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1.  Distinct, Constitutively Active MAPK Phosphatases Function in Xenopus Oocytes: Implications for p42 MAPK Regulation In Vivo 
Molecular Biology of the Cell  1999;10(11):3729-3743.
Xenopus oocyte maturation requires the phosphorylation and activation of p42 mitogen-activated protein kinase (MAPK). Likewise, the dephosphorylation and inactivation of p42 MAPK are critical for the progression of fertilized eggs out of meiosis and through the first mitotic cell cycle. Whereas the kinase responsible for p42 MAPK activation is well characterized, little is known concerning the phosphatases that inactivate p42 MAPK. We designed a microinjection-based assay to examine the mechanism of p42 MAPK dephosphorylation in intact oocytes. We found that p42 MAPK inactivation is mediated by at least two distinct phosphatases, an unidentified tyrosine phosphatase and a protein phosphatase 2A–like threonine phosphatase. The rates of tyrosine and threonine dephosphorylation were high and remained constant throughout meiosis, indicating that the dramatic changes in p42 MAPK activity seen during meiosis are primarily attributable to changes in MAPK kinase activity. The overall control of p42 MAPK dephosphorylation was shared among four partially rate-determining dephosphorylation reactions, with the initial tyrosine dephosphorylation of p42 MAPK being the most critical of the four. Our findings provide biochemical and kinetic insight into the physiological mechanism of p42 MAPK inactivation.
PMCID: PMC25672  PMID: 10564268
2.  Alzheimer’s amyloid-β toxicity and tau hyperphosphorylation are linked via RCAN1 
Amyloid-β peptide (Aβ) toxicity and tau hyperphosphorylation are hallmarks of Alzheimer’s disease (AD). How their molecular relationships may affect the etiology, progression, and severity of the disease, however, has not been elucidated. We now report that incubation of foetal rat cortical neurons with Aβ up-regulates expression of the Regulator of Calcineurin gene RCAN1, and this is mediated by Aβ-induced oxidative stress. Calcineurin (PPP3CA) is a serine-threonine phosphatase that dephosphorylates tau. RCAN1 proteins inhibit this phosphatase activity of calcineurin. Increased expression of RCAN1 also causes up-regulation of glycogen synthase kinase-3beta (GSK3β), a tau kinase. Thus, increased RCAN1 expression might be expected to decrease phospho-tau dephosphorylation (via calcineurin inhibition) and increase tau phosphorylation (via increased GSK3β activity). We find that, indeed, incubation of primary cortical neurons with Aβ results in increased phosphorylation of tau, unless RCAN1 gene expression is silenced, or antioxidants are added. Thus we propose a mechanism to link Aβ toxicity and tau hyperphosphorylation in AD: In our hypothesis, Aβ causes mitochondrial oxidative stress and increases production of reactive oxygen species, which result in an up-regulation of RCAN1 gene expression. RCAN1 proteins then both inhibit calcineurin and induce expression of GSK3β. Both mechanisms shift tau to a hyperphosphorylated state. We also find that lymphocytes from persons whose ApoE genotype is ε4/ε4 (with high risk of developing AD) show higher levels of RCAN1 and phospho-tau than those carrying the ApoE ε3/ε3 or ε3/ε4 genotypes. Thus up-regulation of RCAN1 may be a valuable bio-marker for Alzheimer’s disease risk.
doi:10.3233/JAD-2011-110890
PMCID: PMC3690537  PMID: 21876249
oxidative stress; beta-amyloid toxicity; Alzheimer’s disease; RCAN1
3.  Mitogen-Activated Protein Kinases and Reactive Oxygen Species: How Can ROS Activate MAPK Pathways? 
Journal of Signal Transduction  2011;2011:792639.
Mitogen-activated protein kinases (MAPKs) are serine-threonine protein kinases that play the major role in signal transduction from the cell surface to the nucleus. MAPKs, which consist of growth factor-regulated extracellular signal-related kinases (ERKs), and the stress-activated MAPKs, c-jun NH2-terminal kinases (JNKs) and p38 MAPKs, are part of a three-kinase signaling module composed of the MAPK, an MAPK kinase (MAP2K) and an MAPK kinase (MAP3K). MAP3Ks phosphorylate MAP2Ks, which in turn activate MAPKs. MAPK phosphatases (MKPs), which recognize the TXY amino acid motif present in MAPKs, dephosphorylate and deactivate MAPKs. MAPK pathways are known to be influenced not only by receptor ligand interactions, but also by different stressors placed on the cell. One type of stress that induces potential activation of MAPK pathways is the oxidative stress caused by reactive oxygen species (ROS). Generally, increased ROS production in a cell leads to the activation of ERKs, JNKs, or p38 MAPKs, but the mechanisms by which ROS can activate these kinases are unclear. Oxidative modifications of MAPK signaling proteins and inactivation and/or degradation of MKPs may provide the plausible mechanisms for activation of MAPK pathways by ROS, which will be reviewed in this paper.
doi:10.1155/2011/792639
PMCID: PMC3100083  PMID: 21637379
4.  Stress-activated Protein Kinase-mediated Down-Regulation of the Cell Integrity Pathway Mitogen-activated Protein Kinase Pmk1p by Protein Phosphatases 
Molecular Biology of the Cell  2007;18(11):4405-4419.
Fission yeast mitogen-activated protein kinase (MAPK) Pmk1p is involved in morphogenesis, cytokinesis, and ion homeostasis as part of the cell integrity pathway, and it becomes activated under multiple stresses, including hyper- or hypotonic conditions, glucose deprivation, cell wall-damaging compounds, and oxidative stress. The only protein phosphatase known to dephosphorylate and inactivate Pmk1p is Pmp1p. We show here that the stress-activated protein kinase (SAPK) pathway and its main effector, Sty1p MAPK, are essential for proper deactivation of Pmk1p under hypertonic stress in a process regulated by Atf1p transcription factor. We demonstrate that tyrosine phosphatases Pyp1p and Pyp2p, and serine/threonine phosphatase Ptc1p, that negatively regulate Sty1p activity and whose expression is dependent on Sty1p-Atf1p function, are involved in Pmk1p dephosphorylation under osmostress. Pyp1p and Ptc1p, in addition to Pmp1p, also control the basal level of MAPK Pmk1p activity in growing cells and associate with, and dephosphorylate Pmk1p both in vitro and in vivo. Our results with Ptc1p provide the first biochemical evidence for a PP2C-type phosphatase acting on more than one MAPK in yeast cells. Importantly, the SAPK-dependent down-regulation of Pmk1p through Pyp1p, Pyp2p, and Ptc1p was not complete, and Pyp1p and Ptc1p phosphatases are able to negatively regulate MAPK Pmk1p activity by an alternative regulatory mechanism. Our data also indicate that Pmk1p phosphorylation oscillates as a function of the cell cycle, peaking at cell separation during cytokinesis, and that Pmp1p phosphatase plays a main role in regulating this process.
doi:10.1091/mbc.E07-05-0484
PMCID: PMC2043569  PMID: 17761528
5.  Extracellular Signal-Regulated Kinase (ERK) Activation and Mitogen-Activated Protein Kinase Phosphatase 1 Induction by Pulsatile Gonadotropin-Releasing Hormone in Pituitary Gonadotrophs 
Journal of Signal Transduction  2011;2012:198527.
The frequency of gonadotropin-releasing hormone (GnRH) pulse secreted from the hypothalamus differently regulates the expressions of gonadotropin subunit genes, luteinizing hormone β (LHβ) and follicle-stimulating hormone β (FSHβ), in the pituitary gonadotrophs. FSHβ is preferentially stimulated at slower GnRH pulse frequencies, whereas LHβ is preferentially stimulated at more rapid pulse frequencies. Several signaling pathways are activated, including mitogen-activated protein kinase (MAPK), protein kinase C, calcium influx, and calcium-calmodulin kinases, and these may be preferentially regulated under certain conditions. Previous studies demonstrated that MAPK pathways, especially the extracellular signal-regulated kinase (ERK), play an essential role for induction of gonadotropin subunit gene expression by GnRH, whereas, MAPK phosphatases (MKPs) inactivate MAPKs through dephosphorylation of threonine and/or tyrosine residues. MKPs are also induced by GnRH, and potential feedback regulation between MAPK signaling and MKPs within the GnRH signaling pathway is evident in gonadotrophs. In this paper, we reviewed and mainly focused on our observations of the pattern of ERK activation and the induction of MKP by different frequencies of GnRH stimulation.
doi:10.1155/2012/198527
PMCID: PMC3253478  PMID: 22235371
6.  Regulation of the Saccharomyces cerevisiae HOG1 mitogen-activated protein kinase by the PTP2 and PTP3 protein tyrosine phosphatases. 
Molecular and Cellular Biology  1997;17(3):1289-1297.
In response to increases in extracellular osmolarity, Saccharomyces cerevisiae activates the HOG1 mitogen-activated protein kinase (MAPK) cascade, which is composed of a pair of redundant MAPK kinase kinases, namely, Ssk2p and Ssk22p, the MAPK kinase Pbs2p, and the MAPK Hog1p. Hog1p is activated by Pbs2p through phosphorylation of specific threonine and tyrosine residues. Activated Hog1p is essential for survival of yeast cells at high osmolarity. However, expression of constitutively active mutant kinases, such as those encoded by SSK2deltaN and PBS2(DD), is toxic and results in a lethal level of Hog1p activation. Overexpression of the protein tyrosine phosphatase Ptp2p suppresses the lethality of these mutations by dephosphorylating Hog1p. A catalytically inactive Cys-to-Ser Ptp2p mutant (Ptp2(C/S)p) is tightly bound to tyrosine-phosphorylated Hog1p in vivo. Disruption of PTP2 leads to elevated levels of tyrosine-phosphorylated Hog1p following exposure of cells to high osmolarity. Disruption of both PTP2 and another protein tyrosine phosphatase gene, PTP3, results in constitutive Hog1p tyrosine phosphorylation even in the absence of increased osmolarity. Thus, Ptp2p and Ptp3p are the major phosphatases responsible for the tyrosine dephosphorylation of Hog1p. When catalytically inactive Hog1(K/N)p is expressed in hog1delta cells, it is constitutively tyrosine phosphorylated. In contrast, Hog1(K/N)p, expressed together with wild-type Hog1p, is tyrosine phosphorylated only when cells are exposed to high osmolarity. Thus, the kinase activity of Hog1p is required for its own tyrosine dephosphorylation. Northern blot analyses suggest that Hog1p regulates Ptp2p and/or Ptp3p activity at the posttranscriptional level.
PMCID: PMC231854  PMID: 9032256
7.  Regulation of Angiopoietin‐1/Tie‐2 Receptor Signaling in Endothelial Cells by Dual‐Specificity Phosphatases 1, 4, and 5 
Background
Angiopoietin‐1 (Ang‐1) promotes survival and migration of endothelial cells, in part through the activation of mitogen‐activated protein kinase (MAPK) pathways downstream of Tie‐2 receptors. Dual‐specificity phosphatases (DUSPs) dephosphorylate phosphotyrosine and phosphoserine/phosphothreonine residues on target MAPKs. The mechanisms by which DUSPs modulate MAPK activation in Ang‐1/Tie‐2 receptor signaling are unknown in endothelial cells.
Methods and Results
Expression of various DUSPs in human umbilical vein endothelial cells exposed to Ang‐1 was measured. The functional roles of DUSPs in Ang‐1‐induced regulation of MAPK activation, endothelial cell survival, migration, differentiation, and permeability were measured using selective siRNA oligos. Ang‐1 differentially induces DUSP1, DUSP4, and DUSP5 in human umbilical vein endothelial cells through activation of the PI‐3 kinase, ERK1/2, p38, and SAPK/JNK pathways. Lack‐of‐function siRNA screening revealed that DUSP1 preferentially dephosphorylates p38 protein and is involved in Ang‐1‐induced cell migration and differentiation. DUSP4 preferentially dephosphorylates ERK1/2, p38, and SAPK/JNK proteins and, under conditions of serum deprivation, is involved in Ang‐1‐induced cell migration, several antiapoptotic effects, and differentiation. DUSP5 preferentially dephosphorylates ERK1/2 proteins and is involved in cell survival and inhibition of permeability.
Conclusions
DUSP1, DUSP4, and DUSP5 differentially modulate MAPK signaling pathways downstream of Tie‐2 receptors, thus highlighting the importance of these phosphatases to Ang‐1‐induced angiogenesis.
doi:10.1161/JAHA.113.000571
PMCID: PMC3886752  PMID: 24308939
angiogenesis; angiopoietin‐1; apoptosis; dual‐specificity phosphatases; endothelial cells; mitogen‐activated protein kinases
8.  Mechanism of Okadaic Acid Induced Neuronal Death and the Effect of Estrogens 
Journal of neurochemistry  2008;108(3):732-740.
Serine/threonine protein phosphatases (PPs) are important mediators of general cellular function as well as neurodegenerative processes. We have previously shown inhibition of PPs to be as neurotoxic as glutamate-induced neuronal death but resistant to neuroprotection by estrogens. In this study, the mechanism by which phosphatase inhibition via okadaic acid (OA) induce neurotoxicity is explored. Neurons were exposed to OA or glutamate in the presence or absence of various protein kinases inhibitors, and/or one of four estrogens. Both OA and glutamate induced cell death via increased reactive oxygen species (ROS), protein carbonylation, lipid peroxidation, caspase-3 activity, and mitochondrial dysfunction. All estrogens attenuated glutamate-mediated responses, but not OA-induced responses. In addition, inhibition of PKC and MAPK pathway was neuroprotective against glutamate but not OA toxicity. Interestingly, inhibition of MAPK pathway with PD98096 or U0126 caused a decrease in ROS production suggesting that activation of ERK1/2 could further exacerbate the oxidative stress caused by glutamate-induced toxicity; however, these inhibitors had no effect on OA-induced toxicity. Collectively, these results indicate that both glutamate and OA neurotoxicities are mediated by persistent activation of ERK1/2 and/or PKC and a resulting oxidative stress, and that protein phosphatase activity is an important and necessary aspect of estrogen-mediated neuroprotection.
doi:10.1111/j.1471-4159.2008.05805.x
PMCID: PMC2727740  PMID: 19054278
estradiol; estrogen analogues; okadaic acid; phosphatases; protein kinases; oxidative stress
9.  Negative Regulator of MAP Kinase is Increased in Depression and Is Necessary and Sufficient for Expression of Depressive Behavior 
Nature medicine  2010;16(11):1328-1332.
Lifetime prevalence (~16%)1 and the economic burden ($100 billion annually)2,3 associated with major depressive disorder (MDD) make it one of the most common and debilitating neurobiological illnesses. To date, the exact cellular and molecular mechanisms underlying the pathophysiology of MDD have not been identified. Here we use whole genome expression profiling of postmortem tissue and demonstrate significantly increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the hippocampal subfields of MDD subjects compared to matched controls. MKP-1, also known as DUSP1, is a member of a family of dual-specificity phosphatases (DUSP) that dephosphorylate both threonine and tyrosine residues and thereby serves as a key negative regulator of MAPK cascade4, a major signaling pathway involved in neuronal plasticity, function and survival5,6. The significance of altered MKP-1 was tested in rodent models of depression and demonstrates that increased hippocampal MKP-1 expression, as a result of stress or viral-mediated gene transfer, causes depressive behaviors. Conversely, chronic antidepressant treatment normalizes the stress-induced MKP-1 expression and behavior, and mice lacking MKP-1 are resilient to stress. These postmortem and preclinical studies identify MKP-1 as a critical factor in MDD pathophysiology and as a novel target for therapeutic interventions.
doi:10.1038/nm.2219
PMCID: PMC3066515  PMID: 20953200
depression; hippocampus; postmortem; stress; rat; mouse
10.  Tauroursodeoxycholic Acid Prevents Amyloid-β Peptide–Induced Neuronal Death Via a Phosphatidylinositol 3-Kinase–Dependent Signaling Pathway 
Molecular Medicine  2003;9(9-12):226-234.
Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, modulates cell death by interrupting classic pathways of apoptosis. Amyloid-β (Aβ) peptide has been implicated in the pathogenesis of Alzheimer’s disease, where a significant loss of neuronal cells is thought to occur by apoptosis. In this study, we explored the cell death pathway and signaling mechanisms involved in Aβ-induced toxicity and further investigated the anti-apoptotic effect(s) of TUDCA. Our data show significant induction of apoptosis in isolated cortical neurons incubated with Aβ peptide. Apoptosis was associated with translocation of pro-apoptotic Bax to the mitochondria, followed by cytochrome c release, caspase activation, and DNA and nuclear fragmentation. In addition, there was almost immediate but weak activation of the serine/threonine protein kinase Akt. Inhibition of the phosphatidylinositide 3′-OH kinase (PI3K) pathway with wortmannin did not markedly affect Aβ-induced cell death, suggesting that this signaling pathway is not crucial for Aβ-mediated toxicity. Notably, co-incubation with TUDCA significantly modulated each of the Aβ-induced apoptotic events. Moreover, wortmannin decreased TUDCA protection against Aβ-induced apoptosis, reduced Akt phosphorylation, and increased Bax translocation to mitochondria. Together, these findings indicate that Aβ-induced apoptosis of cortical neurons proceeds through a Bax mitochondrial pathway. Further, the PI3K signaling cascade plays a role in regulating the anti-apoptotic effects of TUDCA.
PMCID: PMC1430980  PMID: 15208744
11.  The Pseudophosphatase MK-STYX Physically and Genetically Interacts with the Mitochondrial Phosphatase PTPMT1 
PLoS ONE  2014;9(4):e93896.
We previously performed an RNA interference (RNAi) screen and found that the knockdown of the catalytically inactive phosphatase, MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein], resulted in potent chemoresistance. Our follow-up studies demonstrated that knockdown of MK-STYX prevents cells from undergoing apoptosis through a block in cytochrome c release, but that MK-STYX does not localize proximal to the molecular machinery currently known to control this process. In an effort to define its molecular mechanism, we utilized an unbiased proteomics approach to identify proteins that interact with MK-STYX. We identified the mitochondrial phosphatase, PTPMT1 (PTP localized to mitochondrion 1), as the most significant and unique interaction partner of MK-STYX. We previously reported that knockdown of PTPMT1, an important component of the cardiolipin biosynthetic pathway, is sufficient to induce apoptosis and increase chemosensitivity. Accordingly, we hypothesized that MK-STYX and PTPMT1 interact and serve opposing functions in mitochondrial-dependent cell death. We confirmed that MK-STYX and PTPMT1 interact in cells and, importantly, found that MK-STYX suppresses PTPMT1 catalytic activity. Furthermore, we found that knockdown of PTPMT1 resensitizes MK-STYX knockdown cells to chemotherapeutics and restores the ability to release cytochrome c. Taken together, our data support a model in which MK-STYX controls apoptosis by negatively regulating PTPMT1. Given the important role of PTPMT1 in the production of cardiolipin and other phospholipids, this raises the possibility that dysregulated mitochondrial lipid metabolism may facilitate chemoresistance.
doi:10.1371/journal.pone.0093896
PMCID: PMC3977970  PMID: 24709986
12.  The Role of Specific Mitogen-Activated Protein Kinase Signaling Cascades in the Regulation of Steroidogenesis 
Journal of Signal Transduction  2011;2011:821615.
Mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine kinases that are activated by a large variety of extracellular stimuli and play integral roles in controlling many cellular processes, from the cell surface to the nucleus. The MAPK family includes four distinct MAPK cascades, that is, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, c-Jun N-terminal kinase or stress-activated protein kinase, and ERK5. These MAPKs are essentially operated through three-tiered consecutive phosphorylation events catalyzed by a MAPK kinase kinase, a MAPK kinase, and a MAPK. MAPKs lie in protein kinase cascades. The MAPK signaling pathways have been demonstrated to be associated with events regulating the expression of the steroidogenic acute regulatory protein (StAR) and steroidogenesis in steroidogenic tissues. However, it has become clear that the regulation of MAPK-dependent StAR expression and steroid synthesis is a complex process and is context dependent. This paper summarizes the current level of understanding concerning the roles of the MAPK signaling cascades in the regulation of StAR expression and steroidogenesis in different steroidogenic cell models.
doi:10.1155/2011/821615
PMCID: PMC3100650  PMID: 21637381
13.  Inhibition of RhoA GTPase and the subsequent activation of PTP1B protects cultured hippocampal neurons against amyloid β toxicity 
Background
Amyloid beta (Aβ) is the main agent responsible for the advent and progression of Alzheimer's disease. This peptide can at least partially antagonize nerve growth factor (NGF) signalling in neurons, which may be responsible for some of the effects produced by Aβ. Accordingly, better understanding the NGF signalling pathway may provide clues as to how to protect neurons from the toxic effects of Aβ.
Results
We show here that Aβ activates the RhoA GTPase by binding to p75NTR, thereby preventing the NGF-induced activation of protein tyrosine phosphatase 1B (PTP1B) that is required for neuron survival. We also show that the inactivation of RhoA GTPase and the activation of PTP1B protect cultured hippocampal neurons against the noxious effects of Aβ. Indeed, either pharmacological inhibition of RhoA with C3 ADP ribosyl transferase or the transfection of cultured neurons with a dominant negative form of RhoA protects cultured hippocampal neurons from the effects of Aβ. In addition, over-expression of PTP1B also prevents the deleterious effects of Aβ on cultured hippocampal neurons.
Conclusion
Our findings indicate that potentiating the activity of NGF at the level of RhoA inactivation and PTP1B activation may represent a new means to combat the noxious effects of Aβ in Alzheimer's disease.
doi:10.1186/1750-1326-6-14
PMCID: PMC3038970  PMID: 21294893
14.  Phosphatase-Dependent Regulation of Epithelial Mitogen-Activated Protein Kinase Responses to Toxin-Induced Membrane Pores 
PLoS ONE  2009;4(11):e8076.
Diverse bacterial species produce pore-forming toxins (PFT) that can puncture eukaryotic cell membranes. Host cells respond to sublytic concentrations of PFT through conserved intracellular signaling pathways, including activation of mitogen-activated protein kinases (MAPK), which are critical to cell survival. Here we demonstrate that in respiratory epithelial cells p38 and JNK MAPK were phosphorylated within 30 min of exposure to pneumolysin, the PFT from Streptococcus pneumoniae. This activation was tightly regulated, and dephosphorylation of both MAPK occurred within 60 min following exposure. Pretreatment of epithelial cells with inhibitors of cellular phosphatases, including sodium orthovanadate, calyculin A, and okadaic acid, prolonged and intensified MAPK activation. Specific inhibition of MAPK phosphatase-1 did not affect the kinetics of MAPK activation in PFT-exposed epithelial cells, but siRNA-mediated knockdown of serine/threonine phosphatases PP1 and PP2A were potent inhibitors of MAPK dephosphorylation. These results indicate an important role for PP1 and PP2A in termination of epithelial responses to PFT and only a minor contribution of dual-specificity phosphatases, such as MAPK phosphatase-1, which are the major regulators of MAPK signals in other cell types. Epithelial regulation of MAPK signaling in response to membrane disruption involves distinct pathways and may require different strategies for therapeutic interventions.
doi:10.1371/journal.pone.0008076
PMCID: PMC2778951  PMID: 19956644
15.  Regulation of JNK and p38 MAPK in the immune system: Signal integration, propagation and termination 
Cytokine  2009;48(3):161-169.
Stress-activated MAP kinases (MAPKs), comprised of JNK and p38, play prominent roles in the innate and adaptive immune systems. Activation of MAPKs is mediated by a three-tiered kinase module comprised of MAPK kinase kinases (MAP3Ks), MAPK kinases (MAP2Ks) and MAPKs through sequential protein phosphorylation. Activated MAPKs, in turn, phosphorylate transcription factors and other targets to regulate gene transcription and immune responses. Recent studies have provided new insight into the upstream and downstream components of the MAPK pathway that facilitate the activation and propagation of MAPK signaling in immune responses. Moreover, MAPK activity is negatively regulated by MAPK phosphatases (MKPs), a group of dual-specificity phosphatases that dephosphorylate and inactivate the MAPKs. Here we discuss the recent advances in our understanding of these regulatory processes in MAPK signaling with a focus on their impacts on immune function.
doi:10.1016/j.cyto.2009.08.002
PMCID: PMC2782697  PMID: 19740675
Kinase; phosphatase; signaling; receptor; immunity
16.  Overproduction, purification and structure determination of human dual-specificity phosphatase 14 
The crystal structure of human dual-specificity phosphatase 14, DUSP14 (MKP6), in complex with a phosphate ion has been determined and refined to 1.88 Å resolution.
Dual-specificity phosphatases (DUSPs) are enzymes that participate in the regulation of biological processes such as cell growth, differentiation, transcription and metabolism. A number of DUSPs are able to dephosphorylate phosphoryl­ated serine, threonine and tyrosine residues on mitogen-activated protein kinases (MAPKs) and thus are also classified as MAPK phosphatases (MKPs). As an increasing number of DUSPs are being identified and characterized, there is a growing need to understand their biological activities at the molecular level. There is also significant interest in identifying DUSPs that could be potential targets for drugs that modulate MAPK-dependent signaling and immune responses, which have been implicated in a variety of maladies including cancer, infectious diseases and inflammatory disorders. Here, the overproduction, purification and crystal structure at 1.88 Å resolution of human dual-specificity phosphatase 14, DUSP14 (MKP6), are reported. This structural information should accelerate the study of DUSP14 at the molecular level and may also accelerate the discovery and development of novel therapeutic agents.
doi:10.1107/S0907444909023762
PMCID: PMC2748964  PMID: 19770498
dual-specificity phosphatases; MAPK phosphatases; DUSP14; MKP6
17.  Dephosphorylation of specific sites in the KIS domain leads to ubiquitin-mediated degradation of the tyrosine phosphatase STEP 
The Biochemical journal  2011;440(1):115-125.
Striatal-enriched phosphatase (STEP) is a non-receptor tyrosine phosphatase that is specifically expressed in neurons of the central nervous system. STEP regulates the activity of several effector molecules involved in synaptic plasticity and neuronal cell survival, including mitogen-activated protein kinases (MAPKs), Src family kinases and N-methyl-D-aspartic acid (NMDA) receptors. The critical role of STEP in regulating these effectors requires that its activity be tightly regulated. Previous studies demonstrated that the activity of STEP is regulated through reversible phosphorylation of a serine residue within the kinase interacting motif (KIM), by cAMP-dependent protein kinase A (PKA). Here we show that STEP is endogenously phosphorylated at two additional sites located within the kinase specificity sequences (KIS). Basal activity of ERK and p38 MAPKs plays an important role in the phosphorylation of these two sites. Dephosphorylation of these two sites leads to poly-ubiquitination and proteolytic degradation of STEP. Conversely, the proteasome inhibitors MG-132 and epoxomicin can stabilize STEP. The active form of STEP is more susceptible to degradation than the inactive form. Taken together our results establish that ubiquitin-dependent proteolysis could be a novel mechanism for terminating the activity of STEP irreversibly.
doi:10.1042/BJ20110240
PMCID: PMC3408964  PMID: 21777200
tyrosine phosphatase; striatal enriched phosphatase (STEP); kinase specificity sequence (KIS); kinase interacting motif (KIM); ubiquitination; proteasomal degradation
18.  Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection 
Journal of Virology  2013;87(1):621-635.
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), and KSHV activation of mitogen-activated protein kinases (MAPKs) initiates a number of key pathogenic determinants of KS. Direct inhibition of signal transduction as a therapeutic approach presents several challenges, and a better understanding of KSHV-induced mechanisms regulating MAPK activation may facilitate the development of new treatment or prevention strategies for KS. MAPK phosphatases, including dual-specificity phosphatase-1 (DUSP1), negatively regulate signal transduction and cytokine activation through MAPK dephosphorylation or interference with effector molecule binding to MAPKs, including the extracellular signal-regulated kinase (ERK). We found that ERK-dependent latent viral gene expression, the induction of promigratory factors, and cell invasiveness following de novo infection of primary human endothelial cells are in part dependent on KSHV suppression of DUSP1 expression during de novo infection. KSHV-encoded miR-K12-11 upregulates the expression of xCT (an amino acid transporter and KSHV fusion/entry receptor), and existing data indicate a role for xCT in the regulation of 14-3-3β, a transcriptional repressor of DUSP1. We found that miR-K12-11 induces endothelial cell secretion of promigratory factors and cell invasiveness through upregulation of xCT-dependent, 14-3-3β-mediated suppression of DUSP1. Finally, proof-of-principle experiments revealed that pharmacologic upregulation of DUSP1 inhibits the induction of promigratory factors and cell invasiveness during de novo KSHV infection. These data reveal an indirect role for miR-K12-11 in the regulation of DUSP1 and downstream pathogenesis.
doi:10.1128/JVI.01441-12
PMCID: PMC3536420  PMID: 23097457
19.  Inability of Serotonin to Activate the c-Jun N-terminal Kinase and p38 Kinase Pathways in Rat Aortic Vascular Smooth Muscle Cells 
BMC Pharmacology  2001;1:8.
Background
Serotonin (5-HT, 5-hydroxytryptamine) activates the Extracellular Signal-Regulated Kinase (ERK)/ Mitogen-Activated Protein Kinase (MAPK) pathways, in vascular smooth muscle cells. Parallel MAPK pathways, the c-Jun N-terminal Kinase (JNK) and p38 pathway, are activated by stimulators of the ERK/MAPK pathway. We hypothesized that 5-HT would activate the JNK and p38 pathways in rat vascular smooth muscle cells.
Results
Results were determined using standard Western analysis and phosphospecific JNK and p38 antibodies. No significant activation by 5-HT (10-9 – 10-5 M; 30 min) of the JNK or p38 pathways, as measured by protein phosphorylation, was observed in any of these experiments. These experiments were repeated in the presence of the serine/threonine phosphatase inhibitor okadaic acid (1 uM) and the tyrosine phosphatase inhibitor sodium orthovanadate (1 uM) to maximize any observable signal. Even under these optimized conditions, no activation of the JNK or p38 pathways by 5-HT was observed. Time course experiments (5-HT 10-5 M; 5 min, 15 min, 30 min and 60 min) showed no significant activation of JNK after incubation with 5-HT at any time point. However, we detected strong activation of JNK p54 and p46 (5- and 7 fold increases in bands p54 and p46, respectively over control levels) by anisomycin (500 ng/ml, 30 min). Similarly, a JNK activity assay failed to reveal activation of JNK by 5-HT, in contrast to the strong stimulation by anisomycin.
Conclusion
Collectively, these data support the conclusion that 5-HT does not activate the JNK or p38 pathways in rat vascular smooth muscle cells.
doi:10.1186/1471-2210-1-8
PMCID: PMC58586  PMID: 11667949
20.  Cross-talk between the p38α and JNK MAPK Pathways Mediated by MAP Kinase Phosphatase-1 Determines Cellular Sensitivity to UV Radiation* 
The Journal of Biological Chemistry  2010;285(34):25928-25940.
MAPK phosphatase-1 (DUSP1/MKP-1) is a mitogen and stress-inducible dual specificity protein phosphatase, which can inactivate all three major classes of MAPK in mammalian cells. DUSP1/MKP-1 is implicated in cellular protection against a variety of genotoxic insults including hydrogen peroxide, ionizing radiation, and cisplatin, but its role in the interplay between different MAPK pathways in determining cell death and survival is not fully understood. We have used pharmacological and genetic tools to demonstrate that DUSP1/MKP-1 is an essential non-redundant regulator of UV-induced cell death in mouse embryo fibroblasts (MEFs). The induction of DUSP1/MKP-1 mRNA and protein in response to UV radiation is mediated by activation of the p38α but not the JNK1 or JNK2 MAPK pathways. Furthermore, we identify MSK1 and -2 and their downstream effectors cAMP-response element-binding protein/ATF1 as mediators of UV-induced p38α-dependent DUSP1/MKP-1 transcription. Dusp1/Mkp-1 null MEFs display increased signaling through both the p38α and JNK MAPK pathways and are acutely sensitive to UV-induced apoptosis. This lethality is rescued by the reintroduction of wild-type DUSP1/MKP-1 and by a mutant of DUSP1/MKP-1, which is unable to bind to either p38α or ERK1/2, but retains full activity toward JNK. Importantly, whereas small interfering RNA-mediated knockdown of DUSP1/MKP-1 sensitizes wild-type MEFs to UV radiation, DUSP1/MKP-1 knockdown in MEFS lacking JNK1 and -2 does not result in increased cell death. Our results demonstrate that cross-talk between the p38α and JNK pathways mediated by induction of DUSP1/MKP-1 regulates the cellular response to UV radiation.
doi:10.1074/jbc.M110.117911
PMCID: PMC2923983  PMID: 20547488
Apoptosis; DNA Damage; Dual Specificity Phosphoprotein Phosphatase; JNK; MAP Kinases (MAPKs); p38 MAPK; Signal Transduction; DUSP1; MKP-1
21.  Role of mitogen-activated protein kinase in cardiac hypertrophy and heart failure 
BACKGROUND:
Mitogen-activated protein kinases (MAPKs) are involved in the regulation of various cellular responses including cell proliferation, differentiation and survival. Although MAPKs are activated by MAPK kinase and inactivated by phosphatases, different types of MAPKs, including extracellular signal-regulated kinases (ERK1 and 2), c-jun N-terminal protein kinases (JNK) and p38 kinases are known to participate in different signalling pathways. This article will review some salient features of the regulation and function of different forms of MAPKs in the heart. Furthermore, the status of cardiac MAPKs under different pathophysiological conditions will be described.
OBSERVATIONS:
A wide variety of external stimuli are known to activate MAPKs, which are then translocated from the cytoplasm to the nucleus and regulate cardiac gene expression by phosphorylating various transcriptional factors. By virtue of the involvement of ERK1/2 in hypertrophic response and of the stress-activated JNKs and p38 kinases in the process of apoptosis, MAPKs are considered to be intimately involved in cardiac remodelling. Both growth factors and phorbol esters have been shown to strongly activate ERK1/2, whereas the activation of JNKs and p38 kinases by these agents is weak. Although ischemia-reperfusion activates all types of MAPKs, JNKs and p38 kinases are mainly proapoptotic, whereas ERK1/2 are antiapoptotic.
CONCLUSIONS:
The activation of ERK1/2 is involved in signal transduction pathways associated with cardiac hypertrophy; however, the exact status of MAPKs in heart failure remains to be clearly defined. While both JNKs and p38 kinases appear to participate in the genesis of ischemia-reperfusion injury, ERK1/2 are considered to be cytoprotective.
PMCID: PMC2719157  PMID: 19649217
Cardiac hypertrophy; c-jun N-terminal protein kinases; Extracellular signal-regulated kinases; Heart failure; Mitogen-activated protein kinase; p38 kinases
22.  Dusp6(Mkp3) is a negative feedback regulator of FGF stimulated ERK signaling during mouse development 
Development (Cambridge, England)  2007;134(1):167-176.
Summary
Mitogen-activated protein kinase (MAPK) pathways are major mediators of extracellular signals that are transduced to the nucleus. MAPK signaling is attenuated at several levels and one class of dual-specificity phosphatases, the MAPK phosphatases (MKPs), inhibit MAPK signaling by dephosphorylating activated MAPKs. Several of the MKPs are themselves induced by the signaling pathways they regulate, forming negative feedback loops that attenuate the signals. We show here that in mouse embryos, Fibroblast growth factor receptors (FGFRs) are required for transcription of Dusp6, which encodes MKP3, an extracellular signal-regulated kinase (ERK)-specific MKP. Targeted inactivation of Dusp6 increases levels of phosphorylated ERK, as well as the pERK target, Erm, and transcripts initiated from the Dusp6 promoter itself. Finally, the Dusp6 mutant allele causes variably penetrant, dominant postnatal lethality, skeletal dwarfism, coronal craniosynostosis and hearing loss; phenotypes that are also characteristic of mutations that activate FGFRs inappropriately. Taken together, these results show that DUSP6 serves in vivo as a negative feedback regulator of FGFR signaling and suggest that mutations in DUSP6 or related genes are candidates for causing or modifying unexplained cases of FGFR-like syndromes.
doi:10.1242/dev.02701
PMCID: PMC2424197  PMID: 17164422
Mkp3; Pyst1; dual specificity phosphatase; craniosynostosis; middle ear; otic capsule
23.  Neuroprotective signaling pathways are modulated by adenosine in the anoxia tolerant turtle 
Cumulative evidence shows a protective role for adenosine A1 receptors (A1R) in hypoxia/ischemia; A1R stimulation reduces neuronal damage, whereas blockade exacerbates damage. The signal transduction pathways may involve the mitogen-activated protein kinase (MAPK) pathways and serine/threonine kinase (AKT), with cell survival depending on the timing and degree of upregulation of these cascades as well as the balance between pro-survival and pro-death pathways. Here, we show in vitro that extracellular signal-regulated kinase (ERK1/2) and phosphatidylinositol 3-kinase (PI3-K/AKT) activation is dependent on A1R stimulation, with further downstream effects that promote neuronal survival. Phosphorylated ERK1/2 (p-ERK) and AKT (p-AKT) as well as Bcl-2 are upregulated in anoxic neuronally enriched primary cultures from turtle brain. This native upregulation is further increased by the selective A1R agonist 2-chloro-N-cyclopentyladenosine (CCPA), whereas the selective antagonist 8-cyclopentyl-1,3-dihydropylxanthine (DPCPX) decreases p-ERK and p-AKT expression. Conversely, A1R antagonism resulted in increases in phosphorylated JNK (p-JNK), p38 (p-p38), and Bax. As pathological and adaptive changes occur simultaneously during anoxia/ischemia in mammalian neurons, the turtle provides an alternative model to analyze protective mechanisms in the absence of evident pathologies.
doi:10.1038/jcbfm.2010.109
PMCID: PMC3049502  PMID: 20648037
adenosine; AKT; anoxia; mitogen-activated protein kinase; turtle
24.  The pseudophosphatase MK-STYX interacts with G3BP and decreases stress granule formation 
Biochemical Journal  2010;427(Pt 3):349-357.
MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein] is a pseudophosphatase member of the dual-specificity phosphatase subfamily of the PTPs (protein tyrosine phosphatases). MK-STYX is catalytically inactive due to the absence of two amino acids from the signature motif that are essential for phosphatase activity. The nucleophilic cysteine residue and the adjacent histidine residue, which are conserved in all active dual-specificity phosphatases, are replaced by serine and phenylalanine residues respectively in MK-STYX. Mutations to introduce histidine and cysteine residues into the active site of MK-STYX generated an active phosphatase. Using MS, we identified G3BP1 [Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding protein-1], a regulator of Ras signalling, as a binding partner of MK-STYX. We observed that G3BP1 bound to native MK-STYX; however, binding to the mutant catalytically active form of MK-STYX was dramatically reduced. G3BP1 is also an RNA-binding protein with endoribonuclease activity that is recruited to ‘stress granules’ after stress stimuli. Stress granules are large subcellular structures that serve as sites of mRNA sorting, in which untranslated mRNAs accumulate. We have shown that expression of MK-STYX inhibited stress granule formation induced either by aresenite or expression of G3BP itself; however, the catalytically active mutant MK-STYX was impaired in its ability to inhibit G3BP-induced stress granule assembly. These results reveal a novel facet of the function of a member of the PTP family, illustrating a role for MK-STYX in regulating the ability of G3BP1 to integrate changes in growth-factor stimulation and environmental stress with the regulation of protein synthesis.
doi:10.1042/BJ20091383
PMCID: PMC2873733  PMID: 20180778
dual-specificity phosphatase; mitogen-activated protein kinase serine-; threonine- and tyrosine-specific phosphatase (MK-STYX); pseudophosphatase; Ras-GTPase-activating protein Src-homology-3-domain-binding protein-1 (G3BP1); stress granule; CMT, Charcot–Marie–Tooth; CRHSP-24, calcium–responsive heat-stable protein with a molecular mass of 24 kDa; Cy3, indocarbocyanine; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; D(U)SP, dual-specificity phosphatase; eIF, eukaryotic translation initiation factor; FBS, fetal bovine serum; FLI1, Friend leukaemia virus integration 1; GAP, GTPase-activating protein; G3BP, Ras-GAP SH3-domain-binding protein; GFP, green fluorescent protein; HEK-293 cell; human embryonic kidney cell; IA2, islet cell antigen 512; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; MKP, MAPK phosphatase; (MK-)STYX, (MAPK) phospho-serine/threonine/tyrosine-binding protein; MTM, myotubularin; MTMR, MTM-related protein; NA, numerical aperture; NTF, nuclear transport factor; p, phospho-; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; RPTP, receptor PTP; SH3, Src homology 3
25.  Mitogen-activated protein kinase phosphatase 1 regulates bone mass, osteoblast gene expression, and responsiveness to parathyroid hormone 
The Journal of endocrinology  2011;211(2):145-156.
Parathyroid hormone (PTH) signaling via PTH 1 receptor (PTH1R) involves mitogen-activated protein kinase (MAPK) pathways. MAPK phosphatase 1 (MKP1) dephosphorylates and inactivates MAPKs in osteoblasts, the bone-forming cells. We previously showed that PTH1R activation in differentiated osteoblasts upregulates MKP1 and downregulates pERK1/2–MAPK and cyclin D1. In this study, we evaluated the skeletal phenotype of Mkp1 knockout (KO) mice and the effects of PTH in vivo and in vitro. Microcomputed tomography analysis of proximal tibiae and distal femora from 12-week-old Mkp1 KO female mice revealed osteopenic phenotype with significant reduction (8–46%) in bone parameters compared with wild-type (WT) controls. Histomorphometric analysis showed decreased trabecular bone area in KO females. Levels of serum osteocalcin (OCN) were lower and serum tartrate-resistant acid phosphatase 5b (TRAP5b) was higher in KO animals. Treatment of neonatal mice with hPTH (1–34) for 3 weeks showed attenuated anabolic responses in the distal femora of KO mice compared with WT mice. Primary osteoblasts derived from KO mice displayed delayed differentiation determined by alkaline phosphatase activity, and reduced expressions of Ocn and Runx2 genes associated with osteoblast maturation and function. Cells from KO females exhibited attenuated PTH response in mineralized nodule formation in vitro. Remarkably, this observation was correlated with decreased PTH response of matrix Gla protein expression. Expressions of pERK1/2 and cyclin D1 were inhibited dramatically by PTH in differentiated osteoblasts from WT mice but much less in osteoblasts from Mkp1 KO mice. In conclusion, MKP1 is important for bone homeostasis, osteoblast differentiation and skeletal responsiveness to PTH.
doi:10.1530/JOE-11-0144
PMCID: PMC3783352  PMID: 21852324

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