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The p53 tumor suppressor protein activates various sets of genes depending on its covalent modifications, which are controlled by the nature and intensity of cellular stress. We observed that actinomycin D and nutlin-3a (A + N) collaborate in inducing activating phosphorylation of p53. Our recent transcriptomic data demonstrated that these substances strongly synergize in the upregulation of DUSP13, a gene with an unusual pattern of expression, coding for obscure phosphatase having two isoforms, one expressed in the testes and the other in skeletal muscles. In cancer cells exposed to A + N, DUSP13 is expressed from an alternative promoter in the intron, resulting in the expression of an isoform named TMDP-L1. Luciferase reporter tests demonstrated that this promoter is activated by both endogenous and ectopically expressed p53. We demonstrated for the first time that mRNA expressed from this promoter actually produces the protein, which can be detected with Western blotting, in all examined cancer cell lines with wild-type p53 exposed to A + N. In some cell lines, it is also induced by clinically relevant camptothecin, by nutlin-3a acting alone, or by a combination of actinomycin D and other antagonists of p53-MDM2 interaction—idasanutlin or RG7112. This isoform, fused with green fluorescent protein, localizes in the perinuclear region of cells. [ABSTRACT FROM AUTHOR]

The p53 tumor suppressor protein activates various sets of genes depending on its covalent modifications, which are controlled by the nature and intensity of cellular stress. We observed that actinomycin D and nutlin-3a (A + N) collaborate in inducing activating phosphorylation of p53. Our recent transcriptomic data demonstrated that these substances strongly synergize in the upregulation of DUSP13, a gene with an unusual pattern of expression, coding for obscure phosphatase having two isoforms, one expressed in the testes and the other in skeletal muscles. In cancer cells exposed to A + N, DUSP13 is expressed from an alternative promoter in the intron, resulting in the expression of an isoform named TMDP-L1. Luciferase reporter tests demonstrated that this promoter is activated by both endogenous and ectopically expressed p53. We demonstrated for the first time that mRNA expressed from this promoter actually produces the protein, which can be detected with Western blotting, in all examined cancer cell lines with wild-type p53 exposed to A + N. In some cell lines, it is also induced by clinically relevant camptothecin, by nutlin-3a acting alone, or by a combination of actinomycin D and other antagonists of p53-MDM2 interaction—idasanutlin or RG7112. This isoform, fused with green fluorescent protein, localizes in the perinuclear region of cells.

Asexual spore serves as essential inoculum of rice blast during the disease cycle, and differentiation of young conidium from conidiophore is intimately regulated by cell cycle. Mih1 encodes a dual-specificity phosphatase that involved in the G2/M transition of the mitotic cell cycle by regulating the Cdk1 activity in eukaryotes. Till now, the roles of Mih1 homologue, however, remain unclear in Magnaporthe oryzae. We here functionally characterized the Mih1 homologue MoMih1 in M. oryzae. MoMih1 is localized to both the cytoplasm and nucleus and can physically interact with the CDK protein MoCdc28 in vivo. Loss of MoMih1 led to delayed nucleus division and a high level of Tyr15 phosphorylation of MoCdc28. The MoMih1 mutants showed retarded mycelial growth with a defective polar growth, less fungal biomass, and shorter distance between diaphragms, compared with the KU80. Asexual reproduction altered in MoMih1 mutants, with both abnormal conidial morphogenesis and decreased conidiation. The MoMih1 mutants severely attenuated the virulence to host plants due to the impaired ability of penetration and biotrophic growth. The incapability of scavenging of host-derived reactive oxygen species, which was possibly ascribed to the severely decreased extracellular enzymes activities, were partially associated with deficiency of pathogenicity. Besides, the MoMih1 mutants displayed also improper localization of retromer protein MoVps26 and polarisome component MoSpa2, and defects of cell wall integrity (CWI), melanin pigmentation, chitin synthesis, and hydrophobicity. In conclusion, our results demonstrate that MoMih1 plays pleiotropic roles during fungal development and plant infection of M. oryzae. [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB) is a heterogeneous cancer of the sympathetic nervous system, which accounts for 7–10% of paediatric malignancies worldwide. Due to the lack of targetable molecular aberrations in NB, most treatment options remain relatively nonspecific. Here, we investigated the therapeutic potential of BCI, an inhibitor of DUSP1 and DUSP6, in cultured NB cells. BCI was cytotoxic in a range of NB cell lines and induced a short‐lived activation of the AKT and stress‐inducible MAP kinases, although ERK phosphorylation was unaffected. Furthermore, a phosphoproteomic screen identified significant upregulation of JNK signalling components and suppression in mTOR and R6K signalling. To assess the specificity of BCI, CRISPR‐Cas9 was employed to introduce insertions and deletions in the DUSP1 and DUSP6 genes. Surprisingly, BCI remained fully cytotoxic in NB cells with complete loss of DUSP6 and partial depletion of DUSP1, suggesting that BCI exerts cytotoxicity in NB cells through a complex mechanism that is unrelated to these phosphatases. Overall, these data highlight the risk of using an inhibitor such as BCI as supposedly specific DUSP1/6, without understanding its full range of targets in cancer cells.

PDZ domains are involved in many cellular processes and are key regulators of the cell physiology. A huge number of studies have investigated the binding specificity of PDZ domains to the carboxyl-terminal sequence of target proteins, while the molecular mechanisms that mediate the recognition of internal binding regions are largely unexplored. In the present study, we describe a ligand motif located in the catalytic domain of the phosphatase Dusp26 which mediates its binding to the PDZ-4 of Scribble. Site-directed mutagenesis identified a conserved tyrosine residue as relevant for the binding. The interaction with the PDZ domain could help the phosphatase to recruit its specific targets.

Background: The dysregulation of melanin production causes skin-disfiguring ultraviolet (UV)-associated hyperpigmented spots. Previously, we found that the activation of c-Jun N-terminal kinase (JNK), a mitogen-activated protein kinase (MAPK), inhibited melanogenesis. Methods: We selected BCI-215 as it may modify MAPK expression via a known function of a dual-specificity phosphatase (DUSP) 1/6 inhibitor. B16F10 melanoma cells, Mel-ab cells, human melanocytes, and a coculture were used to assess the anti-melanogenic activity of BCI-215. The molecular mechanisms were deciphered by assaying the melanin content and cellular tyrosinase activity via immunoblotting and RT-PCR. Results: BCI-215 was found to suppress basal and cAMP-stimulated melanin production and cellular tyrosinase activity in vitro through the downregulation of microphthalmia-associated transcription factor (MITF) protein and its downstream enzymes. The reduction in MITF expression caused by BCI-215 was found to be due to all three types of MAPK activation, including extracellular signal-regulated kinase (ERK), JNK, and p38. The degree of activation was greater in ERK. A phosphorylation of the β-catenin pathway was also demonstrated. The melanin index, expression of MITF, and downstream enzymes were well-reduced in UVB-irradiated ex vivo human skin by BCI-215. Conclusions: As BCI-215 potently inhibits UV-stimulated melanogenesis, small molecules of DUSP-related signaling modulators may provide therapeutic benefits against pigmentation disorders. [ABSTRACT FROM AUTHOR]

Estradiol (E2) and relaxin (Rln) are steroid and polypeptide hormones, respectively, with important roles in the female reproductive tract, including myometrium. Some actions of Rln, which are mediated by its membrane receptor RXFP1, require or are augmented by E2 signaling through its cognate nuclear steroid receptor, estrogen receptor alpha (ERα). In contrast, other actions of Rln act in opposition to the effects of E2. Here we explored the molecular and genomic mechanisms that underlie the functional interplay between E2 and Rln in the myometrium. We used both ovariectomized female mice and immortalized human myometrial cells expressing wild-type or mutant ERα (hTERT-HM-ERα cells). Our results indicate that Rln modulates the genomic actions and biological effects of estrogen in the myometrium and myometrial cells by reducing phosphorylation of ERα on serine 118 (S118), as well as by reducing the E2-dependent binding of ERα across the genome. These effects were associated with changes in the hormone-regulated transcriptome, including a decrease in the E2-dependent expression of some genes and enhanced expression of others. The inhibitory effects of Rln cotreatment on the E2-dependent phosphorylation of ERα required the nuclear dual-specificity phosphatases DUSP1 and DUSP5. Moreover, the inhibitory effects of Rln were reflected in a concomitant inhibition of the E2-dependent contraction of myometrial cells. Collectively, our results identify a pathway that integrates Rln/RXFP1 and E2/ERα signaling, resulting in a convergence of membrane and nuclear signaling pathways to control genomic and biological outcomes., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

Mitogen-activated protein kinase (MAPK) cascades transmit environmental signals and induce stress and defence responses in plants. These signalling cascades are negatively controlled by specific Ser/Thr protein phosphatases of the type 2C (PP2C) and dual-specificity phosphatase (DSP) families that inactivate stress-induced MAPKs; however, the interplay between phosphatases of these different types has remained unknown. This work reveals that different Arabidopsis MAPK phosphatases, the PP2C-type AP2C1 and the DSP-type MKP1, exhibit both specific and overlapping functions in plant stress responses. Each single mutant, ap2c1 and mkp1 , and the ap2c1 mkp1 double mutant displayed enhanced stress-induced activation of the MAPKs MPK3, MPK4, and MPK6, as well as induction of a set of transcription factors. Moreover, ap2c1 mkp1 double mutants showed an autoimmune-like response, associated with increased levels of the stress hormones salicylic acid and ethylene, and of the phytoalexin camalexin. This phenotype was reduced in the ap2c1 mkp1 mpk3 and ap2c1 mkp1 mpk6 triple mutants, suggesting that the autoimmune-like response is due to MAPK misregulation. We conclude that the evolutionarily distant MAPK phosphatases AP2C1 and MKP1 contribute crucially to the tight control of MAPK activities, ensuring appropriately balanced stress signalling and suppression of autoimmune-like responses during plant growth and development. [ABSTRACT FROM AUTHOR]

The sequence-specific backbone assignment of the mitogen-activated protein kinase (MAPK) binding domain of the dual-specificity phosphatase 1 (DUSP1) has been accomplished using a uniformly [13C, 15N]-labeled protein. These assignments will facilitate further studies of DUSP1 in the presence of inhibitors/ligands to target MAPK associated diseases and provide further insights into the function of dual-specificity phosphatase 1 in MAPK regulation. [ABSTRACT FROM AUTHOR]

Background: The dysregulation of melanin production causes skin-disfiguring ultraviolet (UV)-associated hyperpigmented spots. Previously, we found that the activation of c-Jun N-terminal kinase (JNK), a mitogen-activated protein kinase (MAPK), inhibited melanogenesis. Methods: We selected BCI-215 as it may modify MAPK expression via a known function of a dual-specificity phosphatase (DUSP) 1/6 inhibitor. B16F10 melanoma cells, Mel-ab cells, human melanocytes, and a coculture were used to assess the anti-melanogenic activity of BCI-215. The molecular mechanisms were deciphered by assaying the melanin content and cellular tyrosinase activity via immunoblotting and RT-PCR. Results: BCI-215 was found to suppress basal and cAMP-stimulated melanin production and cellular tyrosinase activity in vitro through the downregulation of microphthalmia-associated transcription factor (MITF) protein and its downstream enzymes. The reduction in MITF expression caused by BCI-215 was found to be due to all three types of MAPK activation, including extracellular signal-regulated kinase (ERK), JNK, and p38. The degree of activation was greater in ERK. A phosphorylation of the β-catenin pathway was also demonstrated. The melanin index, expression of MITF, and downstream enzymes were well-reduced in UVB-irradiated ex vivo human skin by BCI-215. Conclusions: As BCI-215 potently inhibits UV-stimulated melanogenesis, small molecules of DUSP-related signaling modulators may provide therapeutic benefits against pigmentation disorders.

Dual-specificity phosphatase 6 (DUSP6) is a key negative feedback regulator of the member of the RAS-ERK MAPK signaling pathway that is associated with cellular proliferation and differentiation. Deterioration of DUSP6 expression could therefore result in deregulated growth activity. We have previously discovered ACA-28, a novel anticancer compound with a unique property to stimulate ERK phosphorylation and induce apoptosis in ERK-active melanoma cells. However, the mechanism of cancer cell-specific-apoptosis by ACA-28 remains obscure. Here, we investigated the involvement of DUSP6 in the mechanisms of the ACA-28-mediated apoptosis by using the NIH/3T3 cells overexpressing HER2/ErbB2 (A4-15 cells), as A4-15 exhibited higher ERK phosphorylation and are more susceptible to ACA-28 than NIH/3T3. We showed that A4-15 exhibited high DUSP6 protein levels, which require ERK activation. Notably, the silencing of the DUDSP6 gene by siRNA inhibited proliferation and induced apoptosis in A4-15, but not in NIH/3T3, indicating that A4-15 requires high DUSP6 expression for growth. Importantly, ACA-28 preferentially down-regulated the DUSP6 protein and proliferation in A4-15 via the proteasome, while it stimulated ERK phosphorylation. Collectively, the up-regulation of DUSP6 may exert a growth-promoting role in cancer cells overexpressing HER2. DUSP6 down-regulation in ERK-active cancer cells might have the potential as a novel cancer measure. [ABSTRACT FROM AUTHOR]

The regulation of the phosphorylation of mitogen‐activated protein kinases (MAPKs) is essential for cellular processes such as proliferation, differentiation, survival, and death. Mutations within the MAPK signaling cascades are implicated in diseases such as cancer, neurodegenerative disorders, arthritis, obesity, and diabetes. MAPK phosphorylation is controlled by an intricate balance between MAPK kinases (enzymes that add phosphate groups) and MAPK phosphatases (MKPs) (enzymes that remove phosphate groups). MKPs are complex negative regulators of the MAPK pathway that control the amplitude and spatiotemporal regulation of MAPKs. MK‐STYX (MAPK phosphoserine/threonine/tyrosine‐binding protein) is a member of the MKP subfamily, which lacks the critical histidine and nucleophilic cysteine residues in the active site required for catalysis. MK‐STYX does not influence the phosphorylation status of MAPK, but even so it adds to the complexity of signal transduction cascades as a signaling regulator. This review highlights the function of MK‐STYX, providing insight into MK‐STYX as a signal regulating molecule in the stress response, HDAC 6 dynamics, apoptosis, and neurite differentiation. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinases (MAPKs) are essential for proper cell functioning as they regulate many molecular effectors. Careful regulation of MAPKs is therefore required to avoid MAPK pathway dysfunctions and pathologies. The mammalian genome encodes about 200 phosphatases, many of which dephosphorylate the MAPKs and bring them back to an inactive state. In this review, we focus on the normal and pathological functions of dual-specificity phosphatase 9 (DUSP9)/MAP kinase phosphatases-4 (MKP-4). This cytoplasmic phosphatase, which belongs to the threonine/tyrosine dual-specific phosphatase family and was first described in 1997, is known to dephosphorylate ERK1/2, p38, JNK and ASK1, and thereby to control various MAPK pathway cascades. As a consequence, DUSP9 plays a major role in human pathologies and more specifically in cardiac dysfunction, liver metabolic syndromes, diabetes, obesity and cancer including drug response and cell stemness. Here, we recapitulate the mechanism of action of DUSP9 in the cell, its levels of regulation and its roles in the most frequent human diseases, and discuss its potential as a therapeutic target.

Breast cancer remains the most lethal type of cancer for women. A significant proportion of breast cancer cases are characterised by overexpression of the human epidermal growth factor receptor 2 protein (HER2). These cancers are commonly treated by Herceptin (Trastuzumab), but resistance to drug treatment frequently develops in tumour cells. Dual-specificity phosphatases (DUSPs) are thought to play a role in the mechanism of resistance, since some of them were reported to be overexpressed in tumours resistant to Herceptin. We used a systems biology approach to investigate how DUSP overexpression could favour cell proliferation and to predict how this mechanism could be reversed by targeted inhibition of selected DUSPs. We measured the expression of 20 DUSP genes in two breast cancer cell lines following long-term (6 months) exposure to Herceptin, after confirming that these cells had become resistant to the drug. We constructed several Boolean models including specific substrates of each DUSP, and showed that our models correctly account for resistance when overexpressed DUSPs were kept activated. We then simulated inhibition of both individual and combinations of DUSPs, and determined conditions under which the resistance could be reversed. These results show how a combination of experimental analysis and modelling help to understand cell survival mechanisms in breast cancer tumours, and crucially enable us to generate testable predictions potentially leading to new treatments of resistant tumours. [ABSTRACT FROM AUTHOR]

Stress-activated MAP kinases (SAPKs) respond to a wide variety of stressors. In most cases, the pathways through which specific stress signals are transmitted to the SAPKs are not known. Our recent findings have begun to address two important and related questions. First, do various stresses activate a SAPK through common pathways initiated at the cell surface, or through alternative, intracellular inputs? Second, how does an activated SAPK mount a specific response appropriate to the particular stress experienced? Our work has uncovered the mechanisms by which two stresses, arsenite treatment and DNA damage, stimulate the yeast SAPKs Hog1 and Mpk1, respectively. We found that these stresses activate the SAPKs through intracellular inputs that modulate their basal phosphorylation, rather than by activation of the protein kinase cascades known to stimulate them. Both stresses act through targeting, in different ways, the tyrosine-specific or dual-specificity protein phosphatases that normally maintain the SAPKs in a low-activity state. Previous work has demonstrated that basal signal flux through SAPK pathways is important for the sensitivity and dynamic response to external signals. Our work reveals that basal activity of SAPKs is additionally important to allow SAPK activation by intracellular inputs that modulate that activity. Additionally, because different stressors may activate SAPKs by modulation of basal signal through inputs at distinct nodes along the canonical activation pathway, stress-specific SAPK outputs may be controlled, in part, by the specific intracellular mechanisms of their activation. Thus, understanding the intracellular pathways through which various stressors activate SAPKs is likely to provide insight into how they elicit physiologically coherent responses to the specific stress experienced. [ABSTRACT FROM AUTHOR]

Dusp22 (dual-specificity phosphatase 22) is considered to regulate various cellular processes through the regulation of protein dephosphorylation. In this study, we prepared a specific antibody against Dusp22, anti-Dusp22, and carried out expression analyses with mouse tissues and cultured cell lines. Western blotting analyses demonstrated a tissue-dependent expression profile of Dusp22 in the adult mouse, and strongly suggested the presence of isoforms with larger molecular masses. In fibroblast NIH3T3 cells, while both endogenous and Myc-tagged Dusp22 was diffusely distributed in the cytoplasm, Myc-Dusp22 was partially colocalized with actin cytoskeleton. From the obtained results, anti-Dusp22 was found to be a useful tool for biochemical and cell biological analyses of Dusp22. [ABSTRACT FROM AUTHOR]

The G2-M transition of the cell cycle requires the activation of members of the Cdc25 dual specificity phosphatase family. Using Xenopus oocyte maturation as a model system, we have previously shown that chelation of transition metals blocks meiosis progression by inhibiting Cdc25C activation. Here, using approaches that allow for the isolation of very pure and active recombinant Cdc25C, we show that Cdc25C does not bind zinc as previously reported. Additionally, we show that mutants in the disordered C-terminal end of Cdc25C are poor initiators of meiosis, likely due to their inability to localize to the proper sub-cellular location.Wefurther demonstrate that the transition metal chelator, TPEN, acts on or upstream of polo-like kinases in the oocyte to block meiosis progression. Together our results provide novel insights into Cdc25C structure-function relationship and the role of transition metals in regulating meiosis. [ABSTRACT FROM AUTHOR]

The mitogen-activated protein kinase (MAPK) cascades are activated in innate immune cells such as macrophages upon the detection of microbial infection, critically regulating the expression of proinflammatory cytokines and chemokines such as TNF-α, IL-6, and MCP-1. As a result, activation of MAPKs is tightly regulated to ensure appropriate and adequate immune responses. Dual-specificity phosphatases (DUSPs) are a family of proteins which specifically dephosphorylates threonine and tyrosine residues essential for MAPK activation to negatively regulate their activation. DUSP12 is a member of atypical DUSPs that lack MAPK-binding domain. Its substrate and function in immune cells are unknown. In this study, we demonstrated that DUSP12 is able to interact with all the three groups of MAPKs, including extracellular signal-regulated protein kinase, JNK, and p38. To investigate the function of DUSP12 in macrophages in response to TLR activation and microbial infection, we established RAW264.7 cell lines stably overexpressing DUSP12 and found that overexpression of DUSP12 inhibited proinflammatory cytokine and chemokine production in response to TLR4 activation, heat-inactivated Mycobacterium tuberculosis stimulation as well as infections by intracellular bacteria including Listeria moncytogenesis and Mycobacterium bovis BCG by specifically inhibiting p38 and JNK. In addition, a scaffold protein known as signal transducing adaptor protein 2 (STAP2), was found to mediate the interaction between DUSP12 and p38. Thus, DUSP12 is a bona fide MAPK phosphatase, playing an important role in MAPK-regulated responses to bacterial infection. Our study provides a model where atypical DUSPs regulate MAPKs via scaffold, thereby regulating immune responses to microbial infection.

DUSP6 is a dual-specificity phosphatase (DUSP) involved in breast cancer progression, recurrence, and metastasis. DUSP6 is predominantly cytoplasmic in HER2+ primary breast cancer cells, but the expression and subcellular localization of DUSPs, especially DUSP6, in HER2-positive circulating tumor cells (CTCs) is unknown. Here we used the DEPArray system to identify and isolate CTCs from metastatic triple negative breast cancer (TNBC) patients and performed single-cell NanoString analysis to quantify cancer pathway gene expression in HER2-positive and HER2-negative CTC populations. All TNBC patients contained HER2-positive CTCs. HER2-positive CTCs were associated with increased ERK1/ERK2 expression, which are direct DUSP6 targets. DUSP6 protein expression was predominantly nuclear in breast CTCs and the brain metastases but not pleura or lung metastases of TNBC patients. Therefore, nuclear DUSP6 may play a role in the association with cancer spreading in TNBC patients, including brain metastasis.

Mitogen-activated protein kinases (MAPKs) are key regulators of signal transduction and cell responses. Abnormalities in MAPKs are associated with multiple diseases. Dual-specificity phosphatases (DUSPs) dephosphorylate many key signaling molecules, including MAPKs, leading to the regulation of duration, magnitude, or spatiotemporal profiles of MAPK activities. Hence, DUSPs need to be properly controlled. Protein post-translational modifications, such as ubiquitination, phosphorylation, methylation, and acetylation, play important roles in the regulation of protein stability and activity. Ubiquitination is critical for controlling protein degradation, activation, and interaction. For DUSPs, ubiquitination induces degradation of eight DUSPs, namely, DUSP1, DUSP4, DUSP5, DUSP6, DUSP7, DUSP8, DUSP9, and DUSP16. In addition, protein stability of DUSP2 and DUSP10 is enhanced by phosphorylation. Methylation-induced ubiquitination of DUSP14 stimulates its phosphatase activity. In this review, we summarize the knowledge of the regulation of DUSP stability and ubiquitination through post-translational modifications.

The mitogen-activated protein kinase (MAPK) cascades are activated in innate immune cells such as macrophages upon the detection of microbial infection, critically regulating the expression of proinflammatory cytokines and chemokines such as TNF-α, IL-6, and MCP-1. As a result, activation of MAPKs is tightly regulated to ensure appropriate and adequate immune responses. Dual-specificity phosphatases (DUSPs) are a family of proteins which specifically dephosphorylates threonine and tyrosine residues essential for MAPK activation to negatively regulate their activation. DUSP12 is a member of atypical DUSPs that lack MAPK-binding domain. Its substrate and function in immune cells are unknown. In this study, we demonstrated that DUSP12 is able to interact with all the three groups of MAPKs, including extracellular signal-regulated protein kinase, JNK, and p38. To investigate the function of DUSP12 in macrophages in response to TLR activation and microbial infection, we established RAW264.7 cell lines stably overexpressing DUSP12 and found that overexpression of DUSP12 inhibited proinflammatory cytokine and chemokine production in response to TLR4 activation, heat-inactivated Mycobacterium tuberculosis stimulation as well as infections by intracellular bacteria including Listeria moncytogenesis and Mycobacterium bovis BCG by specifically inhibiting p38 and JNK. In addition, a scaffold protein known as signal transducing adaptor protein 2 (STAP2), was found to mediate the interaction between DUSP12 and p38. Thus, DUSP12 is a bona fide MAPK phosphatase, playing an important role in MAPKregulated responses to bacterial infection. Our study provides a model where atypical DUSPs regulate MAPKs via scaffold, thereby regulating immune responses to microbial infection. [ABSTRACT FROM AUTHOR]

Dual-specificity MAP kinase (MAPK) phosphatases (DUSPs) are well-established negative modulators in regulating MAPK signaling in mammalian cells and tissues. Our previous studies have shown the involvement of DUSP6 in regulating innate immunity in Japanese flounder Paralichthys olivaceus . In order to gain a better understanding of the role of DUSPs in fish innate immunity, in the present study we identified and characterized three additional DUSP genes including DUSP1, 2 and 5 in P. olivaceus . The three Japanese flounder DUSP proteins share common domain structures composed of a conserved N -terminal Rhodanase/CDC25 domain and a C -terminal catalytic phosphatase domain, while they show only less than 26% sequence identities, indicating that they may have different substrate selectivity. In addition, mRNA transcripts of all the three DUSP genes are detected in all examined Japanese flounder tissues; however, DUSP1 is dominantly expressed in spleen while DUSP2 and 5 are primarily expressed in skin. Furthermore, all the three DUSP genes are constitutively expressed in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood leucocytes (PBLs) with unequal distribution patterns. Moreover, all the three DUSPs gene expression was induced differently in response to the LPS and double-stranded RNA mimic poly(I:C) stimulations both in the Japanese flounder HKMs and PBLs, suggesting an association of DUSPs with TLR signaling in fish. Taken together, the co-expression of various DUSPs members together with their different responses to the immune challenges indicate that the DUSP members may operate coordinately in regulating the MAPK-dependent immune responses in the Japanese flounder. [ABSTRACT FROM AUTHOR]

Phospho-cofilin ( p -cofilin), which has a phosphate group on Ser-3, is involved in actin polymerization. Its dephosphorylated form promotes filopodia formation and cell migration by enhancing actin depolymerization. Protein phosphatase slingshot homologs (SSHs), known as dual-specificity phosphatases, catalyze hydrolytic removal of the Ser-3 phosphate group from phospho-cofilin. Aberrant SSH activity results in cancer metastasis, implicating SSHs as potential therapeutic targets for cancer metastasis. In this study, we screened 658 natural products purified from traditional oriental medicinal plants to identify three potent SSH inhibitors with submicromolar or single-digit micromolar K i values: gossypol, hypericin, and sennoside A. The three compounds were purified from cottonseed, Saint John’s wort, and rhubarb, respectively. Sennoside A markedly increased cofilin phosphorylation in pancreatic cancer cells, leading to impaired actin dynamics in pancreatic cancer cells with or without EGF stimulation and reduced motility and invasiveness in vitro and in vivo . Collaboratively, these results demonstrate that sennoside A is a novel inhibitor of SSHs and suggest that it may be valuable in the development of pharmaceutical drugs for treating cancer metastasis. [ABSTRACT FROM AUTHOR]

Asexual spore serves as essential inoculum of rice blast during the disease cycle, and differentiation of young conidium from conidiophore is intimately regulated by cell cycle. Mih1 encodes a dual-specificity phosphatase that involved in the G2/M transition of the mitotic cell cycle by regulating the Cdk1 activity in eukaryotes. Till now, the roles of Mih1 homologue, however, remain unclear in Magnaporthe oryzae . We here functionally characterized the Mih1 homologue MoMih1 in M. oryzae . MoMih1 is localized to both the cytoplasm and nucleus and can physically interact with the CDK protein MoCdc28 in vivo . Loss of MoMih1 led to delayed nucleus division and a high level of Tyr15 phosphorylation of MoCdc28. The MoMih1 mutants showed retarded mycelial growth with a defective polar growth, less fungal biomass, and shorter distance between diaphragms, compared with the KU80 . Asexual reproduction altered in MoMih1 mutants, with both abnormal conidial morphogenesis and decreased conidiation. The MoMih1 mutants severely attenuated the virulence to host plants due to the impaired ability of penetration and biotrophic growth. The incapability of scavenging of host-derived reactive oxygen species, which was possibly ascribed to the severely decreased extracellular enzymes activities, were partially associated with deficiency of pathogenicity. Besides, the MoMih1 mutants displayed also improper localization of retromer protein MoVps26 and polarisome component MoSpa2, and defects of cell wall integrity (CWI), melanin pigmentation, chitin synthesis, and hydrophobicity. In conclusion, our results demonstrate that MoMih1 plays pleiotropic roles during fungal development and plant infection of M. oryzae ., Competing Interests: 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., (Copyright © 2023 Liu, Gong, Ma, Wang and Guo.)

Tribbles homolog 2 (TRIB2) is a member of the mammalian Tribbles family of serine/threonine pseudokinases (TRIB1-3). Studies of TRIB2 indicate that many of the molecular interactions between the single Drosophila Tribbles (Trbl) protein and interacting partners are evolutionary conserved. In this study, we examined the relationship between TRIB2 and cell division cycle 25 (CDC25) family of dual-specificity protein phosphatases (mammalian homologues of Drosophila String), which are key physiological cell cycle regulators. Using co-immunoprecipitation we demonstrate that TRIB2 interacts with CDC25B and CDC25C selectively. Forced overexpression of TRIB2 caused a marked decrease in total CDC25C protein levels. Following inhibition of the proteasome, CDC25C was stabilized in the nuclear compartment. This implicates TRIB2 as a regulator of nuclear CDC25C turnover. In complementary ubiquitination assays, we show that TRIB2-mediated degradation of CDC25C is associated with lysine-48-linked CDC25C polyubiquitination driven by the TRIB2 kinase-like domain. A cell cycle associated role for TRIB2 is further supported by the cell cycle regulated expression of TRIB2 protein levels. Our findings reveal mitotic CDC25C as a new target of TRIB2 that is degraded via the ubiquitin proteasome system. Inappropriate CDC25C regulation could mechanistically underlie TRIB2 mediated regulation of cellular proliferation in neoplastic cells. [ABSTRACT FROM AUTHOR]

Glucan phosphatases are a recently discovered class of enzymes that dephosphorylate starch and glycogen, thereby regulating energy metabolism. Plant genomes encode two glucan phosphatases, called Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), that regulate starch metabolism by selectively dephosphorylating glucose moieties within starch glucan chains. Recently, the structures of both SEX4 and LSF2 were determined, with and without phosphoglucan products bound, revealing the mechanism for their unique activities. This review explores the structural and enzymatic features of the plant glucan phosphatases, and outlines how they are uniquely adapted to perform their cellular functions. We outline the physical mechanisms used by SEX4 and LSF2 to interact with starch glucans: SEX4 binds glucan chains via a continuous glucan-binding platform comprising its dual-specificity phosphatase domain and carbohydrate-binding module, while LSF2 utilizes surface binding sites. SEX4 and LSF2 both contain a unique network of aromatic residues in their catalytic dualspecificity phosphatase domains that serve as glucan engagement platforms and are unique to the glucan phosphatases. We also discuss the phosphoglucan substrate specificities inherent to SEX4 and LSF2, and outline structural features within the active site that govern glucan orientation. This review defines the structural mechanism of the plant glucan phosphatases with respect to phosphatases, starch metabolism and protein-glucan interaction, thereby providing a framework for their application in both agricultural and industrial settings. [ABSTRACT FROM AUTHOR]

Dual-specificity MAP kinase (MAPK) phosphatases (MKPs or DUSPs) are well-established negative regulators of MAPK signalling in mammalian cells and tissues. By virtue of their differential subcellular localisation and ability to specifically recognise, dephosphorylate and inactivate different MAPK isoforms, they are key spatiotemporal regulators of pathway activity. Furthermore, as they are transcriptionally regulated as downstream targets of MAPK signalling they can either act as classical negative feedback regulators or mediate cross talk between distinct MAPK pathways. Because MAPKs and particularly Ras/ERK signalling are implicated in cancer initiation and development, the observation that MKPs are abnormally regulated in human tumours has been interpreted as evidence that these enzymes can either suppress or promote carcinogenesis. However, definitive evidence of such roles has been lacking. Here we review recent work based on the use of mouse models, biochemical studies and clinical data that demonstrate key roles for MKPs in modulating the oncogenic potential of Ras/ERK signalling and also indicate that these enzymes may play a role in the response of tumours to certain anticancer drugs. Overall, this work reinforces the importance of negative regulatory mechanisms in modulating the activity of oncogenic MAPK signalling and indicates that MKPs may provide novel targets for therapeutic intervention in cancer. [ABSTRACT FROM AUTHOR]