Cells multiply through a cell division cycle that has distinct phases. In a phase called mitosis, a cell splits its genetic material, which was duplicated in a preceding phase, into two identical sets. Each of these sets will form the genetic material of daughter cells. If this process goes wrong, then cells can die or become cancerous, and so cells have evolved a complex regulatory process to ensure that mitosis begins and ends at the correct time.

For mitosis to begin, an enzyme adds tags called phosphate groups to hundreds of target proteins. These phosphate groups are then removed again to end mitosis. PP2A-B55 is an enzyme that removes these phosphate groups and is needed to complete mitosis, but must remain inactive before this point. This inactivation occurs because a protein called Greatwall activates two other proteins that inhibit PP2A-B55. To reactivate PP2A-B55 at the end of mitosis, Greatwall must be inactivated, but it was not known how cells do this.

Della Monica, Visconti et al. have now investigated this process in human cells. The experiments show that towards the end of mitosis, another enzyme called Fcp1 inactivates Greatwall by removing phosphate groups from it. This allows PP2A-B55 to reactivate.

These studies reveal that Fcp1 is a key factor that is needed to complete mitosis. The next challenge is to determine how Fcp1 activity is regulated at the end of mitosis.

We recently reported a critical, transcription-independent, role for the essential RNA polymerase II-carboxy-terminal domain (RNAP II-CTD) phosphatase Fcp1 in Cdk1 inactivation at the end of mitosis (Visconti et al., 2012). Indeed, depleting Fcp1 from living HeLa cells as well as from mitotic HeLa cell extracts, that are nuclei-free thus non-transcribing, substantially impaired cyclin B degradation, Cdk1 inactivation and mitosis exit (Visconti et al., 2012). In that study, we also noticed that Fcp1 depletion impaired bulk mitotic protein dephosphorylation also upon chemical inhibition of Cdk1 in non-transcribing mitotic cell extracts (Visconti et al., 2012). This observation suggested that Fcp1 was required for crucial mitosis exit dephosphorylations even downstream Cdk1 inactivation in a transcription-independent manner. However, bulk dephosphorylation at mitosis exit are likely due to action of major phosphatases like PP1 or PP2A, rather than Fcp1 itself (Ferrigno et al., 1993; Qian et al., 2013). The PP2A-B55 isoform, in particular, has relevant roles for late mitotic events like spindle breakdown, chromatin decondensation, nuclear membrane and Golgi reassembly and cytokinesis (Schmitz et al., 2010; Cundell et al., 2013). In addition, PP2A-B55 has been shown to reverse bulk mitotic phosphorylations detectable by a commercially available anti-Cdk1 substrate antibody, recognizing the K/HpSP motif (where pS is phosphorylated Ser), and the phosphorylation of PRC1, a crucial cytokinesis protein, at T481 (Schmitz et al., 2010; Cundell et al., 2013; Qian et al., 2013). In preliminary experiments, in which Fcp1 expression was downregulated in HeLa cells by small interfering RNAs (siRNAs), we found that dephosphorylations of bulk K/HpSP motif and pT481-PRC1 at mitosis exit were indeed dependent on Fcp1 (Figure 1—figure supplement 1). However, given the role for Fcp1 in inactivation of the spindle assembly checkpoint and of Cdk1 (Visconti et al., 2012; Visconti et al., 2013), delayed dephosphorylations could be due to persistance of Cdk1 kinase activity rather than impaired PP2A-B55 phosphatase activation at the end of mitosis. To know whether Fcp1 controlled PP2A-B55 activation downstream Cdk1 inactivation, we determined whether Fcp1 depletion impaired bulk K/HpSP motif and pT481-PRC1 dephosphorylation upon chemical inhibition of Cdk1 activity in mitotic cells and cell extracts. Control and Fcp1 siRNAs-depleted, as well as Fcp1 depleted complemented with siRNAs-resistant wild type Fcp1 (Fcp1WT) expression vector, HeLa cells were arrested at pro-metaphase and further treated with the Cdk1 inhibitor RO-3306 (Figures 1A,B). Nuclei-free, mitotic HeLa cell extracts were, instead, either mock immunodepleted, as control, or immunodepleted of Fcp1 or immunodepleted of Fcp1 and reconstituted with purified, active, Fcp1 wild type (Fcp1WT) protein before treatment with RO-3306 (Figures 1C,D). The results showed that, in cells and cell extracts, Fcp1 was indeed required for timely PP2A-B55-dependent dephosphorylations following Cdk1 inactivation (Figure 1A,C). Fcp1 is relatively resistant to the potent PP2A inhibitor okadaic acid (OA); nevertheless, we confirmed that pT481-PRC1 and bulk K/HpSP motif dephosphorylations were OA sensitive (Figure 1—figure supplement 2) (Schmitz et al., 2010; Cundell et al., 2013).

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Considering that pT481-PRC1 and bulk K/HpSP motif dephosphorylations at mitosis exit have been shown to depend on PP2A-B55 activity (Schmitz et al., 2010; Cundell et al., 2013), together, our data suggested that PP2A-B55 activation might require Fcp1 downstream Cdk1 inactivation.

Whether pT481-PRC1 dephosphorylation is necessary for PRC1 localization and spindle midzone organization is debated (Cundell et al., 2013; Hu et al., 2012). Nevertheless, we found that in Fcp1-depleted cells PRC1 poorly concentrated at spindle midzone, even upon Cdk1 inhibition (Figure 1—figure supplements 3A,3B), and that Fcp1 depletion induced several mitotic phenotypes in asynchronously growing cells, in addition to cell death, including accumulation of binucleated cells, suggesting that Fcp1 was indeed required also for proficient cytokinesis (Figure 1—figure supplement 4).

During mitosis onset, PP2A-B55 is inhibited by a recently elucidated pathway: Cdk1 phosphorylates and stimulates Gwl kinase that, in turn, represses PP2A-B55 activity by phosphorylating Ensa/ARPP19 and converting these proteins into potent PP2A-B55 inhibitors (Schmitz et al., 2010; Castilho et al., 2009; Vigneron et al. 2009; Lorca and Castro, 2013; Mochida and Hunt, 2012). How this condition is reversed at the end of mitosis is still unclear.

The Fcp1 phosphatase has already been called in the mechanism for PP2A-B55 reactivation at mitosis exit (Hegarat et al., 2014). Indeed, Fcp1-depleted, mitotic cells were found to maintain Gwl-dependent phosphorylation of Ensa (at S67 in human Ensa; pS67-Ensa) upon Cdk1 inhibition, and some evidence was provided that Fcp1 directly dephosphorylated pS67-Ensa to allow PP2A-B55 reactivation (Hegarat et al., 2014). However, this evidence was subsequently challenged by Williams and co-workers who showed that PP2A-B55 itself, rather than Fcp1, dephosphorylated pS67-Ensa and autoactivated, although this slow reaction could be rapidly reversed as long as Gwl stays active (Williams et al., 2014). We tested the ability of Fcp1 to dephosphorylate specifically pS67-Ensa in vitro and found that, in agreement with Williams and coworkers conclusions (Williams et al., 2014), Fcp1 was unable to do so (Figure 2A).

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To address mechanistically the Fcp1 dependency of PP2A-B55 activation, we hypothesized that Fcp1 was required to inactivate the Ensa/ARPP19 kinase ability of Gwl and allow PP2A-B55 autoactivation. Gwl is higly phosphorylated in mitosis and several observations indicate that Cdk1 directly phosphorylates Gwl stimulating its kinase activity (Vigneron et al., 2011; Blake-Hodek et al., 2012; Dephoure et al., 2008). Once activated by Cdk1, Gwl can autophosphorylate and autophosphorylation appears to contribute to its own overall activity (Blake-Hodek et al. 2012). A study in Xenopus laevis egg extracts has very recently provided compelling evidence that PP1 is the phosphatase that dephosphorylates Gwl at autophosphorylation sites, contributing this way to Gwl inactivation at the end of mitosis. However, the same study showed that dephosphorylation of Gwl at other sites, most likely also those phosphorylated by Cdk1, is PP1-independent (Heim et al., 2015). As Fcp1 is known to be able to reverse Cdk-dependent phosphorylation (Ghosh et al., 2008; Visconti et al., 2012), we hypothesised that Fcp1 was required for Gwl inactivation by removing Cdk1-dependent, activatory, phosphorylations of Gwl at the end of mitosis. First, we established a way to monitor changes at potential Cdk1-dependent Gwl phosphorylation sites during mitosis exit. Two commercially available anti-Cdk1 substrate antibodies, the previously mentioned anti-K/HpSP motif and an anti-pSPXR/K (where pS is phosphorylated serine and X any aminoacid) motif, can in principle recognize serine 90 and serine 453 in human Gwl (S90-Gwl and S453-Gwl), respectively, being S90-Gwl (89-KSP-91) and S453-Gwl (453-SPCK-456), the only human Gwl serine residues in those contexts. While S453-Gwl has been shown to be specifically phosphorylated in mitosis by proteomic approaches, S90-Gwl has not (Dephoure et al., 2008; Blake-Hodek et al., 2012). Nevertheless, both antibodies reacted against V5-tagged wild-type Gwl (V5-GwlWT) but not against V5-Gwl versions in which serine 90 and serine 453 were respectively mutated into non-phosphorylatable alanine (V5-GwlS90A; V5-GwlS453A) when the tagged proteins were isolated from transfected, mitotic, HeLa cells, indicating that both residues are phosphorylated in mitosis (Figure 2—figure supplement 1A). In addition, these antibodies did not react against V5-GwlWT isolated from HeLa cells in G1, unless it was treated with purified, active, Cdk1 in vitro (Figure 2—figure supplement 1B). To analyse potential changes in Gwl phosphorylation at S90 and S453 during mitosis exit, we probed endogenous Gwl isolated from HeLa cells taken at various time points during mitosis exit with anti-K/HpSP and pSPXR/K antibodies (Figure 2B). PS90- and pS453-Gwl signals were readly detected in prometaphase but were progressively lost as cells transited out of mitosis (Figure 2B). Importantly, dephosphorylation at both sites was resistant to OA at a dose (2 μM) that potently inhibited PP1 (Figure 2C), as indicated by persistance, despite Cdk1 inactivation by cyclin B degradation, of Cdk1-dependent inhibitory phosphorylation of PP1 catalytic subunit a (PP1cα) at T320 (pT320-PP1cα), a site that PP1 autodephosphorylates upon Cdk1 inactivation (Qian et al., 2013; Heim et al., 2015) (Figure 2C). However, OA significantly delayed the kinetics of Gwl migration shift on SDS/PAGE, in agreement with the notion that also OA-sensitive phosphatase(s) dephosphorylate Gwl at several other sites during mitosis exit (Hegarat et al., 2014; Williams et al., 2014; Heim et al., 2015). Conversely, depletion of Fcp1 delayed Gwl dephosphorylation at both S90 and S453 as well as Gwl migration shift and pS67-Ensa dephosphorylation (Figure 2D). In addition, a 15-min treatment with RO-3306 promptly induced pS90- and pS453-Gwl dephosphorylation and Gwl downshift in prometaphase-arrested control and Fcp1 re-expressing cells but not in prometaphase-arrested Fcp1-depleted cells, indicating that Fcp1 was required for these dephosphorylations downstream Cdk1 inactivation (Figure 2E). Prolonging Cdk1 inhibitor treatment up to 30 min resulted in some Gwl dephosphorylation also in Fcp1 siRNAs-treated cells (Figure 2—figure supplement 2); however, we could not rule out whether this was caused by the action of other phosphatases or of residual Fcp1 after substantial time from Cdk1 inactivation. Nevertheless, the 15-min treatment with RO-3306 was able to potently induce autoactivatory pT320-PP1cα dephosphorylation in Fcp1-depleted as in control cells (Figure 2E). Thus, Fcp1 is required for timely dephosphorylation of at least two Cdk1-dependent sites of Gwl, S90 and S453, at mitosis exit.

We set out to determine whether Fcp1 directly dephosphorylated Gwl at S90 and S453. As Fcp1 can be found in complexes with its substrates (Visconti et al., 2015), we first asked whether Fcp1 and Gwl physically interacted during mitosis exit. Indeed, by co-immunoprecipitation (coIP) of endogenous proteins, we found that Gwl transiently bound Fcp1 during mitosis exit (Figure 3A). Binding was lower in prometaphase, increased during the period of spindle assembly (20-30 min) to decrease thereafter (Figure 3A; Fcp1 and Gwl appear to be similarly abundant proteins in HeLa cells and we estimate that approximately 8-14% of Fcp1 interacts with Gwl in cells at the peak of interaction as, routinely, ~3-5% of total lysate Fcp1 was found in Gwl IP that contained ~30-40% of total lysate Gwl). In Fcp1-depleted cells complemented with exogenous Fcp1, Gwl interacted with the exogenous protein with similar kinetics observed with the endogenous Fcp1 (Figure 3B). Transient Gwl-Fcp1 interaction was also detected during mitosis exit in non-transformed, telomerase-immortalized, human retinal pigment epithelium cells hTERT-RPE1 (Figure 3—figure supplement 1).

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Taken together, these data suggest that Fcp1 bound and dephosphorylated Gwl at S90 and S453, and possibly at other Cdk1-dependent sites, during mitosis exit and that Fcp1-catalyzed dephosphorylation lowered Gwl activity towards Ensa/ARPP19, allowing PP2A-B55 to autoactivate.

To investigate this hypothesis, we first asked whether purified Fcp1 could dephosphorylate Gwl at S90 and S453 in vitro. Indeed, purified active Fcp1 (Fcp1WT), but not an inactive, catalytic dead, Fcp1 version (Fcp1CD), was able to dephosphorylate Gwl isolated from mitotic cells at both sites (Figure 3C). Next, we asked whether Fcp1-dependent dephosphorylation of Gwl in vitro lowered its kinase activity towards Ensa/ARPP19. To this end, endogenous Gwl, isolated from mitotic cells, was mock-treated or treated with active Fcp1WT or inactive Fcp1CD proteins as described in Figure 3C. Subsequently, Gwl activity was assessed on recombinant X. laevis Ensa protein as substrate (Figure 4A). The results clearly show that pre-treatment of mitotic Gwl with Fcp1WT, but not with Fcp1CD, substantially reduced Gwl ability to phosphorylate S67-Ensa (Figure 4A). Similar results were obtained with V5-GwlWT isolated from transfected, mitotic, cells or using ARPP19 as Gwl substrate (Figure 4—figure supplement 1A,1B). We cannot exclude that, in addition to S90 and S453, other Cdk1 phosphorylation sites in Gwl are dephosphorylated by Fcp1; nevertheless, assaying S67-Ensa kinase activity of V5-GwlS90A and V5-GwlS453A mutant proteins, isolated from transfected and prometaphase-arrested HeLa cells, revealed that both mutants had significantly reduced S67-Ensa kinase activity compared to V5-GwlWT (Figure 4B). The S90A mutation of human Gwl showed highest kinase reduction similarly to what reported for a non-phosphorylatable mutant at equivalent residues in X. laevis Gwl (Blake-Hodek et al., 2012). Moreover, a non-phosphorylatable Gwl mutant at S452 (V5-GwlS452A), just preceding serine S453, that is not in a Cdk1 phosphorylation consensus but is found phosphorylated in mitotic cells (Dephoure et al., 2008), had S67-Ensa kinase activity similar to GwlWT (Figure 4—figure supplement 1C). In addition, we assessed the S67-Ensa kinase activity of Gwl isolated from the same lysates of the experiment described in Figure 2E in which control, Fcp1-depleted and Fcp1-depleted and complemented prometaphase-arrested cells were treated with the Cdk1 inhibitor for 15 min, and found that while this treatment completely abolished Gwl activity in control cells it had negligible effects in Fcp1-depleted cells (Figure 4C; see also Figure 2E). Like for Gwl dephosphorylation, prolonging Cdk1 inhibitor treatment up to 30 min resulted in a reduction of Ensa kinase activity of Gwl also in Fcp1-depleted cells and, again, we cannot rule out whether this is due to the fact that other phosphatases or residual Fcp1 dampened Gwl activity after substantial time from Cdk1 inactivation (Figure 4—figure supplement 2). Nevertheless, it is important to remark that the 15-mintreatment with RO-3306 induced full pT320-PP1cα dephosphorylation in Fcp1-depleted cells without significantly affecting Gwl kinase activity towards Ensa (Figures 2E and 4C).

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Together, these data indicate that Fcp1-dependent dephosphorylation greatly reduces S67-Ensa kinase activity of Gwl and that, downstream inactivation of Cdk1, Fcp1 deficit substantially blunts inactivation of Gwl.

We previosly reported that the Fcp1 phosphatase is required to perform dephosphorylations that ultimately bring about Cdk1 inactivation at the end of mitosis (Visconti et al., 2012). We report now that, downstream Cdk1 inactivation, Fcp1 dephosphorylates Gwl at sites probably phosphorylated by Cdk1 and downregulates Gwl kinase activity towards Ensa/ARPP19 (Figure 4D). This ultimately leads PP2A-B55 to take the upper hand in dephosphorylating and releasing Ensa/ARPP19 as competitive inhibitors, getting free to dephosphorylate other substrates to complete mitosis (Williams et al., 2014). Recently, PP1 has been involved in the mechanism of Gwl inactivation at the end of mitosis by reversing Gwl activatory autophosphorylation (Heim et al., 2015). We found that, upon Cdk1 inactivation, Gwl inactivation is strongly delayed if Fcp1 is downregulated, despite potentially active PP1 (see Figures 2E and 4C). As Cdk1-dependent phosphorylation stimulates Gwl activity not only towards Ensa/ARPP19 but also towards Gwl itself at autoactivatory sites (Blake-Hodek et al., 2012), by reversing Cdk1-dependent phosphorylation Fcp1 could also reduce Gwl autoactivatory strength, favouring this way PP1 action to stably switch off Gwl autoactivation and, along with directly reducing Gwl activity towards Ensa/ARPP19, allow PP1 and PP2A-B55 to shut Gwl activity off definitively (Hegarat et al., 2014; Williams et al., 2014; Heim et al., 2015). Thus, by controlling Cdk1 and Gwl inactivation, Fcp1 appears to be at the apex of a phosphorylation cascade required to exit mitosis (Figure 4D). Along with other recently described phosphatase activation networks (Lorca and Castro, 2013; Porter et al., 2013; Nijenhuis et al., 2014; Grallert et al., 2015), this pathway contributes to ensure coordinated reversal of mitotic phosphorylations to grant correct completion of mitosis.

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Author details

1. Rosa Della Monica

   1. CEINGE Biotecnologie Avanzate, Naples, Italy
   2. Dipartimento di Medicina molecolare e Biotecnologie mediche, University of Naples Federico II, Naples, Italy

   Contribution

   RDM, Made initial observations on the Fcp1-Gwl interaction and designed experiments. Performed IP/blot experiments. Performed subcloning and site directed mutagenesis. Performed phosphatase and kinase assays. Analysed and discussed all data.

   Contributed equally with

   Roberta Visconti

   Competing interests

   The authors declare that no competing interests exist.

2. Roberta Visconti

   Istituto per l'endocrinologia e l'oncologia "Gaetano Salvatore", Consiglio Nazionale delle Ricerche, Naples, Italy

   Contribution

   RV, Made initial observations on the Fcp1-Gwl interaction and designed experiments. Performed IP/blot experiments. Performed subcloning and site directed mutagenesis. Analysed and discussed all data.

   Contributed equally with

   Rosa Della Monica

   Competing interests

   The authors declare that no competing interests exist.

3. Nando Cervone

   1. CEINGE Biotecnologie Avanzate, Naples, Italy
   2. Dipartimento di Medicina molecolare e Biotecnologie mediche, University of Naples Federico II, Naples, Italy

   Contribution

   NC, Performd IP/blot experiments, subcloning and site directed mutagenesis. Analysed and discussed all data.

   Competing interests

   The authors declare that no competing interests exist.

4. Angela Flavia Serpico

   1. CEINGE Biotecnologie Avanzate, Naples, Italy
   2. Dipartimento di Medicina molecolare e Biotecnologie mediche, University of Naples Federico II, Naples, Italy

   Contribution

   AFS, Performed IP/blot experiments. Performed subcloning and site directed mutagenesis. Performed phosphatase and kinase assays. Analysed and discussed all data.

   Competing interests

   The authors declare that no competing interests exist.

5. Domenico Grieco

   1. CEINGE Biotecnologie Avanzate, Naples, Italy
   2. Dipartimento di Medicina molecolare e Biotecnologie mediche, University of Naples Federico II, Naples, Italy

   Contribution

   DG, Made initial observations on the Fcp1-Gwl interaction and designed experiments. Performed phosphatase and kinase assays. Analysed and discussed all data. Conceived and wrote the manuscript, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.

   For correspondence

   domenico.grieco@unina.it

   Competing interests

   The authors declare that no competing interests exist.

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