Identification of protein phosphatase 4 catalytic subunit as a Wnt promoting factor in pan-cancer and Xenopus early embryogenesis

Wang, YiLi; Han, WonHee; Yun, SeokMin; Han, JinKwan

doi:10.1038/s41598-023-35719-y

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Published: 23 June 2023

Identification of protein phosphatase 4 catalytic subunit as a Wnt promoting factor in pan-cancer and Xenopus early embryogenesis

YiLi Wang¹,
WonHee Han^1,2,
SeokMin Yun¹ &
…
JinKwan Han¹

Scientific Reports volume 13, Article number: 10240 (2023) Cite this article

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Abstract

Protein Phosphatase 4 Catalytic Subunit (PPP4C) is an evolutionarily conserved protein involved in multiple biological and pathological events, including embryogenesis, organogenesis, cellular homeostasis, and oncogenesis. However, the detailed mechanisms underlying these processes remain largely unknown. Thus, we investigated the potential correlation between PPP4C and biological processes (BPs) and canonical Wnt signaling using pan-cancer analysis and Xenopus laevis (X. laevis) embryo model. Our results indicate that PPP4C is a potential biomarker for specific cancer types due to its high diagnostic accuracy and significant prognostic correlation. Furthermore, in multiple cancer types, PPP4C-related differentially expressed genes (DEGs) were significantly enriched in pattern specification, morphogenesis, and canonical Wnt activation. Consistently, perturbation of Ppp4c in X. laevis embryos interfered with normal embryogenesis and canonical Wnt responses. Moreover, biochemical analysis of X. laevis embryos demonstrated that both endogenous and exogenous Ppp4c negatively regulated AXIN1 (Wnt inhibitor) abundance. This study provides novel insights into PPP4C roles in pattern specification and Wnt activation. The similarities in BPs and Wnt signaling regulation regarding PPP4C support the intrinsic link between tumorigenesis and early embryogenesis.

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Introduction

In gene regulatory networks, reversible phosphorylation is a highly dynamic protein modification that incorporates signals from specific pathway(s) and modulates target pathway(s), including itself, without de novo protein synthesis¹. Thus, dephosphorylation of serine/threonine residues, by protein phosphatase (PPM and PPP) families, regulates multiple signaling pathways and cellular processes. The highly conserved protein phosphatase 4 (PP4) complex, belonging to the type 2A PPP family, is composed of one catalytic subunit (PPP4C) and five regulatory subunits (PPP4R1, PPP4R2, PPP4R3\(\upalpha\), PPP4R3\(\upbeta\), and PPP4R4). The PP4 complex exists in heterodimeric (PPP4C/PPP4R1; PPP4C/PPP4R4) or heterotrimeric (PPP4C/PPP4R2/PPP4R3\(\upalpha\); PPP4C/PPP4R2/PPP4R3\(\upbeta\)) forms. Its catalytic activity is contributed by PPP4C, while the substrate specificity is mainly determined by the binding regulatory subunits^2,3,4. PP4 is reported to regulate multiple cellular events, such as DNA damage response, tumorigenesis, cell migration, immune response, stem cell development, and glucose metabolism^5,6. The fundamental function of PPP4C in embryogenesis is evidenced by the lethality and dorsal-ventral (DV) patterning defects in mouse and zebrafish PPP4C-deficient embryos, respectively^7,8. In tumorigenesis, PPP4C levels are associated with metastasis and poor prognosis in breast, lung, pancreatic, and colon cancer^{9,10,11,12,13,14,15}. However, the detailed mechanisms underlying embryogenesis and oncogenesis are still limited.

Canonical Wnt signaling, induced by secreted cysteine-rich Wnt glycoproteins, governs multiple cellular processes, including cell cycle, proliferation, cell fate determination, and tissue patterning. The destruction complex, composed of AXIN, glycogen synthase kinase 3 (GSK3), casein kinase 1 (CK1), and adenomatous polyposis coli tumor-suppressor protein (APC), regulates the stability and abundance of cytosolic \(\upbeta\)-catenin, which is intimately associated with canonical Wnt signaling “ON/OFF” status¹⁶. Wnts are essential for organogenesis and tissue homeostasis in multiple tissues, such as skeleton, intestine, hair follicle, skin, hematopoietic and neural systems^17,18,19. Therefore, perturbation of canonical Wnt signaling induces congenital malformations and cancer, given its substantial impact on organ development and tissue homeostasis¹⁷. Moreover, in multiple cancer types, high mutation frequencies in the Wnt components and regulators highlight its function in tumorigenesis regarding cell maintenance, proliferation, and metastasis²⁰. In embryogenesis, the most fundamental role of Wnt is as a morphogen shaping DV and anterior–posterior (AP) axes formation¹⁹. In 1989, McMahon first reported the X. laevis axis duplication induced by mouse Wnt1 mRNA, providing a rapid and convenient assay for estimating Wnt regulation in vertebrates²¹. Genetic interactions between PPP4C and Wnt signaling have been reported. In Drosophila, PP4 enhanced Wnt ligand wg transcription, while in mouse embryonic stem cells, it promoted neuronal differentiation through the Wnt/RYK pathway^22,23.

In this study, we investigated PPP4C-related BPs and potential correlation between PPP4C and Wnt signaling in human pan-cancer RNA-seq profiles and X. laevis model. In both species RNA-seq data, PPP4C was ubiquitously transcribed in all adult tissues, with relatively lower levels in muscle, brain, heart, and pancreas tissues. In X. laevis embryos, ppp4c was enriched in the neural crest and head regions during neurula and tailbud stages, respectively. In the cohort study of The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx), PPP4C exhibited transcriptional imbalance between normal and tumor tissues in 28 cancer types, with high diagnostic accuracy in 14 and certain accuracy in the remaining. Additionally, PPP4C levels were significantly associated with overall survival (OS) in 15 cancer types. Gene Ontology (GO) enrichment analysis of PPP4C-related DEGs in pan-cancer revealed that PPP4C is linked with pattern specification, morphogenesis, and tissue development, which are essential for embryogenesis. Consistently, overexpression of Ppp4c in X. laevis embryos interfered with AP patterning, and loss of Ppp4c impaired anterior structures and melanocytes. Gene Set Enrichment Analysis (GSEA) of the DEGs presented a significant correlation between PPP4C and canonical Wnt activation in nine tumor types. This positive correlation was validated in X. laevis model, as Wnt responses were compromised in Ppp4c knock-down (Ppp4c-KD) embryos. Additionally, both endogenous and exogenous Ppp4c inhibited AXIN1 abundance, indicating that Ppp4c promotes canonical Wnt signaling at the destruction complex level. Our results highlight the biomarker role of PPP4C in multiple cancers, provide novel insights into its function in pattern specification and Wnt signaling regulation, and support the intrinsic link between embryogenesis and oncogenesis.

Results

Highly conserved PPP4C is expressed ubiquitously among vertebrates

Amino acid (AA) sequence alignment in Fig. 1a reveals that PPP4C is highly conserved among vertebrate species. Specifically, X. laevis Ppp4c exhibits 99% identity with PPP4C in human (NP_001290432.1), mouse (NP_001347393.1), and rat (NP_599186.1) species. The expression pattern of PPP4C was explored in adult human and X. laevis transcriptome profiles and X. laevis embryos. Human single-cell and tissue RNA-seq analyses from The Human Protein Atlas (HPA) and GTEx showed that PPP4C was detected in all cell types and tissues, with higher transcriptional levels in reproductive, blood, and skin systems, and lower in muscle, brain, heart, and pancreas (Fig. 1b, Fig. S1). Likewise, X. laevis ppp4c were highly transcribed in the reproductive systems and lowly in muscle, pancreas, heart, and brain tissues (Fig. 1c), indicating its transcriptional conservatism among vertebrate species. X. laevis embryonic ppp4c expression was explored by RT-PCR, western blot, and whole-mount in situ hybridization (WMISH). Both the RNA and protein of ppp4c were ubiquitously detected in all embryonic stages (Fig. 1d,e). Notably, Ppp4c protein was slightly elevated between late gastrula and mid neurula stages (Fig. 1e). WMISH in Fig. 1f presents the ppp4c spatial information. Maternal ppp4c transcripts were localized in the animal hemisphere during cleavage (stage 3) and blastula (stage 8) stages. During gastrula stages (stage 10 and 11.5), the ppp4c-positive region extended to the dorsal and ventral marginal zone. In the neurula stage (stage 15), ppp4c was detected in the anterior part of neural plate and the neural plate border in the trunk region. As development proceeded to the tailbud stage (stage 32), ppp4c was enriched in the head region but almost undetectable in others. The PPP4C expression differences observed among different adult tissues and between adult and embryonic stages, together with its structural conservatism, promoted us to question its potential roles in embryogenesis and oncogenesis.

PPP4C is a potential biomarker in pan-cancer

With the cohort study of TCGA and GTEx, we compared PPP4C transcription levels between 33 tumor and normal tissues. Except for mesothelioma (MESO) and uveal melanoma (UVM), which lacked normal tissue data, PPP4C transcripts were significantly increased in most (27/31) tumor types and decreased in acute myeloid leukemia (LAML), while no statistical difference was observed in kidney chromophobe (KICH), pheochromocytoma and paraganglioma (PCPG), or sarcoma (SARC) (Fig. S2a). Paired comparison of tumor and adjacent non-tumor tissue (n > 3) further confirmed the significant elevation of PPP4C in bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung squamous cell carcinoma (LUSC), stomach adenocarcinoma (STAD), and thyroid carcinoma (THCA) (Fig. S2b).

The transcriptional differences between tumor and normal tissues were further supported by the diagnostic index factor receiver operating characteristic (ROC) curve. ROC results in Table 1 display that PPP4C levels had high predictive accuracy in 14 tumor types, including BLCA, BRCA, cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), CHOL, COAD, glioblastoma multiforme (GBM), LAML, brain lower grade glioma (LGG), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), rectum adenocarcinoma (READ), STAD, uterine corpus endometrial carcinoma (UCES), and uterine carcinosarcoma (USC). PPP4C levels had certain diagnostic accuracy in adrenocortical carcinoma (ACC), lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), ESCA, HNSC, KIRC, KIRP, LIHC, lung adenocarcinoma (LUAD), LUSC, prostate adenocarcinoma (PRAD), skin cutaneous melanoma (SKCM), PCPG, THCA, and thymoma (THYM) for tumor diagnoses.

Table 1 ROC results of PPP4C in pan-cancer.

Full size table

Next, the prognostic potential of PPP4C was evaluated by the Kaplan–Meier (KM) estimate in TCGA. The transcription levels of PPP4C significantly correlated with patient OS in 15 tumor types, of which PPP4C was a risk factor for poor prognosis in ACC, BRCA, HNSC, KIRC, KIRP, LAML, LGG, LIHC, LUAD, MESO, PAAD, and UVM, whereas a protective factor for better OS in CESC, READ, and THYM (Fig. S3). Altogether, the distinct expression pattern between normal and tumor tissues, and the close correlation between PPP4C and OS indicate that PPP4C is a potential biomarker for tumorigenesis.

PPP4C-related DEGs are involved in multiple BPs

To uncover PPP4C-related BPs in tumorigenesis, we analyzed the DEGs between PPP4C high (50%–100%) and low (0%–50%) expression tissues in each tumor. The criteria and numbers of DEGs selected for GO analysis in each tumor are listed in Table S1. The volcano plot of DEGs in LGG is shown in Fig. 2a. Interestingly, besides immune or DNA damage responses that facilitate or impede oncogenesis, PPP4C-related BPs focused on regionalization, pattern specification, morphogenesis, metabolism, and the development of skin, keratinocyte, epidermis, and muscle tissues (Fig. 2b,c). Notably, in BLCA, BRCA, CHOL, COAD, ESCA, LGG, MESO, PCPG, PRAD, SARC, THCA, and UCES, elevation of PPP4C correlated with activation of genes involved in the skin, epidermis, or keratinocyte-related processes (Fig. 2b,d, Supplementary Dataset File 1). In BLCA, BRCA, CESC, CHOL, DLBC, ESCA, GBM, HNSC, KICH, KIRP, LAML, LIHC, LUSC, MESO, OV, PCPG, PRAD, SARC, STAD, TGCT, THYM, and UCES, the DEGs that negatively correlated with PPP4C were enriched in muscle-related processes (Fig. 2c,e, Supplementary Dataset File 2). PPP4C levels in ACC, CHOL, GBM, KICH, KIRC, LAML, LGG, LIHC, LUSC, MESO, PCPG, SARC, STAD, THCA, UCES, and UVM positively correlated with pattern specification, regionalization, and morphogenesis (Fig. 2b,f, Supplementary Dataset File 3), which are crucial for early embryogenesis. Besides, in 26 out of 33 tumor types, either high or low, PPP4C were significantly correlated with synaptic or synapse-related DEGs (Fig. 2g, Supplementary Dataset File 4, 5).

PPP4C correlates with canonical Wnt signaling in tumors and X. laevis embryos

Given the fundamental function of Wnt in AP and DV pattern specification, and the correlation between PPP4C and pattern specification in certain tumor types, we explored the relationship between PPP4C and canonical Wnt activation through GSEA of DEGs in these tumors. We observed a positive correlation between PPP4C and canonical Wnt target genes at the transcriptional level in ACC, CHOL, LAML, LGG, LIHC, PCPG, SARC, THCA, and UVM (Fig. 3a, Supplementary Dataset File 6). Additionally, the Wnt-down gene set was negatively associated with PPP4C levels in CHOL and LGG (Fig. 3b, Supplementary Dataset File 6). While in GBM, a negative relationship between PPP4C and canonical Wnt activation was observed (Supplementary Dataset File 6).

Furthermore, we validated the correlation between Ppp4c and pattern specification, morphogenesis, and canonical Wnt signaling in the X. laevis embryo model, a fast and easy in vitro culture system. In this model, activation of canonical Wnt signaling in the ventral vegetal region or nearly totipotent animal cap (AC) region by ectopic \(\upbeta\)-catenin induced axis duplication or target gene transcription, respectively (Fig. 3c–g). KD of Ppp4c in X. laevis embryos was achieved through injection of ppp4c morpholino (ppp4c MO), which was designed to block ppp4c translation by binding to its 5\('\) untranslated region (5\('\) UTR). The effectiveness and specificity of MO were confirmed by its ability to inhibit the translation of 5\('\) UTR containing ppp4c-ha (5\('\) UTR-ppp4c-ha) rather than MO-resistant non-5\('\) UTR containing ppp4c-ha (ppp4c-ha or Re.ppp4c-ha) (Fig. S4a,b). In Ppp4c-deficient embryos, the Wnt responses induced by wild-type \(\upbeta\)-catenin (WT-\(\upbeta\)-catenin or WT-BC) were compromised but could be rescued by co-injection of Re.ppp4c (Fig. 3e–g).

To further identify which hierarchy level in Wnt signaling was affected by Ppp4c, we compared the Wnt responses induced by WT-\(\upbeta\)-catenin with those induced by constitutively active \(\upbeta\)-catenin (CA-\(\upbeta\)-catenin) in X. laevis embryos. CA-\(\upbeta\)-catenin (S37A mutant) cannot be fully phosphorylated by GSK-3\(\upbeta\) and is therefore less susceptible to proteasome degradation²⁴. The titration of CA-\(\upbeta\)-catenin to a comparable WT-\(\upbeta\)-catenin dose in axis duplication is shown in Fig. S5. Unlike WT-\(\upbeta\)-catenin-induced axis duplication and nodal3.1 transcription, which were reversibly inhibited upon loss of Ppp4c (Fig. 3f,g), both responses induced by CA-\(\upbeta\)-catenin were not affected by Ppp4c perturbation (Fig. 3h,i). Collectively, our pan-cancer data determined a correlation between PPP4C and canonical Wnt activation. In the X. laevis embryo model, Ppp4c promoted canonical Wnt responses at or upstream of \(\upbeta\)-catenin stability regulation.

X. laevis Ppp4c interacts with AXIN1 in vivo

Co-immunoprecipitation (co-IP) assay of Ppp4c-HA with \(\upbeta\)-catenin and AXIN1 supported previous observations in X. laevis embryos. As shown in Fig. 4a, immunoprecipitates of Ppp4c-HA with an anti-HA antibody retrieved Myc-AXIN1, but not \(\upbeta\)-catenin-Myc in X. laevis embryo animal cap tissue. Consistently, we were able to detect Ppp4c-HA in the reciprocal immunoprecipitates of Myc-AXIN1. As the association between Ppp4c-HA and \(\upbeta\)-catenin was undetectable, we questioned whether Ppp4c regulated endogenous \(\upbeta\)-catenin through destruction complex. In the presence of Myc-AXIN1, endogenous \(\upbeta\)-catenin was detected in the immunoprecipitates of Ppp4c-HA (Fig. 4b), suggesting that Ppp4c indirectly associates with \(\upbeta\)-catenin through AXIN1. Moreover, co-IP of Ppp4c-HA with several truncated Myc-AXIN1 mutants showed that Ppp4c-HA interacted with several AXIN1 mutants lacking N-terminal parts, rather than the ones lacking C-terminal halves (Fig. 4c–e), suggesting that Ppp4c interacts with AXIN1 through the C-terminal halves.

X. laevis Ppp4c antagonizes AXIN1 abundance

Considering that the phosphorylation status of AXIN1 is closely associated with its stability and scaffold function, we postulate that Ppp4c may regulate AXIN1 abundance or scaffold function through dephosphorylation. As the endogenous Axin1 abundance in X. laevis embryos is below the western blot detection threshold, we evaluated ectopic Myc-AXIN1 regarding protein abundance and its interaction with endogenous \(\upbeta\)-catenin in embryos. In the lysate of stage 11 embryos, ectopic Myc-AXIN1 abundance was dose-dependently decreased by co-injection of ppp4c-ha mRNA (Fig. 5a), and reversibly accumulated in Ppp4c-deficient embryos (Fig. 5b,c), conveying that Ppp4c inhibits AXIN1 at the protein level. Furthermore, as ectopic Myc-AXIN1 in Ppp4c-KD embryos shifted behind the normal ones (Fig. 5c, lanes 2 and 3), and two catalytic-dead Ppp4c mutants (D82A, H115Q) failed to inhibit Myc-AXIN1 abundance (Fig. 5d, lanes 1–5), AXIN1 inhibition is most likely to be correlated with its protein modification, such as dephosphorylation. Notably, Ppp4c could partially rescue AXIN1 band shift in the presence of Wnt8 (Fig. 5c, Lane 2–4 and 7), suggesting that exogenous Wnt ligand promotes Ppp4c’s function in AXIN1 protein modification. Additionally, we observed that the tail motif deletion Ppp4c mutant (\(\Delta\)DYFL) expressed enhanced Myc-AXIN1 inhibition compared to WT-Ppp4c (Fig. 5d, lanes 2, 3, and 6), implying that the tail motif may negatively regulate Ppp4c activity in AXIN inhibition during gastrula stages. In the scaffold function study, to normalize precipitated AXIN1 abundance within groups, we utilized a TANKs domain truncated AXIN1 mutant (Myc-AXIN1-C1), which possessed interaction sites with both \(\upbeta\)-catenin and Ppp4c (Fig. 4c,d), and its abundance was not Ppp4c-dependent (Fig. 5e,f). Perturbation of Ppp4c rarely affected the abundance of endogenous \(\upbeta\)-catenin that immunoprecipitated with Myc-AXIN1-C1 (Fig. 5f,g). Collectively, these observations indicate that Ppp4c inhibits AXIN1 abundance in a catalytic-dependent manner.

Perturbation of Ppp4c interferes with normal embryogenesis in X. laevis

In pan-cancer study, PPP4C levels correlated with multiple BPs, such as regionalization, pattern specification, morphogenesis, metabolism, and the development of skin, keratinocyte, epidermis, and muscle tissues. In both X. laevis embryos and certain tumor tissues, PPP4C levels significantly correlated with canonical Wnt activation. Therefore, we questioned whether interference with Ppp4c could affect normal embryogenesis. As activation of canonical Wnt signaling in dorsal animal (DA) or ventral vegetal regions of embryos could induce head truncation or axis duplication respectively, we wondered whether excess Ppp4c could generate similar phenotypes. Overexpression of Ppp4c in the DA region, where the future head structure originates from, dose-dependently reduced the eye field, cement gland, and overall head structures in tailbud embryos (Fig. 6a,b). In addition, WMISH of several markers of head structure shows that Ppp4c DA overexpression reduced bf1 (forebrain) and pax6 (eye), rather than krox20 (hindbrain) (Fig. 6c), indicating excess Ppp4c alters AP patterning in embryogenesis. Unlike \(\upbeta\)-catenin (50 pg), injection of ppp4c or ppp4c-\(\Delta\)DYFL (up to 2 ng) failed to induce axis duplication when injected ventral-vegetally (Fig. S6a), implying that Ppp4c enhances rather than directly induces canonical Wnt activation in X. laevis embryos and may require the cooperation from other Wnt axis levels.

In KD studies, we first validated the efficiency of injection sites, and the most efficient and consistent KD phenotype was observed in 1-cell stage AC injected embryo (Fig. S6b). Injection of ppp4c MO into animal hemisphere of 1-cell stage embryos dose-dependently impaired anterior structures and melanocytes located on dorsal and lateral regions (Fig. 6d,e). The head size, retinal pigment epithelium (RPE), and melanocyte defects in Ppp4c-KD embryos can be partially rescued by co-injection of Re.ppp4c mRNA (Fig. 6f,g), suggesting that Ppp4c is essential for proper development of anterior structures and melanocytes during X. laevis embryogenesis. Notably, anterior structure defects in Ppp4c-KD embryos differ from those in Ppp4c overexpression embryos. In Ppp4c-KD embryos, the cement gland size was unaffected and anterior structures were not fully eliminated, indicating that the anterior deficiencies in the Ppp4c-KD embryos are more likely caused by tissue-specific rather than global AP patterning defects. Collectively, perturbation of Ppp4c in early embryos interferes with normal embryogenesis.

Discussion

Like other serine/threonine phosphatases, PP4 participates in multiple cellular processes, including DNA damage response, cell cycle regulation, tumorigenesis, immune response, stem cell pluripotency maintenance, and differentiation^{11,25,26,27,28,29}. In human cancer RNA-seq profiles and X. laevis embryos, we explored the function of PPP4C, the PP4 catalytic subunit. PPP4C levels correlated with pattern specification and canonical Wnt activation in both fields. Specifically, we propose that Ppp4c promotes canonical Wnt activity at the destruction complex level, as X. laevis Ppp4c interacted with AXIN1 and catalytically inhibited its protein abundance. Furthermore, anterior structure and melanocyte defects in Ppp4c-deficient X. laevis embryos highlight its developmental significance.

Perturbation of certain genes which are active in embryos and silent in adult tissue can tip the balance between proliferation and differentiation, inducing congenital diseases or cancer. Scattered evidence of PPP4C elevation has been documented in breast, lung, ovarian, colorectal, and pancreatic ductal tumors^{10,11,12,13,14}. In lung cancer and colorectal carcinoma, PPP4C promoted tumorigenesis by facilitating tumor cell survival, proliferation, migration, and invasion^10,13. In ovarian cancer cells, PP4 inhibition by fostriecin or KD enhanced immune response and reduced homologous recombination in tumor cells, leading to tumor repression¹⁴. In breast tumor cells, PPP4C can act as either an anti-tumor factor promoting cell apoptosis through PEA15 regulation or an oncogenic factor facilitating proliferation and migration in a cell-content dependent manner^9,30. However, to our knowledge, no research has addressed its diagnostic or prognostic potential in pan-cancer. Consistent with previous reports, PPP4C mRNA levels were significantly upregulated in most cancers (27/31) and decreased in LAML. Moreover, the unbalanced transcriptional levels of PPP4C between normal and tumor tissues contributed to its high diagnostic accuracy in 14 tumor types. In tumorigenesis, PPP4C probably behaves as an oncogenic or protective factor cell-content-dependently, as its transcription levels corresponded to different OS outcomes in various cancer types. PPP4C-related DEGs in specific tumors were also consistent with the existing roles of PPP4C, as the DEGs closely correlated with immune response and DNA replication-related processes.

Additionally, PPP4C levels in multiple cancers were closely associated with various BPs, including morphogenesis, regionalization, and pattern specification. The DV patterning role of Ppp4c has been reported in zebrafish model. Depletion of Ppp4ca or Ppp4cb resulted in embryo dorsalization, as indicated by the expansion of organizer-specific marker chordin, and reduction of ventral marker gata2 at the shield stage⁸. However, injection of ppp4c MO into the future dorsal marginal zone of 4-cell stage X. laevis embryos, rarely affected the dorsal organizer (gsc) domain at the gastrula stage (data not shown). As the ppp4c domain was not spatially overlapped with the organizer, we postulated that zygotic Ppp4c is not essential for organizer formation. However, due to MO limitations, we cannot preclude that maternally inherited Ppp4c protein participates in organizer shaping processes. In X. laevis model, Ppp4c correlated with AP pattern specification, as overexpression of Ppp4c in DA dose-dependently reduced anterior structures at the tailbud stage. Reduction of forebrain (bf1) and eye (pax6) markers in neurula embryos, without impact on hindbrain marker (krox20), further corroborated its anterior-inhibition effect. The discrepancies between zebrafish and X. laevis models may be due to: (1) other phosphatases functional redundancy; (2) protein modifications affecting PP4 complex assembly; and (3) zebrafish and higher vertebrate PPP4C structural differences at N\('\) terminal 9 AA, which may lead to binding regulatory subunits and further substrates differences. The developmental role of Ppp4c in anterior structures and melanocytes was supported by the KD studies, and the ppp4c positive domain overlapped with neural crest tissue, which has potential to develop into future sensory organs, melanocytes, ganglia, and facial skeleton structures. Interestingly, DEGs in GBM, UVM, and LAML were significantly enriched in retina, melanosome, or pigment-related BPs (Supplementary Dataset File 4), further supporting the similarities between embryogenesis and tumorigenesis. We also observed that PPP4C-related DEGs in specific tumors were involved in epidermis, skin, keratinocyte, and muscle-related BPs. As PPP4C was transcribed unevenly between skin and muscle in both human and X. laevis tissue RNA-seq profiles, exploring the potential of PPP4C in epidermis/muscle cell fate determination in embryonic and adult tissue will benefit future studies. These cellular response similarities imply that the tumors mentioned above may share similar cell content with embryos concerning each biological process, and clues from cancer studies may provide alternative views for embryonic research. Therefore, X. laevis embryos may serve as an alternative model for cancer research and vice versa.

PPP4C is reported to regulate Wnt, BMP, and Hedgehog signaling pathways^8,22,31. We specifically explored the relationship between PPP4C and Wnt signaling due to its fundamental function in AP patterning and neural crest formation. The positive genetic interaction between PP4 and Wnt signaling was initially described in Drosophila through an RNAi screen³². In further studies, PP4 enhanced Notch-driven wnt ligand wg transcription or initiated the nuclear translocation of the receptor-like tyrosine kinase (Ryk) intracellular domain to promote Wnt signaling^22,23. A Wnt inhibition role was reported in embryonic stem cells rather than cancer cells. The PP4 complex promoted histone deacetylation, inhibiting Wnt target gene brachyury transcription²⁵. In our study, PPP4C levels correlated with canonical Wnt activation in multiple cancers, and X. laevis Ppp4c was required for canonical Wnt responses in embryos. Overexpression of Ppp4c in the DA region reduced the most anterior structures, phenocopying weak Wnt activation in embryogenesis. Unlike neural crest-derived melanocytes necessitating persistent Wnt for specification, RPE pigment cells originate from the forebrain and require dynamic Wnt regulation. Although we did not observe apparent DV defects by ppp4c MO as seen in zebrafish, the BMP regulatory activity may also be required for Ppp4c-mediated anterior structure formation in X. laevis embryos. Therefore, the functional time-point, region, and the cooperation between its Wnt and BMP regulation in Ppp4c-mediated RPE and melanocyte development, are yet to be determined in future studies.

AXIN abundance is several magnitudes lower than other components in the destruction complex, thus, regulation of AXIN abundance and scaffold function is essential for canonical Wnt signaling regulation. AXIN phosphorylation, by GSK3 and CK1, stabilizes it and enhances its binding affinity for \(\upbeta\)-catenin and GSK3 in the destruction complex^33,34,35. In Wnt “ON” status, AXIN is dephosphorylated, leading to the dissociation of \(\upbeta\)-catenin from the destruction complex and subsequent AXIN degradation³⁶. Several PP complexes (PP1, PP2A, and PP2C) have been reported to interact with AXIN. PP1 antagonizes CK1 on the phosphorylation status of AXIN, releasing it from GSK3³⁷. Moreover, PP1 changes AXIN conformation into “inactivation” mode upon LRP6 phosphorylation, diminishing its binding to phospho-LRP6, leaving it unperturbed for continuous signaling³⁸. PP2C decreases AXIN half-life, eliciting a synergistic response with \(\upbeta\)-catenin and Wnt1 in cells³⁶. Another AXIN interactor, PP2A, contributes to Wnt inhibition possibly through dephosphorylation of GSK3\(\upbeta\)³⁹. In this study, Ppp4c was identified as a Wnt-promoting factor in X. laevis. Unlike previously reported Wnt-promoting roles, Ppp4c was proposed to target AXIN1 in \(\upbeta\)-catenin regulation, which was supported by the following evidence: (1) unlike WT-\(\upbeta\)-catenin, stable CA-\(\upbeta\)-catenin-induced Wnt responses were Ppp4c perturbation-resistant; (2) AXIN1, particularly the C-terminal halves, co-precipitated with Ppp4c; (3) \(\upbeta\)-catenin could not be detected in the immunoprecipitates of Ppp4c unless AXIN1 was present; and (4) X. laevis Ppp4c inhibited AXIN1 abundance in a catalytic-dependent manner.

AXIN can be subjected to either PARsylation/ubiquitination by Tankyrase/RNF146 or ubiquitination by Smurf2 for proteasome degradation^40,41,42. In our study, TANKs domain truncated AXIN1 mutant was resistant to Ppp4c perturbation, suggesting that PARsylation by Tankyrase may be involved in Ppp4c-mediated AXIN1 degradation. Interestingly, DYFL motif deletion in Ppp4c enhanced its AXIN1 inhibition in gastrula embryos. The tail motif, which is highly conserved in PP2A family catalytic subunit factors, is subject to methylation and phosphorylation modification^43,44,45. For PP2AC, DYFL methylation or phosphorylation within or upstream was found to differentially affected subunit affinity, thereby tipping the balance between different PP2A complexes⁴³. Likewise, demethylation in the PPP4C DYFL motif diminished its interaction with PP4R1, PP4R2, PP4R3\(\upalpha\), and PP4R3\(\upbeta\)⁴⁵. Considering that the substrate specificity is mainly determined by binding subunits and the DYFL motif modifications affect PP complex assemble, we postulate that DYFL modification during the gastrula stage is unfavorable for AXIN-inhibitory PP4 complex formation. Notably, Ppp4c or Ppp4c-\(\Delta\)DYFL failed to induce axis duplication when injected ventral-vegetally. Based on the existing literature and our data, we postulate that this may be due to three possibilities as below: (1) abundance limitation of the PP4 regulatory subunits or other co-factors. In ventral vegetal overexpression studies, these proteins may be the limiting factors for the formation of Wnt-promoting PP4 machinery. In this case, insufficient co-factors or PP4 complex may compromise the Wnt responses in the ventral vegetal region when excess Ppp4c present. (2) BMP inhibition. In C2C12 cells, PPP4C was found to interact with Smad1 without affecting its linker or C-terminal phosphorylation, and the nuclear translocation of p-Smad1 shuttled PPP4C from the cytosol to the nucleus⁸. Therefore, in the ventral region, the accumulation of p-Smad1 and its nuclear translocation upon BMP activation may favor the nuclear rather than cytosolic PP4 complex formation, reduce the interaction between Ppp4c and AXIN1, and ultimately affect the Wnt regulatory activity of Ppp4c. (3) Canonical Wnt activity requirements. Ppp4c could partially rescue AXIN1 band shift in KD embryos with exogenous Wnt8. However, endogenous Wnt activity is not completely blocked in X. laevis embryos, therefore, whether Wnt activation is required for Ppp4c-mediated AXIN1 inhibition, and if Ppp4c dephosphorylates other Wnt signaling components to degrade AXIN, are yet to be determined.

These findings collectively provide a novel Ppp4c function in regulating canonical Wnt signaling. However, this research had some limitations due to technical issues. In embryo model, as massive amounts of \(\upbeta\)-catenin exist on the plasma membrane, perturbation of cytosolic \(\upbeta\)-catenin, by loss or gain of Ppp4c, was not sensitively detected by western blot. Additionally, in our unpublished data, excess of Wnt8 enhanced the AXIN1 inhibitory activity of Ppp4c in Xenopus embryos. In future investigations, it would be fascinating to investigate the following: (1) whether Ppp4c promotes canonical Wnt signaling through endogenous Axin1 or not; (2) if so, whether this mechanism can be applied to the tumors identified in pan-cancer analysis; (3) the binding regulatory subunit(s) for Wnt activation and the PP4 assembly regulation; (4) if and how Ppp4c dephosphorylation correlates with Wnt/Tankyrase/RNF146-mediated AXIN degradation; and (5) whether Ppp4c regulates other Wnt axis levels and how these regulations interact. A detailed analysis will uncover the precise mechanism of Ppp4c in canonical Wnt signaling and the involvement of other pathways in this regulation.

Materials and methods

Data acquisition

The RNA-seq data of normal human tissues [GTEx Analysis V8 (dbGaP Accession phs000424.v8.p2)] and single cell were downloaded from GTEx (https://www.gtexportal.org/home/datasets) and HPA (https://www.proteinatlas.org/about/download) respectively. The expression feature of PPP4C in pan-cancer and its diagnostic value were explored in a combined cohort study of TCGA and GTEx downloaded from UCSC Xena (https://xenabrowser.net/datapages/). The analysis of prognostic value, DEGs, GO enrichment, and GSEA were performed in the transcriptome data across 33 tumor types downloaded from the TCGA (https://portal.gdc.cancer.gov) together with the relevant clinical profile.

Diagnostic and prognostic value analysis

The diagnostic potential of PPP4C in each cancer type was assessed by the ROC curve in the cohort of TCGA and GTEx data. The accuracy of prediction was presented by the area under the curve (AUC), which was calculated by the package pROC (v 1.18.0). AUC values were classified as high accuracy (> 0.9), certain accuracy (> 0.7 and \(\le\) 0.9), and low accuracy (> 0.5 and \(\le\) 0.7).

KM plots were used to investigate the association between PPP4C level and patients OS in pan-cancer. The cutoff value for PPP4C high and low expression groups was determined using the minimum p value method⁴⁶. To avoid extreme imbalance in group numbers, the cutoff value in each cancer was restricted between the upper and lower quartile of PPP4C values. The survminer package (v 0.4.9) was used for visualization, and the survival package (v 3.4-0) was used for statistical analysis. p < 0.05 was considered as statistically significant.

GO enrichment analysis and GSEA of the DEGs

DEGs were analyzed using the DESeq2 package (v 1.36.0) based on the gene counts data between PPP4C high and low groups (divided by the median). The minimal values of |log2FoldChange| and numbers of DEGs (p.adjust < 0.05) in each tumor type selected for GO enrichment analysis are listed in Table S1. GO enrichment analysis of the DEGs was conducted using the clusterProfiler package (v 4.4.4). In each tumor, the top 25 GO biological process items with a p.adjust value < 0.05 were collected for word cloud analysis using the wordcloud2 (v 0.2.1) and jiebaR (0.11) packages.

GSEA was performed on DEGs to determine the correlation between canonical Wnt activation and PPP4C level. The clusterProfiler package was used for statistical analysis, and the enrichplot package (v 1.16.1) was used for visualization. The C2 curated gene sets (c2.all.v7.5.symbols.gmt) in the MSigDB Collection were used as the reference gene collection. p.adjust value < 0.05, false discovery rate (qvalue) < 0.25, and normalized enrichment score (NES) > 1 were considered significant enrichment.

Embryos manipulation and microinjection

X. laevis eggs were obtained, fertilized, and staged as previously described^47,48. Embryos were cultured in 0.33\(\times\)Modified Ringer (MR) until the mid-blastula transition, after which transferred to 0.1\(\times\)MR until harvest. Embryo injection was performed using HARVARD Injector in 0.33\(\times\)MR containing 4% Ficoll-PM400 (GE Healthcare) at indicated stages. Injected embryos were cultured in 4% Ficoll-400 in 0.33\(\times\)MR before the mid-blastula transition. For the axis duplication assay, mRNA or morpholino was injected into the ventral vegetal region at the 4-cell stage, and the embryos were cultured until the tailbud stage (Fig. 3c)⁴⁹. In the animal cap explant induction experiment, animal caps were dissected at stage 8 and cultured until the sibling embryos reached stage 10.5 (Fig. 3d)⁴⁹.

Plasmid constructs, mRNA synthesis, and MO

The open reading frame of ppp4c was amplified by PCR from stage 9 X. laevis cDNA using Q5\(^{\circledR }\) Hot Start High-Fidelity DNA Polymerase (NEB), cloned into pGEM\(^{\circledR }\)-T Easy Vector (Promega), and subcloned into the BamHI and XbaI sites of pCS2+, pCS2+HA, and pCS2+6Myc vectors. Full length of mouse Axin1 and human AXIN1 constructs were kindly provided by Dr. Frank Costantini. The \(\upbeta\)-catenin CTNNB1 constructs were obtained as described previously⁵⁰. Point and deletion mutagenesis were used to generate catalytic-dead (D82A, H115Q) and deletion (\(\Delta\)DYFL) mutants of ppp4c, and the truncated forms (1–433, 434–826, 65–826, 65–545, and 396–826) of mouse Axin1. Capped mRNA was synthesized from the linearized constructs using the SP6 mMessage mMachine kit (Ambion) following the manufacturer’s instructions. Antisense MO for X. laevis ppp4c and control MO were obtained from Gene Tools, LLC, with the sequences 5\('\)-CATGGTCCCTCCTCAATGAGCCTGT-3\('\) and 5\('\)-CCTCTTACCTCAGTTACAATTTATA-3\('\), respectively.

RT-PCR

The target gene abundance was analyzed semi-quantitatively by RT-PCR. 5 or 10 whole embryos or 30 explants were harvested as described and snapped in liquid nitrogen. Total RNA was extracted from the samples using TRIZOL reagent (Invitrogen) according to the manufacturer’s protocol. Purified total RNA (5 \(\upmu\)g) was reverse transcribed into cDNA using the M-MLV reverse transcriptase (Promega). odc1 was used as an internal control for normalization. Amplification of each target gene was performed in a 25 \(\upmu\)l system using Taq polymerase (Solgent). Primers used in RT-PCR are listed in Table S2.

WMISH and lacZ \(\upbeta\)-galactosidase Staining

WMISH was performed as previously described and stained with NBT/BCIP (Sigma)⁵¹. The antisense in situ probe labeled with digoxigenin (DIG) against ppp4c was transcribed using T7 RNA polymerase with the template generated by linearization of ppp4c-T-easy construct with SpeI. The probes against bf1, pax6, and krox20 were obtained as previously described^52,53. Indication of injection site was performed by lacZ \(\upbeta\)-galactosidase staining. 250 pg of lacZ mRNA was coinjected into the embryos. \(\upbeta\)-galactosidase-expressing embryos at the indicated stage were fixed in MEMFA solution (10% formaldehyde, 0.1 M MOPS (pH 7.4), 2 mM EGTA, 1 mM \(\hbox {MgSO}_{4}\)) for 30 min at room temperature after being rinsed with PBS (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM \(\hbox {Na}_{2}\hbox {HPO}_{4}\), 1.8 mM \(\hbox {KH}_{2}\hbox {PO}_{4}\)). Then, the embryos were stained for 1–2 h at room temperature in PBS containing 5 mM \(\hbox {K}_{3}\hbox {Fe(CN)}_{6}\), 5 mM \(\hbox {K}_{4}\hbox {Fe(CN)}_{6}\), 1 mg/ml X-gal (red), 2 mM MgCl\({}_{2}\). The stained embryos were fixed in MEMFA solution for 4 h for further WMISH.

Western blot, immunoprecipitation, and antibodies

Western blot assays and co-IP were carried out as previously described^49,54. Briefly, embryos or animal cap explants were homogenized in Triton-X 100 lysis buffer [20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 140 mM NaCl, 10% glycerol, 1 mM EGTA, 1.5 mM \(\hbox {MgCl}_{2}\), 1 mM DTT, 1 mM sodium vanadate (\(\hbox {Na}_{3}\hbox {VO}_{4}\)), 50 mM NaF, 20 \(\upmu\)g/ml aprotinin, 40 \(\upmu\)g/ml leupeptin, and 0.75 mM phenylmethylsulfonyl fluoride (PMSF)] for western blot assay, or IP buffer [20 mM Tris-Cl (pH 7.5), 150 mM NaCl, 2 mM EDTA (pH 7.5), 1% TritonX-100, 1 mM \(\hbox {Na}_{3}\hbox {VO}_{4}\), 100 mM NaF, 25 mM \(\upbeta\)-glycerophosphate, 20 \(\upmu\)g/ml aprotinin, 40 \(\upmu\)g/ml leupeptin, and 0.75 mM PMSF] for co-IP assay. The lysate was centrifuged to remove insoluble debris and yolk. In western blot, an equivalent amount of 1/4 whole embryo protein was loaded for each well of the SDS-PAGE gel, and \(\upbeta\)-Actin was used as the internal control. In co-IP assay, the indicated antibodies were added to the supernatants and incubated at \(4\,^{\circ }\hbox {C}\) for 6 h. Rec-Protein G-Sepharose\(^{\circledR }\) 4B Conjugate (Invitrogen) was added, and the mixture was incubated at \(4^{\circ }\hbox {C}\) overnight. The immune complexes bound to Protein-G beads were washed six times with IP buffer and then subjected to SDS-PAGE gel for western blot. The mobility shift assay was performed on a 6.5% SDS-PAGE gel. The antibodies used in western blot and co-IP were listed in Table S3. Immunoreactivity was visualized by incubation with SuperSignal\(^{\textrm{TM}}\) West Femto Maximum Sensitivity Substrate (Thermo) following the manufacturer’s instructions or with ECL solution unless specified. For the blots in western blot that required equal or more than 3 antibodies hybridization, and co-IP experiments, the membranes were cropped at indicated region before antibody hybridization to reduce bleaching times and non-specific staining interference .

Statistical analyses and image process

Statistical analysis was conducted using R software (v 4.2.1) downloaded from R website (https://www.r-project.org/). The Wilcoxon rank sum test was used to compare the difference of PPP4C between normal and tumor tissues in non-paired and paired samples. For the phenotype and western blot analysis, the comparison between groups was assessed by Student’s t-test. The p value was calculated using the rstatix package (v 0.7.0). The ggplot2 package (v 3.3.6) was used for visualization unless specified. p < 0.05 was considered statistically significant (ns, p \(\ge\) 0.05; *p < 0.05; **p < 0.01; ***p < 0.001). PPP4C AA sequence blast was performed in SnapGene (v 6.1). The figures were processed and combined using Adobe Photoshop (v 23.2.1) and Adobe Illustrator (v 26.1).

Ethical approval

This study was approved by the Institutional Animal Care and Use Committee of Pohang University of Science and Technology. All methods and procedures were carried out in accordance with relevant guidelines and regulations. The study is reported in accordance with ARRIVE guidelines.

Data availability

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

References

Day, E. K., Sosale, N. G. & Lazzara, M. J. Cell signaling regulation by protein phosphorylation: A multivariate, heterogeneous, and context-dependent process. Curr. Opin. Biotechnol. 40, 185–192 (2016).
Article CAS PubMed PubMed Central Google Scholar
Chen, G. I. et al. Pp4r4/kiaa1622 forms a novel stable cytosolic complex with phosphoprotein phosphatase 4. J. Biol. Chem. 283, 29273–29284 (2008).
Article CAS PubMed PubMed Central Google Scholar
Lee, D.-H. et al. A pp4 phosphatase complex dephosphorylates rpa2 to facilitate dna repair via homologous recombination. Nat. Struct. Mol. Biol. 17, 365–372 (2010).
Article CAS PubMed PubMed Central Google Scholar
Hwang, J., Lee, J. A. & Pallas, D. C. Leucine carboxyl methyltransferase 1 (lcmt-1) methylates protein phosphatase 4 (pp4) and protein phosphatase 6 (pp6) and differentially regulates the stable formation of different pp4 holoenzymes. J. Biol. Chem. 291, 21008–21019 (2016).
Article CAS PubMed PubMed Central Google Scholar
Ramos, F., Villoria, M. T., Alonso-Rodríguez, E. & Clemente-Blanco, A. Role of protein phosphatases pp1, pp2a, pp4 and cdc14 in the dna damage response. Cell Stress 3, 70 (2019).
Article CAS PubMed PubMed Central Google Scholar
Park, J. & Lee, D.-H. Functional roles of protein phosphatase 4 in multiple aspects of cellular physiology: A friend and a foe. BMB Rep. 53, 181 (2020).
Article CAS PubMed PubMed Central Google Scholar
Shui, J.-W., Hu, M.C.-T. & Tan, T.-H. Conditional knockout mice reveal an essential role of protein phosphatase 4 in thymocyte development and pre-t-cell receptor signaling. Mol. Cell. Biol. 27, 79–91 (2007).
Article CAS PubMed Google Scholar
Jia, S. et al. Protein phosphatase 4 cooperates with smads to promote bmp signaling in dorsoventral patterning of zebrafish embryos. Dev. Cell 22, 1065–1078 (2012).
Article ADS CAS PubMed Google Scholar
Xie, W. et al. Comprehensive analysis of pppcs family reveals the clinical significance of ppp1ca and ppp4c in breast cancer. Bioengineered 13, 190–205 (2022).
Article CAS PubMed Google Scholar
Wang, B., Zhu, X.-X., Pan, L.-Y., Chen, H.-F. & Shen, X.-Y. Pp4c facilitates lung cancer proliferation and inhibits apoptosis via activating mapk/erk pathway. Pathol. Res. Pract. 216, 152910 (2020).
Article CAS PubMed Google Scholar
Wang, B. et al. Protein phosphatase pp4 is overexpressed in human breast and lung tumors. Cell Res. 18, 974–977 (2008).
Article PubMed Google Scholar
Weng, S. et al. Overexpression of protein phosphatase 4 correlates with poor prognosis in patients with stage ii pancreatic ductal adenocarcinomapp4 in pancreatic ductal adenocarcinoma. Cancer Epidemiol. Biomark. Prev. 21, 1336–1343 (2012).
Article CAS Google Scholar
Li, X. et al. High expression of protein phosphatase 4 is associated with the aggressive malignant behavior of colorectal carcinoma. Mol. Cancer 14, 1–13 (2015).
Article Google Scholar
Raja, R. et al. Pp4 inhibition sensitizes ovarian cancer to nk cell-mediated cytotoxicity via stat1 activation and inflammatory signaling. J. Immunother. Cancer 10, e005026 (2022).
Article PubMed PubMed Central Google Scholar
Hang, J. et al. The role of phosphoprotein phosphatases catalytic subunit genes in pancreatic cancer. Biosci. Rep. 41, 25 (2021).
Article Google Scholar
Clevers, H. & Nusse, R. Wnt/\(\beta\)-catenin signaling and disease. Cell 149, 1192–1205 (2012).
Article CAS PubMed Google Scholar
Clevers, H. Wnt/\(\beta\)-catenin signaling in development and disease. Cell 127, 469–480 (2006).
Article CAS PubMed Google Scholar
Mulligan, K. A. & Cheyette, B. N. Wnt signaling in vertebrate neural development and function. J. Neuroimmune Pharmacol. 7, 774–787 (2012).
Article PubMed PubMed Central Google Scholar
Steinhart, Z. & Angers, S. Wnt signaling in development and tissue homeostasis. Development 145, dev146589 (2018).
Article PubMed Google Scholar
Yang, K. et al. The evolving roles of canonical wnt signaling in stem cells and tumorigenesis: Implications in targeted cancer therapies. Lab. Invest. 96, 116–136 (2016).
Article CAS PubMed Google Scholar
McMahon, A. P. & Moon, R. T. Ectopic expression of the proto-oncogene int-1 in xenopus embryos leads to duplication of the embryonic axis. Cell 58, 1075–1084 (1989).
Article CAS PubMed Google Scholar
Hall, E. T., Pradhan-Sundd, T., Samnani, F. & Verheyen, E. M. The protein phosphatase 4 complex promotes the notch pathway and wingless transcription. Biol. Open 6, 1165–1173 (2017).
CAS PubMed PubMed Central Google Scholar
Chang, W.-H. et al. Smek1/2 is a nuclear chaperone and cofactor for cleaved wnt receptor ryk, regulating cortical neurogenesis. Proc. Natl. Acad. Sci. 114, E10717–E10725 (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Björklund, P., Lindberg, D., Åkerström, G. & Westin, G. Stabilizing mutation of ctnnb1/beta-catenin and protein accumulation analyzed in a large series of parathyroid tumors of swedish patients. Mol. Cancer 7, 1–8 (2008).
Article Google Scholar
Lyu, J., Jho, E.-H. & Lu, W. Smek promotes histone deacetylation to suppress transcription of wnt target gene brachyury in pluripotent embryonic stem cells. Cell Res. 21, 911–921 (2011).
Article CAS PubMed PubMed Central Google Scholar
Lyu, J. et al. Protein phosphatase 4 and smek complex negatively regulate par3 and promote neuronal differentiation of neural stem/progenitor cells. Cell Rep. 5, 593–600 (2013).
Article CAS PubMed Google Scholar
Brechmann, M. et al. A pp4 holoenzyme balances physiological and oncogenic nuclear factor-kappa b signaling in t lymphocytes. Immunity 37, 697–708 (2012).
Article CAS PubMed Google Scholar
Lipinszki, Z. et al. Centromeric binding and activity of protein phosphatase 4. Nat. Commun. 6, 1–13 (2015).
Article Google Scholar
Villoria, M. T. et al. Pp4 phosphatase cooperates in recombinational dna repair by enhancing double-strand break end resection. Nucleic Acids Res. 47, 10706–10727 (2019).
Article CAS PubMed PubMed Central Google Scholar
Mohammed, H. N., Pickard, M. R. & Mourtada-Maarabouni, M. The protein phosphatase 4-pea15 axis regulates the survival of breast cancer cells. Cell. Signal. 28, 1389–1400 (2016).
Article CAS PubMed Google Scholar
Liao, H. et al. Protein phosphatase 4 promotes hedgehog signaling through dephosphorylation of suppressor of fused. Cell Death Dis. 11, 1–14 (2020).
Article Google Scholar
Swarup, S., Pradhan-Sundd, T. & Verheyen, E. M. Genome-wide identification of phospho-regulators of wnt signaling in drosophila. Development 142, 1502–1515 (2015).
Article CAS PubMed Google Scholar
Yamamoto, H. et al. Phosphorylation of axin, a wnt signal negative regulator, by glycogen synthase kinase-3\(\beta\) regulates its stability. J. Biol. Chem. 274, 10681–10684 (1999).
Article CAS PubMed Google Scholar
Jho, E.-H., Lomvardas, S. & Costantini, F. A gsk3\(\beta\) phosphorylation site in axin modulates interaction with \(\beta\)-catenin and tcf-mediated gene expression. Biochem. Biophys. Res. Commun. 266, 28–35 (1999).
Article CAS PubMed Google Scholar
Tacchelly-Benites, O., Wang, Z., Yang, E., Lee, E. & Ahmed, Y. Toggling a conformational switch in wnt/\(\beta\)-catenin signaling: Regulation of axin phosphorylation: The phosphorylation state of axin controls its scaffold function in two wnt pathway protein complexes. BioEssays 35, 1063–1070 (2013).
Article CAS PubMed Google Scholar
Strovel, E. T., Wu, D. & Sussman, D. J. Protein phosphatase 2c\(\alpha\) dephosphorylates axin and activates lef-1-dependent transcription. J. Biol. Chem. 275, 2399–2403 (2000).
Article CAS PubMed Google Scholar
Luo, W. et al. Protein phosphatase 1 regulates assembly and function of the \(\beta\)-catenin degradation complex. EMBO J. 26, 1511–1521 (2007).
Article CAS PubMed PubMed Central Google Scholar
Kim, S.-E. et al. Wnt stabilization of \(\beta\)-catenin reveals principles for morphogen receptor-scaffold assemblies. Science 340, 867–870 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Chu, D. et al. Gsk-3\(\beta\) is dephosphorylated by pp2a in a leu309 methylation-independent manner. J. Alzheimers Dis. 49, 365–375 (2016).
Article CAS PubMed Google Scholar
Huang, S.-M.A. et al. Tankyrase inhibition stabilizes axin and antagonizes wnt signalling. Nature 461, 614–620 (2009).
Article ADS CAS PubMed Google Scholar
Zhang, Y. et al. Rnf146 is a poly (adp-ribose)-directed e3 ligase that regulates axin degradation and wnt signalling. Nat. Cell Biol. 13, 623–629 (2011).
Article CAS PubMed Google Scholar
Kim, S. & Jho, E.-H. The protein stability of axin, a negative regulator of wnt signaling, is regulated by smad ubiquitination regulatory factor 2 (smurf2). J. Biol. Chem. 285, 36420–36426 (2010).
Article CAS PubMed PubMed Central Google Scholar
Longin, S. et al. Selection of protein phosphatase 2a regulatory subunits is mediated by the c terminus of the catalytic subunit. J. Biol. Chem. 282, 26971–26980 (2007).
Article CAS PubMed Google Scholar
Martin, M., Kettmann, R. & Dequiedt, F. Recent insights into protein phosphatase 2a structure and regulation: The reason why pp2a is no longer considered as a lazy passive housekeeping enzyme. Biotechnol. Agron. Soc. Environ. 14, 243–252 (2010).
Google Scholar
Lee, J. & Lee, D.-H. Leucine methylation of protein phosphatase pp4c at c-terminal is critical for its cellular functions. Biochem. Biophys. Res. Commun. 452, 42–47 (2014).
Article CAS PubMed Google Scholar
Mizuno, H., Kitada, K., Nakai, K. & Sarai, A. Prognoscan: A new database for meta-analysis of the prognostic value of genes. BMC Med. Genom. 2, 1–11 (2009).
Article Google Scholar
Newport, J. & Kirschner, M. A major developmental transition in early xenopus embryos: I. Characterization and timing of cellular changes at the midblastula stage. Cell 30, 675–686 (1982).
Article CAS PubMed Google Scholar
Nieuwkoop, P. D. & Faber, J. Normal table of. Xenopus laevis 252 (1994).
Choi, S.-C. & Han, J.-K. Rap2 is required for wnt/\(\beta\)-catenin signaling pathway in xenopus early development. EMBO J. 24, 985–996 (2005).
Article CAS PubMed PubMed Central Google Scholar
Han, W., Koo, Y., Chaieb, L., Keum, B.-R. & Han, J.-K. Uchl5 controls \(\beta\)-catenin destruction complex function through axin1 regulation. Sci. Rep. 12, 1–13 (2022).
Google Scholar
Harland, R. M. In situ hybridization: An improved whole-mount method for xenopus embryos. Methods Cell Biol. 36, 685–695 (1991).
Article CAS PubMed Google Scholar
Kim, H. et al. Xenopus wntless and the retromer complex cooperate to regulate xwnt4 secretion. Mol. Cell. Biol. 29, 2118–2128 (2009).
Article CAS PubMed PubMed Central Google Scholar
Lee, H. et al. Head formation requires dishevelled degradation that is mediated by march2 in concert with dapper1. Development 145, dev143107 (2018).
Article PubMed Google Scholar
Cheong, S.-M., Kim, H. & Han, J.-K. Identification of a novel negative regulator of activin/nodal signaling in mesendodermal formation of xenopus embryos. J. Biol. Chem. 284, 17052–17060 (2009).
Article CAS PubMed PubMed Central Google Scholar

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Acknowledgements

We sincerely thank the frogs (Korean Xenopus Resource Center for Research) kindly provided by Prof. Jaebong Kim (Hallym University), and the AXIN1 plasmids provided by Prof. Frank Costantini (Columbia University). We thank all the lab members for their daily support. We also appreciate Dr. Xiaojuan Jiang (Shanghai Jiao Tong University) for her assistance in LaTex formatting.

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Laboratory of Developmental Biology, Department of Life Sciences, Pohang University of Science and Technology, Pohang, 37673, Korea
YiLi Wang, WonHee Han, SeokMin Yun & JinKwan Han
Department of Neurology, F. M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston, MA, 02115, USA
WonHee Han

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Contributions

Y.L.W. designed, performed most of the experiments and data analysis; Y.L.W. and S.M.Y. performed gene cloning and co-IP experiment. Y.L.W. and W.H.H. carried out the WMISH analysis. J.K.H. conceived and supervised the project. Y.L.W. and J.K.H. wrote the manuscript. All authors reviewed and approved the manuscript.

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Correspondence to JinKwan Han.

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This work was supported by the Grant from the National Research Foundation of Korea (NRF) (2021R1F1A1055713) and the BK21 PLUS Research Fellowship from the Ministry of Education, Science and Technology, Republic of Korea. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Wang, Y., Han, W., Yun, S. et al. Identification of protein phosphatase 4 catalytic subunit as a Wnt promoting factor in pan-cancer and Xenopus early embryogenesis. Sci Rep 13, 10240 (2023). https://doi.org/10.1038/s41598-023-35719-y

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Received: 11 January 2023
Accepted: 23 May 2023
Published: 23 June 2023
DOI: https://doi.org/10.1038/s41598-023-35719-y

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