Human CPPED1 belongs to calcineurin‐like metallophosphoesterase superfamily and dephosphorylates PI3K‐AKT pathway component PAK4

Abstract Protein kinases and phosphatases regulate cellular processes by reversible phosphorylation and dephosphorylation events. CPPED1 is a recently identified serine/threonine protein phosphatase that dephosphorylates AKT1 of the PI3K‐AKT signalling pathway. We previously showed that CPPED1 levels are down‐regulated in the human placenta during spontaneous term birth. In this study, based on sequence comparisons, we propose that CPPED1 is a member of the class III phosphodiesterase (PDE) subfamily within the calcineurin‐like metallophosphoesterase (MPE) superfamily rather than a member of the phosphoprotein phosphatase (PPP) or metal‐dependent protein phosphatase (PPM) protein families. We used a human proteome microarray to identify 36 proteins that putatively interact with CPPED1. Of these, GRB2, PAK4 and PIK3R2 are known to regulate the PI3K‐AKT pathway. We further confirmed CPPED1 interactions with PAK4 and PIK3R2 by coimmunoprecipitation analyses. We characterized the effect of CPPED1 on phosphorylation of PAK4 and PIK3R2 in vitro by mass spectrometry. CPPED1 dephosphorylated specific serine residues in PAK4, while phosphorylation levels in PIK3R2 remained unchanged. Our findings indicate that CPPED1 may regulate PI3K‐AKT pathway activity at multiple levels. Higher CPPED1 levels may inhibit PI3K‐AKT pathway maintaining pregnancy. Consequences of decreased CPPED1 expression during labour remain to be elucidated.

| 6305 HAAPALAINEN Et AL. mammalian phosphoproteome, phosphoserine is the most prevalent of the three phosphoamino acids. 2 Based on substrate specificity and catalytic mechanisms, protein phosphatases are classified as protein serine/threonine phosphatases (PSTPs or PSPs) or protein tyrosine phosphatases (PTPs).
PSPs are further categorized into three classes: phosphoprotein phosphatases (PPPs), metal-dependent protein phosphatases (PPMs) and aspartate-based phosphatases. 3 The PPP family is divided into PP1, PP2A, PP2B (also known as calcineurin and Ca 2+ stimulated), PP4, PP5, PP6 and PP7 subclasses. 3,4 PPMs comprise protein phosphatases dependent upon Mn 2+ /Mg 2+ ions for catalytic activity; PP2C is the largest PPM class. In aspartate-based phosphatases, the catalytically important nucleophilic water molecule is replaced by the side chain of an aspartate residue. 5 The catalytic subunits of several members of the PPP family associate with a wide variety of regulatory subunits. 3 One active site can contain two of the same cations or two different cations. For example, in PAPs, Fe 3+ is the first cation of the metallic centre of the catalytic site, while the second metal can be Fe 2+ , Zn 2+ , Mn 2+ or Mg 2+ . 7,8 Cyclic nucleotide PDEs hydrolyse both cAMPs and cGMPs, and class III PDEs can accept Fe 2+ , Mn 2+ Mg 2+ , or Co 2+ as catalysts. 9 The phosphoinositide 3-kinase (PI3K)-AKT signalling pathway is evolutionarily conserved and is involved in cell metabolism, innate and adaptive immunity, and diseases such as cancer. 10 Calcineurinlike phosphoesterase domain-containing protein 1 (CPPED1) dephosphorylates AKT1 at Ser473, thereby preventing cancer progression in bladder cancer. 11 CPPED1 expression levels are down-regulated in non-invasive bladder cancer tissue, whereas overexpression is associated with regression in tumour size. Additionally, CPPED1 regulates uptake of glucose in adipose tissue. Increased glucose uptake is associated with decreased CPPED1 expression. 12 Treatment with the PI3K-specific inhibitor wortmannin decreases glucose uptake in CPPED1 knockdown cells; this suggests that CPPED1 mediates glucose metabolism via the PI3K-AKT signalling pathway. 12 Moreover, silencing of CPPED1 expression in the human trophoblast cell line HTR8/SVneo leads to up-regulation of negative regulators of the PI3K pathway. 13 In the present study, we used human proteome microarray technology to identify proteins that can interact with CPPED1 and to identify potential functions of CPPED1, particularly in the human placenta. A protein interactome study yielded a list of proteins, some of which have previously reported associations with the PI3K-AKT signalling pathway; for example, p21 [RAC1] activated kinase 4 (PAK4) and phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2). Coimmunoprecipitation (CoIP) and bimolecular fluorescence complementation (BiFC) methods revealed that CPPED1 interacts with PAK4 and PIK3R2 in vivo. Further, mass spectrometry analysis of CPPED1 with PAK4 and PIK3R2 revealed that CPPED1 dephosphorylated PAK4 at serine residues, whereas PIK3R2 phosphorylation levels remained unaffected. These findings suggest that CPPED1 not only inhibits the activity of AKT1 but also affects the function of PI3K. We also show that CPPED1 mediates the effect of progesterone on the expression of certain genes in human placental HTR8/SVneo trophoblast cells.

| Homology search and sequence alignment
We used the human CPPED1 amino acid sequence and Network Protein Sequence Analysis (NPS@) software 14

| Construction of plasmids for protein microarray, coimmunoprecipitation and bimolecular fluorescence complementation experiments
Primers used for cloning into different plasmids are listed in Table S1. pBiFC-VN173 (Addgene plasmid #22010) and pBiFC-VC155 (Addgene plasmid #22011) were gifts from Chang-Deng Hu. 16 Further details are presented in the Supporting Information.

| Recombinant CPPED1 expression and purification
CPPED1 cloned into the PSF-OXB20-NH2-6HIS-V5-TEV constitutive expression plasmid was transformed and expressed in E coli BL21(DE3) cells at 37°C for 7-8 h. Cells were then harvested. After cell lysis by sonication, most of the CPPED1 was in a soluble form.

| Identification of recombinant CPPED1 by western blot
CPPED1 was expressed as a recombinant protein with an N-terminal His-V5 dual tag for protein microarray experiments. To confirm that the purified protein was CPPED1 and had the V5 tag in the N terminus, we used anti-CPPED1 and anti-V5 antibodies (Fig. S2). Further details are presented in the Supporting Information.

| Detection of CPPED1 phosphatase activity by western blot
Previous studies have shown that CPPED1 dephosphorylates AKT1. 11 To confirm that our purified human recombinant CPPED1 was active, we followed the in vitro phosphatase assay protocol of Gao et al. 17 Dephosphorylation reactions were carried out in a reaction buffer that contained 50 mM Tris (pH 7.4), 1 mM dithiothreitol and 5 mM MnCl 2 or CaCl 2 at 30°C for 30 min ( Figure 1B). The final concentration of CPPED1 and AKT1 (009-001-P21, Rockland) in the reactions was 0.1 µM and 0.13 µM, respectively. Significant differences were estimated with the nonparametric Mann-Whitney U test (n = 3 per sample type). Further details are presented in the Supporting Information.

| Circular dichroism and static light scattering
CD spectroscopy was performed with a Chirascan CD spectrometer (Applied Photophysics). CD data were collected between 280 and 200 nm at 22°C with a 0.1-cm path length quartz cuvette (Fig. S3). The Superdex 200 HR 5/150 GL (GE Healthcare) size-exclusion column and SLS instrument (Wyatt Technology) were used to determine the monodispersity of the purified recombinant human CPPED1 (Fig. S4).
Further details are presented in the Supporting Information.

| Human proteome microarray
The HuProt TM v3.1-Human Proteome Microarray (Cambridge Protein Arrays Ltd.) was used to identify protein interactions on an immobilized array. The protein microarray contains >20 000 human recombinant proteins and covers approximately 75% of the annotated protein-coding genome. Recombinant proteins on the microarray were expressed in Saccharomyces cerevisiae and purified with the GST tag purification system. Further details are presented in the Supporting Information.

| Fluorescence colocalization analysis
Fluorescence colocalization analysis was performed with trophoblast cell and human placenta tissue samples. HTR8/SVneo cells were seeded into the CELLview Cell Culture Dish (35 mm; Greiner Bio-One), which has a glass bottom, and the cells were grown as described previously. 13 Immunofluorescence staining on cell culture dishes was done as described previously, 18 using 0.1% saponin for cell permeabilization. Human placental tissue samples were treated as described previously. 13 Colocalization of the following complexes was analysed: CPPED1-AKT1, CPPED1-PAK4 and CPPED1-PIK3R2 ( Figure 2A,B, Fig. S5). Antibodies and detection methods are presented in the Supporting Information.

| Bimolecular fluorescence complementation assay
HEK293T cells were seeded into the CELLview Cell Culture Dish  Prior to CoIP, both anti-c-Myc agarose and control agarose resins were blocked with 1% BSA at 4°C for 1 h.

| Coimmunoprecipitation
CoIP samples were separated by SDS-PAGE and immunoblotted as described previously. 13,19 The detection was done as presented in the Supporting Information.

| Phosphatase assay detection by mass spectrometry
In vitro dephosphorylation reactions with CPPED1, PAK4 and PIK3R2 were performed as described earlier for AKT1, 17

| Sequence homology places CPPED1 in the cAMP phosphodiesterase family
CPPED1 has a calcineurin-like phosphoesterase domain and is inhibited by trifluoperazine, which suggests that CPPED1 could belong to the PP2A or PP2B families of PPPs. 11 This classification prompted us to perform sequence homology searches with human CPPED1. We did not detect the conserved amino acid sequence motifs of PPP and PPM family members in the CPPED1 sequence. 3  In particular, the tyrosine that is replaced by histidine in CPPED1 is important for Fe (III) binding; this tyrosinate-Fe (III) charge transfer results in the purple colour at 560 nm. Instead, we found that CPPED1 has the characteristic sequence motif of the class III cAMP PDE family ( Figure 1A). Class III cAMP PDEs belong to a family of calcineurin-like metallophosphoesterases (MPE). 1 These findings suggest that human CPPED1 belongs to the class III PDE of MPE superfamily rather than to the PPP or PPM families.

| Purification and characterization of human recombinant CPPED1 for human proteome microarray
To overexpress and purify recombinant CPPED1 protein, we cloned CPPED1 into the constitutively active expression plasmid pSFOXB20 and expressed the recombinant protein with the N-terminal His-V5 dual tag in E coli BL21 (DE3) cells. CPPED1 was expressed as the soluble form and purified to apparent homogeneity by affinity, hydrophobic interaction and size-exclusion chromatography. Protein purity was analysed by SDS-gel electrophoresis (Fig. S1).
To ensure that the purified recombinant CPPED1 was folded properly, monodispersed and enzymatically active, we carried out circular dichroism (CD) spectroscopy, static light scattering (SLS) and in vitro activity measurements, respectively. CD spectroscopy confirmed that human recombinant CPPED1 was folded properly F I G U R E 1 Alignment of CPPED1 with 3′,5′-cyclic adenosine monophosphate phosphodiesterase, and in vitro phosphatase activity of CPPED1. (A) CPPED1 homologous sequences were searched with NPS@ software. 14 CPPED1 showed similarities with 3′,5′-cyclic adenosine monophosphate phosphodiesterase (CpdA). Seven eukaryotic CPPED1 sequences are aligned with the two closest CpdA homologs from Synechococcus sp and Streptomyces avermitilis. Amino acid residues conserved throughout the sequences are highlighted in red. Similar residues are denoted by red letters. Residues that belong to the class III cAMP phosphodiesterase (PDE) sequence motif are framed. Framed residues are involved in metal coordination or phosphate binding. Amino acid residue and the interacting group (Fe 3+ , Mn 2+ or PO 4 ) are shown above the sequence alignment. These interactions were obtained from the crystal structure of Mycobacterium tuberculosis PDE (Protein Data Bank code 3IB8) and Matange. 9 Conserved class III cAMP PDE motif: Figure prepared with ESPript 3.0. 15 (B) Phosphorylation of Ser473 of AKT1 was quantified and compared with total AKT1 with and without addition of CPPED1. Effects of Mn 2+ and Ca 2+ on CPPED1 activity were also tested. Reaction mixture consisting of AKT1 alone was used as the reference to which other reaction mixtures were compared. pS473/S473 ratio values are shown. Significant differences were estimated with the nonparametric Mann-Whitney U test (n = 6 per group). Statistically significant change is indicated by an asterisk. (P <.05) CPPED1 was previously shown to dephosphorylate phospho-Ser473 of AKT1. 11,17 To confirm that our purified recombinant CPPED1 was enzymatically active, we carried out in vitro phosphatase assays with Mn 2+ in accordance with the methods of Gao et al. 17 We also incubated CPPED1 and AKT1 together in reaction buffer with Ca 2+ and without cations (Mn 2+ or Ca 2+ ) to determine whether CPPED1-mediated dephosphorylation is cation dependent.
Ca 2+ was added because CPPED1 was observed to have PP2B-like protein phosphatase activity, 11 and PP2B-like phosphatases are stimulated by Ca 2+ . 3,4 CPPED1 phosphatase activity was cation dependent and showed higher enzymatic activity in the presence of Mn 2+ ( Figure 1B). Once we had confirmed that purified recombinant CPPED1 was folded properly, enzymatically active and monomeric in solution, we used it in the HuProt TM human proteome microarray chip platform to identify target proteins for CPPED1.

| Identification of human CPPED1-interacting proteins by protein microarray
Protein microarrays can be used to identify protein-protein interactions, posttranslational protein modifications and modulations in protein expression levels under different conditions. To identify novel targets for CPPED1, we used the HuProt ™ v3.1 human proteome microarray to screen for protein-protein interactions with CPPED1. The HuProt ™ v3.1 microarray covers approximately 75% of the annotated human protein-coding genome. Analysis yielded a list of 36 proteins ( Table 1) that interact with CPPED1 in vitro. Although CPPED1 has been shown to dephosphorylate AKT1, 11 AKT1 was not in the list of CPPED1 interacting proteins. We then performed a DAVID functional annotation study that included all of the identified binding partners of CPPED1 (Table 2); this study identified growth factor receptor bound protein 2 (GRB2), PAK4 and PIK3R2 as enriched in the pathway analysis. In a previous study, we showed that knockdown of CPPED1 in HTR8/SVneo trophoblasts leads to enhanced expression of negative regulatory genes of the PI3K pathway, such as phosphoinositide-3-kinase interacting protein 1 (PIK3IP1) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PIK3CG). 13 PAK4 binds to phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) 22 and activates the PI3K pathway, whereas CPPED1, according to our protein microarray analysis, bound to PIK3R2 and PAK4. Based on these findings, we continued to investigate interactions between CPPED1 and PAK4 and PIK3R2.
Namely, the signal from AKT1 mostly originated from the network structure within cells (Fig. S5C). By contrast, CPPED1, PAK4 and PIK3R2 localization resembled individual spot patterns (Fig. S5A,   Fig. S5B). In placental tissue samples, CPPED1, PAK4, PIK3R2 and AKT1 all localized in the cytosol. However, CPPED1 and PIK3R2 were found more on the cell membranes, and CPPED1 and PAK4 were found more around nucleus and close to cell membrane ( Figure 2A,B).
Next, we visualized protein-protein interactions within a cell by bimolecular fluorescence complementation (BiFC). In BiFC, proteins fused to the N-and C-terminal fragments of yellow fluorescent protein (YFP) or variants 23,24 are coexpressed. Protein-protein interactions between the two putative protein-binding partners bring together the two halves of YFP, which results in a fluorescence signal. BiFC is used to investigate interactions between proteins in a cellular milieu as well as subcellular localization. 16,25 We used BiFC to verify interactions between CPPED1 and PAK4 and PIK3R2 and to determine the subcellular localization of the pro- When single plasmids, either VN or VC variants, were transfected into HEK293T cells, we did not observe a fluorescence signal (Fig. S6A,B). After cotransfection with JUN-VN and FOS-VC, there was a strong fluorescence signal in the nucleus, which confirmed the validity of the method for the identification of protein interactions and subcellular localization of protein complexes (Fig. S6C). Cells expressing CPPED1 and PIK3R2 showed a fluorescence signal in the cytosol, with high-intensity spots at the cell membrane ( Figure 2C We conclude that this method could not be used to demonstrate whether two proteins interact under normal conditions, as it appears to be too sensitive; when highly expressed, the YFP halves have a tendency to self-assemble, resulting in a stable YFP independent of proteinprotein interactions between the two proteins under investigation. 23,24 However, it is feasible to use BiFC to investigate the subcellular localization of known interactions. In summary, in our experimental setting, the BiFC assay could not confirm interactions between CPPED1 and its putative binding partners PAK4 and PIK3R2. However, it did indicate that CPPED1-PIK3R2 and CPPED1-PAK4 ( Figure 2C) complexes were cell membrane associated and cytosolic, respectively.

| Coimmunoprecipitation of PAK4 and PIK3R2 with CPPED1
Since PAK4 and PIK3R2 colocalized with CPPED1 in cells, we carried out CoIP analysis to investigate whether these protein complexes formed in vivo. HEK293T cells were cotransfected with plasmids expressing CPPED1 and PAK4 or CPPED1 and PIK3R2.
PAK4 or PIK3R2 were expressed as recombinant proteins with an N-terminal myc-tag, and anti-myc antibody was used for the pulldown; afterwards, protein complexes were analysed by Western blot. Successful pulldown of the bait was verified with anti-myc, anti-PAK4, or anti-PIK3R2 antibodies (Figure 3, Fig. S8, Fig. S9). Antiflag and anti-CPPED1 antibodies were used to detect CPPED1 in the complexes. Overexpressed CPPED1 coimmunoprecipitated with PAK4 or PIK3R2 (Figure 3, Fig. S8, Fig. S9). These results provided further evidence that CPPED1 interacts with PAK4 and PIK3R2.

| CPPED1 dephosphorylates PAK4 in vitro
CPPED1 is known to dephosphorylate AKT1. 11 According to the SwissProt database, PAK4 and PIK3R2 are phosphorylated at various sites including serine and threonine residues. We investigated whether CPPED1 dephosphorylates any of the phosphorylated TA B L E 1 CPPED1-interacting proteins obtained by human proteome microarray. Above-threshold interactions are listed in order of highest to lowest binding affinity with CPPED1 with PAK4 or PIK3R2 and looked for changes in the phosphorylated residues by mass spectrometry. We detected numerous phosphorylated serines, threonines, and tyrosines in PAK4 (Fig. S10) and PIK3R2 (Fig. S11). None of the identified phosphorylations decreased in PIK3R2 in the presence of CPPED1. In the PAK4 assay, there were two phosphopeptides in which the number of phosphoserines decreased when CPPED1 was included in the reaction

| CPPED1 mediates the effect of progesterone on gene expression in HTR8/SVneo cells
To determine whether CPPED1 mediates the effect of progesterone on gene expression, we treated CPPED1-silenced and CPPED1unsilenced HTR8/SVneo cells with progesterone (P4). Silencing was accomplished with small interfering RNA (siRNA), as described previously. 13 Based on qRT-PCR results, silencing of CPPED1 at the mRNA level was 68%. 13 When gene expression levels of HTR-8/SVneo cells were analysed by RNA sequencing, expression levels of 98 genes were either down (n = 46; Table S2) or up (n = 52; Table S3) in P4treated vs. untreated control cells. When we compared this gene list with the transcriptomes of cells in which there was simultaneous silencing of CPPED1 expression and P4 treatment (Table S4), we found that silencing of CPPED1 expression removed the effect of P4 on the expression of a subset of genes (n = 71; Table S4, Fig. S12). Out of these 71 genes, the expression levels of 36 genes change more than 50% after silencing of CPPED1 expression (Table S4). This suggests that in HTR8/SVneo cells, CPPED1 mediates the effect of P4 on the expression of certain genes.

| D ISCUSS I ON
A previous report placed CPPED1 in both the PP2A and PP2B subclasses of the PPP protein family. 11 Based on the amino acid

F I G U R E 4
In vitro phosphatase activity of CPPED1 determined by mass spectrometry. Human recombinant PAK4 incubated with and without human recombinant CPPED1. Effect of cations Mn 2+ , Ca 2+ or Mg 2+ on phosphatase activity was also measured. Phosphopeptides of PAK4 determined by mass spectrometry. Two phosphopeptides showed a decrease in their phosphorylation content. The changes were seen at Ser104 (A) and Ser167, Ser173, Ser174, Ser181 and Ser195 (B) F I G U R E 5 CPPED1 involvement with PI3K-AKT pathway. Schematic view of activated (A) and inactivated (B) PI3K-AKT pathway. PI3K-AKT is a conserved pathway that is involved in many types of cellular processes. When growth factor (GF) binds to receptor tyrosine kinase (RTK), RTK tyrosine residues are phosphorylated. Regulatory subunit p85 binds to phosphorylated tyrosine and brings catalytic subunit p110 adjacent to substrate PIP2. Once PIP3 is formed from PIP2 by PI3K, pleckstrin homology (PH) domains of PDK1, PDK2 and AKT1 bind to PIP3, which results in phosphorylation and activation of AKT1. 30 PDK1 phosphorylates Thr-308. Ser-473 is phosphorylated by various proteins such as PDK2, integrin-linked kinase (ILK), mechanistic target of rapamycin complex (mTORC2) and DNA-dependent protein kinase (DNA-PK). 52,53 PAK4 has been shown to bind to p85α (also known as PIK3R1) and is required for maximal phosphorylation of AKT1. 22 By contrast, protein phosphatase 2A (PP2A), 54 protein phosphatase 1 (PP1), 55 PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) 56 and CPPED1 11 are known to dephosphorylate Ser-473 and inactivate AKT1. Among other functions, activated AKT1 phosphorylates transcription factor FOXO1, which results in nuclear exclusion of phosphorylated FOXO1. 57 This might also prevent formation of the FOXO-progesterone receptor (PR) transcription factor complex. [48][49][50] By contrast, PI3K-AKT pathway inhibition allows for formation of the FOXO-PR complex. We found that CPPED1 bound p85β (also known as PIK3R2) and PAK4, which could have an effect on the PI3K-AKT Pathway. We also found that CPPED1 mediated the effect of progesterone on the expression of a set of genes.  44 There are different classes of PI3Ks.
Class I PI3Ks comprise p110 catalytic and p85 regulatory subunits ( Figure 5A). There are different forms of p110 and p85 subunits, such as p110α or p110β and p85α (PIK3R1) and p85β (PIK3R2). 45 p110α and p110β regulate cell cycle entry, 46 and regulatory subunits PIK3R1 and PIK3R2 stabilize these catalytic subunits. 45 When bound to the catalytic subunits, the regulatory subunits inhibit the activity of PI3K. Upon activation of receptor tyrosine kinase (RTK), the regulatory subunits bind to RTK and liberate PI3K, which results in PI3K activation and localization of PI3K adjacent to its substrate PIP2 at the cell membrane 30,45 ( Figure 5A). One other way to activate PI3K is via RAS in which RAS activates p110 catalytic subunit independently of p85. 47 In the current study, we found that the CPPED1-PIK3R2 complex was located on the cytosolic side of the cell membrane. Formation of the CPPED1-PIK3R2 complex could affect localization of PI3K at the cell membrane, thereby inactivating the PI3K-AKT pathway ( Figure 5B), or the complex could bring CPPED1 to an as-yet unidentified target on the cell membrane. In addition, we identified a CPPED1-PAK4 complex. This complex could also affect activity of the PI3K-AKT pathway, because PAK4 is required for maximal phosphorylation of AKT1. 22 If CPPED1 inhibits the PI3K-AKT pathway by binding to PIK3R2 or PAK4, or by dephosphorylating AKT1, this could enable nuclear localization of FOXO1, formation of the FOXO1-PR transcription complex, and expression of genes that maintain pregnancy ( Figure 5B).
Namely, FOXO1 and progesterone receptor (PR) are located in the same transcriptional complex, which results in the activation of genes coding for senescence-associated cell cycle inhibitors and decidualization. 48,49 Progesterone binds to PR and maintains pregnancy. 50 In addition, androgen, oestrogen and follicle-stimulating hormone receptors are FOXO1 transcription factor-binding partners. 51 During spontaneous term labour, the amount of CPPED1 decreases. 13 This could result in the activation of the PI3K-AKT pathway ( Figure 5A), nuclear exclusion of FOXO1, disassembly of the FOXO1-PR complex, and initiation of labour. When we treated HTR8/SVneo cells with progesterone, we found that expression levels of certain genes either increased or decreased. By simultaneously silencing CPPED1 expression and treating with progesterone, we identified a subset of genes that no longer responded to progesterone treatment. These findings suggest that CPPED1 is required to mediate the effect of progesterone on the expression of certain genes in human placental HTR8/ SVneo trophoblast cells.
A growing body of evidence indicates that CPPED1 is an important regulator of the PI3K-AKT signalling pathway. In the placenta, CPPED1 levels are associated with the timing of birth.
However, the exact role of CPPED1 in placental trophoblast cells is still unclear. The exact downstream effects of decreased CPPED1 expression during spontaneous labour remain to be established but the changes could be mediated via the PI3K-AKT-FOXO/PR axis.

ACK N OWLED G EM ENTS
We acknowledge Maarit Haarala for technical assistance. We also acknowledge the Biocenter Oulu Tissue Imaging Center (BCO-TIC) light microscopy core facility, Biocenter Oulu Proteomics and protein analysis mass spectrometry core facility, and Biocenter Oulu Proteomics and protein analysis, protein biophysical measurement and amino acid analysis core facility. We thank Finnish

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.