Since cancer is the leading cause of death worldwide, there is an urgent need to understand the mechanisms underlying cancer progression and the development of cancer inhibitors. Signal transducer and activator of transcription 3 (STAT3) is a major transcription factor that regulates the proliferation and survival of various cancer cells. Here, dual-specificity phosphatase 3 (DUSP3) was identified as a regulator of STAT3 based on an interaction screening performed using the protein tyrosine phosphatase library. DUSP3 interacted with the C-terminal domain of STAT3 and dephosphorylated p-Y705 of STAT3. In vitro dephosphorylation assay revealed that DUSP3 directly dephosphorylated p-STAT3. The suppressive effects of DUSP3 on STAT3 were evaluated by a decreased STAT3-specific promoter activity, which in turn reduced the expression of the downstream target genes of STAT3. In summary, DUSP3 downregulated the transcriptional activity of STAT3 via dephosphorylation at Y705 and also suppressed the migratory activity of cancer cells. This study demonstrated that DUSP3 inhibits interleukin 6 (IL-6)/STAT3 signaling and is expected to regulate cancer development. Novel functions of DUSP3 discovered in IL-6/STAT3 signaling regulation would help expand the understanding of cancer development mechanisms. [BMB Reports 2020; 53(6): 335-340].

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic hepatic pathology in Western countries. It encompasses a spectrum of conditions ranging from simple steatosis to more severe and progressive non-alcoholic steatohepatitis (NASH) that can lead to hepatocellular carcinoma (HCC). Obesity and related metabolic syndrome are important risk factors for the development of NAFLD, NASH and HCC. DUSP3 is a small dual-specificity protein phosphatase with a poorly known physiological function. We investigated its role in metabolic syndrome manifestations and in HCC using a mouse knockout (KO) model. While aging, DUSP3-KO mice became obese, exhibited insulin resistance, NAFLD and associated liver damage. These phenotypes were exacerbated under high fat diet (HFD). In addition, DEN administration combined to HFD led to rapid HCC development in DUSP3-KO compared to wild type (WT) mice. DUSP3-KO mice had more serum triglycerides, cholesterol, AST and ALT compared to control WT mice under both regular chow diet (CD) and HFD. The level of fasting insulin was higher compared to WT mice, though, fasting glucose as well as glucose tolerance were normal. At the molecular level, HFD led to decreased expression of DUSP3 in WT mice. DUSP3 deletion was associated with increased and consistent phosphorylation of the insulin receptor (IR) and with higher activation of the downstream signaling pathway. In conclusion, our results support a new role for DUSP3 in obesity, insulin resistance, NAFLD and liver damage.

Glial cells missing homolog 1 (GCM1) is a transcription factor essential for placental development. GCM1 promotes syncytiotrophoblast formation and placental vasculogenesis by activating fusogenic and proangiogenic gene expression in placenta. GCM1 activity is regulated by multiple post-translational modifications. The cAMP/PKA-signaling pathway promotes CBP-mediated GCM1 acetylation and stabilizes GCM1, whereas hypoxia-induced GSK-3β-mediated phosphorylation of Ser322 causes GCM1 ubiquitination and degradation. How and whether complex modifications of GCM1 are coordinated is not known. Here we show that the interaction of GCM1 and dual-specificity phosphatase 23 (DUSP23) is enhanced by PKA-dependent phosphorylation of GCM1 on Ser269 and Ser275. The recruitment of DUSP23 reverses GSK-3β-mediated Ser322 phosphorylation, which in turn promotes GCM1 acetylation, stabilization and activation. Supporting a central role in coordinating GCM1 modifications, knockdown of DUSP23 suppressed GCM1 target gene expression and placental cell fusion. Our study identifies DUSP23 as a novel factor that promotes placental cell fusion and reveals a complex regulation of GCM1 activity by coordinated phosphorylation, dephosphorylation and acetylation.

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.

Background Heart pathological hypertrophy has been recognized as a predisposing risk factor for heart failure and arrhythmia. DUSP (dual-specificity phosphatase) 26 is a member of the DUSP family of proteins, which has a significant effect on nonalcoholic fatty liver disease, neuroblastoma, glioma, and so on. However, the involvement of DUSP26 in cardiac hypertrophy remains unclear. Methods and Results Our study showed that DUSP26 expression was significantly increased in mouse hearts in response to pressure overload as well as in angiotensin II-treated cardiomyocytes. Cardiac-specific overexpression of DUSP26 mice showed attenuated cardiac hypertrophy and fibrosis, while deficiency of DUSP26 in mouse hearts resulted in increased cardiac hypertrophy and deteriorated cardiac function. Similar effects were also observed in cellular hypertrophy induced by angiotensin II. Importantly, we showed that DUSP26 bound to transforming growth factor-β activated kinase 1 and inhibited transforming growth factor-β activated kinase 1 phosphorylation, which led to suppression of the mitogen-activated protein kinase signaling pathway. In addition, transforming growth factor-β activated kinase 1-specific inhibitor inhibited cardiomyocyte hypertrophy induced by angiotensin II and attenuated the exaggerated hypertrophic response in DUSP26 conditional knockout mice. Conclusions Taken together, DUSP26 was induced in cardiac hypertrophy and protected against pressure overload induced cardiac hypertrophy by modulating transforming growth factor-β activated kinase 1-p38/ c-Jun N-terminal kinase-signaling axis. Therefore, DUSP26 may provide a therapeutic target for treatment of cardiac hypertrophy and heart failure.

Type-3 cardiorenal syndrome (CRS-3) is acute kidney injury followed by cardiac injury/dysfunction. Mitochondrial injury may impair myocardial function during CRS-3. Since dual-specificity phosphatase 1 (DUSP1) and prohibitin 2 (PHB2) both promote cardiac mitochondrial quality control, we assessed whether these proteins were dysregulated during CRS-3-related cardiac depression. We found that DUSP1 was downregulated in heart tissues from a mouse model of CRS-3. DUSP1 transgenic (DUSP1Tg) mice were protected from CRS-3-induced myocardial damage, as evidenced by their improved heart function and myocardial structure. CRS-3 induced the inflammatory response, oxidative stress and mitochondrial dysfunction in wild-type hearts, but not in DUSP1Tg hearts. DUSP1 overexpression normalized cardiac mitochondrial quality control during CRS-3 by suppressing mitochondrial fission, restoring mitochondrial fusion, re-activating mitophagy and augmenting mitochondrial biogenesis. We found that DUSP1 sustained cardiac mitochondrial quality control by binding directly to PHB2 and maintaining PHB2 phosphorylation, while CRS-3 disrupted this physiological interaction. Transgenic knock-in mice carrying the Phb2S91D variant were less susceptible to cardiac depression upon CRS-3, due to a reduced inflammatory response, suppressed oxidative stress and improved mitochondrial quality control in their heart tissues. Thus, CRS-3-induced myocardial dysfunction can be attributed to reduced DUSP1 expression and disrupted DUSP1/PHB2 binding, leading to defective cardiac mitochondrial quality control.

DUSP4 is a member of the DUSP (Dual-Specificity Phosphatase) subfamily that is selective to the mitogen-activated protein kinases (MAPK) and has been implicated in a range of biological processes and functions in Alzheimer's disease (AD). In this study, we utilized stereotactic delivery of adeno-associated virus (AAV)-DUSP4 to overexpress DUSP4 in the dorsal hippocampus of 5xFAD and wildtype (WT) mice, then used mass spectrometry (MS)-based proteomics along with label-free quantification to profile the proteome and phosphoproteome in the hippocampus. We identified patterns of protein expression and phosphorylation that are modulated in 5xFAD mice and examined the sex-specific impact of DUSP4 overexpression on the 5xFAD proteome/phosphoproteome. In 5xFAD mice, a substantial number of proteins were up- or down-regulated in both male and female mice in comparison to age and sex-matched WT mice, many of which are involved in AD-related biological processes, such as the activated immune response or suppression of synaptic activities. Upon DUSP4 overexpression, significantly regulated proteins were found in pathways that were suppressed, such as the immune response, in male 5xFAD mice. In contrast, such a shift was absent in female mice. For the phosphoproteome, we detected an array of phosphorylation sites that are regulated in 5xFAD compared to WT, and are modulated by DUSP4 overexpression in each sex. Interestingly, the changes in 5xFAD- and DUSP4-associated phosphorylation occurred in opposite directions. Strikingly, both the 5xFAD- and DUSP4-associated phosphorylation changes were found for the most part in neurons, and play key roles in neuronal processes and synaptic function. Site-centric pathway analysis revealed that both the 5xFAD- and DUSP4-associated phosphorylation sites were enriched for a number of kinase sets in female, but only a limited number of sets of kinases in male mice. Taken together, our results suggest that male and female 5xFAD mice respond to DUSP4 overexpression via shared and sex-specific molecular mechanisms, which might underly similar reductions in amyloid pathology in both sexes, while learning deficits were reduced in only females with DUSP4 overexpression. Finally, we validated our findings with the sex-specific AD-associated proteomes in human cohorts and further developed DUSP4-centric proteomic network models and signaling maps for each sex.

CDC25 enzymes are dual-specificity phosphatases involved in the regulation of the cell cycle. No CDC25 enzymes have been described in higher plant organisms. We report here the characterization of an Arabidopsis thaliana CDC25 enzyme, constituted by a sole catalytic domain and devoid of the N-terminal regulatory region found in the human CDC25. We describe the recombinant expression in Escherichia coli of the Arath;CDC25 and its purification for activity assay and structure determination by NMR. The recombinant enzyme has a tyrosine phosphatase activity towards an artificial substrate, a NMR characterization equally concludes to its correct folding. The secondary structure of the protein was predicted on the basis of the assigned chemical shift of (1)H, (15)N, and (13)C backbone atoms of the protein. The presence of a metal ion in the C-terminus of this new protein points to a zinc finger, and sequence homology indicates that this new structural element might be conserved in related plant homologs.

The functions of dual-specificity phosphatase 9 (DUSP9) in hepatic steatosis and metabolic disturbance during nonalcoholic fatty liver disease were discussed in our prior study. However, its roles in the pathophysiology of pressure overload-induced cardiac hypertrophy remain to be illustrated. This study attempted to uncover the potential contributions and underpinning mechanisms of DUSP9 in cardiac hypertrophy. Utilizing the gain-and-loss-of-functional approaches of DUSP9 the cardiac phenotypes arising from the pathological, echocardiographic, and molecular analysis were quantified. The results showed increased levels of DUSP9 in hypertrophic mice heart and angiotensin II treated cardiomyocytes. In accordance with the results of cellular hypertrophy in response to angiotensin II, cardiac hypertrophy exaggeration, fibrosis, and malfunction triggered by pressure overload was evident in the case of cardiac-specific conditional knockout of DUSP9. In contrast, transgenic mice hearts with DUSP9 overexpression portrayed restoration of the hypertrophic phenotypes. Further explorations of molecular mechanisms indicated the direct interaction of DUSP9 with ASK1, which further repressed p38 and JNK signaling pathways. Moreover, blocking ASK1 with ASK1-specific inhibitor compensated the pro-hypertrophic effects induced by DUSP9 deficiency in cardiomyocytes. The main findings of this study suggest the potential of DUSP9 in alleviating cardiac hypertrophy at least partially by repressing ASK1, thereby looks promising as a prospective target against cardiac hypertrophy.

It was reported that dual specificity phosphatase 1 (DUSP1) is specifically upregulated in the liver of patients with chronic hetpatitis C virus (HCV) infection who do not respond to peginterferon (PegIFN) treatment. Here, we have investigated the role of DUSP1 in HCV replication in hepatoma cells stably expressing the full HCV replicon (FK). DUSP1 was silenced in cells harboring the FK replicon using a lentiviral vector encoding a DUSP1-specific short hairpin RNA (LV-shDUSP1). We demonstrated that knock-down of DUSP1 significantly inhibited HCV RNA and protein expression. Also, DUSP1 silencing enhanced the expression of phosphorylated signal transducer and activator of transcription 1 (phosho-STAT1) and facilitated the translocation of STAT1 into the nucleus. The mRNA expression levels of myxovirus resistance protein A (MxA), 2'-5'-oligoadenylate synthetase 1 (OAS1), ISG15 ubiquitin-like modifier (ISG15), chemokine C-X-C motif ligand 10 (CXCL10), and ubiquitin-specific protease 18 (USP18) were also accelerated by silencing of DUSP1. Furthermore, combined with the IFN treatment, DUSP1 silencing synergistically decreased the levels of HCV RNA. These results suggest that suppression of DUSP1 expression enhances phosphorylation and nuclear translocation of STAT1, resulting in increasing expression of interferon-stimulated genes (ISGs), which synergizes with IFN's antiviral effect against HCV. In conclusion, DUSP1 is involved in the antiviral host defense mechanism against a HCV infection and thus DUSP1 might be a target to treat chronic HCV infection.

Integrin-linked kinase (ILK) is an intracellular scaffold protein with critical cell-specific functions in the embryonic and mature mammalian kidney. Previously, we demonstrated a requirement for Ilk during ureteric branching and cell cycle regulation in collecting duct cells in vivo Although in vitro data indicate that ILK controls p38 mitogen-activated protein kinase (p38MAPK) activity, the contribution of ILK-p38MAPK signaling to branching morphogenesis in vivo is not defined. Here, we identified genes that are regulated by Ilk in ureteric cells using a whole-genome expression analysis of whole-kidney mRNA in mice with Ilk deficiency in the ureteric cell lineage. Six genes with expression in ureteric tip cells, including Wnt11, were downregulated, whereas the expression of dual-specificity phosphatase 8 (DUSP8) was upregulated. Phosphorylation of p38MAPK was decreased in kidney tissue with Ilk deficiency, but no significant decrease in the phosphorylation of other intracellular effectors previously shown to control renal morphogenesis was observed. Pharmacologic inhibition of p38MAPK activity in murine inner medullary collecting duct 3 (mIMCD3) cells decreased expression of Wnt11, Krt23, and Slo4c1 DUSP8 overexpression in mIMCD3 cells significantly inhibited p38MAPK activation and the expression of Wnt11 and Slo4c1. Adenovirus-mediated overexpression of DUSP8 in cultured embryonic murine kidneys decreased ureteric branching and p38MAPK activation. Together, these data demonstrate that Ilk controls branching morphogenesis by regulating the expression of DUSP8, which inhibits p38MAPK activity and decreases branching morphogenesis.

Immune responses are critically regulated by the functions of CD4 helper T cells. Based on their secreted cytokines, helper T cells are further categorized into different subsets like Treg or Th17 cells, which suppress or promote inflammatory responses, respectively. Signals from IL-2 activate the transcription factor STAT5 to promote Treg but suppress Th17 cell differentiation. Our previous results found that the deficiency of a dual-specificity phosphatase, DUSP4, induced STAT5 hyper-activation, enhanced IL-2 signaling, and increased T cell proliferation. In this report, we examined the effects of DUSP4 deficiency on helper T cell differentiation and STAT5 regulation. Our in vivo data showed that DUSP4 mice were more resistant to the induction of autoimmune encephalitis, while in vitro differentiations revealed enhanced iTreg and reduced Th17 polarization in DUSP4-deficient T cells. To study the cause of this altered helper T cell polarization, we performed luciferase reporter assays and confirmed that, as predicted by our previous report, DUSP4 over-expression suppressed the transcription factor activity of STAT5. Surprisingly, we also found that DUSP4-deficient T but not B cells exhibited elevated STAT5 protein levels, and over-expressed DUSP4 destabilized STAT5 in vitro; moreover, this destabilization required the phosphatase activity of DUSP4, and was insensitive to MG132 treatment. Finally, domain-mapping results showed that both the substrate-interacting and the phosphatase domains of DUSP4 were required for its optimal interaction with STAT5, while the coiled-coil domain of STAT5 appeared to hinder this interaction. Our data thus provide the first genetic evidence that DUSP4 is important for helper T cell development. In addition, they also help uncover the novel, DUSP4-mediated regulation of STAT5 protein stability.

Lafora Disease (LD) is a fatal neurodegenerative epileptic disorder that presents as a neurological deterioration with the accumulation of insoluble, intracellular, hyperphosphorylated carbohydrates called Lafora bodies (LBs). LD is caused by mutations in either the gene encoding laforin or malin. Laforin contains a dual specificity phosphatase domain and a carbohydrate-binding module, and is a member of the recently described family of glucan phosphatases. In the current study, we investigated the functional and physiological relevance of laforin dimerization. We purified recombinant human laforin and subjected the monomer and dimer fractions to denaturing gel electrophoresis, mass spectrometry, phosphatase assays, protein-protein interaction assays, and glucan binding assays. Our results demonstrate that laforin prevalently exists as a monomer with a small dimer fraction both in vitro and in vivo. Of mechanistic importance, laforin monomer and dimer possess equal phosphatase activity, and they both associate with malin and bind glucans to a similar extent. However, we found differences between the two states' ability to interact simultaneously with malin and carbohydrates. Furthermore, we tested other members of the glucan phosphatase family. Cumulatively, our data suggest that laforin monomer is the dominant form of the protein and that it contains phosphatase activity.

Protein phosphorylation plays critical roles in the regulation of protein activity and cell signaling. The level of protein phosphorylation is controlled by protein kinases and protein tyrosine phosphatases (PTPs). Disturbance of the equilibrium between protein kinase and PTP activities results in abnormal protein phosphorylation, which has been linked to the etiology of several diseases, including cancer. In this study, we screened protein tyrosine phosphatases (PTPs) by in vitro phosphatase assays to identify PTPs that are inhibited by bis (4-trifluoromethyl-sulfonamidophenyl, TFMS)-1,4-diisopropylbenzene (PTP inhibitor IV). PTP inhibitor IV inhibited DUSP14 phosphatase activity. Kinetic studies with PTP inhibitor IV and DUSP14 revealed a competitive inhibition, suggesting that PTP inhibitor IV binds to the catalytic site of DUSP14. PTP inhibitor IV effectively and specifically inhibited DUSP14-mediated dephosphorylation of JNK, a member of the mitogen-activated protein kinase (MAPK) family.

Dual specificity phosphatase 1 (DUSP1) is regarded as an anti-inflammatory factor in cardiovascular disorders. Mitophagy removes damaged mitochondria and thus promotes mitochondrial regeneration. We investigated whether DUSP1 could attenuate inflammation-induced cardiomyopathy by improving mitophagy.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) play a central role in the intracellular signaling of P2X7 nucleotide receptors in neurons and glial cells. Fine spatio-temporal tuning of mitogen-activated protein (MAP) kinases is essential to regulate their biological activity. MAP kinase phosphatases (MKPs) are dual specificity protein phosphatases (DUSPs) that dephosphorylate phosphothreonine and phosphotyrosine residues in MAP kinases. This study focuses on how DUSP, DUSP6/MKP3, a phosphatase specific for ERK1/2 is regulated by the P2X7 nucleotide receptor in cerebellar granule neurons and astrocytes. Stimulation with the specific P2X7 agonist, BzATP, or epidermal growth factor (EGF) (positive control for ERK activation) regulates the levels of DUSP6 in a time dependent manner. Both agonists promote a decline in DUSP6 protein, reaching minimal levels after 30 min yet recovering to basal levels after 1 h. The initial loss of protein occurs through proteasomal degradation, as confirmed in experiments with the proteasome inhibitor, MG-132. Studies carried out with Actinomycin D demonstrated that the enhanced transcription of the Dusp6 gene is responsible for recovering the DUSP6 protein levels. Interestingly, ERK1/2 proteins are involved in the biphasic regulation of the protein phosphatase, being required for both the degradation and the recovery phase. We show that direct Ser197 phosphorylation of DUSP6 by ERK1/2 proteins could be part of the mechanism regulating their cytosolic levels, at least in glial cells. Thus, the ERK1/2 activated by P2X7 receptors exerts positive feedback on these kinase's own activity, promoting the degradation of one of their major inactivators in the cytosolic compartment, DUSP6, both in granule neurons and astrocytes. This feedback loop seems to function as a common universal mechanism to regulate ERK signaling in neural and non-neural cells.

Protein phosphorylation/dephosphorylation is an important regulatory mechanism that controls many key physiological processes. Numerous pathogens successfully use kinases and phosphatases to internalize, replicate, and survive, modifying the host's phosphorylation profile or signal transduction pathways. Multiple phosphatases and kinases from diverse bacterial pathogens have been implicated in human infections before. In this work, we have identified and characterized the dual specificity protein/lipid phosphatase LmDUSP1 as a novel virulence factor governing Leishmania mexicana infection. The LmDUSP1-encoding gene (LmxM.22.0250 in L. mexicana) has been acquired from bacteria via horizontal gene transfer. Importantly, its orthologues have been associated with virulence in several bacterial species, such as Mycobacterium tuberculosis and Listeria monocytogenes. Leishmania mexicana with ablated LmxM.22.0250 demonstrated severely attenuated virulence in the experimental infection of primary mouse macrophages, suggesting that this gene facilitates Leishmania pathogenicity in vertebrates. Despite significant upregulation of LmxM.22.0250 expression in metacyclic promastigotes, its ablation did not affect the ability of mutant cells to differentiate into virulent stages in insects. It remains to be further investigated which specific biochemical pathways involve LmDUSP1 and how this facilitates the parasite's survival in the host. One of the interesting possibilities is that LmDUSP1 may target host's substrate(s), thereby affecting its signal transduction pathways.

Acquisition of resistance to anti-cancer drugs is a significant obstacle to effective cancer treatment. Although several efforts have been made to overcome drug resistance in cancer cells, the detailed mechanisms have not been fully elucidated. Here, we investigated whether microRNAs (miRNAs) function as pivotal regulators in the acquisition of anti-cancer drug resistance to 5-fluorouracil (5-FU). A survey using a lentivirus library containing 572 precursor miRNAs revealed that five miRNAs promoted cell survival after 5-FU treatment in human hepatocellular carcinoma Hep3B cells. Among the five different clones, the clone expressing miR-200a-3p (Hep3B-miR-200a-3p) was further characterized as a 5-FU-resistant cell line. The cell viability and growth rate of Hep3B-miR-200a-3p cells were higher than those of control cells after 5-FU treatment. Ectopic expression of a miR-200a-3p mimic increased, while inhibition of miR-200a-3p downregulated, cell viability in response to 5-FU, doxorubicin, and CDDP (cisplatin). We also showed that dual-specificity phosphatase 6 (DUSP6) is a novel target of miR-200a-3p and regulates resistance to 5-FU. Ectopic expression of DUSP6 mitigated the pro-survival effects of miR-200a-3p. Taken together, these results lead us to propose that miR-200a-3p enhances anti-cancer drug resistance by decreasing DUSP6 expression.

Hepatocellular carcinoma (CC) is a common and deadly cancer with complex molecular pathogenesis. Little is known about dual-specificity phosphatases (DUSPs) in HCC. We investigated DUSP9 expression in human HCC, associations between DUSP9 and patient outcomes, and effects of altered DUSP9 expression on HCC biology. We studied public data sets as well as 196 patients at our institution who had HCC resections. Quantitative real-time reverse transcription polymerase chain reaction and western blot demonstrated that DUSP9 expression was increased >10-fold in HCC compared to adjacent liver and healthy controls (P = 0.005). Kaplan-Meier and multivariable regression analyses revealed that higher DUSP9 expression was associated with shorter disease-free survival (high DUSP9, 1.6; 95% confidence interval, 0.9-2.3 vs. low DUSP9, 3.4; 95% confidence interval, 1.8-5.0 years; P = 0.04) and increased risk of recurrence (hazard ratio 1.55; 95% confidence interval, 1.01-2.67; P = 0.05) after resection. DUSP9 complementary DNA (cDNA) was cloned using rapid amplification of cDNA ends, revealing two DUSP9 isoforms in human HCC cells. Studies of transcriptional regulation using promoter-luciferase reporter constructs suggested that DUSP9 transcription is regulated by E26 transformation-specific transcription factors. Proliferation of hepatic cells in vitro was enhanced by lentiviral-mediated overexpression of DUSP9. In contrast, DUSP9 knockout HCC cells generated using clustered regularly interspaced short palindromic repeats (CRISPR) demonstrated decreased HCC proliferation and doxorubicin resistance in vitro and impaired xenograft growth in vivo. RNA sequencing, gene set enrichment, and network/pathway analysis revealed that DUSP9 knockout is associated with activation of protein kinase activity and apoptosis. Conclusion: DUSP9 regulates cell proliferation and predicts recurrence after surgery in HCC. DUSP9 may represent a novel prognostic candidate and therapeutic target. Additional studies are warranted to further explore the role and regulation of DUSP9 in HCC.

Specific outcomes upon activation of the c-Jun N-terminal kinase (JNK) pathway critically depend on the intensity and duration of signal transmission. Dual-specificity phosphatases (DUSPs) play a very important role in these events by modulating the extent of JNK phosphorylation and activation and thus regulating cellular responses to stress. M3/6 (DUSP8) is one of the dual-specificity protein phosphatases with distinct specificity towards JNK. It has been shown that M3/6 itself is phosphorylated by JNK upon stimulation with arsenite, but the role of this phosphorylation has not been investigated. In this study, we mapped JNK-induced phosphorylation sites on M3/6 using mass spectrometry. Phosphorylated residues Ser 515, Thr 518 and Ser 520 were identified and site-directed mutagenesis was employed to investigate their role. Upon arsenite stimulation, M3/6 mutated at these sites exhibited decreased phosphorylation compared to the wild-type protein. No difference was observed in terms of the enzyme's in vitro phosphatase activity, its substrate specificity towards JNK isoforms, its interactions with JNK and the scaffold family of JNK-interacting proteins (JIPs), its stability or its subcellular localization. Interestingly, expression of M3/6 phosphorylation mutants delayed the time-course of JNK phosphorylation and activation by arsenite. We propose that phosphorylation of the M3/6 phosphatase by JNK in response to stress stimuli results in attenuation of phosphatase activity and acceleration of JNK activation.