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The PP1 regulator PPP1R2 coordinately regulates AURKA and PP1 to control centrosome phosphorylation and maintain central spindle architecture.

ABSTRACT: BACKGROUND:Maintenance of centrosome number in cells is essential for accurate distribution of chromosomes at mitosis and is dependent on both proper centrosome duplication during interphase and their accurate distribution to daughter cells at cytokinesis. Two essential regulators of cell cycle progression are protein phosphatase 1 (PP1) and Aurora A kinase (AURKA), and their activities are each regulated by the PP1 regulatory subunit, protein phosphatase 1 regulatory subunit 2 (PPP1R2). We observed an increase in centrosome number after overexpression of these proteins in cells. Each of these proteins is found on the midbody in telophase and overexpression of PPP1R2 and its mutants increased cell ploidy and disrupted cytokinesis. This suggests that the increase in centrosome number we observed in PPP1R2 overexpressing cells was a consequence of errors in cell division. Furthermore, overexpression of PPP1R2 and its mutants increased midbody length and disrupted midbody architecture. Additionally, we show that overexpression of PPP1R2 alters activity of AURKA and PP1 and their phosphorylation state at the centrosome. RESULTS:Overexpression of PPP1R2 caused an increase in the frequency of supernumerary centrosomes in cells corresponding to aberrant cytokinesis reflected by increased nuclear content and cellular ploidy. Furthermore, AURKA, PP1, phospho PPP1R2, and PPP1R2 were all localized to the midbody at telophase, and PP1 localization there was dependent on binding of PPP1R2 with PP1 and AURKA as well as its phosphorylation state. Additionally, overexpression of both PPP1R2 and its C-terminal AURKA binding site altered enzymatic activity of AURKA and PP1 at the centrosome and disrupted central spindle structure. CONCLUSIONS:Results from our study reveal the involvement of PPP1R2 in coordinating PP1 and AURKA activity during cytokinesis. Overexpression of PPP1R2 or its mutants disrupted the midbody at cytokinesis causing accumulation of centrosomes in cells. PPP1R2 recruited PP1 to the midbody and interference with its targeting resulted in elongated and severely disrupted central spindles supporting an important role for PPP1R2 in cytokinesis.

SUBMITTER: Bresch AM

PROVIDER: S-EPMC7687763 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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The PP1 regulator PPP1R2 coordinately regulates AURKA and PP1 to control centrosome phosphorylation and maintain central spindle architecture.

Bresch Alan-Michael AM Yerich Nadiya N Wang Rong R Sperry Ann O AO

BMC molecular and cell biology 20201125 1

<h4>Background</h4>Maintenance of centrosome number in cells is essential for accurate distribution of chromosomes at mitosis and is dependent on both proper centrosome duplication during interphase and their accurate distribution to daughter cells at cytokinesis. Two essential regulators of cell cycle progression are protein phosphatase 1 (PP1) and Aurora A kinase (AURKA), and their activities are each regulated by the PP1 regulatory subunit, protein phosphatase 1 regulatory subunit 2 (PPP1R2). ...[more]

PMID: 33238888

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Project description:BackgroundThe centrosome is the primary site for microtubule nucleation in cells and orchestrates reorganisation of the microtubule cytoskeleton during the cell cycle. The activities of the centrosome must be closely aligned with progression of the cell cycle; misregulation of centrosome separation and duplication is a hallmark of cancer. In a subset of cells, including the developing spermatid, the centrosome becomes specialised to form the basal body thereby supporting growth of the axoneme in morphogenesis of cilia and flagella, structures critical for signalling and motility. Mammalian spermatogenesis is an excellent model system to investigate the transformations in cellular architecture that accompany these changes including formation of the flagellum. We have previously identified a leucine-rich repeat protein (PPP1R42) that contains a protein phosphatase-1 binding site and translocates from the apical nucleus to the centrosome at the base of the flagellum during spermiogenesis. In this manuscript, we examine localisation and function of PPP1R42 in a ciliated epithelial cell model as a first step in understanding the role of this protein in centrosome function and flagellar formation.ResultsWe demonstrate that PPP1R42 localises to the basal body in ARPE-19 retinal epithelial cells. Co-localisation and co-immunoprecipitation experiments further show that PPP1R42 interacts with γ-tubulin. Inhibition of PPP1R42 with small interfering RNAs causes accumulation of centrosomes indicating premature centrosome separation. Importantly, the activity of two signalling molecules that regulate centrosome separation, PP1 phosphatase and NEK2 kinase, changes when PPP1R42 is inhibited: PP1 activity is reduced with a corresponding increase in NEK2 activity.ConclusionsWe have identified a role for the PP1-binding protein, PPP1R42, in centrosome separation in ciliated ARPE-19 cells. Our finding that inhibition of PPP1R42 expression increases the number of centrosomes per cell is consistent with our model that PPP1R42 is a positive regulator of PP1. PPP1R42 depletion reduces the activity of PP1 leading to activation of NEK2, the kinase responsible for phosphorylation of centrosomal linker proteins promoting centrosome separation. This work identifies a new molecule localised to the centrosome and basal body with a role in the complex signalling network responsible for controlling centrosome activities.

Dataset Information

The PP1 regulator PPP1R2 coordinately regulates AURKA and PP1 to control centrosome phosphorylation and maintain central spindle architecture.

Publications

The PP1 regulator PPP1R2 coordinately regulates AURKA and PP1 to control centrosome phosphorylation and maintain central spindle architecture.

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