Abstract
Metazoan mitosis requires remodelling of sub-cellular structures to ensure proper division of cellular and genetic material. Faults often lead to genomic instability, cell cycle arrests and disease onset. These key structural changes are under tight spatial-temporal and post-translational control, with crucial roles for reversible protein phosphorylation. The phosphoprotein phosphatases PP1 and PP2A are paramount for the timely execution of mitotic entry and exit but their interaction partners and substrates are still largely unresolved. High throughput, mass-spectrometry based studies have limited sensitivity for the detection of low-abundance and transient complexes, a typical feature of many protein phosphatase complexes. Moreover, the limited timeframe during which mitosis takes place reduces the likelihood of identifying mitotic phosphatase complexes in asynchronous cells. Hence, numerous mitotic protein phosphatase complexes still await identification. Here we present a strategy to enrich and identify serine/threonine protein phosphatase complexes at the mitotic spindle. We thus identified a nucleolar RNA helicase, Ddx21/Gu, as a novel, direct PP1 interactor. Furthermore, our results place PP1 within the toposome, a Topoisomerase II alpha (TOPOIIα) containing complex with a key role in mitotic chromatin regulation and cell cycle progression, possibly via regulated protein phosphorylation. This study provides a strategy for the identification of further mitotic PP1 partners and the unravelling of PP1 functions during mitosis.
Highlights
Initiation, execution and successful termination of metazoan mitosis require extensive remodelling of subcellular structures, including breakdown of the nuclear envelope, nuclear pore complex and the nucleolus
Mitotic spindles must be formed, condensed chromosomes aligned and separated and the nucleolus and nucleus re-assembled [1]. Key processes such as DNA transcription and RNA splicing are generally downregulated during mitosis, yet some nuclear pore complex proteins and splicing factors were recently found to relocate to the spindle and kinetochores during metazoan mitosis where they are essential for proper mitotic progression [2]
Protein phosphatase complexes at transient structures such as the mitotic spindle apparatus are restricted in time and place
Summary
Initiation, execution and successful termination of metazoan mitosis require extensive remodelling of subcellular structures, including breakdown of the nuclear envelope, nuclear pore complex and the nucleolus. Mitotic spindles must be formed, condensed chromosomes aligned and separated and the nucleolus and nucleus re-assembled [1] Key processes such as DNA transcription and RNA splicing are generally downregulated during mitosis, yet some nuclear pore complex proteins and splicing factors were recently found to relocate to the spindle and kinetochores during metazoan mitosis where they are essential for proper mitotic progression [2]. Mitotic kinases, including the cyclin dependent kinase 1 (Cdk1) and Aurora kinases, have been studied extensively, leading to an in-depth understanding of their key roles in mitotic phosphorylation and progression [3] Protein phosphatases, their counteracting enzymes, were only recently recognized as crucial regulators of metazoan mitotic progression [4,5]. The identity of mitotic metazoan PPP complexes and their interaction partners and substrates remains largely unknown [4,5]
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