1. Cell Biology
  2. Chromosomes and Gene Expression
Download icon

Fcp1 phosphatase controls Greatwall kinase to promote PP2A-B55 activation and mitotic progression

  1. Rosa Della Monica
  2. Roberta Visconti
  3. Nando Cervone
  4. Angela Flavia Serpico
  5. Domenico Grieco  Is a corresponding author
  1. CEINGE Biotecnologie Avanzate, Italy
  2. University of Naples Federico II, Italy
  3. Consiglio Nazionale delle Ricerche, Italy
Short Report
  • Cited 18
  • Views 984
  • Annotations
Cite this article as: eLife 2015;4:e10399 doi: 10.7554/eLife.10399

Abstract

During cell division, progression through mitosis is driven by a protein phosphorylation wave. This wave namely depends on an activation-inactivation cycle of cyclin B-dependent kinase (Cdk) 1 while activities of major protein phosphatases, like PP1 and PP2A, appear directly or indirectly repressed by Cdk1. However, how Cdk1 inactivation is coordinated with reactivation of major phosphatases at mitosis exit still lacks substantial knowledge. We show here that activation of PP2A-B55, a major mitosis exit phosphatase, required the phosphatase Fcp1 downstream Cdk1 inactivation in human cells. During mitosis exit, Fcp1 bound Greatwall (Gwl), a Cdk1-stimulated kinase that phosphorylates Ensa/ARPP19 and converts these proteins into potent PP2A-B55 inhibitors during mitosis onset, and dephosphorylated it at Cdk1 phosphorylation sites. Fcp1-catalyzed dephosphorylation drastically reduced Gwl kinase activity towards Ensa/ARPP19 promoting PP2A-B55 activation. Thus, Fcp1 coordinates Cdk1 and Gwl inactivation to derepress PP2A-B55, generating a dephosphorylation switch that drives mitosis progression.

https://doi.org/10.7554/eLife.10399.001

eLife digest

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.

https://doi.org/10.7554/eLife.10399.002

Results and discussion

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).

Figure 1 with 4 supplements see all
Fcp1 affects PP2A-B55-dependent dephosphorylations at mitosis exit.

(A, B) Control (Cont.) or Fcp1-depleted (Fcp1) by siRNAs, as well as Fcp1-depleted complemented with wild type Fcp1 (Fcp1WT), HeLa cells were arrested at prometaphase. (A) Cells were collected and split into two samples, one sample received vehicle (-), the other RO3306 (+), and they were further incubated for 15 min, lysed and lysates probed for indicated antigens. (B) Cells were lysed and lysates probed for indicated antigens. (C, D) Mitotic HeLa cell extracts were mock-depleted (Cont. dep.), Fcp1-depleted (Fcp1 dep.) or Fcp1-depleted and reconsituted with Fcp1WT. (C) Each set was split into two portions, one received just vehicle (-), the other RO3306 (+), and they were incubated for 30 min at 23°C and probed for indicated antigens. (D) Before incubation, extracts samples were also probed for Fcp1 and Cdk1. The data shown are representative of three independent experiments per type.

https://doi.org/10.7554/eLife.10399.003

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).

Figure 2 with 2 supplements see all
Fcp1-dependency of Gwl dephosphorylation at S90 and S453 during mitosis exit.

(A) Ensa IP from prometaphase-arrested HeLa cells, previously transfected with a Flag-hEnsa expression vector, was divided into four sets and incubated as substrate in phosphatase reactions with either just buffer (Mk) or with purified active Fcp1 (WT), or an inactive, catalytic dead, Fcp1 version (CD), or active PP2A for 60 min at 30°C (lanes 1, 2, 3 and 4, respectively). The reactions were then probed for pS67Ensa and Ensa content (the Fcp1WT and Fcp1CD protein preparations were the same used in the experiment depicted in Figure 3C; see below). (B) Prometaphase-arrested HeLa cells were released into fresh medium and sampled at indicated time points of further incubation, lysed and processed for mock (Mk) or Gwl IP. IPs were separated in parallel SDS/PAGE and probed for indicated antigens. (C) Prometaphase-arrested HeLa cells were released into fresh medium and split into two portions; then, immediately after taking the time 0 sample, vehicle (Control) or 2 μM okadaic acid (+ OA) were added. Cells were further sampled at indicated time points of incubation. Upper panels, cell lysates were processed for mock (Mk) or Gwl IP that were probed for indicated antigens; lower panels, total cell lysate samples (Tot.) were probed for indicated antigens. (D) Control (Cont.) or Fcp1-depleted (Fcp1), as well as Fcp1-depleted complemented with wild type Fcp1 (Fcp1WT), HeLa cells were arrested at prometaphase, released and sampled at the indicated time points of incubation. Upper panels, cell lysates were processed for mock (Mk) or Gwl IP that were probed for indicated antigens; lower panels, total cell lysate samples (Tot.) were probed for indicated antigens. (E) Control (Cont.) or Fcp1-depleted (Fcp1), as well as Fcp1-depleted complemented with wild type Fcp1 (Fcp1WT), HeLa cells were arrested at prometaphase and split into two samples, one sample received vehicle (-) the other RO3306 (+), further incubated for 15 min. Left panels, cell lysates were processed for mock (Mk) or Gwl IP that were probed for indicated antigens; right panels, total cell lysate samples (Tot.) were probed for indicated antigens. The levels of Fcp1 depletion and complementation were similar to those shown in Figure 1B. Data shown are representative of at least four independent experiments per type.

https://doi.org/10.7554/eLife.10399.008

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).

Figure 3 with 1 supplement see all
Fcp1 binds and dephosphorylates Gwl during mitosis exit.

(A) Prometaphase-arrested HeLa cells were released into fresh medium and sampled at indicated time points of incubation. Upper panels, cell lysates were processed for mock (Mk) or Gwl IP that were probed for indicated antigens; lower panels, total cell lysate samples (Tot.) were probed for indicated antigens. (B) Prometaphase-arrested, Fcp1-siRNAs-transfected and complemented with siRNAs-resistant Fcp1WT (Fcp1-siRNAs +Fcp1 comp.) HeLa cells were released into fresh medium and sampled at indicated time points of incubation. Total lysates (Tot.), mock (Mk) or Gwl IPs were probed for indicated antigens. Data shown are representative of at least three independent experiments per type. (C) Gwl IP from prometaphase-arrested HeLa cell lysates was divided into three sets and incubated in phosphatase reactions with either just buffer (Cont.), Fcp1WT or Fcp1CD proteins (Mk IP; 1/3 mock IP incubated with buffer) for 60 min at 30°C. Then, IPs were washed and probed for indicated antigens. Data shown are representative of three independent experiments per type.

https://doi.org/10.7554/eLife.10399.011

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).

Figure 4 with 2 supplements see all
Fcp1-dependent dephosphorylation reduces Gwl kinase activity towards Ensa.

(A) Gwl IP from prometaphase-arrested HeLa cell lysates were divided into three sets and incubated for phosphatase reactions with either just buffer (Cont.), Fcp1WT or Fcp1CD proteins (Mk IP; 1/3 mock IP incubated with buffer) for 60 min at 30°C. Then, each IP set was split into three portions and incubated in kinase reactions with recombinant Ensa protein. (B) V5 IP from lysates of prometaphase-arrested HeLa cells, previously transfected with V5-GwlWT and V5-GwlS90A or V5-GwlS453A, were split into three portions and incubated at 37°C in kinase reactions with recombinant Ensa protein. (C) Gwl IPs from the same cell lysates of the experiment described in Figure 2E, in which prometaphase-arrested control (Cont.) and Fcp1 siRNAs-treated (Fcp1), as well as Fcp1 siRNAs-treated complemented with wild type Fcp1 (Fcp1WT), HeLa cells were treated – or + RO3306 for 15 min, split into three portions and incubated in kinase reactions at 37°C and with recombinant Ensa protein. Kinase reactions were stopped at indicated time points of incubation and probed for indicated antigens (Mk IPs were incubated for 15 min). Graphs show quantitation (arbitrary units) of pS67-Ensa optical density. Data shown are representative of three independent experiments per type. (D) Schematic for the proposed model of PP2A-B55 control.

https://doi.org/10.7554/eLife.10399.013

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.

Materials and methods

Cell culture and treatments

Request a detailed protocol

HeLa and hTERT-RPE1 cells were grown and maintained as previously described (Visconti et al., 2012; Visconti et al., 2010). Prometaphase-arrested cells were obtained by performing a double thymidine (4 mM; Sigma-Aldrich, St. Louis, MO) block (18 hr each, separated by a 6 hr incubation in fresh medium) followed by release into fresh medium containing nocodazole (500 nM; Calbiochem, Billerica, MA) and incubation for 12 or 14 hr for HeLa and hTERT-RPE1, respectively. Release from prometaphase arrest was obtained by washing detached cells twice with PBS and twice with fresh medium, followed by incubation in fresh medium. Cells in G1 were obtained after 120 min incubation from prometaphase release.

For asynchronous siRNAs treatment, Hela cells were transfected with non-targeting or human Fcp1 3’ UTR-targeting (5’-guaagugacagguguuaaa-3’) siRNAs (Dharmacon Inc., Lafayette, CO). For siRNAs treatment and complementation experiments, HeLa cells were first transfected with 3XFlag-Fcp1WT expression vector (or empty vector for mock transfections; Visconti et al., 2012). Eight hours post transfection, cells were treated with thymidine (4 mM; Sigma-Aldrich) for 18 hr. Cells were released from thymidine block into fresh medium and transfected with non-targeting or human Fcp1-targeting siRNAs as above. Cells were treated again with thymidine 6 hr after siRNAs transfections, and incubated for further 18 hr. Cells were then released from the second thymidine block into fresh medium containing nocodazole (500 nM; Calbiochem) for 12 hr. Mitotic extracts from prometaphase-arrested HeLa cells (checkpoint extracts) were produced, Fcp1-immunodepleted and complementated exactly as previously described (Visconti et al., 2012). Recombinat Fcp1WT and Fcp1CD proteins were produced and stored in EXB (20 mM HEPES pH 7.6, 5 mM KCl, 1mM DTT, 100 μg/ml 3XFLAG peptide, 10% glycerol; Sigma-Aldrich) as previously described (Visconti et al., 2012). V5-GwlWT expression vector was obtained by subcloning pENTR221-Gwl clone into V5-tagged pcDNA3.1 vector (Invitrogen, Carlsbad, CA). To generate the V5-Gwl-S90A mutant, V5-Gwl-S453A mutant and V5-Gwl-S452A mutant, serine 90, serine 453 and serine 452 of human Gwl were mutagenized into alanine by QuikChange II XL site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) using the V5-GwlWT expression construct as template. 3XFlag-Fcp1WT or 3XFlag-Fcp1CD expression vectors have been previously described (Visconti et al., 2012). Flag-hEnsa expression vector was purchased from Origene (Rockville, MD). Eukaryotic expression vectors transfections were performed using Linear Polyethlenimine (Polysciences Inc., Warrington, PA). Recombinant 6His-tagged X. laevis Ensa and ARPP19 proteins were expressed in BL21 E. coli cells and purified using Ni-NTA agarose kit (Qiagen, Germany) according to the manufacturer’s instructions. The Cdk1 inhibitor RO3306 (Calbiochem) was used at 5 and 50 μM in cells and mitotic cell extracts, respectively. Okadaic acid (Calbiochem) was used at 500 nM or 2 μM as indicated, MG132 (Calbiochem) at 10 μM. Cell viability was assessed by trypan blue (EuroClone, Italy) exclusion and immunoblots for cleaved caspase 3.

Immunological procedures

Request a detailed protocol

Anti-phospho-serine Cdk Substrates (P-S2-100; recognizing K/HpSP), anti phosphorylated MAPK/CDK substrates (recognizing PXpSP or pSPXK/R) and anti phospho-ENSA/ARPP19 (pS67/pS62), anti phosphoT320-PP1cα and Cleaved Caspase-3 antibodies were purchased from Cell Signaling Technology (Danvers, MA); anti-MASTL antibodies from Bethyl Laboratories (Montgomery, TX) and NOVUS (Littleton, CO); anti-Fcp1 antibodies from Bethyl Laboratories and Santa Cruz Biotechnology (Dallas, TX). Other antibodies were from Santa Cruz Biotechnology. Immunoprecipitations and immunoblots were performed as previously described (Visconti et al., 2012). For immunofluorescence, cells were grown or spun on microscopy slides, washed in PBS, fixed with 4% formaldehyde in PBS for 10 min and permeabilized with 0.2% Triton X-100 in PBS for further 10 min. After blocking with 3% BSA in PBS for 1 hr, samples were incubated with primary antibodies in PBS + 1% BSA for 3 hr. After 3 PBS washes, samples were incubated with secondary antibodies (Jackson ImmunoResearch Laboratories Inc., Westgrove, PA) in PBS + 1% BSA for 1 hr at room temperature. DNA was stained by incubation with Hoechst 33258 (10 µg/ml; Santa Cruz Biotechnology) in PBS. Samples were observed and photographed using an Axiovert 200M inverted microscope equipped with the Apotome slider module with 63X or 40X objectives (Zeiss, Germany).

In vitro treatments and assays

Request a detailed protocol

For in vitro dephosphorylation assays, endogenous Gwl IP or V5 IP or Flag IP, from previously V5-GwlWT- or Flag-hEnsa-transfected cells respectively, from 3 ml lysates, of ~1.5 mg/ml of protein concentration, of prometaphase-arrested cells were washed in phosphatase assay buffer (PAB: 20 mM HEPES, pH 7.6, 10 mM MgCl2, 1 mM dithiothreitol), split into three portions, each containing approximately 500 ng of Gwl, and incubated at 30°C for 1 hr in 10 μl of either PAB + 1/10 volume of EXB, as control, PAB + 1/10 volume of Fcp1WT (50 ng/μl; final protein conc.) or PAB + 1/10 volume of Fcp1CD (50 ng/μl; final protein conc.) or PAB + 1/10 volume of purified PP2A (0.1 unit per reaction; Merck Millipore, Billerica, MA). After phosphatase reaction, samples were separated on SDS/PAGE and probed for the indicated antigens or, where indicated, further processed for Gwl kinase activity assays. For Gwl kinase, after phosphatase reactions, each IP set was washed with EB (80 mM β-glycerophosphate, 10 mM MgCl2 and 20 mM EGTA), divided into three aliquots and incubated for indicated time points in EB buffer supplemented with 1 mM ATP, 10 mM phosphocreatine, 0.1 mg/ml creatine phosphokinase (kinase buffer, KB) and recombinant X. laevis, Ensa or ARPP19 proteins (1 μg per sample). One-tenth of each reaction was probed on separate blots for total Ensa or ARPP19 proteins, the remaining was probed for pS67/62-Ensa/ARPP19 and Gwl. Total Ensa was also visualized by re-probing blots previosly probed for pS67-Ensa. For in vitro Gwl rephosphorylation assays, V5 IPs from mock- or V5-GwlWT-transfected, G1-synchronised, HeLa cells were split in two portion and incubated at 37°C for 20 min in KB − or + active Cyclin A2-CDK1 (126 ng/reaction, ProQuinase, Germany).

References

  1. 1
  2. 2
  3. 3
  4. 4
    A quantitative atlas of mitotic phosphorylation
    1. N Dephoure
    2. C Zhou
    3. J Villen
    4. SA Beausoleil
    5. CE Bakalarski
    6. SJ Elledge
    7. SP Gygi
    (2008)
    Proceedings of the National Academy of Sciences of the United States of America 105:10762–10767.
    https://doi.org/10.1073/pnas.0805139105
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23

Decision letter

  1. Tony Hunter
    Reviewing Editor; Salk Institute, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for submitting your work entitled "Fcp1 controls Greatwall kinase to promote PP2A-B55 activation and mitotic progression" for peer review at eLife. Your submission has been favorably evaluated by Tony Hunter (Senior Editor) and three reviewers.

The reviewers have discussed the reviews with one another and the editor has drafted this decision to help you prepare a revised submission.

The authors have previously reported that the Fcp1, a metal-dependent RNA polymerase CTD phosphatase, is required for dephosphorylation of CDK1 substrates and exit from mitosis in mammalian cells. Here, they provide evidence that Fcp1 mediates dephosphorylation of CDK1 targets indirectly by dephosphorylating two CDK1 sites on Greatwall (Gwl), a kinase that through phosphorylation of endosulfine creates a potent inhibitor of PP2A/B55, the phosphatase that is responsible for dephosphorylating the majority of the CDK1 substrates, an event needed for exit from mitosis. In summary, here the authors propose a new link between Fcp1 and the Greatwall/Ensa/B55 pathway and suggest that Fcp1 lies at the top of this cascade and triggers mitotic exit by dephosphorylating and inactivating Greatwall kinase.

The reviewers each found strength in the paper, but were not fully convinced that your data completely justify your conclusions, and find that several aspects of your model have to be tested more rigorously. The following major points will need to be addressed in a revised version.

Essential revisions:

1) The authors' model places Fcp1 at the top of the mitotic exit cascade and one would expect a strong mitotic phenotype in Fcp1 knockdown cells, and certainly at least a significant delay in mitotic exit and an increase in mitotic index and/or polyploidy related to cytokinesis problems. Most of the experiments that support a role for Fcp1 were done using nocodazole release, and there is only a single picture shown of a binucleated cell without any quantification of the Fcp1 mitotic phenotypes. It is essential to show quantitative data that Fcp1 knockdown in asynchronous cells causes a significant increase in mitotic index and delays progression from M-phase into G1 phase resulting in binucleation to support the idea that Fcp1 is the key to this cell cycle transition. This is especially important since other siRNA screens (e.g. mitocheck) have not picked up any mitotic phenotypes with Fcp1.

2) The experiments in Figure 1A show clearly that Fcp1 depletion caused a block in cyclin B degradation in cells released from nocodazole. Thus, most of the subsequent data on Greatwall dephosphorylation that were carried out using the nocodazole-release synchronization method could simply be a consequence of Cdk1 activity remaining high because of cyclin B stabilization. The authors did carry out one set of experiments using the RO3306 Cdk inhibitor (Figure 1C and D), but Gwl phosphorylation was not assessed here, and the authors relied on measuring downstream events. The effect of RO3306 Cdk inhibition on Gwl phosphorylation should be tested.

3) The authors failed to cite the Hégarat paper (PLoS Genet. 10:e1004004, 2014), which showed that Fcp1 depletion caused a block in Ensa dephosphorylation, but did not affect Gwl T-loop dephosphorylation. Are the conclusions of the authors' study compatible with those in the Hégarat et al. paper? It is essential to discuss this point (e.g. are there experimental differences?) to avoid confusing readers. In this regard, there is a critical experiment in the Hégarat paper that the authors should take into account: in Okadaic Acid treated cells, that are forced to exit mitosis by Cdk1 inhibition, Gwl retains approximately 60% of its activity as measured by IP kinase assays using Gwl purified before and after mitotic release. This would suggest that an OA-sensitive phosphatase is the major Gwl phosphatase at relevant activating phosphosites. The authors should perform a similar experiment in which they purify Gwl from Fcp1-depleted mitotic cells before and after Cdk1 inhibition, and measure its kinase activity in vitro. This would definitively determine how much Fcp1 does indeed contribute to Greatwall inactivation during mitotic exit.

4) Given the alternative hypothesis that PP1 rather than Fcp1 is responsible for Gwl inactivation, the Okadaic Acid experiments shown in Figure 2C and Figure 1—figure supplement 1 take on an added significance. In Figure 2C, the dephosphorylation of pS90 and pS453 was not blocked by OA, implying that these dephosphorylations are catalyzed by Fcp1, a phosphatase not sensitive to OA. However, the concentration of OA used in these experiments (500 nM) is uncomfortably close to the borderline. PP2A-like enzymes are almost certainly nearly completely inhibited by this dose of OA, but the drug binds significantly less tightly to the active site of PP1-like enzymes. For complete inhibition of all the PP1-like enzymes, the stoichiometric concentration of the inhibitor must be in excess of the intracellular concentration of all proteins that bind it with Ki values equal to or lower than the dissociation constant for PP1. In other words, it remains possible that 500 nM of Okadaic Acid is sufficient to achieve quantitative inhibition of PP2A but not of PP1. Thus, the experiment done in Figure 2C needs to be repeated with a higher dose of OA, preferably 2 micromolar or above, but any type of evidence that PPI is not involved would suffice.

5) Figure 2D and part of Figure 3B: In the experiments evaluating Fcp1's role in cells, the authors expressed and compared Fcp1-WT and Fcp1-CD (catalytically-dead mutant). Ideally, however, they should compare cells siRNA-depleted of endogenous Fcp1 with mock-treated cells (and possibly siRNA-depleted cells re-expressing siRNA-resistant WT), because it is difficult to control expression level of an exogenous gene similar to the endogenous one (as shown in Figure 1B), and because the transfection efficiency of an exogenous gene never reaches 100%, making the cell population more heterogeneous. In addition, exogenous overexpression of a gene can cause artificial effects that cannot be predicted. As the authors have already done this in Figure 1C, it is technically doable. To support the main conclusion it would be important that Figure 2D and the bottom half of Figure 3B (at least for pS67-Ensa) are re-tested in this context (mock/Fcp1-knockdown/rescued).

https://doi.org/10.7554/eLife.10399.016

Author response

Essential revisions:

1) The authors' model places Fcp1 at the top of the mitotic exit cascade and one would expect a strong mitotic phenotype in Fcp1 knockdown cells, and certainly at least a significant delay in mitotic exit and an increase in mitotic index and/or polyploidy related to cytokinesis problems. Most of the experiments that support a role for Fcp1 were done using nocodazole release, and there is only a single picture shown of a binucleated cell without any quantification of the Fcp1 mitotic phenotypes. It is essential to show quantitative data that Fcp1 knockdown in asynchronous cells causes a significant increase in mitotic index and delays progression from M-phase into G1 phase resulting in binucleation to support the idea that Fcp1 is the key to this cell cycle transition. This is especially important since other siRNA screens (e.g. mitocheck) have not picked up any mitotic phenotypes with Fcp1.

Quantitative data of the phenotypes induced by Fcp1 knockdown in asynchronous cells, that show a significant increase in mitotic index, delayed progression through M-phase, binucleation and multinucleation in addition to cell death, have now been provided in Figure 1—figure supplement 4. The reason why these very evident phenotypes induced by Fcp1 downregulation, in our hands, were not picked up in previous large siRNA screens (e.g. mitocheck) is obscure to us. It is worth noting, however, that while the previously mentioned large siRNA screens did not score any phenotype upon Fcp1 downregulation, in other very recent large genetic knockdown screens, Fcp1 was classified as an essential gene in human cells (Science 27 November 2015: 1092-1096 and: 1096-1101. Published online 15 October 2015). Perhaps large screens may sometimes mislead to inaccurate conclusions.

2) The experiments in Figure 1A show clearly that Fcp1 depletion caused a block in cyclin B degradation in cells released from nocodazole. Thus, most of the subsequent data on Greatwall dephosphorylation that were carried out using the nocodazole-release synchronization method could simply be a consequence of Cdk1 activity remaining high because of cyclin B stabilization. The authors did carry out one set of experiments using the RO3306 Cdk inhibitor (Figure 1C and D), but Gwl phosphorylation was not assessed here, and the authors relied on measuring downstream events. The effect of RO3306 Cdk inhibition on Gwl phosphorylation should be tested.

The experiments suggested have now been performed, and the effects of Cdk1 inhibitor RO3306 on Gwl phosphorylation in control and Fcp1-depleted cells are now shown in Figure 2E and Figure 2—figure supplement 2. In addition, previous Figure 1A has now been removed from the main text and is shown as Figure 1—figure supplement 1. Moreover, following the objection expressed in Essential Revision point 5, we have removed data concerning experiments involving re-expression of Fcp1 catalytic dead (Fcp1 CD) in Fcp1-siRNAs-treated cells.Previous Figures 1C and D were changed to Figures 1A–1D.

3) The authors failed to cite the Hégaratpaper (PLoS Genet. 10:e1004004, 2014), which showed that Fcp1 depletion caused a block in Ensa dephosphorylation, but did not affect Gwl T-loop dephosphorylation. Are the conclusions of the authors' study compatible with those in the Hégarat et al. paper? It is essential to discuss this point (e.g. are there experimental differences?) to avoid confusing readers. In this regard, there is a critical experiment in the Hégarat paper that the authors should take into account: in Okadaic Acid treated cells, that are forced to exit mitosis by Cdk1 inhibition, Gwl retains approximately 60% of its activity as measured by IP kinase assays using Gwl purified before and after mitotic release. This would suggest that an OA-sensitive phosphatase is the major Gwl phosphatase at relevant activating phosphosites. The authors should perform a similar experiment in which they purify Gwl from Fcp1-depleted mitotic cells before and after Cdk1 inhibition, and measure its kinase activity in vitro. This would definitively determine how much Fcp1 does indeed contribute to Greatwall inactivation during mitotic exit.

The Hégarat paper has now been discussed and cited (Results and Discussion, fourth and last paragraphs). In addition, the experiments suggested to measure activity of Gwl isolated from control and Fcp1-depleted cells before and after Cdk1 inhibition have been performed, and the results are shown in Figure 4C and Figure 4—figure supplement 2. Furthermore, we have included in this revised version a set of data that we already had, but did not include in the previous version, showing that active Fcp1 is not able to dephosphorylate pS67-Ensa in vitro under our experimental conditions, now shown in Figure 2A. Since a paper showing the relevance of PP1 in Gwl inactivation was published during the preparation of this revised manuscript (Heim et al., 2015), we have discussed and cited that paper too and performed experiments related to PP1 under conditions of Fcp1 depletion and Cdk1 inhibition, now shown in Figure 2C, 2E and 4C. We tried to homogeneously integrate all previous information with ours (please see the last paragraph of the Results and Discussion).

4) Given the alternative hypothesis that PP1 rather than Fcp1 is responsible for Gwl inactivation, the Okadaic Acid experiments shown in Figure 2C and Figure 1—figure supplement 1 take on an added significance. In Figure 2C, the dephosphorylation of pS90 and pS453 was not blocked by OA, implying that these dephosphorylations are catalyzed by Fcp1, a phosphatase not sensitive to OA. However, the concentration of OA used in these experiments (500 nM) is uncomfortably close to the borderline. PP2A-like enzymes are almost certainly nearly completely inhibited by this dose of OA, but the drug binds significantly less tightly to the active site of PP1-like enzymes. For complete inhibition of all the PP1-like enzymes, the stoichiometric concentration of the inhibitor must be in excess of the intracellular concentration of all proteins that bind it with Ki values equal to or lower than the dissociation constant for PP1. In other words, it remains possible that 500 nM of Okadaic Acid is sufficient to achieve quantitative inhibition of PP2A but not of PP1. Thus, the experiment done in Figure 2C needs to be repeated with a higher dose of OA, preferably 2 micromolar or above, but any type of evidence that PPI is not involved would suffice.

The experiment shown in previous Figure 2C has been repeated with a higher dose of OA, 2 μM, as recommended. The results are now shown in new Figure 2C. In addition, new Figure 2C, lower panels, shows that at this concentration OA is able to completely prevent auto-dephosphorylation of PP1 catalytic subunit alpha at T320, the inhibitory Cdk1-phosphorylated site in mitosis. These data indicate that this concentration of OA is indeed able to bind tightly to the active site of PP1 and inhibit the enzyme but, despite maintenance of Cdk1-dependent, inhibitory, phosphorylation of PP1, it is not able to prevent Gwl dephosphorylation at pS90 and pS453. Previous Figure 1—figure supplement 1, now Figure 1—figure supplement 2, was not changed since OA was used at 500 nM to essentially inhibit PP2A action and as you mentioned “PP2A-like enzymes are almost certainly nearly completely inhibited by this dose of OA.

5) Figure 2D and part of Figure 3B: In the experiments evaluating Fcp1's role in cells, the authors expressed and compared Fcp1-WT and Fcp1-CD (catalytically-dead mutant). Ideally, however, they should compare cells siRNA-depleted of endogenous Fcp1 with mock-treated cells (and possibly siRNA-depleted cells re-expressing siRNA-resistant WT), because it is difficult to control expression level of an exogenous gene similar to the endogenous one (as shown in Figure 1B), and because the transfection efficiency of an exogenous gene never reaches 100%, making the cell population more heterogeneous. In addition, exogenous overexpression of a gene can cause artificial effects that cannot be predicted. As the authors have already done this in Figure 1C, it is technically doable. To support the main conclusion it would be important that Figure 2D and the bottom half of Figure 3B (at least for pS67-Ensa) are re-tested in this context (mock/Fcp1-knockdown/rescued).

Figures 2D and 3B have been changed following the suggested experiments and recommendations. Gwl and Ensa dephosphorylation have been compared in experiments with cells siRNA-depleted of endogenous Fcp1, mock-treated and siRNA-depleted re-expressing siRNA-resistant Fcp1 WT in new Figure 2D. Following the reviewers’ suggestions, data concerning re-expression of Fcp1CD have also been removed from present Figure 3B, in addition to present Figure 1 and Figure 1—figure supplement 1.

https://doi.org/10.7554/eLife.10399.017

Article and author information

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.

Funding

Associazione Italiana per la Ricerca sul Cancro (IG 2014 Id.15476)

  • Domenico Grieco

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication

Acknowledgements

The authors wish to thank VE Avvedimento and RM Melillo for helpful suggestions, S Mochida for providing X. laevis ENSA and ARPP19 expression vectors. Supported by a grant of Associazione Italiana per la Ricerca sul Cancro (AIRC) N. IG 2014 Id.15476 to DG.

Reviewing Editor

  1. Tony Hunter, Salk Institute, United States

Publication history

  1. Received: July 29, 2015
  2. Accepted: December 14, 2015
  3. Accepted Manuscript published: December 14, 2015 (version 1)
  4. Version of Record published: January 29, 2016 (version 2)

Copyright

© 2015, Della Monica et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 984
    Page views
  • 368
    Downloads
  • 18
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)