Inactivation of dual-specificity phosphatases is involved in the regulation of extracellular signal-regulated kinases by heat shock and hsp72

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) dramatically enhance survival of cells exposed to heat shock. Using Cos-7 cells and primary human fibroblasts (IMR90 cells), we demonstrated that heat shock activates ERKs via two distinct mechanisms: stimulation of the ERK-activating kinases, MEK1/2, and inhibition of ERK dephosphorylation. Under milder heat shock conditions, activation of ERKs proceeded mainly through stimulation of MEK1/2, whereas under more severe heat shock MEK1/2 could no longer be activated and the inhibition of ERK phosphatases became critical. In Cos-7 cells, nontoxic heat shock caused rapid inactivation of the major ERK phosphatase, MKP-3, by promoting its aggregation, so that in cells exposed to 45 degrees C for 20 min, 90% of MKP-3 became insoluble. MKP-3 aggregation was reversible and, 1 h after heat shock, MKP-3 partially resolubilized. The redistribution of MKP-3 correlated with an increased rate of ERK dephosphorylation. Similar heat-induced aggregation, followed by partial resolubilization, was found with a distinct dual-specificity phosphatase MKP-1 but not with MKP-2. Therefore, MKP-3 and MKP-1 appeared to be critical heat-labile phosphatases involved in the activation of ERKs by heat shock. Expression of the major heat shock protein Hsp72 inhibited activation of MEK1/2 and prevented inactivation of MKP-3 and MKP-1. Hsp72DeltaEEVD mutant lacking a chaperone activity was unable to protect MKP-3 from heat inactivation but interfered with MEK1/2 activation similar to normal Hsp72. Hence, Hsp72 suppressed ERK activation by both protecting dual-specificity phosphatases, which was dependent on the chaperone activity, and suppressing MEK1/2, which was independent of the chaperone activity.     

Endoplasmic reticulum stress associated with extracellular aggregates. Evidence from transthyretin deposition in familial amyloid polyneuropathy

The hallmark of familial amyloid polyneuropathy (FAP) is the presence of extracellular deposits of transthyretin (TTR) aggregates and amyloid fibers in several tissues, particularly in the peripheral nervous system. The molecular pathways to neurodegeneration in FAP still remain elusive; activation of nuclear factor kappaB, pro-inflammatory cytokines, oxidative stress, and pro-apoptotic caspase-3 has been demonstrated "in vivo" in clinical samples and in cell culture systems. In this study, we investigated the involvement of endoplasmic reticulum (ER) stress response in FAP by showing activation of the classical unfolded protein response pathways in tissues not specialized in TTR synthesis but presenting extracellular TTR aggregate and fibril deposition. We also proved cytotoxicity by Ca2+ efflux from the ER in cell cultures incubated with TTR oligomers. Taken together, these studies evidence ER stress associated with a extracellular signal in a misfolding disorder.     

Impact of IFNα2b upon pSTAT3 and the MEK/ERK MAPK Pathway in Melanoma

PURPOSE: High-dose IFNalpha2b (HDI) was established as the first effective adjuvant therapy for patients with high-risk resected melanoma more than a decade ago, but its fundamental molecular mechanism of action remains unclear. STAT3 and the mitogen activated protein kinases (MAPKs), especially ERK (extracellular signal-regulating kinase) and MEK (MAPK/ERK kinase), play roles in melanoma progression and host immunity. We have therefore evaluated STAT3 and MEK/ERK MAP kinases in patients with regional lymph node metastasis (stage IIIB) of melanoma in the context of a prospective neoadjuvant trial of HDI (UPCI 00-008). PATIENTS AND METHODS: In the context of this trial, HDI was administered daily for 20 doses following diagnostic biopsy, and prior to definitive surgery. Immunohistochemistry for pSTAT3, phospho-MEK1/2, phospho-ERK1/2, and EGFR was performed on paired fixed (nine patients) biopsies. RESULTS: HDI was found to down-regulate pSTAT3 (P = 0.008) and phospho-MEK1/2 (P = 0.008) levels significantly in tumor cells. Phospho-ERK1/2 was down-regulated by HDI in tumor cells (P = 0.015), but not in lymphoid cells. HDI down-regulated EGFR (P = 0.013), but pSTAT3 activation appeared not to be associated with EGFR expression and the MEK/ERK MAPK pathway. CONCLUSION: We conclude that HDI regulates MAPK signaling differentially in melanoma tumor cells and host lymphoid cells in vivo. STAT3 activation is independent of the EGFR/MEK/ERK signaling pathway.     

ERK1/2-driven and MKP-mediated inhibition of EGF-induced ERK5 signaling in human proximal tubular cells

The MEK1-ERK1/2 signaling pathway has been implicated in the regulation of renal epithelial cell proliferation, epithelial-to-mesenchymal transition and the induction of an invasive cell phenotype. Much less information is available about the MEK5-ERK5 module and its role in renal epithelial cell proliferation and differentiation. In the present study we have investigated the regulation of these two families of extracellular signal-regulated kinases in epidermal growth factor (EGF)-stimulated human kidney-2 (HK-2) cells and a possible interaction between ERK1/2 and ERK5. Here we report that 5 ng/ml EGF led to a strong stimulation of HK-2 cell proliferation, which was largely U0126-sensitive. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at 10 and 1 microM, respectively, inhibited basal and EGF-induced ERK1/2 phosphorylation but not ERK5 phosphorylation. Long-term inhibition of MEK1/2-ERK1/2 signaling and/or vanadate-sensitive protein phosphatases enhanced and prolonged EGF-induced ERK5 phosphorylation, while transient expression of an adenoviral constitutively active MEK1 (Ad-caMEK1) construct completely blocked EGF-induced ERK5 phosphorylation. Expression of Ad-caMEK1 in HK-2 cells resulted in the upregulation of the dual-specificity phosphatases MKP-3/DUSP6, MKP-1/DUSP1, and DUSP5. The EGF-mediated time-dependent induction of MKP-3, MKP-1 and DUSP5 mRNA levels was U0126-sensitive at a concentration, which blocked EGF-mediated ERK1/2 phosphorylation but not ERK5 phosphorylation. Furthermore, U0126 inhibited EGF-induced MKP-3 and MKP-1 protein expression. Both MKP-3 and MKP-1 co-immunoprecipitated with ERK5 in unstimulated as well as in EGF-stimulated HK-2 cells. These results suggest the existence of an ERK1/2-driven negative feed-back regulation of ERK5 signaling in EGF-stimulated HK-2 cells, which is mediated by MKP-3, DUSP5 and/or MKP-1.     

Extracellular signal-regulated kinases phosphorylate mitogen-activated protein kinase phosphatase 3/DUSP6 at serines 159 and 197, two sites critical for its proteasomal degradation

Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.     

The mechanism by which MEK/ERK regulates JNK and p38 activity in polyamine depleted IEC-6 cells during apoptosis

Polyamine-depletion inhibited apoptosis by activating ERK1/2, while, preventing JNK1/2 activation. MKP-1 knockdown by SiRNA increased ERK1/2, JNK1/2, and p38 phosphorylation and apoptosis. Therefore, we predicted that polyamines might regulate MKP1 via MEK/ERK and thereby apoptosis. We examined the role of MEK/ERK in the regulation of MKP1 and JNK, and p38 activities and apoptosis. Inhibition of MKP-1 activity with a pharmacological inhibitor, sanguinarine (SA), increased JNK1/2, p38, and ERK1/2 activities without causing apoptosis. However, pre-activation of these kinases by SA significantly increased camptothecin (CPT)-induced apoptosis suggesting different roles for MAPKs during survival and apoptosis. Inhibition of MEK1 activity prevented the expression of MKP-1 protein and augmented CPT-induced apoptosis, which correlated with increased activities of JNK1/2, caspases, and DNA fragmentation. Polyamine depleted cells had higher levels of MKP-1 protein and decreased JNK1/2 activity and apoptosis. Inhibition of MEK1 prevented MKP-1 expression and increased JNK1/2 and apoptosis. Phospho-JNK1/2, phospho-ERK2, MKP-1, and the catalytic subunit of PP2Ac formed a complex in response to TNF/CPT. Inactivation of PP2Ac had no effect on the association of MKP-1 and JNK1. However, inhibition of MKP-1 activity decreased the formation of the MKP-1, PP2Ac and JNK complex. Following inhibition by SA, MKP-1 localized in the cytoplasm, while basal and CPT-induced MKP-1 remained in the nuclear fraction. These results suggest that nuclear MKP-1 translocates to the cytoplasm, binds phosphorylated JNK and p38 resulting in dephosphorylation and decreased activity. Thus, MEK/ERK activity controls the levels of MKP-1 and, thereby, regulates JNK activity in polyamine-depleted cells.     

Glutamine up-regulates MAPK phosphatase-1 induction via activation of Ca2+→ ERK cascade pathway

The non-essential amino acid L-glutamine (Gln) displays potent anti-inflammatory activity by deactivating p38 mitogen activating protein kinase and cytosolic phospholipase A₂ via induction of MAPK phosphatase-1 (MKP-1) in an extracellular signal-regulated kinase (ERK)-dependent way. In this study, the mechanism of Gln-mediated ERK-dependency in MKP-1 induction was investigated. Gln increased ERK phosphorylation and activity, and phosphorylations of Ras, c-Raf, and MEK, located in the upstream pathway of ERK, in response to lipopolysaccharidein vitro and in vivo. Gln-induced dose-dependent transient increases in intracellular calcium ([Ca²⁺]_i) in MHS macrophage cells. Ionomycin increased [Ca²⁺]_i and activation of Ras → ERK pathway, and MKP-1 induction, in the presence, but not in the absence, of LPS. The Gln-induced pathways involving Ca²⁺→ MKP-1 induction were abrogated by a calcium blocker. Besides Gln, other amino acids including L-phenylalanine and l-cysteine (Cys) also induced Ca²⁺ response, activation of Ras → ERK, and MKP-1 induction, albeit to a lesser degree. Gln and Cys were comparable in suppression against 2, 4-dinitrofluorobenzene-induced contact dermatitis. Gln-mediated, but not Cys-mediated, suppression was abolished by MKP-1 small interfering RNA. These data indicate that Gln induces MKP-1 by activating Ca²⁺→ ERK pathway, which plays a key role in suppression of inflammatory reactions.     

Positive regulation of ERK activation and MKP-1 expression by peroxovanadium complex bpV (phen)

Lower micromolar concentrations of peroxovanadium compound potassium bisperoxo(1,10-phenanthroline)oxovanadate (V) [bpV (phen)] stimulate RINm5F cell metabolic activity. 1 and 3 mol/L bpV (phen) induces strong and sustained activation of extracellular signal-regulated kinase (ERK). However, it seems that bpV (phen) does not effect c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, bpV (phen) induces mitogen-activated protein kinase phosphatase-1 (MKP-1) expression. We found that ERK activation could be completely abolished if RINm5F cells were incubated with both bpV (phen) and PD 98059, a specific inhibitor of upstream ERK kinase MEK1. On the other hand, this combined treatment up-regulated activation of stress kinases, JNK and p38 MAPK, significantly suppressed MKP-1 expression and induced cell death. Thus, our results suggest that the mechanism underlying bpV (phen) survival-enhancing effect could be associated with induced ERK activation and MKP-1 expression.     

Possible involvement of mitogen-activated protein kinase phosphatase-1 (MKP-1) in thyrotropin-releasing hormone (TRH)-induced prolactin gene expression

The role of extracellular signal-regulated kinase (ERK) in mediating the ability of thyrotropin-releasing hormone (TRH) to stimulate the prolactin gene has been well elucidated. ERK is inactivated by a dual specificity phosphatase, mitogen-activated protein kinase phosphatase (MKP). In this study, we examined the induction of MKP-1 protein by thyrotropin-releasing hormone (TRH) in pituitary GH3 cells, and investigated the possible role for MKP-1 in TRH-induced prolactin gene expression. MKP-1 protein was induced significantly from 60 min after TRH stimulation, and remained elevated at 4 h. The effect of TRH on MKP-1 expression was completely prevented in the presence of the MEK inhibitor, U0126. In the experiments using triptolide, a potent blocker for MKP-1, MKP-1 induction by TRH was completely inhibited in a dose-dependent manner. TRH-induced ERK activation was significantly enhanced in this condition. Prolactin promoter activity, activated by TRH, was reduced to the control level in the presence of triptolide in a dose-dependent manner. In GH3 cells, which were transfected with MKP-1 specific siRNA, both the basal and TRH-stimulated activities of the prolactin promoter were significantly reduced compared to the cells transfected with negative control siRNA. Our present results support a critical role of MKP-1 in TRH-induced, ERK-dependent, prolactin gene expression.     

Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase.

The Ras guanylnucleotide exchange protein SOS undergoes feedback phosphorylation and dissociation from Grb2 following insulin receptor kinase activation of Ras. To determine the serine/threonine kinase(s) responsible for SOS phosphorylation in vivo, we assessed the role of mitogen-activated, extracellular-signal-regulated protein kinase kinase (MEK), extracellular-signal-regulated protein kinase (ERK), and the c-JUN protein kinase (JNK) in this phosphorylation event. Expression of a dominant-interfering MEK mutant, in which lysine 97 was replaced with arginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively active MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Although expression of the mitogen-activated protein kinase-specific phosphatase (MKP-1) completely inhibited the insulin stimulation of ERK activity both in vitro and in vivo, SOS phosphorylation and the dissociation of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specificity of insulin for the ERK pathway. The insulin-stimulated and MKP-1-insensitive SOS-phosphorylating activity was reconstituted in whole-cell extracts and did not bind to a MonoQ anion-exchange column. In contrast, ERK1/2 protein was retained by the MonoQ column, eluted with approximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also does not bind to MonoQ, immunodepletion analysis demonstrated that MEK is not the insulin-stimulated SOS-phosphorylating activity. Together, these data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.     

De-differentiation of primary human hepatocytes depends on the composition of specialized liver basement membrane

Despite the understanding of the importance of mitogen-activated protein (MAP) kinase activation in the stimulation of growth, little is known about the role of MAP kinase regulation during contact inhibited growth control. To investigate the role of the MAP kinase extracellular signal-regulated kinase (ERK) during the transition to a contact inhibited state, cultures of normal fibroblasts (BJ) were grown to different stages of confluency. The levels of MAP kinase phosphatase (MKP) expression and the amount of active ERK and MAP ERK kinase (MEK) in these cultures were assessed through western blot analysis and were compared to fibrosarcoma cell cultures (HT-1080), which lack contact inhibition. In normal fibroblasts, the amounts of active MEK and ERK decline at contact inhibition, concurrently with a rise in MKP-1, MKP-2, and MKP-3 protein levels. In contrast, fibrosarcoma cells appear to lack density-dependent regulation of the ERK pathway. Additionally, altering the redox environment of fibrosarcoma cells to a less reducing state, as seen during contact inhibition, results in increased MKP-1 expression. Taken together, these results suggest that the altered redox environment upon contact inhibition may contribute to the regulation of ERK inactivation by MKPs.     

Interleukin-17A stimulates cardiac fibroblast proliferation and migration via negative regulation of the dual-specificity phosphatase MKP-1/DUSP-1

The dual-specificity mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) inactivates MAP kinases by dephosphorylation. Here we show that the proinflammatory cytokine interleukin (IL)-17A induces adult mouse primary cardiac fibroblast (CF) proliferation and migration via IL-17 receptor A//IL-17 receptor C-dependent MKP-1 suppression, and activation of p38 MAPK and ERK1/2. IL-17A mediated p38 MAPK and ERK1/2 activation is inhibited by MKP-1 overexpression, but prolonged by MKP-1 knockdown. IL-17A induced miR-101 expression via PI3K/Akt, and miR-101 inhibitor reversed MKP-1 down regulation. Importantly, MKP-1 knockdown, pharmacological inhibition of p38 MAPK and ERK1/2, or overexpression of dominant negative MEK1, each markedly attenuated IL-17A-mediated CF proliferation and migration. Similarly, IL-17F and IL-17A/F heterodimer that also signal via IL-17RA/IL-17RC, stimulated CF proliferation and migration. These results indicate that IL-17A stimulates CF proliferation and migration via Akt/miR-101/MKP-1-dependent p38 MAPK and ERK1/2 activation. These studies support a potential role for IL-17 in cardiac fibrosis and adverse myocardial remodeling.     

ERK1/2 achieves sustained activation by stimulating MAPK phosphatase-1 degradation via the ubiquitin-proteasome pathway

Sustained extracellular signal-regulated kinase 1/2 (ERK1/2) activation does not always correlate with its upstream Ras-Raf-mitogen-activated protein kinase kinase 1/2 (MKK1/2) signal cascade in cancer cells, and the mechanism remains elusive. Here we report a novel mechanism by which sustained ERK1/2 activation is established. We demonstrate that Pb(II), a carcinogenic metal, persistently induces ERK1/2 activity in CL3 human lung cancer cells and that Ras-Raf-MKK1/2 signaling cannot fully account for such activation. It is intriguing that Pb(II) treatment reduces mitogen-activated protein kinase phosphatase 1 (MKP-1) protein levels in time- and dose-dependent manners, which correlates with sustained ERK1/2 activation, and that Pb(II) also induces mRNA and de novo protein synthesis of MKP-1. In Pb(II)-treated cells, MKP-1 is polyubiquitinated, and proteasome inhibitors markedly alleviate the ubiquitination and degradation of MKP-1. Inhibiting the Pb(II)-induced ERK1/2 activation by PD98059 greatly suppresses MKP-1 ubiquitination and degradation. It is remarkable that constitutive activation of MKK1/2 triggers endogenous MKP-1 ubiquitination and degradation in various mammalian cell lines. Furthermore, expression of functional MKP-1 decreases ERK1/2 activation and the c-Fos protein level and enhances cytotoxicity under Pb(II) exposure. Taken together, these results demonstrate that activated ERK1/2 can trigger MKP-1 degradation via the ubiquitin-proteasome pathway, thus facilitating long-term activation of ERK1/2 against cytotoxicity.     

Mitogen-activated protein kinase phosphatase-3 is a tumor promoter target in initiated cells that express oncogenic Ras

We have capitalized on the unique properties of the skin tumor promoter palytoxin, which does not activate protein kinase C, to investigate alternative mechanisms by which major signaling molecules can be modulated during carcinogenesis. We report here that palytoxin activates extracellular signal-regulated kinase (ERK) through a novel mechanism that involves inactivation of an ERK phosphatase in keratinocytes derived from initiated mouse skin (308 cells). Use of U0126 revealed that palytoxin requires the ERK kinase MEK to stimulate ERK activity, although palytoxin did not activate MEK. We found that 308 keratinocytes highly express mitogen-activated protein kinase phosphatase-3 (MKP-3), which selectively inactivates ERK. Palytoxin induced the loss of MKP-3 in a manner that corresponded to increased ERK phosphorylation. Complementary studies showed that sustained expression of exogenous MKP-3 inhibited palytoxin-stimulated ERK activation. As is characteristic of initiated keratinocytes, 308 cells express activated H-Ras. To investigate whether expression of oncogenic Ras is key to palytoxin-stimulated ERK activation, we determined how palytoxin affected ERK and MKP-3 in MCF10A human breast epithelial cells and in H-ras MCF10A cells, which stably express activated H-Ras. Palytoxin did not affect ERK activity in MCF10A cells, which had no detectable MKP-3. Like 308 cells, H-ras MCF10A cells highly express MKP-3. Strikingly, palytoxin stimulated ERK activity and induced a corresponding loss of MKP-3 in H-ras MCF10A cells. These studies indicate that in initiated cells palytoxin unleashes ERK activity by down-regulating MKP-3, an ERK inhibitor, and further suggest that MKP-3 may be a vulnerable target in cells that express oncogenic Ras.     

Pancreatic tumor cells with mutant K-ras suppress ERK activity by MEK-dependent induction of MAP kinase phosphatase-2

Activating mutations within the K-ras gene occur in a high percentage of human pancreatic carcinomas. We reported previously that the presence of oncogenic, activated K-ras in human pancreatic carcinoma cell lines did not result in constitutive activation of the extracellular signal-regulated kinases (ERK1 and ERK2). In the present study, we further characterized the ERK signaling pathway in pancreatic tumor cell lines in order to determine whether the ERK pathway is subject to a compensatory downregulation. We found that the attenuation of serum-induced ERK activation was not due to a delay in the kinetics of ERK phosphorylation. Treatment with the tyrosine phosphatase inhibitor orthovanadate increased the level of ERK phosphorylation, implicating a vanadate-sensitive tyrosine phosphatase in the negative regulation of ERK. Furthermore, expression of a dual specificity phosphatase capable of inactivating ERK known as mitogen-activated protein (MAP) kinase phosphatase-2 (MKP-2) was elevated in most of the pancreatic tumor cell lines and correlated with the presence of active MAP kinase kinase (MEK). Taken together, these results suggest that pancreatic tumor cells expressing oncogenic K-ras compensate, in part, by upregulating the expression of MKP-2 to repress the ERK signaling pathway.