Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death.

The dual specificity protein phosphatases (Dusps) control dephosphorylation of mitogen-activated protein kinases (MAPKs) as well as other substrates. Here, we report that Dusp26, which is highly expressed in neuroblastoma cells and primary neurons is targeted to the mitochondrial outer membrane via its NH2-terminal mitochondrial targeting sequence. Loss of Dusp26 has a significant impact on mitochondrial function that is associated with increased levels of reactive oxygen species (ROS), reduction in ATP generation, reduction in mitochondria motility and release of mitochondrial HtrA2 protease into the cytoplasm. The mitochondrial dysregulation in dusp26-deficient neuroblastoma cells leads to the inhibition of cell proliferation and cell death. In vivo, Dusp26 is highly expressed in neurons in different brain regions, including cortex and midbrain (MB). Ablation of Dusp26 in mouse model leads to dopaminergic (DA) neuronal cell loss in the substantia nigra par compacta (SNpc), inflammatory response in MB and striatum, and phenotypes that are normally associated with Neurodegenerative diseases. Consistent with the data from our mouse model, Dusp26 expressing cells are significantly reduced in the SNpc of Parkinson's Disease patients. The underlying mechanism of DA neuronal death is that loss of Dusp26 in neurons increases mitochondrial ROS and concurrent activation of MAPK/p38 signaling pathway and inflammatory response. Our results suggest that regulation of mitochondrial-associated protein phosphorylation is essential for the maintenance of mitochondrial homeostasis and dysregulation of this process may contribute to the initiation and development of neurodegenerative diseases.

DUSP4 promotes the carcinogenesis of CCRCC via negative regulation of autophagic death.

DUSP4 is considered as an oncogenic gene. However, the effect of DUSP4 on the carcinogenesis of clear cell Renal cell carcinoma (CCRCC) is still unclear. In this study, DUSP4 mRNA levels were significantly increased in CCRCC tissues and cell lines. Furthermore, DUSP4 overexpression promotes the proliferation, migration, and tumorigenicity of CCRCC cells while DUSP4 silencing showed the opposite effects. Importantly, both autophagic activity (LC3 conversion rate and LC3 puncta formation) and total death level promoted by DUSP4 silencing were reversed by treatment with 3-MA in CCRCC cells. Moreover, the proliferation and migration of CCRCC cells inhibited by DUSP4 silencing were also recovered by suppression of autophagy with 3-MA. In conclusion, DUSP4 serves as an oncogenic gene in CCRCC carcinogenesis due to its inhibitory effect on autophagic death, indicating the potential value of DUSP4 in the diagnosis and treatment of CCRCC.

GFAP alternative splicing regulates glioma cell-ECM interaction in a DUSP4-dependent manner.

Gliomas are the most common primary brain tumors. Their highly invasive character and the heterogeneity of active oncogenic pathways within single tumors complicate the development of curative therapies and cause poor patient prognosis. Glioma cells express the intermediate filament protein glial fibrillary acidic protein (GFAP), and the level of its alternative splice variant GFAP-δ, relative to its canonical splice variant GFAP-α, is higher in grade IV compared with lower-grade and lower malignant glioma. In this study we show that a high GFAP-δ/α ratio induces the expression of the dual-specificity phosphatase 4 (DUSP4) in focal adhesions. By focusing on pathways up- and downstream of DUSP4 that are involved in the cell-extracellular matrix interaction, we show that a high GFAP-δ/α ratio equips glioma cells to better invade the brain. This study supports the hypothesis that glioma cells with a high GFAP-δ/α ratio are highly invasive and more malignant cells, thus making GFAP alternative splicing a potential therapeutic target.-Van Bodegraven, E. J., van Asperen, J. V., Sluijs, J. A., van Deursen, C. B. J., van Strien, M. E., Stassen, O. M. J. A., Robe, P. A. J., Hol, E. M. GFAP alternative splicing regulates glioma cell-ECM interaction in a DUSP4-dependent manner.

The protective role of the MKP-5-JNK/P38 pathway in glucolipotoxicity-induced islet ß-cell dysfunction and apoptosis.

Hyperglycemia and hyperlipidemia (glycolipotoxicity)-triggered islet ß-cell dysfunction is known to drive the progression of obesity-related type 2 diabetes, however the underlying mechanisms have not been clearly elucidated. The current study aimed to investigate the role of mitogen-activated protein kinase phosphatase 5 (MKP-5) in islet cells under glucolipotoxic conditions. Using gene overexpression and knockdown approaches, we demonstrated that MKP-5 could alleviate glucolipotoxicity-induced apoptosis via the endoplasmic reticulum (ER) stress and mitochondrial apoptosis pathways owing to the altered regulation of caspase family members and ER stress-related molecules in MIN6 and primary islet cells. Overexpression of MKP-5 reversed the glucose and palmitic acid (GP)-induced impairment of insulin secretion as well as the abnormal decreases in the expression of islet functional genes, thereby maintaining the normal insulin secretory functionality, whereas the absence of MKP-5 aggravated islet cell dysfunction. In parallel, the production of ROS and increased inflammation-associated genes in response to GP were also reduced upon MKP-5 overexpression. Further, inhibition of JNK or P38 MAPK pathways resisted to glucolipotoxicity observed in MKP-5 knockdown MIN6 cells. These findings indicate that MKP-5 is an important mediator for glucolipotoxicity-induced islet cell dysfunction and apoptosis, with JNK and P38 as the critical downstream pathways.

JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Osteoblasts are versatile cells involved in multiple whole-body processes, including bone formation and immune response. Secretory amounts and patterns of osteoblast-derived proteins such as osteopontin (OPN) and osteocalcin (OCN) modulate osteoblast function. However, the regulatory mechanism of OPN and OCN expression remains unknown. Here, we demonstrate that p54/p46 c-jun N-terminal kinase (JNK) inhibition suppresses matrix mineralization and OCN expression but increases OPN expression in MC3T3-E1 cells and primary osteoblasts treated with differentiation inducers, including ascorbic acid, bone morphogenic protein-2, or fibroblast growth factor 2. Preinhibition of JNK before the onset of differentiation increased the number of osteoblasts that highly express OPN but not OCN (OPN-OBs), indicating that JNK affects OPN secretory phenotype at the early stage of osteogenic differentiation. Additionally, we identified JNK2 isoform as being critically involved in OPN-OB differentiation. Microarray analysis revealed that OPN-OBs express characteristic transcription factors, cell surface markers, and cytokines, including glycoprotein hormone α2 and endothelial cell-specific molecule 1. Moreover, we found that inhibitor of DNA binding 4 is an important regulator of OPN-OB differentiation and that dual-specificity phosphatase 16, a JNK-specific phosphatase, functions as an endogenous regulator of OPN-OB induction. OPN-OB phenotype was also observed following LPS from Porphyromonas gingivalis stimulation during osteogenic differentiation. Collectively, these results suggest that the JNK-Id4 signaling axis is crucial in the control of OPN and OCN expression during osteoblastic differentiation.-Kusuyama, J., Amir, M. S., Albertson, B. G., Bandow, K., Ohnishi, T., Nakamura, T., Noguchi, K., Shima, K., Semba, I., Matsuguchi, T. JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Dual-specificity MAP kinase phosphatases in health and disease.

It is well established that a family of dual-specificity MAP kinase phosphatases (MKPs) play key roles in the regulated dephosphorylation and inactivation of MAP kinase isoforms in mammalian cells and tissues. MKPs provide a mechanism of spatiotemporal feedback control of these key signalling pathways, but can also mediate crosstalk between distinct MAP kinase cascades and facilitate interactions between MAP kinase pathways and other key signalling modules. As our knowledge of the regulation, substrate specificity and catalytic mechanisms of MKPs has matured, more recent work using genetic models has revealed key physiological functions for MKPs and also uncovered potentially important roles in regulating the pathophysiological outcome of signalling with relevance to human diseases. These include cancer, diabetes, inflammatory and neurodegenerative disorders. It is hoped that this understanding will reveal novel therapeutic targets and biomarkers for disease, thus contributing to more effective diagnosis and treatment for these debilitating and often fatal conditions.

A mitogen-activated protein kinase phosphatase influences grain size and weight in rice.

Grain size and weight are directly associated with grain yield in crops. However, the molecular mechanisms that set final grain size and weight remain largely unknown. Here, we characterize two large grain mutants, large grain8-1 (large8-1) and large grain8-2 (large8-2). LARGE8 encodes the mitogen-activated protein kinase phosphatase1 (OsMKP1). Loss of function mutations in OsMKP1 results in large grains, while overexpression of OsMKP1 leads to small grains. OsMKP1 determines grain size by restricting cell proliferation in grain hulls. OsMKP1 directly interacts with and deactivates the mitogen-activated protein kinase 6 (OsMAPK6). Taken together, we identify OsMKP1 as a crucial factor that influences grain size by deactivating OsMAPK6, indicating that the reversible phosphorylation of OsMAPK6 plays important roles in determining grain size in rice.

NEAP/DUSP26 suppresses receptor tyrosine kinases and regulates neuronal development in zebrafish.

Expression of neuroendocrine-associated phosphatase (NEAP, also named as dual specificity phosphatase 26, [DUSP26]) is restricted to neuroendocrine tissues. We found that NEAP, but not its phosphatase-defective mutant, suppressed nerve growth factor (NGF) receptor TrkA and fibroblast growth factor receptor 1 (FGFR1) activation in PC12 cells upon NGF stimulation. Conversely, suppressing NEAP expression by RNA interference enhanced TrkA and FGFR1 phosphorylation. NEAP was capable of de-phosphorylating TrkA and FGFR1 directly in vitro. NEAP-orthologous gene existed in zebrafish. Morpholino (MO) suppression of NEAP in zebrafish resulted in hyper-phosphorylation of TrkA and FGFR1 as well as abnormal body postures and small eyes. Differentiation of retina in zebrafishes with NEAP MO treatment was severely defective, so were cranial motor neurons. Taken together, our data indicated that NEAP/DUSP26 have a critical role in regulating TrkA and FGFR1 signaling as well as proper development of retina and neuronal system in zebrafish.

NADPH oxidase-generated reactive oxygen species are required for stromal cell-derived factor-1α-stimulated angiogenesis.

OBJECTIVE: Reactive oxygen species (ROS) act as signaling molecules during angiogenesis; however, the mechanisms used for such signaling events remain unclear. Stromal cell-derived factor-1α (SDF-1α) is one of the most potent angiogenic chemokines. Here, we examined the role of ROS in the regulation of SDF-1α-dependent angiogenesis. APPROACH AND RESULTS: Bovine aortic endothelial cells were treated with SDF-1α, and intracellular ROS generation was monitored. SDF-1α treatment induced bovine aortic endothelial cell migration and ROS generation, with the majority of ROS generated by bovine aortic endothelial cells at the leading edge of the migratory cells. Antioxidants and nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitors blocked SDF-1α-induced endothelial migration. Furthermore, knockdown of either NOX5 or p22phox (a requisite subunit for NOX1/2/4 activation) significantly impaired endothelial motility and tube formation, suggesting that multiple NOXs regulate SDF-1α-dependent angiogenesis. Our previous study demonstrated that c-Jun N-terminal kinase 3 activity is essential for SDF-1α-dependent angiogenesis. Here, we identified that NOX5 is the dominant NOX required for SDF-1α-induced c-Jun N-terminal kinase 3 activation and that NOX5 and MAP kinase phosphatase 7 (MKP7; the c-Jun N-terminal kinase 3 phosphatase) associate with one another but decrease this interaction on SDF-1α treatment. Furthermore, MKP7 activity was inhibited by SDF-1α, and this inhibition was relieved by NOX5 knockdown, indicating that NOX5 promotes c-Jun N-terminal kinase 3 activation by blocking MKP7 activity. CONCLUSIONS: We conclude that NOX is required for SDF-1α signaling and that intracellular redox balance is critical for SDF-1α-induced endothelial migration and angiogenesis.

Inhibition of the TNF-α-induced serine phosphorylation of IRS-1 at 636/639 by AICAR.

AMP-activated protein kinase (AMPK) contributes to the acceleration of insulin signaling. However, the mechanism by which AMPK regulates insulin signaling remains unclear. Serine phosphorylation of insulin receptor substrate (IRS)-1 negatively regulates insulin signaling. Here we investigated the role of AMPK in serine phosphorylation of IRS-1 at 636/639 and 307, which is induced by tumor necrosis factor (TNF)-α in 3T3L1 adipocytes. We demonstrated that the AMPK activator 5-aminoimidazole-4-carboxamide-1-d-ribofuranoside (AICAR) significantly inhibited the TNF-α-induced serine phosphorylation of IRS-1 at 636/639 and 307 by suppression of extracellular signal-regulated kinase (ERK) phosphorylation but not c-Jun-NH2-terminal kinase (JNK) phosphorylation. In addition, AICAR stimulation resulted in enhanced interaction between ERK and MAP kinase phosphatase-4 (DUSP9/MKP-4) without affecting DUSP9/MPK4 mRNA synthesis. Moreover, intraperitoneal administration (0.25 g/kg) of AICAR to db/db mice improved blood glucose levels and inhibited the phosphorylation of ERK in adipose tissue. In conclusion, we propose a new mechanism in which AICAR suppresses TNF-α-induced serine phosphorylation of IRS-1 at 636/639 and 307 by enhancing the interaction between ERK and DUSP9/MKP-4. Taken together, these findings provide evidence that AMPK plays a crucial role in improving of type 2 diabetes.

The dual-specificity phosphatase DUSP14 negatively regulates tumor necrosis factor- and interleukin-1-induced nuclear factor-κB activation by dephosphorylating the protein kinase TAK1.

The transcription factor NF-κB is critically involved in the inflammatory response triggered by the proinflammatory cytokines TNF and IL-1. Various studies have demonstrated that activation of TAK1 (TGF-ß-activated kinase 1) is an essential step in TNF- and IL-1-induced NF-κB activation pathways. In this study, we identified a member of the dual-specificity phosphatase family, DUSP14, as a negative regulator of TNF- and IL-1-triggered NF-κB activation by expression screens. We found that DUSP14 interacted with TAK1 and that this interaction was enhanced by TNF or IL-1 stimulation. Overexpression of DUSP14 dephosphorylated TAK1 at Thr-187, a residue in the activation loop critically involved in TAK1 activation. Knockdown of DUSP14 increased basal as well as TNF- and IL-1-induced TAK1 phosphorylation at Thr-187. Overexpression of DUSP14, but not its phosphatase-deficient mutant, inhibited TNF- and IL-1-induced as well as TAK1-mediated NF-κB activation, whereas knockdown of DUSP14 had opposite effects. These findings suggest that DUSP14 negatively regulates TNF- or IL-1-induced NF-κB activation by dephosphorylating TAK1 at Thr-187. Our study reveals a new post-translational regulatory mechanism of NF-κB activation triggered by the proinflammatory cytokines.

Dual specificity phosphatases 10 and 16 are positive regulators of EGF-stimulated ERK activity: indirect regulation of ERK signals by JNK/p38 selective MAPK phosphatases.

We have explored the possible role of dual specificity phosphatases (DUSPs) on acute EGF-mediated ERK signalling using high content imaging and a delayed MEK inhibition protocol to distinguish direct and indirect effects of the phosphatases on ERK activity. Using siRNAs, we were unable to find evidence that any of the MAPK phosphatases (MKPs) expressed in HeLa cells acts directly to dephosphorylate ppERK1/2 (dual phosphorylated ERKs 1 and/or 2) in the acute time-frame tested (0-14 min). Nevertheless, siRNAs against two p38/JNK MKPs (DUSPs 10 and 16) inhibited acute EGF-stimulated ERK activation. No such effect was seen for acute effects of the protein kinase C activator PDBu (phorbol 12,13 dibutyrate) on ERK activity, although effects of EGF and PDBu on ERK-dependent transcription (Egr-1 luciferase activity) were both reduced by siRNA targeting DUSPs 10 and 16. Inhibition of EGF-stimulated ERK activity by these siRNAs was reversed by pharmacological inhibition of p38 MAPK and single cell analysis revealed that the siRNAs did not influence the nuclear-cytoplasmic distribution of ppERK1/2. Thus, DUSPs 10 and 16 are positive regulators of activation, apparently acting by modulating cross-talk between the p38 and ERK pathways. A simplified mathematical model of this scenario accurately predicted the experimental data, supporting the conclusion that the major mechanism by which MKPs influence acute EGF-stimulated ERK responses is the negative regulation of p38, resulting in the positive regulation of ERK phosphorylation and activity.

The dual-specificity MAP kinase phosphatases: critical roles in development and cancer.

Intracellular signaling by mitogen-activated protein (MAP) kinases (MAPK) is involved in many cellular responses and in the regulation of various physiological and pathological conditions. Tight control of the localization and duration of extracellular-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), or p38 MAPK activity is thus a fundamental aspect of cell biology. Several members of the dual-specificity phosphatase (DUSPs) family are able to dephosphorylate MAPK isoforms with different specificity, cellular, and tissue localization. Understanding how these phosphatases are themselves regulated during development or in physiological and pathological conditions is therefore fundamental. Over the years, gene deletion and knockdown studies have completed initial in vitro studies and shed a new light on the global and specific roles of DUSPs in vivo. Whereas DUSP1, DUSP2, and DUSP10 appear as crucial players in the regulation of immune responses, other members of the family, like the ERK-specific DUSP6, were shown to play a major role in development. Recent findings on the involvement of DUSPs in cancer progression and resistance will also be discussed.

DUSP26 negatively affects the proliferation of epithelial cells, an effect not mediated by dephosphorylation of MAPKs.

Dual specificity phosphatases are characterised by their ability to dephosphorylate both phosphotyrosine and phosphoserine/threonine residues within the one substrate. The aim of this study was to characterise the phosphatase activity of the atypical dual specificity phosphatase, DUSP26 on MAP kinases, and to determine its expression, regulation and function in cancer cells. Overexpression and knockdown of DUSP26 in epithelial cells and in vitro phosphatase assays were used to demonstrate that, contrary to several published reports, DUSP26 does not act as a dual specificity phosphatase on ERK, JNK or p38 MAPKs. However, overexpression of DUSP26 in MCF10A epithelial cells suppressed colony formation and acinar growth in 3D culture, effects dependent on its phosphatase activity, while knockdown of DUSP26 in HOSE17.1 cells enhanced colony formation and cellular proliferation. DUSP26 mRNA expression was reduced in neuroblastoma, brain and ovarian cancer cell lines. Consistent with epigenetic silencing of DUSP26, expression was enhanced by treatment of cells with 5-aza-2-deoxycitidine and trichostatin A, and a CpG island upstream of the DUSP26 transcriptional start site was variably methylated in cancer cell lines. Together, these results help to clarify confusion in the literature relating to DUSP26 substrate specificity and support recent reports that substrates other than MAPKs are the primary substrates of this phosphatase. In addition, they indicate that DUSP26 may function as a tumour suppressor in particular cancers.

Epigenetic downregulation of mitogen-activated protein kinase phosphatase MKP-2 relieves its growth suppressive activity in glioma cells.

Critical tumor suppression pathways in brain tumors have yet to be fully defined. Along with mutational analyses, genome-wide epigenetic investigations may reveal novel suppressor elements. Using differential methylation hybridization, we identified a CpG-rich region of the promoter of the dual-specificity mitogen-activated protein kinase phosphatase-2 gene (DUSP4/MKP-2) that is hypermethylated in gliomas. In 83 astrocytic gliomas and 5 glioma cell lines examined, hypermethylation of the MKP-2 promoter was found to occur relatively more frequently in diffuse or anaplastic astrocytomas and secondary glioblastomas relative to primary glioblastomas. MKP-2 hypermethylation was associated with mutations in TP53 and IDH1, exclusive of EGFR amplification, and with prolonged survival of patients with primary glioblastoma. Expression analysis established that promoter hypermethylation correlated with reduced expression of MKP-2 mRNA and protein. Consistent with a regulatory role, reversing promoter hypermethylation by treating cells with 5-aza-2'-deoxycytidine increased MKP-2 mRNA levels. Furthermore, we found that glioblastoma cell growth was inhibited by overexpression of exogenous MKP-2. Our findings reveal MKP-2 as a common epigenetically silenced gene in glioma, the inactivation of which may play a significant role in glioma development.

MAPK phosphatase AP2C3 induces ectopic proliferation of epidermal cells leading to stomata development in Arabidopsis.

In plant post-embryonic epidermis mitogen-activated protein kinase (MAPK) signaling promotes differentiation of pavement cells and inhibits initiation of stomata. Stomata are cells specialized to modulate gas exchange and water loss. Arabidopsis MAPKs MPK3 and MPK6 are at the core of the signaling cascade; however, it is not well understood how the activity of these pleiotropic MAPKs is constrained spatially so that pavement cell differentiation is promoted only outside the stomata lineage. Here we identified a PP2C-type phosphatase termed AP2C3 (Arabidopsis protein phosphatase 2C) that is expressed distinctively during stomata development as well as interacts and inactivates MPK3, MPK4 and MPK6. AP2C3 co-localizes with MAPKs within the nucleus and this localization depends on its N-terminal extension. We show that other closely related phosphatases AP2C2 and AP2C4 are also MAPK phosphatases acting on MPK6, but have a distinct expression pattern from AP2C3. In accordance with this, only AP2C3 ectopic expression is able to stimulate cell proliferation leading to excess stomata development. This function of AP2C3 relies on the domains required for MAPK docking and intracellular localization. Concomitantly, the constitutive and inducible AP2C3 expression deregulates E2F-RB pathway, promotes the abundance and activity of CDKA, as well as changes of CDKB1;1 forms. We suggest that AP2C3 downregulates the MAPK signaling activity to help maintain the balance between differentiation of stomata and pavement cells.

Cannabinoid receptor type 2 activation induces a microglial anti-inflammatory phenotype and reduces migration via MKP induction and ERK dephosphorylation.

BACKGROUND: Cannabinoid receptor type 2 (CBR2) inhibits microglial reactivity through a molecular mechanism yet to be elucidated. We hypothesized that CBR2 activation induces an anti-inflammatory phenotype in microglia by inhibiting extracellular signal-regulated kinase (ERK) pathway, via mitogen-activated protein kinase-phosphatase (MKP) induction. MKPs regulate mitogen activated protein kinases, but their role in the modulation of microglial phenotype is not fully understood. RESULTS: JWH015 (a CBR2 agonist) increased MKP-1 and MKP-3 expression, which in turn reduced p-ERK1/2 in LPS-stimulated primary microglia. These effects resulted in a significant reduction of tumor necrosis factor-alpha (TNF) expression and microglial migration. We confirmed the causative link of these findings by using MKP inhibitors. We found that the selective inhibition of MKP-1 by Ro-31-8220 and PSI2106, did not affect p-ERK expression in LPS+JWH015-treated microglia. However, the inhibition of both MKP-1 and MKP-3 by triptolide induced an increase in p-ERK expression and in microglial migration using LPS+JWH015-treated microglia. CONCLUSION: Our results uncover a cellular microglial pathway triggered by CBR2 activation. These data suggest that the reduction of pro-inflammatory factors and microglial migration via MKP-3 induction is part of the mechanism of action of CBR2 agonists. These findings may have clinical implications for further drug development.

Transcriptional profiling of endogenous germ layer precursor cells identifies dusp4 as an essential gene in zebrafish endoderm specification.

A major goal for developmental biologists is to define the behaviors and molecular contents of differentiating cells. We have devised a strategy for isolating cells from diverse embryonic regions and stages in the zebrafish, using computer-guided laser photoconversion of injected Kaede protein and flow cytometry. This strategy enabled us to perform a genome-wide transcriptome comparison of germ layer precursor cells. Mesendoderm and ectoderm precursors cells isolated by this method differentiated appropriately in transplantation assays. Microarray analysis of these cells reidentified known genes at least as efficiently as previously reported strategies that relied on artificial mesendoderm activation or inhibition. We also identified a large set of uncharacterized mesendoderm-enriched genes as well as ectoderm-enriched genes. Loss-of-function studies revealed that one of these genes, the MAP kinase inhibitor dusp4, is essential for early development. Embryos injected with antisense morpholino oligonucleotides that targeted Dusp4 displayed necrosis of head tissues. Marker analysis during late gastrulation revealed a specific loss of sox17, but not of other endoderm markers, and analysis at later stages revealed a loss of foregut and pancreatic endoderm. This specific loss of sox17 establishes a new class of endoderm specification defect.

Microtubule disruption and tumor suppression by mitogen-activated protein kinase phosphatase 4.

The extracellular signal-regulated kinase (Erk) is one of the downstream effectors of the Ras pathway whose activation is essential for the proliferation and survival of cancer cells. Erk activation is negatively regulated by mitogen-activated protein kinase (MAPK) phosphatases (MKP), which are generally up-regulated by Erk activation, thus forming a feedback loop for regulation of Erk activity. In searching for early alterations in the Ras pathway in epidermal carcinogenesis, we identified MKP4, a cytosolic MKP with specificity to not only Erk, but also, to a lesser extent, c-jun-NH(2)-kinase and p38. MKP4 is down-regulated at initiation and lost at malignant conversion in a clonal model of epidermal carcinogenesis that lacks Ras mutation. The loss of MKP4 was associated with squamous cell carcinoma (SCC) but not benign papilloma clonal lineages and with independently induced SCC relative to benign tumors in mouse skin. Reconstitution of MKP4 expression in malignant tumor cells leads to cell death and tumor suppression. Unlike Erk inhibition that blocks cell cycle entry, MKP4 reconstitution resulted in G(2)-M associated cell death and microtubule disruption. Thus, microtubule disruption by MKP4 provides a novel mechanism for tumor suppression by a cytosolic MKP and implies a novel therapeutic strategy through combined MAPK inhibitions that mimic the function of MKP4.

Role of mitogen-activated protein kinase phosphatases (MKPs) in cancer.

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are a family of dual-specificity protein phosphatases that dephosphorylate both phospho-threonine and phospho-tyrosine residues in MAP kinases, including the c-Jun N-terminal protein kinase (JNK)/stress-activated protein kinase (SAPK), the p38 MAPK, and the extracellular signal-related kinase (ERK). Since phosphorylation is required for the activation of MAP kinases, dephosphorylation by MKPs inhibits MAPK activity, thereby negatively regulating MAPK signaling. It is known that deregulation of MAPK signaling is the most common alteration in human cancers. Recent studies have suggested that MKPs play an important role not only in the development of cancers, but also in the response of cancer cells to chemotherapy. Thus, understanding the roles of MKPs in the development of cancer and their impact on chemotherapy can be exploited for therapeutic benefits for the treatment of human cancer.