BMAL1 dephosphorylation determines the pace of the circadian clock.

In mammals, virtually all body cells harbor cell-autonomous and self-sustained circadian oscillators that rely on delayed negative feedback loops in gene expression. Transcriptional activation and repression play a major role in keeping these clocks ticking, but numerous post-translational mechanisms-and particularly the phosphorylation of core clock components by protein kinases-are also critically involved in setting the pace of these timekeepers. In this issue of Genes & Development, Klemz and colleagues (pp. 1161-1174) now show how dephosphorylation of BMAL1 by protein phosphatase 4 (PPP4) participates in the modulation of circadian timing.

Protein phosphatase 4 controls circadian clock dynamics by modulating CLOCK/BMAL1 activity.

In all organisms with circadian clocks, post-translational modifications of clock proteins control the dynamics of circadian rhythms, with phosphorylation playing a dominant role. All major clock proteins are highly phosphorylated, and many kinases have been described to be responsible. In contrast, it is largely unclear whether and to what extent their counterparts, the phosphatases, play an equally crucial role. To investigate this, we performed a systematic RNAi screen in human cells and identified protein phosphatase 4 (PPP4) with its regulatory subunit PPP4R2 as critical components of the circadian system in both mammals and Drosophila Genetic depletion of PPP4 shortens the circadian period, whereas overexpression lengthens it. PPP4 inhibits CLOCK/BMAL1 transactivation activity by binding to BMAL1 and counteracting its phosphorylation. This leads to increased CLOCK/BMAL1 DNA occupancy and decreased transcriptional activity, which counteracts the "kamikaze" properties of CLOCK/BMAL1. Through this mechanism, PPP4 contributes to the critical delay of negative feedback by retarding PER/CRY/CK1δ-mediated inhibition of CLOCK/BMAL1.

Protein phosphatases in the RNAPII transcription cycle: erasers, sculptors, gatekeepers, and potential drug targets.

The transcription cycle of RNA polymerase II (RNAPII) is governed at multiple points by opposing actions of cyclin-dependent kinases (CDKs) and protein phosphatases, in a process with similarities to the cell division cycle. While important roles of the kinases have been established, phosphatases have emerged more slowly as key players in transcription, and large gaps remain in understanding of their precise functions and targets. Much of the earlier work focused on the roles and regulation of sui generis and often atypical phosphatases-FCP1, Rtr1/RPAP2, and SSU72-with seemingly dedicated functions in RNAPII transcription. Decisive roles in the transcription cycle have now been uncovered for members of the major phosphoprotein phosphatase (PPP) family, including PP1, PP2A, and PP4-abundant enzymes with pleiotropic roles in cellular signaling pathways. These phosphatases appear to act principally at the transitions between transcription cycle phases, ensuring fine control of elongation and termination. Much is still unknown, however, about the division of labor among the PPP family members, and their possible regulation by or of the transcriptional kinases. CDKs active in transcription have recently drawn attention as potential therapeutic targets in cancer and other diseases, raising the prospect that the phosphatases might also present opportunities for new drug development. Here we review the current knowledge and outstanding questions about phosphatases in the context of the RNAPII transcription cycle.

Protein phosphatase 4 mediates palmitic acid-induced endothelial dysfunction by decreasing eNOS phosphorylation at serine 633 in HUVECs.

Plasma saturated free fatty acid (FFA)-induced endothelial dysfunction (ED) contributes to the pathogenesis of atherosclerosis and cardiovascular diseases. However, the mechanism underlying saturated FFA-induced ED remains unclear. This study demonstrated that palmitic acid (PA) induced ED by activating the NADPH oxidase (NOX)/ROS signaling pathway to activate protein phosphatase 4 (PP4) and protein phosphatase 2A (PP2A), thereby reducing endothelial nitric oxide synthase (eNOS) phosphorylation at Ser633 and Ser1177, respectively. Okadaic acid (OA) and fostriecin (FST), which are inhibitors of PP2A, inhibited the PA-induced decreases in eNOS phosphorylation at Ser633 and Ser1177. The antioxidants N-acetylcysteine (NAC) and apocynin (APO) or knockdown of gp91phox or p67phox (NOX subunits) restored PA-mediated downregulation of PP4R2 protein expression and eNOS Ser633 phosphorylation. Knockdown of the PP4 catalytic subunit (PP4c) specifically increased eNOS Ser633 phosphorylation, while silencing the PP2A catalytic subunit (PP2Ac) restored only eNOS Ser1177 phosphorylation. Furthermore, PA dramatically decreased the protein expression of the PP4 regulatory subunit R2 (PP4R2) but not the other regulatory subunits. PP4R2 overexpression increased eNOS Ser633 phosphorylation, nitric oxide (NO) production, cell migration and tube formation but did not change eNOS Ser1177 phosphorylation levels. Coimmunoprecipitation (Co-IP) suggested that PP4R2 and PP4c interacted with the PP4R3α and eNOS proteins. In summary, PA decreases PP4R2 protein expression through the Nox/ROS pathway to activate PP4, which contributes to ED by dephosphorylating eNOS at Ser633. The results of this study suggest that PP4 is a novel therapeutic target for ED and ED-associated vascular diseases.

Meiotic DNA double-strand break-independent role of protein phosphatase 4 in Hop1 assembly to promote meiotic chromosome axis formation in budding yeast.

Dynamic changes in chromosomal structure that occur during meiotic prophase play an important role in the progression of meiosis. Among them, meiosis-specific chromosomal axis-loop structures are important as a scaffold for integrated control between the meiotic recombination reaction and the associated checkpoint system to ensure accurate chromosome segregation. However, the molecular mechanism of the initial step of chromosome axis-loop construction is not well understood. Here, we showed that, in budding yeast, protein phosphatase 4 (PP4) that primarily counteracts Mec1/Tel1 phosphorylation is required to promote the assembly of a chromosomal axis component Hop1 and Red1 onto meiotic chromatin via interaction with Hop1. PP4, on the other hand, less affects Rec8 assembly. Notably, unlike the previously known function of PP4, this PP4 function in Hop1/Red1 assembly was independent of meiotic DSB-dependent Tel1/Mec1 kinase activities. The defect in Hop1/Red1 assembly in the absence of PP4 function was not suppressed by dysfunction of Pch2, which removes Hop1 protein from the chromosome axis, suggesting that PP4 is required for the initial step of chromatin loading of Hop1 rather than stabilization of Hop1 on axes. These results indicate phosphorylation/dephosphorylation-mediated regulation of Hop1 recruitment onto chromatin during chromosome axis construction before meiotic double-strand break formation.

A second Wpl1 anti-cohesion pathway requires dephosphorylation of fission yeast kleisin Rad21 by PP4.

Cohesin mediates sister chromatid cohesion which is essential for chromosome segregation and repair. Sister chromatid cohesion requires an acetyl-transferase (Eso1 in fission yeast) counteracting Wpl1, promoting cohesin release from DNA We report here that Wpl1 anti-cohesion function includes an additional mechanism. A genetic screen uncovered that Protein Phosphatase 4 (PP4) mutants allowed cell survival in the complete absence of Eso1. PP4 co-immunoprecipitated Wpl1 and cohesin and Wpl1 triggered Rad21 de-phosphorylation in a PP4-dependent manner. Relevant residues were identified and mapped within the central domain of Rad21. Phospho-mimicking alleles dampened Wpl1 anti-cohesion activity, while alanine mutants were neutral indicating that Rad21 phosphorylation would shelter cohesin from Wpl1 unless erased by PP4. Experiments in post-replicative cells lacking Eso1 revealed two cohesin populations. Type 1 was released from DNA by Wpl1 in a PP4-independent manner. Type 2 cohesin, however, remained DNA-bound and lost its cohesiveness in a manner depending on Wpl1- and PP4-mediated Rad21 de-phosphorylation. These results reveal that Wpl1 antagonizes sister chromatid cohesion by a novel pathway regulated by the phosphorylation status of the cohesin kleisin subunit.

Oligoasthenoteratospermia and sperm tail bending in PPP4C-deficient mice.

Protein phosphatase 4 (PPP4) is a protein phosphatase that, although highly expressed in the testis, currently has an unclear physiological role in this tissue. Here, we show that deletion of PPP4 catalytic subunit gene Ppp4c in the mouse causes male-specific infertility. Loss of PPP4C, when assessed by light microscopy, did not obviously affect many aspects of the morphology of spermatogenesis, including acrosome formation, nuclear condensation and elongation, mitochondrial sheaths arrangement and '9 + 2' flagellar structure assembly. However, the PPP4C mutant had sperm tail bending defects (head-bent-back), low sperm count, poor sperm motility and had cytoplasmic remnants attached to the middle piece of the tail. The cytoplasmic remnants were further investigated by transmission electron microscopy to reveal that a defect in cytoplasm removal appeared to play a significant role in the observed spermiogenesis failure and resulting male infertility. A lack of PPP4 during spermatogenesis causes defects that are reminiscent of oligoasthenoteratospermia (OAT), which is a common cause of male infertility in humans. Like the lack of functional PPP4 in the mouse model, OAT is characterized by abnormal sperm morphology, low sperm count and poor sperm motility. Although the causes of OAT are probably heterogeneous, including mutation of various genes and environmentally induced defects, the detailed molecular mechanism(s) has remained unclear. Our discovery that the PPP4C-deficient mouse model shares features with human OAT might offer a useful model for further studies of this currently poorly understood disorder.

Genetic interaction between Ptc2 and protein phosphatase 4 (PP4) in the regulation of DNA damage response and virulence in Candida albicans.

In the pathogenic fungus Candida albicans, phosphoregulation of the checkpoint kinase Rad53 plays a crucial role in the filamentous growth response to genotoxic stresses. The protein phosphatase 4 (PP4) complex, containing Pph3 and either Psy2 or Psy4, is proved to play a critical role in Rad53 dephosphorylation. In previous studies, we characterized CaPtc2 (the ortholog of both Ptc2 and Ptc3 in Saccharomyces cerevisiae) as a potential DNA-damage-related protein phosphatase. In this study, we checked the genetic interaction of PTC2 with the PP4 complex in the DNA damage response pathway. The results suggest that Ptc2 shows a negative genetic interaction with Pph3, but positive genetic interaction with either Psy2 or Psy4 in response to genotoxic stress. Deletion of PTC2 alone resulted in no significant change in cell virulence, but double deletion of PTC2 PPH3 significantly decreased virulence, while double deletions of either PTC2 PSY2 or PTC2 PSY4 caused virulence levels similar to that shown by PSY2 or PSY4 single-gene deletion cells. Taken together, we propose that Ptc2 in C. albicans plays a compensatory role for Pph3 but is dependent on Psy2 and Psy4 in regulation of DNA damage and cell virulence.

Leucine methylation of protein phosphatase PP4C at C-terminal is critical for its cellular functions.

BACKGROUND: Protein phosphatase 4 (PP4) has been known to have critical functions in DNA double strand break (DSB) repair and cell cycle by the regulation of phosphorylation of its target proteins, such as H2AX, RPA2, KAP-1, 53BP1. However, it is largely unknown how PP4 itself is regulated. METHODS: We examined the PP4C methylation on L307 at C-terminal by using methylated-leucine specific antibody. Then with PP4C L307A mutant, we explored that how nonmethylated form of PP4C affects its known cellular functions by immunoprecipitation, immunofluorescence, and DNA DSB repair assays. RESULTS: Here we show that PP4C is methylated on its C-terminal leucine residue in vivo and this methylation is important for cellular functions mediated by PP4. In the cells PP4C L307A mutant has significantly low activity of dephosphorylation against its known target proteins, and the loss of interaction between L307A PP4 mutant and regulatory subunits, R1, R2, or R3α/ß causes the dissociation from its target proteins. Moreover, PP4C L307A mutant loses its role in both DSB repair pathways, HR (homologous recombination) and NHEJ (non-homologous end joining), which phenocopies PP4C depletion. CONCLUSION: Our results demonstrate the key site of PP4C methylation and establish the physiological importance of this regulation.

Kin17 regulates proper cortical localization of Miranda in Drosophila neuroblasts by regulating Flfl expression.

During asymmetric division of Drosophila larval neuroblasts, the fate determinant Prospero (Pros) and its adaptor Miranda (Mira) are segregated to the basal cortex through atypical protein kinase C (aPKC) phosphorylation of Mira and displacement from the apical cortex, but Mira localization after aPKC phosphorylation is not well understood. We identify Kin17, a DNA replication and repair protein, as a regulator of Mira localization during asymmetric cell division. Loss of Kin17 leads to aberrant localization of Mira and Pros to the centrosome, cytoplasm, and nucleus. We provide evidence to show that the mislocalization of Mira and Pros is likely due to reduced expression of Falafel (Flfl), a component of protein phosphatase 4 (PP4), and defects in dephosphorylation of serine-96 of Mira. Our work reveals that Mira is likely dephosphorylated by PP4 at the centrosome to ensure proper basal localization of Mira after aPKC phosphorylation and that Kin17 regulates PP4 activity by regulating Flfl expression.

The diagnostic, prognostic, drug sensitivity and ceRNA internet of PPP4R1 in liver hepatocellular carcinoma (LIHC).

Background: Recent studies have reported a role of protein phosphatase 4 regulatory subunit 1 (PPP4R1) in cancer development. However, its expression, diagnostic significance, prognostic value and biological function in liver hepatocellular carcinoma (LIHC) are not known. Methods: The expression level of PPP4R1 in pan-cancer was evaluated by analyzing publicly accessible data from the University of California Santa Cruz (UCSC) Xena database. The diagnostic value of PPP4R1 for tumors was assessed using receiver operating characteristic (ROC) curves, whereas the impact of PPP4R1 on tumor prognosis was determined using Kaplan-Meier survival curves, and a prognostic model for LIHC was established using cox regression analysis. In addition, analysis of the correlation between PPP4R1 and anti-cancer drugs using Spearman's correlation coefficient was carried out. Four databases, miRWalk (mRNA-miRNA interactions), MicroT-CDS (mRNA-miRNA interactions), LncBase (miRNA-lncRNA interactions) and Encyclopedia of RNA Interactomes (ENCORI), were used to predict the competitive endogenous RNA (ceRNA) regulatory network of PPP4R1. Finally, the expression of PPP4R1 protein levels was verified using experiments. Results: The findings indicated that the PPP4R1 expression level in cancerous tissues was notably greater than in adjacent tissues (P<0.05). PPP4R1 showed diagnostic significance for 14 tumors based on the ROC curves results area under the curve >0.7. Furthermore, the Kaplan-Meier survival plots demonstrated that PPP4R1 exhibited prognostic significance for all five tumors (P<0.05). According to the cox regression analysis, LIHC patients' prognosis was independently influenced by pathological stage, M stage, and PPP4R1 (P<0.05). The drug sensitivity analysis revealed a positive correlation between the expression level of PPP4R1 and the half maximal inhibitory concentration (IC50) of fludarabine. Additionally, the ceRNA network prediction indicated that the FGD5 antisense RNA 1 (FGD5-AS1)-hsa-miR-22-3p-PPP4R1 ceRNA network could potentially contribute to the progression of LIHC. The experimental results showed that the expression level of PPP4R1 protein was higher in cancer tissues than in paracancerous tissues. Conclusions: PPP4R1 has diagnostic value in most cancers, and high expression of PPP4R1 is associated with poor prognosis, drug resistance and natural killer cell-mediated toxicity, particularly in LIHC. Therefore, PPP4R1 may be a prognostic biomarker and a potential target for immunotherapy in LIHC.

Expression of protein phosphatase 4 in different tissues under hypoxia.

Relevant research data shows that there is a certain degree of energy metabolism imbalance in highland residents. Protein phosphatase 4 (PP4) has been found as a new factor in the regulation of sugar and lipid metabolism. Here, we investigate the differential expression of PP4 at a simulated altitude of 4,500 m in the heart, lung, and brain tissues of rats. A hypoxic plateau rat model was established using an animal decompression chamber. A blood routine test was performed by an animal blood cell analyzer on rats cultured for different hypoxia periods at 4,500 m above sea level. Quantitative polymerase chain reaction (qPCR) and western blot were used to detect the changes of protein phosphatase 4 catalytic subunit (PP4C) gene and protein in heart, lung, and brain tissues. The PP4C gene with the highest expression level found in rats slowly entering the high altitude area (20 m-2200 m-7 d-4500 m-3 d) was about twice as high as the low elevation group (20 m above sea level). The simulated high-altitude hypoxia induced an increase of PP4C expression level in all tissues, and the expression in the lung tissue was twice as expressed as heart and brain tissue at high altitude (P < 0.05). These results suggest that the PP4 phosphatase complex is ubiquitously expressed in rat tissues and likely involved in adaptation to or disease associated with high-altitude hypoxia.

Protein Phosphatase 4 Is Required for Centrobin Function in DNA Damage Repair.

Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, which is mainly achieved by homologous recombination (HR). Among the regulators of various cellular functions, Protein phosphatase 4 (PP4) plays a pivotal role in coordinating cellular response to DNA damage. Meanwhile, Centrobin (CNTRB), initially recognized for its association with centrosomal function and microtubule dynamics, has sparked interest due to its potential contribution to DNA repair processes. In this study, we investigate the involvement of PP4 and its interaction with CNTRB in HR-mediated DNA repair in human cells. Employing a range of experimental strategies, we investigate the physical interaction between PP4 and CNTRB and shed light on the importance of two specific motifs in CNTRB, the PP4-binding FRVP and the ATR kinase recognition SQ sequences, in the DNA repair process. Moreover, we examine cells depleted of PP4 or CNTRB and cells harboring FRVP and SQ mutations in CNTRB, which result in similar abnormal chromosome morphologies. This phenomenon likely results from the impaired resolution of Holliday junctions, which serve as crucial intermediates in HR. Taken together, our results provide new insights into the intricate mechanisms of PP4 and CNTRB-regulated HR repair and their interrelation.

Dual-Specificity Protein Phosphatase 4 (DUSP4) Overexpression Improves Learning Behavior Selectively in Female 5xFAD Mice, and Reduces ß-Amyloid Load in Males and Females.

Recent multiscale network analyses of banked brains from subjects who died of late-onset sporadic Alzheimer's disease converged on VGF (non-acronymic) as a key hub or driver. Within this computational VGF network, we identified the dual-specificity protein phosphatase 4 (DUSP4) [also known as mitogen-activated protein kinase (MAPK) phosphatase 2] as an important node. Importantly, DUSP4 gene expression, like that of VGF, is downregulated in postmortem Alzheimer's disease (AD) brains. We investigated the roles that this VGF/DUSP4 network plays in the development of learning behavior impairment and neuropathology in the 5xFAD amyloidopathy mouse model. We found reductions in DUSP4 expression in the hippocampi of male AD subjects, correlating with increased CDR scores, and in 4-month-old female and 12-18-month-old male 5xFAD hippocampi. Adeno-associated virus (AAV5)-mediated overexpression of DUSP4 in 5xFAD mouse dorsal hippocampi (dHc) rescued impaired Barnes maze performance in females but not in males, while amyloid loads were reduced in both females and males. Bulk RNA sequencing of the dHc from 5-month-old mice overexpressing DUSP4, and Ingenuity Pathway and Enrichr analyses of differentially expressed genes (DEGs), revealed that DUSP4 reduced gene expression in female 5xFAD mice in neuroinflammatory, interferon-gamma (IFNγ), programmed cell death protein-ligand 1/programmed cell death protein 1 (PD-L1/PD-1), and extracellular signal-regulated kinase (ERK)/MAPK pathways, via which DUSP4 may modulate AD phenotype with gender-specificity.

Low DUSP4 Expression Is Associated With Aggressive Phenotypes and Poor Prognosis in Gastric Cancer.

BACKGROUND/AIM: Dual-specificity protein phosphatase 4 (DUSP4) negatively regulates MAPK signaling and is involved in various cellular processes. We herein evaluated the relationship between DUSP4 expression and clinicopathological characteristics in a large series of gastric cancer samples. MATERIALS AND METHODS: DUSP4 expression was examined by immunohistochemistry in 508 gastric cancer samples. Cases were classified according to the TCGA molecular classification and HER2 amplification. Kaplan-Meier plots were used to predict the relationship between mRNA expression of DUSP4 and survival. RESULTS: Low expression of DUSP4 was significantly correlated with larger tumor size, higher pT category, positive nodal status, higher stage, lymphovascular invasion, perineural invasion, worse overall survival, and worse recurrence-free survival. No correlation was observed between DUSP4 expression and molecular characteristics. Bioinformatics analysis showed that low mRNA expression was associated with a poor prognosis. CONCLUSION: Low expression of DUSP4 is associated with aggressive phenotypes of gastric cancer and a poor prognosis.

Loss of DUSP4 Expression as a Prognostic Biomarker in Clear Cell Renal Cell Carcinoma.

Dual-specificity protein phosphatase 4 (DUSP4) is a negative regulator of mitogen-activated protein kinases. The prognostic impact of DUSP4 expression in renal cell carcinoma is not well studied. Therefore, we evaluated the clinicopathological implications of DUSP4 expression in clear cell renal cell carcinoma by performing immunohistochemistry (IHC). The clinical outcome according to DUSP4 expression was evaluated through survival analyses, and the association between mRNA expression and prognosis was confirmed by online analysis (Kaplan-Meier plotter). Loss of DUSP4 expression was noted in most histological subtypes of renal cell carcinoma. Loss of DUSP4 expression in clear cell renal cell carcinoma was significantly correlated with old age (p = 0.033), high histologic grade (p < 0.001), tumor necrosis (p < 0.001), and high pT category (p < 0.001). In survival analysis, loss of DUSP4 expression was associated with poor clinical outcomes in cancer-specific survival and recurrence-free survival (p = 0.010 and p = 0.007, respectively). Upon TCGA data analysis, patients with low DUSP4 mRNA expression showed a shorter overall survival (p = 0.023). These results suggest that loss of DUSP4 expression can be used as a potential biomarker for predicting clinical outcomes in clear cell renal cell carcinoma patients.

DUSP4 alleviates LPS-induced chondrocyte injury in knee osteoarthritis via the MAPK signaling pathway.

Knee osteoarthritis (KOA) is characterized by cartilage damage, and the associated pathogenesis is complex. The expression of dual specificity protein phosphatase 4 (DUSP4) is significantly decreased in osteoarthritis (OA); however, the specific role and mechanism underlying DUSP4 in OA are yet to be elucidated. ATDC5 cells were treated with lipopolysaccharide (LPS) to establish the cell injury model. The expression levels of DUSP4 were decreased in OA chondrocytes, demonstrated by reverse transcription-quantitative PCR and western blot analysis. Following overexpression of DUSP4 by cell transfection, Cell Counting Kit-8, ELISA, TUNEL and western blotting assays were used to detect the cell viability, oxidative stress, inflammation and apoptosis levels of LPS-induced ATDC5 cells. Overexpression of DUSP4 inhibited the activation of the MAPK signaling pathway, thereby reducing oxidative stress levels, inflammatory response and apoptosis in the OA cell model. The mechanisms underlying DUSP4 in OA were further explored following the addition of MAPK signaling pathway agonist, phorbol 12-myristate 13-acetate (PMA). The addition of PMA reversed the inhibitory effects of DUSP4 overexpression on oxidative stress, inflammatory response and apoptosis in cells. In summary, DUSP4 alleviated LPS-induced chondrocyte injury in KOA via the MAPK signaling pathway.

Identification of Protein Phosphatase 4 Inhibitory Protein That Plays an Indispensable Role in DNA Damage Response.

Protein phosphatase 4 (PP4) is a crucial protein complex that plays an important role in DNA damage response (DDR), including DNA repair, cell cycle arrest and apoptosis. Despite the significance of PP4, the mechanism by which PP4 is regulated remains to be elucidated. Here, we identified a novel PP4 inhibitor, protein phosphatase 4 inhibitory protein (PP4IP) and elucidated its cellular functions. PP4IP-knockout cells were generated using the CRISPR/Cas9 system, and the phosphorylation status of PP4 substrates (H2AX, KAP1, and RPA2) was analyzed. Then we investigated that how PP4IP affects the cellular functions of PP4 by immunoprecipitation, immunofluorescence, and DNA double-strand break (DSB) repair assays. PP4IP interacts with PP4 complex, which is affected by DNA damage and cell cycle progression and decreases the dephosphorylational activity of PP4. Both overexpression and depletion of PP4IP impairs DSB repairs and sensitizes cells to genotoxic stress, suggesting timely inhibition of PP4 to be indispensable for cells in responding to DNA damage. Our results identify a novel inhibitor of PP4 that inhibits PP4-mediated cellular functions and establish the physiological importance of this regulation. In addition, PP4IP might be developed as potential therapeutic reagents for targeting tumors particularly with high level of PP4C expression.

Protein phosphatase 4 catalytic subunit (PP4C) increases hepatitis B virus X protein levels and promotes its biological functions / 中华微生物学和免疫学杂志

Objective:To investigate the role of protein phosphatase 4 catalytic subunit (PP4C) in regulating hepatitis B virus X protein (HBx) levels and its effects on the biological functions of HBx, thus to provide a potential therapeutic targets for hepatitis B virus (HBV)-related hepatocellular carcinoma.Methods:In vivo and in vitro interactions between HBx and PP4C were analyzed by co-immunoprecipitation (Co-IP) and GST pull-down assay. Recombinant plasmids of PP4C and HBx were co-transfected with Lipofectamine 3000 reagents into hepatoma cells to detect the protein levels of HBx by Western blot. The half-life of HBx in the transfected cells treated with cycloheximide (CHX) were detected. The phosphorylation assay was used to evaluate the effects of PP4C on HBx phosphorylation. CCK8 assay, wound healing assay and Matrigel invasion chamber assay were used to analyze the effects of PP4C on the biological functions of HBx. Results:PP4C interacted with HBx in vivo and in vitro. PP4C overexpression significantly increased the protein level and stability of HBx and the phosphorylation assay confirmed that PP4C overexpression decreased the serine phosphorylation of HBx in hepatoma cells. PP4C overexpression enhanced the migration and invasion of hepatoma cells, but had no significant effects on the proliferation. Conclusions:The interactions between HBx and PP4C promoted the stability of HBx and ultimately enhanced the migration and invasion of hepatoma cells, and the mechanisms might be related to the decrease of HBx serine phosphorylation by PP4C. This study provided a theoretical basis for further investigation of the pathogenic mechanisms of HBx, and targeting PP4C and HBx interaction might provide insights for developing novel treatment for HBV-related hepatocellular carcinoma.

PATZ1 induces PP4R2 to form a negative feedback loop on IKK/NF-κB signaling in lung cancer.

Activation of IKK enhances NF-κB signaling to facilitate cancer cell migration, invasion and metastasis. Here, we uncover the existence of a negative feedback loop of IKK. The transcription factor PATZ1 induces protein phosphatase-4 (PP4) regulatory subunit 2 (PP4R2) in an IKK-dependent manner. PP4R2 enhances the binding of PP4 to phosphorylated IKK to inactivate IKK/NF-κB signaling during sustained stimulation by cellular stimuli such as growth factors and inflammatory mediators. Matched pair studies reveal that primary lung cancers express more PATZ1 and PP4R2 than lymph node metastases in patients. Ectopic PATZ1 decreases invasion/colonization of lung cancers and prolongs the survival of xenograft mice. These effects of PATZ1 are reversed by downregulating PP4R2. Our results suggest that PATZ1 and PP4R2 provide negative feedback on IKK/NF-κB signaling to prevent cancer cells from over-stimulation from cellular stimuli; a decline in PATZ1 and PP4R2 is functionally associated with cancer migration/invasion and agents enhancing PATZ1 and PP4R2 are worth exploring to prevent invasion/metastasis of lung cancers.