ERK/MAPK pathway regulates GABAA receptors

The GABAA receptor is a ligand-gated ion channel whose function and activity can be regulated by ligand binding or alternatively may be influenced indirectly through the phosphorylation of specific subunits that comprise the GABAA receptor pentamer. With respect to phosphorylation, most studies have focused on either beta or gamma subunits, whereas the role of the alpha subunit as a relevant target of signaling kinases is largely unknown. Interestingly, we found a putative phosphorylation site for extracellular-signal regulated kinase (ERK), a key effector of the MAPK pathway, in almost all known alpha subunits of the GABAA receptor, including the ubiquitously expressed alpha1 subunit. To determine whether this putative ERK phosphorylation site was functionally relevant, we evaluated if ERK inhibition (through pharmacological inhibition of its upstream kinase, MEK) altered GABA-gated currents. Using HEK293 cells stably transfected with the alpha1beta2gamma2 form of the GABAA receptor, we found that UO126 reduced basal ERK phosphorylation and resulted in an enhancement of GABA-induced peak current amplitudes. Further, the enhancement of GABA-gated currents required an intact intracellular environment as it was robust in perforated patch recordings (which preserves the intracellular milieu), but absent in conventional whole-cell recordings (which dialyzes the cytosolic contents), supporting the involvement of an intracellular signaling pathway. Finally, mutation of the ERK phosphorylation site (T375-->A) prevented the UO126-induced enhancement of GABA-gated currents. Collectively, our results implicate the MAPK pathway as a negative modulator of GABAA receptor function, whose influence on GABA-gated currents may be mediated by phosphorylation of the alpha subunit.     

WNK4 inhibits NCC protein expression through MAPK ERK1/2 signaling pathway

WNK [with no lysine (K)] kinase is a subfamily of serine/threonine kinases. Mutations in two members of this family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia, and metabolic acidosis. WNK1 and WNK4 were shown to regulate sodium chloride cotransporter (NCC) activity through phosphorylating SPAK and OSR1. Previous studies including ours have also shown that WNK4 inhibits NCC function and its protein expression. A recent study reported that a phorbol ester inhibits NCC function via activation of extracellular signal-regulated kinase (ERK) 1/2 kinase. In the current study, we investigated whether WNK4 affects NCC via the MAPK ERK1/2 signaling pathway. We found that WNK4 increased ERK1/2 phosphorylation in a dose-dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) lost the ability to increase the ERK1/2 phosphorylation. Hypertonicity significantly increased ERK1/2 phosphorylation in mDCT cells. Knock-down of WNK4 expression by siRNA resulted in a decrease of ERK1/2 phosphorylation. We further showed that WNK4 knock-down significantly increases the cell surface and total NCC protein expressions and ERK1/2 knock-down also significantly increases cell surface and total NCC expression. These data suggest that WNK4 inhibits NCC through activating the MAPK ERK1/2 signaling pathway.     

HIV-1 Tat regulates endothelial cell cycle progression via activation of the Ras/ERK MAPK signaling pathway

Tat, the transactivator of HIV-1 gene expression, is released by acutely HIV-1-infected T-cells and promotes adhesion, migration, and growth of inflammatory cytokine-activated endothelial and Kaposi's sarcoma cells. It has been previously demonstrated that these effects of Tat are due to its ability to bind through its arginine-glycine-aspartic (RGD) region to the alpha5beta1 and alphavbeta3 integrins. However, the signaling pathways linking Tat to the regulation of cellular functions are incompletely understood. Here, we report that Tat ligation on human endothelial cells results in the activation of the small GTPases Ras and Rac and the mitogen-activated protein kinase ERK, specifically through its RGD region. In addition, we demonstrated that Tat activation of Ras, but not of Rac, induces ERK phosphorylation. We also found that the receptor proximal events accompanying Tat-induced Ras activation are mediated by tyrosine phosphorylation of Shc and recruitment of Grb2. Moreover, Tat enabled endothelial cells to progress through the G1 phase in response to bFGF, and the process is linked to ERK activation. Taken together, these data provide novel evidence about the ability of Tat to activate the Ras-ERK cascade which may be relevant for endothelial cell proliferation and for Kaposi's sarcoma progression.     

Trihydrophobin 1 phosphorylation by c-Src regulates MAPK/ERK signaling and cell migration

c-Src activates Ras-MAPK/ERK signaling pathway and regulates cell migration, while trihydrophobin 1 (TH1) inhibits MAPK/ERK activation and cell migration through interaction with A-Raf and PAK1 and inhibiting their kinase activities. Here we show that c-Src interacts with TH1 by GST-pull down assay, coimmunoprecipitation and confocal microscopy assay. The interaction leads to phosphorylation of TH1 at Tyr-6 in vivo and in vitro. Phosphorylation of TH1 decreases its association with A-Raf and PAK1. Further study reveals that Tyr-6 phosphorylation of TH1 reduces its inhibition on MAPK/ERK signaling, enhances c-Src mediated cell migration. Moreover, induced tyrosine phosphorylation of TH1 has been found by EGF and estrogen treatments. Taken together, our findings demonstrate a novel mechanism for the comprehensive regulation of Ras/Raf/MEK/ERK signaling and cell migration involving tyrosine phosphorylation of TH1 by c-Src.     

Plasminogen/plasmin regulates c-fos and egr-1 expression via the MEK/ERK pathway

In this study, we showed that plasminogen (Plg) and plasmin (Pla) bind to lysine-binding sites on cell surface and trigger a signaling pathway that activates the mitogen-activated protein kinase (MAPK) MEK and ERK1/2, which in turn leads to the expression of the primary response genes c-fos and early growth response gene egr-1. Our data show that the Plg/Pla-stimulated steady-state mRNA levels of both genes reached a maximum by 30 min and then returned to basal levels by 1h. The gene induction was sensitive to both pharmacological and genetic inhibition of MEK. Leupeptin, a serine protease inhibitor, suppressed Pla but not Plg-induced c-fos and egr-1 expression, emphasizing the role played by the serine protease activity associated with Pla. Pre-incubation with cholera toxin completely blocked the Plg/Pla-induced gene expression, suggesting that another signaling pathway, which recruits G protein-coupled receptors, may also be involved. Furthermore, Plg/Pla also stimulated AP-1 and EGR-1 DNA-binding activities, which were abrogated by pharmacological inhibition of MEK. Altogether, these results suggest that Plg/Pla stimulates c-fos and egr-1 expression via activation of the MEK/ERK pathway.     

MAPK signalling: ERK5 versus ERK1/2

Extracellular-signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and, similar to ERK1/2, has the Thr-Glu-Tyr (TEY) activation motif. Both ERK5 and ERK1/2 are activated by growth factors and have an important role in the regulation of cell proliferation and cell differentiation. Moreover, both the ERK5 and the ERK1/2 pathways are sensitive to PD98059 and U0126, which are two well-known inhibitors of the ERK pathway. Despite these similarities, recent studies have revealed distinctive features of the ERK5 pathway: ERK5 has a key role in cardiovascular development and neural differentiation; ERK5 nuclear translocation is controlled by its own nuclear localizing and nuclear export activities; and the carboxy-terminal half of ERK5, which follows its kinase catalytic domain, has a unique function.     

MEKK2 associates with the adapter protein Lad/RIBP and regulates the MEK5-BMK1/ERK5 pathway

MEKK2 and MEKK3 are two closely related mitogen-activated protein kinase (MAPK) kinase kinases. The kinase domains of MEKK2 and MEKK3 are nearly identical, although their N-terminal regulatory domains are significantly divergent. By yeast two-hybrid library screening, we have identified MEK5, the MAPK kinase in the big mitogen-activated protein kinase 1 (BMK1)/ERK5 pathway, as a binding partner for MEKK2. MEKK2 expression stimulates BMK1/ERK5 activity, the downstream substrate for MEK5. Compared with MEKK3, MEKK2 activated BMK1/ERK5 to a greater extent, which might correlate with a higher affinity MEKK2-MEK5 interaction. A dominant negative form of MEK5 blocked the activation of BMK1/ERK5 by MEKK2, whereas activation of c-Jun N-terminal kinase (JNK) was unaffected, showing that MEK5 is a specific downstream effector of MEKK2 in the BMK1/ERK5 pathway. Activation of BMK1/ERK5 by epidermal growth factor and H2O2 in Cos7 and HEK293 cells was completely blocked by a kinase-inactive MEKK3 (MEKK3kin(-)), whereas MEKK2kin(-) had no effect. However, in D10 T cells, expression of MEKK2kin(-) but not MEKK3kin(-) inhibited BMK1/ERK5 activity. Two-hybrid screening also identified Lck-associated adapter/Rlk- and Itk-binding protein (Lad/RIBP), a T cell adapter protein, as a binding partner for MEKK2. MEKK2 and Lad/RIBP colocalize at the T cell contact site with antigen-loaded presenting cells, demonstrating cotranslocation of MEKK2 and Lad/RIBP during T cell activation. MEKK3 neither binds Lad/RIBP nor is recruited to the T cell contact with antigen presenting cell. MEKK2 and MEKK3 are differentially associated with signaling from specific upstream receptor systems, whereas both activate the MEK5-BMK1/ERK5 pathway.     

Mitogen-activated protein kinase ERK1/2 regulates the class II transactivator

The expression of major histocompatibility class II genes is necessary for proper antigen presentation and induction of an immune response. This expression is initiated by the class II transactivator, CIITA. The establishment of the active form of CIITA is controlled by a series of post-translational events, including GTP binding, ubiquitination, and dimerization. However, the role of phosphorylation is less clearly defined as are the consequences of phosphorylation on CIITA activity and the identity of the kinases involved. In this study we show that the extracellular signal-regulated kinases 1 and 2 (ERK1/2) interact directly with CIITA, targeting serine residues in the amino terminus of the protein, including serine 288. Inhibition of this phosphorylation by dominant-negative forms of ERK or by treatment of cells with the ERK inhibitor PD98059 resulted in the increase in CIITA-mediated gene expression from a class II promoter, enhanced the nuclear concentration of CIITA, and impaired its ability to bind to the nuclear export factor, CRM1. In contrast, inhibition of ERK1/2 activity had little effect on serine-to-alanine mutant forms of CIITA. These data suggest a model whereby ERK1/2-mediated phosphorylation of CIITA down-regulates CIITA activity by priming it for nuclear export, thus providing a means for cells to tightly regulate the extent of antigen presentation.     

Annexin1 regulates the erythroid differentiation through ERK signaling pathway

K562 cells can be used as a model of erythroid differentiation on being induced by hemin. We found that the level of annexin1 gene expression was notably increased during this indicated process. To test the hypothesis that annexin1 can regulate erythropoiesis, K562 cell clones in which annexin1 was stably increased and was knocked down by RNAi were established, respectively. With analysis by hemoglobin quantification, benzidine staining, and marker gene expression profile determination, we confirmed that hemin-induced erythroid differentiation of K562 cells was modestly stimulated by overexpression of annexin1 while it was significantly blocked by knock down of annexin1. Further studies revealed that the mechanisms of annexin1 regulation of the erythroid differentiation was partially related to the increased ERK phosphorylation and expression of p21(cip/waf), since specific inhibitor of MEK blocked the function of annexin1 in erythroid differentiation. We concluded that annexin1 exerted its erythropoiesis regulating effect by ERK pathway.     

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.     

Chlamydia inhibit host cell apoptosis by inducing Bag-1 via the MAPK/ERK survival pathway

Chlamydia are obligate intracellular bacteria that frequently cause human disease. Host cells infected with Chlamydia are profoundly resistant to diverse apoptotic stimuli. The inhibition of apoptosis is thought to be an important immune escape mechanism allowing Chlamydia to productively complete their obligate intracellular growth cycle. Chlamydial antiapoptotic activity involves activation of the MAPK/ERK survival pathway. However, the molecular mechanisms are not well understood. Here we show that Bag-1 is up-regulated in Chlamydia-infected cells. U0126 and GW5074 suppress the induction of Bag-1 by Chlamydia, implying that Chlamydia may up-regulate Bag-1 via the MAPK/ERK survival pathway. Overexpression of Bag-1 is sufficient to protect against apoptosis, while depletion of Bag-1 suppresses the antiapoptotic effect of Chlamydia. The data indicate Chlamydia may up-regulate Bag-1 through the MAPK/ERK survival pathway to suppress apoptosis.     

USP7 regulates the ERK1/2 signaling pathway through deubiquitinating Raf-1 in lung adenocarcinoma

Ubiquitin-specific protease 7 (USP7) is one of the deubiquitinating enzymes (DUBs) in the ubiquitin-specific protease (USP) family. It is a key regulator of numerous cellular functions including immune response, cell cycle, DNA damage and repair, epigenetics, and several signaling pathways. USP7 acts by removing ubiquitin from the substrate proteins. USP7 also binds to a specific binding motif of substrate proteins having the [P/A/E]-X-X-S or K-X-X-X-K protein sequences. To date, numerous substrate proteins of USP7 have been identified, but no studies have been conducted using the binding motif that USP7 binds. In the current study, we analyzed putative substrate proteins of USP7 through the [P/A/E]-X-X-S and K-X-X-X-K binding motifs using bioinformatics tools, and confirmed that Raf-1 is one of the substrates for USP7. USP7 binds to the Pro-Val-Asp-Ser (PVDS) motif of the conserved region 2 (CR2) which contains phosphorylation sites of Raf-1 and decreased M1-, K6-, K11-, K27-, K33-, and K48-linked polyubiquitination of Raf-1. We further identified that the DUB activity of USP7 decreases the threonine phosphorylation level of Raf-1 and inhibits signaling transduction through Raf activation. This regulatory mechanism inhibits the activation of the ERK1/2 signaling pathway, thereby inhibiting the G2/M transition and the cell proliferation of lung adenocarcinoma cells. In summary, our results indicate that USP7 deubiquitinates Raf-1 and is a new regulator of the ERK1/2 signaling pathway in lung adenocarcinoma.Subject terms: Ubiquitylation, Non-small-cell lung cancer, Protein-protein interaction networks     

Melanoma metabolism contributes to the cellular responses to MAPK/ERK pathway inhibitors

Background

Besides its influence on survival, growth, proliferation, invasion and metastasis, cancer cell metabolism also greatly influences the cellular responses to molecular-targeted therapies.