Cloning and Functional Characterization of Ptpcd 2 as a Novel Cell Cycle Related Protein Tyrosine Phosphatase that Regulates Mitotic Exit

Alternating dynamic activities of protein kinases and phosphatases ensure tight cell cycle regulation and DNA damage response control (Maya-Mendoza and Jackson, 2009; Shimada and Nakanishi, 2013). Correct execution of mitosis in eukaryotes relies on timely activation and inactivation of cyclin dependent kinase1 (Cdk1) and its activating subunit, cyclin B (CycB) (Visconti et al., 2012). Anti-mitotic drugs are current drugs used to treat cancer by selectively perturbing progression through mitosis. Cancer cells treated with anti-mitotic drugs will delay mitotic progression due mitotic checkpoint (Janssen and Medema, 2011). This prolonged mitotic delay is often followed by mitotic cell death (Gascoigne and Taylor, 2008). However, a subset of cells can escape and exit mitosis. Blocking mitotic exit might circumvent drug resistance seen in those cells and enhance the efficacy of anti-mitotic drugs (Huang et


Introduction
Alternating dynamic activities of protein kinases and phosphatases ensure tight cell cycle regulation and DNA damage response control (Maya-Mendoza and Jackson, 2009;Shimada and Nakanishi, 2013).
Correct execution of mitosis in eukaryotes relies on timely activation and inactivation of cyclin dependent kinase1 (Cdk1) and its activating subunit, cyclin B (CycB) (Visconti et al., 2012).Anti-mitotic drugs are current drugs used to treat cancer by selectively perturbing progression through mitosis.Cancer cells treated with anti-mitotic drugs will delay mitotic progression due mitotic checkpoint (Janssen and Medema, 2011).This prolonged mitotic delay is often followed by mitotic cell death (Gascoigne and Taylor, 2008).However, a subset of cells can escape and exit mitosis.Blocking mitotic exit might circumvent drug resistance seen in those cells and enhance the efficacy of anti-mitotic drugs (Huang et

Cloning and Functional Characterization of Ptpcd2 as a Novel
Cell Cycle Related Protein Tyrosine Phosphatase that Regulates Mitotic Exit Doaa H Zineldeen 1 *, Ayman A Wagih 1 , Makoto Nakanishi 2 al., 2009;Manchado et al., 2010;Hunt, 2013).Mitotic exit comprises all events that occur after completion of the spindle assembly checkpoint, including chromosome segregation, cytokinesis and reassembly of interphase cell structures.This is regulated through degradation of key mitotic factors and removal of phosphorylations from mitotic substrates (Wurzenberger and Gerlich, 2011;Gascoigne and Cheeseman, 2013).The Cdc14 family of dual specificity phosphatases (DSP) has been extensively studied in the context of yeast via its role in regulating late mitotic events, promoting Cdk substrate (s) dephosphorylation and mitotic exit (Bremmer et al., 2012;Hancioglu and Tyson, 2012;Sanchez-Diaz et al., 2012).Mammalian cells possess two orthologs, hCdc14A and hCdc14B (Vázquez-Novelle et al., 2005;Mocciaro and Schiebel, 2010).Interestingly, the essential role of Cdc14 in mitotic exit control is not conserved among species, where knockout studies of human Cdc14 orthologs in established cell lines have shown no obvious mitotic defects (Mocciaro and Schiebel, 2010).This strengthens the notion that alternative phosphatase (s) might function during mitosis (Berdougo et al., 2008;Mocciaro et al., 2010).We have previously identified the centrosomal protein tyrosine phosphatase containing domain 1 (Ptpcd-1) as a mitotic phosphatase involved in cytokinesis and regulation of centrosomal duplication (Zineldeen et al., 2009).Ptpcd1 is a Cdc14B homologue that is colocalized with Polo like kinase (Plk1), an oncogene overexpressed in many cancers (Zineldeen et al., 2009).In mammalian cells, the phosphatase (s) that counteracts Cdk1-CycB is yet to be identified, but is likely to impact mitotic exit regulation (Skoufias et al., 2007;Bremmer et al., 2012), here we functionally characterized a non-centrosomal isozyme of Ptpcd1, that we named; Ptpcd2 which is a cell cycle regulated non-centrosomal phosphatase involved in mitotic exit.

Cloning of mPtpcd2
The data base of the NCBI (National Center for Biotechnology Information) was searched for sequences that bear homology to a protein sequence corresponding to the entire region of mouse Ptpcd1 No. NM 207232 (Zineldeen et al., 2009).
A mouse eyeball cDNA clone: E130020P12, from the RIKEN full-length enriched library was identified and we named it Ptpcd2 (with gene bank accession No. AK053480).The complete ORF of Ptpcd2 was amplified by RT-PCR.Total RNA was extracted from MEF (mouse embryonic fibroblast) using Isogene (Nippon Gene, Japan) and reverse transcribed using RevertAid H Minus First Strand cDNA Synthesis kit (Thermo Scientific).
For Transient plasmids transfection, Lipofectamine Plus reagent (Life Technologies, Inc.) was used according to manufacturer's protocol.Cells were also co-transfected with a green fluorescent protein (GFP) expression vector pEGFP-C1 (Clontech, CA, USA) as a marker of transfection efficiency.

Flow cytometry assessment of cell cycle
Cells were cultured in 6-well plates and transfected with plasmid DNA 48h later, assessment of cellular DNA content by flow cytometry was performed as described elsewhere (Massey et al., 2010).Cells were analyzed with a BD flow cytometer (FACScan, Becton-Dickinson).Cell cycle distribution was determined by Cyflogic software.

Immunoblotting
Whole cell extract and immunoblotting were performed as previously reported (Méndez and Stillman, 2000;Shimada et al., 2008).Subcellular fractionation and preparation of cytosolic, nuclear fractions and insoluble nuclear pellet were according to (Méndez and Stillman, 2000;Niida et al., 2007).Protein concentrations were determined by protein assay kit (Biodiagnostic., Egypt).Equal amounts of Proteins were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes.Membranes were probed with the corresponding antibodies as in figure legends; β-actin was used as a loading control.Protein expression levels were determined using ECL detection kit (Amersham Biosciences) and chemiluminescence was detected by gel documentation (Biometra, Goettingen, Germany).

Bioinformatic analysis
Multiple sequence alignments were performed using Clustal W and Clustal X programs (Larkin et al., 2007).Motif analysis was performed according to (Dinkel et al., 2012) and for coiled domains prediction was according to (Lupas et al., 1991).

Statistical analysis
Data were expressed as mean±SD (standard deviation).Comparisons were achieved using, unpaired t -test according to the Graph Pad PRISM software version 5 (GraphPad Software, Inc., San Diego, CA).

Identification and cloning of mouse Ptpcd2
Loss of function studies of human Cdc14A and B revealed that neither of them is essential for cell viability nor cell proliferation (Berdougo et al., 2008;Mocciaro and Schiebel, 2010;Mocciaro et al., 2010).Thus, we speculated that other mammalian homolog (s) of Cdc14 phosphatases may exist and participate in regulation of late mitotic events such as mitotic exit.We previously identified Ptpcd1 as a centrosomal phosphatase that regulates its duplication and cytokinesis (Zineldeen et al., 2009).

Ptpcd2 has protein phosphatase catalytic activity
To functionally characterize our newly identified Ptpcd2, we first examined for the catalytic activity of ectopically expressed Ptpcd2 protein, HeLa cells were transfected with Ptpcd2-pcDNA3.1/Myc-His or empty vector, followed by immunoprecipitation with increasing amounts of HeLa cell lysates.PTP activity of the precipitated Ptpcd2 was then assayed using pNPP as a phosphatase substrate.PTP activity was recovered in the immune complex in a dose-dependent manner (Figure 2 A).Furthermore, the PTP activity in the immune complex was completely abolished by 1 mM sodium vanadate, an inhibitor of PTP activity (Figure 2A).Overexpression of Ptpcd2 in U2OS cells was performed with transfection efficiency of about 60% when the efficiency was monitored by co-transfection with EGFP vector (Figure 2B).Ptpcd2 encoded a 525 amino acid protein with a predicated molecular weight of 58.7 kDa.A band corresponding to 60 kDa was detected by western blotting (Figure 2C).Since sequence analysis of Ptpcd2 identified multiple nuclear localizing and export signals (Figure 1B), therefore we went further for subcellular fractionation of asynchronous HeLa cells expressing Ptpcd2 followed by western blotting.Ptpcd2 was almost detected in all cellular compartments (Figure 2 D), with enrichment at cytosolic fraction and insoluble nuclear pellet.

Overexpression of Ptpcd2 drives mitotic exit
To investigate the effect of Ptpcd2 overexpression on protein levels of various cell cycle key players, western blotting of U2OS cells expressing Ptpcd2 or empty vector (control) was performed.We found minor decrease in cyclin B1 levels relative to control.As for inhibitory Cdk1 phosphorylation at tyrosine 15, we noted mild increase in Cdk1-cyclin B1 activity in cells expressing Ptpcd2 when compared to control cells, probably due to rapid exit into the next G1 phase of the cell cycle.Sirtuin 2 (SIRT2), a human class III histone deacetylase which is phosphorylated by Cdk1 during mitosis and was reported to be dephosphorylated by Cdc14 A and B during mitosis (North and Verdin, 2007).Interestingly, overexpression of Ptpcd2 resulted in a decreased protein level of the mitotic SIRT2.(Figure 2B).To examine the effect of Ptpcd2 overexpression on cell cycle progression, we performed flow cytometric analysis of HeLa cells expressing either Ptpcd2 or control vector 48h after transfection.The majority of Ptpcd2 expressing cells accumulated at G1 phase with few >4N cells (Figure 3A-a), probably due to compared to mock transfectants (Figure 3 A(b) and A(c)).

Ptpcd2 spatiotemporal regulation throughout the cell cycle
We then determined the subcellular localization of Ptpcd2 by immunofluorescence analysis of HeLa cells expressing Ptpcd2 that revealed a cell cycle dependent spatiotemporal localization (Figure 5C).Ptpcd2 intensity was maximal during mitosis particularly in metaphase cells (Figure 5A, C) with some cells exhibiting micronuclei and multinucleation (Figure 5B a, b respectively) that may be due to mitotic slippage.Ptpcd2 signal was decreased in cytoplasm of interphase cells (Figure 5C) and contained in the nuclei in the form of dots like signals (Figure 5C, white arrows), with onset of mitosis, Ptpcd2 signal was mainly detected in the cytoplasm of metaphase, anaphase and telophase cells (Figure 5C).The observed immunostaining pattern of Ptpcd2 characterizes it as a cell cycle regulated kinase with possible mitotic functions.

Ptpcd2 is a non centrosomal variant of Ptpcd1
Ptpcd1 was previously identified as a centrosomal phosphatase that controls proper centrosomal duplication during S phase and cytokinesis (Zineldeen et al., 2009).To investigate whether Ptpcd2 has a similar centrosomal function, HeLa cells expressing Ptpcd2/Myc were double immunostained with c-myc antibody and an antibody that recognizes γ-tubulin, a known centrosomal marker (Zineldeen et al., 2009).Interestingly, Ptpcd2 immunoreactivity was mainly cytoplasmic with small scattered nuclear dots (Figure 5D upper panel).Colocalization of Ptpcd2 was not detected with γ-tubulin, suggesting that Ptpcd2 is a non-centrosomal protein.We confirmed our results by bioinformatic analysis of the C-terminal part of Ptpcd2 using the algorithm by Lupas et al. (1991) that showed lack of the coiled structures which are required for anchoring proteins to the centrosome (Figure 5E, a).In contrast, centrosomal Ptpcd1 exhibited multiple coiled structures at its C-terminal (Figure 5E,b).Taken together, our current data identify Ptpcd2 as a non-centrosomal protein.

Discussion
Targeting mitotic exit is of great hope for anti-cancer modalities (Chan et al., 2012;Russell et al., 2012;Hunt, 2013).Hence, mammalian mitotic exit remained largely elusive, the present study characterizes Ptpcd2 as a new member of the family of DSPs (Patterson et al., 2009) with possible function during mitotic exit.It shares homology to yeast and human Cdc14 and calcineurin; a phosphatase that has been reported to control meiotic M phase exit in xenopus (Mochida and Hunt, 2007) and completion of mitosis in drosophila (Takeo et al., 2010).Although mitotic functions of mammalian Cdc14B has been described (Cho et al., 2005;Wu et al., 2008), Cdc14B deficient cells were viable and lacked apparent mitotic defects (Mocciaro and Schiebel, 2010;Bremmer et al., 2012), suggesting that alternative phosphatase (s) is being able to complement mitotic functions of Cdc14B.
In the current study Ptpcd2 is sequestered in the nucleus during interphase and is exported to the cytoplasm with mitotic onset and almost detected in all cellular compartments, with enrichment at cytosolic fraction and insoluble nuclear pellet.The nuclear pellet contains, in addition to nuclei, mitochondria and sheets of plasma membrane (Graham, 2002).
These data concluded that Ptpcd2 is enriched in various cellular compartments that might be subjected to regulation by nucleocytoplasmic shuttling as predicted from its sequence analysis.Similarly, Cdc14B has been shown to be sequestered in the nucleolus then released in to the cytoplasm to induce mitotic exit (Wei and Zhang, 2011).Our identified Ptpcd2, thus strengthens the notion that highly structured intra-nuclear phosphatase (s) network might exist in mammalian cells.In accordance, previous report in living cells had revealed tight association of human Cdc14B with long filaments starting from nucleolar periphery to the nuclear envelope, and making connections with the nuclear pore complexes (Nalepa and Harper, 2004).In the present study we describe Ptpcd2 as a novel non-centrosomal isozyme of Ptpcd1 (Zineldeen et al., 2009) that does not associate with centrosomes, and is sequestered in the nucleus during interphase and is exported to the cytoplasm with mitotic onset conveying a cell cycle dependent nucleocytoplasmic shuttle.Unscheduled overexpression of Ptpcd2 induces exit into the next G1 phase with some cells showing multinucleation that may be due to mitotic slippage, where cells exit mitosis in a tetraploid state despite an active mitotic checkpoint (Janssen and Medema, 2011).Similarly, mitotic slippage in budding yeast was in part mediated by Cdc14p (Rossio et al., 2010).Forced Ptpcd2 overexpression in HeLa cells is associated with micronuclei formation (Figure 5 B, a) indicating that some DOI:http://dx.doi.org/10.7314/APJCP.2013.14.6.3669 Cloning and Functional Characterization of Ptpcd2 as a Novel Tyrosine Phosphatase cells exit mitosis in a tetraploid stat and are subjected to mitotic catastrophe that is characterized micronuclei (Galán-Malo et al., 2012).Targeting Ptpcd2 thus could be of great help in combination with microtubule poisons and other anti-mitotic drugs to delay mitosis and induce apoptosis and tumor death.In line with this notion, suppression of ser/thr phosphatase 4 induced tetraploid cell death (Theobald et al., 2013).Moreover, inhibition of mitotic exit has led to tumor regression in conditional Cdc20 null mouse model (Manchado et al., 2010).
In the current work, Ptpcd2 overexpression associates with altered cell cycle key players with decrease of Cdk1 phosphorylation of at tyrosine residue 15 (cdc2, Y15), and lowered protein levels of SIRT2, which could be due to Ptpcd2 mediated proteasomal degradation of SIRT2 via its RXXL motif.
SIRT2 has been shown to be overexpressed in a subset of tumors like hepatocellular carcinoma and squamous cell carcinoma (Chen et al., 2013;Lai et al., 2013).In line with our findings, SIRT2 function has been described to be modulated by Cdc14B during mitotic exit, where Cdc14B overexpression induced dephosphorylation and decreased protein levels of SIRT2 (Dryden et al., 2003).SIRT2 has been shown to be phosphorylated by Cdk1 during mitosis and its overexpression causes delay of cells to exit mitosis (North and Verdin, 2007).The fact that Ptpcd2 overexpression, caused loss of SIRT2 protein suggests that there is a functional link between Ptpcd2 phosphatase activity, SIRT2 phosphorylation, and SIRT2 abundance, consistent with a role for these proteins in mitosis.Although Cdk1 activity was not completely abolished in cells overexpressing Ptpcd2, this could be attributed to a low Cdk1-CycB activity at G1 phase, after exiting from mitosis (Novak et al., 2007).Furthermore, we noted that the percentage of mitotic cells expressing Ptpcd2 has decreased S10-pHH3 immunoreactivity, which could be due to mitotic exit of greater proportion of cells or due to unknown functions of Ptpcd2.Specifically, bioinformatic analysis of Ptpcd2 sequence identifies two STAT5 Src Homology 2 (SH2) domains as well as PDZ protein-protein interaction binding domain; both are implicated in signal transduction cascades and tumor suppression by some phosphatases (van den Berk et al., 2005;Bard-Chapeau et al., 2011).
This could define Ptpcd2 as an unidentified tumor suppressor phosphatase, which might be mutated in a subset of cancer cells.Interestingly, Cdc14 B has been proved to be of oncogenic potential which is an unusual feature for mammalian phosphatases (Chiesa et al., 2011;Wei and Zhang, 2011).Furtherly, Cdc14 down regulation has been proved to suppress glioblastoma growth (Galeano et al., 2013).An emerging role of phosphatases in cancer development has been recently highlighted (Zhang and Claret, 2012), that drives us to unravel new phosphatases and their role in cell cycle control and tumorgenesis.Conclusively, Ptpcd2 is a novel cell cycle related noncentrosomal isozyme of Ptpcd1 with functional homology to Cdc14.
It is implicated in mitotic exit and mitotic slippage.Ptpcd2 nuclear localization during interphase, may specifically affect transcriptional regulation, although this hypothesis deserve further investigations.Insights into substrate specificity of Ptpcd2 are needed to explore its role in cell cycle, where targeting mitotic exit in malignant cells could open new avenues for anti-cancer therapeutics.

Figure 1 .
Figure 1.Sequence Analysis of Ptpcd2.(A) Amino acid sequence analysis of Ptpcd2 showing PTP and DSP catalytic domains and PDZ binding domain.(B) Alignment of the amino acid sequences of the mouse Ptpcd2, mouse Ptpcd1 and human Ptpcd1.Black and gray backgrounds indicate residues that are identical or conservative substitutions, respectively.Hyphens within the sequences represent gaps introduced to optimize alignment, and residue numbers are shown on the left.(Generated by Clustal X 2.1; computer program).The conserved nuclear export signals (NES), bipartitite nuclear localizing signal (BNLS), RXXL motif that binds to APC/C (anaphase promoting complex/ cyclosome) and SH2 (Src Homology 2) domains are boxed.(C) Multiple alignment of the amino acid sequences of mouse Ptpcd2, mouse Ptpcd1, human Ptpcd1, human Cdc14A, human Cdc14B, S. cereviseae (Cdc14p) and mouse calcineurin.Identical and conservative residues are colored.Hyphens within the sequences represent gaps introduced to optimize alignment, and residue numbers are shown on the left.(D), a phylogenic tree of the aligned proteins (generated by Calsustal x 2.1, a computer program).The accession numbers of the compared proteins were as follows: mouse Ptpcd1 (NP_997115.1),human Ptpcd1 (NP_689635.3),hCdc14 A (NP_003663.2), hCdc14B (NP_001070649.1),Cdc14p (NP_116684.3) and mouse calcineurin (NP_032939.1)