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.
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.
oxidative stress; beta-amyloid toxicity; Alzheimer’s disease; RCAN1
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.
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.
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.
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.
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.
estradiol; estrogen analogues; okadaic acid; phosphatases; protein kinases; oxidative stress
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.
depression; hippocampus; postmortem; stress; rat; mouse
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.
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.
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.
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.
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.
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.
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.
dual-specificity phosphatases; MAPK phosphatases; DUSP14; MKP6
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.
Kinase; phosphatase; signaling; receptor; immunity
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.
tyrosine phosphatase; striatal enriched phosphatase (STEP); kinase specificity sequence (KIS); kinase interacting motif (KIM); ubiquitination; proteasomal degradation
Oxidative stress induced by glutathione depletion in the mouse HT22 neuroblastoma cell line and embryonic rat immature cortical neurons causes a delayed, sustained activation of extracellular signal-regulated kinases-1/2 (ERK1/2), which is required for cell death. This sustained activation of ERK1/2 is mediated primarily by a selective inhibition of distinct ERK1/2-directed phosphatases either by enhanced degradation (i.e. for Mitogen activated protein kinase [MAPK] Phosphatase-1) or as shown here by reductions in enzymatic activity (i.e. for Protein Phosphatase type 2A [PP-2A]). The inhibition of ERK1/2 phosphatases in HT22 cells and immature neurons subjected to glutathione depletion results from oxidative stress as phosphatase activity is restored in cells treated with the antioxidant butylated hydroxyanisole (BHA). This leads to reduced ERK1/2 activation and neuroprotection. Furthermore, an increase in free intracellular zinc that accompanies glutathione-induced oxidative stress in HT22 cells and immature neurons contributes to selective inhibition of ERK1/2 phosphatase activity and cell death. Finally, ERK1/2 also functions to maintain elevated levels of zinc. Thus the elevation of intracellular zinc within neurons subjected to oxidative stress can trigger a robust positive feedback loop operating through activated ERK1/2 that rapidly sets into motion a zinc-dependent pathway of cell death.
Background: Dual specificity protein phosphatases (DSPs) bind MAPKs through specific interaction motifs.
Results: A novel motif (IYT) in Msg5 mediates a common docking domain-independent interaction with the yeast cell integrity kinase Slt2.
Conclusion: Distinct mechanisms allow Msg5 to differentially bind Slt2 and Mlp1 or mating/pseudohyphal MAPKs Fus3 and Kss1.
Significance: Elucidation of the mechanisms by which MAPKs interact with key regulators is vital to understanding cell signaling.
MAPK phosphatases (MKPs) are negative regulators of signaling pathways with distinct MAPK substrate specificities. For example, the yeast dual specificity phosphatase Msg5 dephosphorylates the Fus3 and Slt2 MAPKs operating in the mating and cell wall integrity pathways, respectively. Like other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains. These include D-motifs, which contain basic residues that interact with acidic residues in the common docking (CD) domain of MAPKs. Here we show that Msg5 interacts not only with Fus3, Kss1, and Slt2 but also with the pseudokinase Slt2 paralog Mlp1. Using yeast two-hybrid and in vitro interaction assays, we have identified distinct regions within the N-terminal domain of Msg5 that differentially bind either the MAPKs Fus3 and Kss1 or Slt2 and Mlp1. Whereas a canonical D-site within Msg5 mediates interaction with the CD domains of Fus3 and Kss1, a novel motif (102IYT104) within Msg5 is involved in binding to Slt2 and Mlp1. Furthermore, mutation of this site prevents the phosphorylation of Msg5 by Slt2. This motif is conserved in Sdp1, another MKP that dephosphorylates Slt2, as well as in Msg5 orthologs from other yeast species. A region spanning amino acids 274–373 within Slt2 and Mlp1 mediates binding to this Msg5 motif in a CD domain-independent manner. In contrast, Slt2 uses its CD domain to bind to its upstream activator Mkk1. This binding flexibility may allow MAPK pathways to exploit additional regulatory controls in order to provide fine modulation of both pathway activity and specificity.
Cell Signaling; Dual Specificity Phosphoprotein Phosphatase; MAP Kinases (MAPKs); Protein-Protein Interactions; Yeast; Binding Motif; Mlp1; Slt2
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.
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.
Apoptosis; DNA Damage; Dual Specificity Phosphoprotein Phosphatase; JNK; MAP Kinases (MAPKs); p38 MAPK; Signal Transduction; DUSP1; MKP-1
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 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.
Collectively, these data support the conclusion that 5-HT does not activate the JNK or p38 pathways in rat vascular smooth muscle cells.
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.
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.
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.
Cardiac hypertrophy; c-jun N-terminal protein kinases; Extracellular signal-regulated kinases; Heart failure; Mitogen-activated protein kinase; p38 kinases
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.
adenosine; AKT; anoxia; mitogen-activated protein kinase; turtle
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.
Mkp3; Pyst1; dual specificity phosphatase; craniosynostosis; middle ear; otic capsule
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.
Amyloid precursor protein cleavage through β- and γ-secretases produces β-amyloid peptide, which is believed to be responsible for death of neurons and dementia in Alzheimer’s disease. Levels of β- and γ-secretase are increased in sensitive areas of the Alzheimer’s disease brain, but the mechanism of this process is unknown. In this review, we prove that brain ischemia generates expression and activity of both β- and γ-secretases. These secretases are induced in association with oxidative stress following brain ischemia. Data suggest that ischemia promotes overproduction and aggregation of β-amyloid peptide in brain, which is toxic for ischemic neuronal cells. In our review, we demonstrated the role of brain ischemia as a molecular link between the β- and the γ-secretase activities and provided a molecular explanation of the possible neuropathogenesis of sporadic Alzheimer’s disease.
Brain ischemia; Amyloid precursor protein; β-Amyloid peptide; α-Secretase; β-Secretase; γ-Secretase; Oxidative stress; Neuronal death; Alzheimer’s disease