation of how PNUTS governs the mitotic activation and function of Aurora kinases will improve the understanding of the complex phospho-regulation in mitotic progression, and suggest new options to enhance the therapeutic efficacy of Aurora inhibitors.
Highlights
Mitotic kinases, cyclin-dependent kinase 1 (CDK1), polo-like kinase 1 (Plk1), and Aurora A/B, are well established as central regulators of mitosis
Antibodies and other reagents Cdc27 antibody was purchased from BD Transduction Laboratories; Aurora A, Aurora B, Bub1, Cenp-E, phosphatase 1 (PP1), and PNUTS antibodies were purchased from Bethyl Laboratories; histone H3, phospho-H3 Ser-10, phospho-Aurora A/B/C, Aurora B, and b-actin antibodies were purchased from Cell Signaling Technology; PP1 antibody was purchased from Santa Cruz Biotechnology
PNUTS regulates the mitotic progression of mammalian cells To investigate the expression level of PNUTS during the cell cycle, HeLa cells were released into cell-cycle progression from thymidine arrest
Summary
Cyclin-dependent kinase 1 (CDK1), polo-like kinase 1 (Plk1), and Aurora A/B, are well established as central regulators of mitosis. Activation of these kinases triggers the phosphorylation of various substrates to modulate all aspects of mitotic cell reorganization and progression [1,2,3,4]. Regulated activation and deactivation of these kinases are defined as molecular events that dictate M-phase entry and exit. Aurora A and Aurora B kinases phosphorylate a number of substrates to regulate the dynamics of centrosomes, microtubules, and chromatin during mitotic progression. Owing to the crucial role of mitotic kinases in cell proliferation, and the profound toxicity of their inhibition, mitotic kinases are appreciated as potential drug targets for cancer
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
