Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Much effort has focused on the identification of MAPK cascades that are activated by the MEKK family of protein kinases. However, direct phosphorylation and regulation of the MEKK proteins has not been shown. To address this question, we have expressed recombinant (His)6FLAG.MEKK3 in Sf9 insect cells and tethered the purified protein to Ni-Sepharose so that we could precipitate interacting proteins and then identify such proteins by liquid chromatography and mass spectrometry (LC-MS). We identified 14-3-3 proteins as interacting with MEKK3, which suggested that (His)6FLAG.MEKK3 was phosphorylated on serine since 14-3-3 proteins are known to associate with phosphorylated proteins. We identified two phosphorylated amino acids at Ser166 and Ser337 of tryptic peptides derived from (His)6FLAG.MEKK3 by using LC-MS. Antibodies were developed that recognize the specific phosphorylated amino acid and with these antibodies, we demonstrate that various stimuli (tumor necrosis factor, arsenite, forskolin, and serum) promote phosphorylation of Ser166 and Ser337. However, neither of these phosphorylated amino acids is required for association with 14-3-3 protein or regulation of MEKK3-dependent ERK and JNK activity. Nonetheless, these results suggest that MEKK3 is a convergence point of multiple upstream signaling pathways.
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PMID:Phosphorylation of the stress-activated protein kinase, MEKK3, at serine 166. 1239 20

To investigate regulation of D2 receptor (D2R) gene expression by protein kinases, we evaluated effects of constitutively active MAPK kinase kinase (MEKK), Ca2+/calmodulin-dependent protein kinase (CaMK) II, CaMKIV and cyclic AMP-dependent protein kinase (PKA) on D2R promoter activity using luciferase reporter gene assays. A 1.5-kbp fragment containing the rat D2R promoter was cloned upstream of the reporter and transfected into D2R-expressing NB2A cells or nonexpressing NG108-15 and C6 glioma cells. MEKK and CaMKII, but not CaMKIV and PKA, increased promoter activity 4.5- and 1.5-fold, respectively, in NB2A cells. Inhibitory effects of a MEK inhibitor and lack of effect by dominant negative (DN)-JNK1 or DN-p38MAPK revealed that ERK but not JNK and p38MAPK is involved in MEKK-induced promoter activation. Deletion and mutation of the promoter revealed that the MEKK-responsive region was Sp1 site B between nucleotides -56 and -47. Overexpression of Sp1 suppressed promoter activity without affecting MEKK-induced activation. Interestingly, overexpression of Zif268 increased promoter activity through the region between nucleotides -56 and -36. Increased activity by Zif268 was additive with CaMKII-induced activation but not with activation induced by MEKK. Co-transfection with CaMKII stimulated nuclear translocation of Zif268. These results suggest that ERK and CaMKII positively regulate the D2R promoter and that Zif268 is a potential transcription factor for the CaMKII-dependent pathway.
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PMID:Activation of the rat dopamine D2 receptor promoter by mitogen-activated protein kinase and Ca2+/calmodulin-dependent protein kinase II pathways. 1242 50

Whilst many studies have examined the role of the MAP Kinases in regulating the G1-->S transition, much less is known about the function of these pathways in regulating other cell cycle transitions. Stimulation of the conditional mutant Delta MEKK3:ER* in asynchronous hamster (CCl39) and rat (Rat-1) fibroblasts resulted in the strong activation of endogenous JNK and p38 but only a weak activation of ERK. Activation of Delta MEKK3:ER* inhibited cell proliferation through a combination of an initial G1 and G2 cell cycle arrest, followed by a delayed onset of apoptosis. When cells were synchronized in S phase with aphidicolin and then released, activation of Delta MEKK3:ER* resulted in the up-regulation of p21(CIP1) and a pronounced inhibition of cyclin A/CDK2 and cyclin B1/CDK1 kinase activity. Analysis of mitotic figures indicated that cells failed to enter mitosis, arresting late in G2. Delta MEKK3:ER*-mediated CDK inhibition and G2 arrest did not absolutely require p21(CIP1), since both events were observed in Rat-1 cells in which p21(CIP1) is transcriptionally silenced due to promoter methylation. Rather, CDK inhibition was associated with a down-regulation of cyclin A and B1 expression. Finally, application of the p38 inhibitor SB203580 partially restored cyclin B associated kinase activity and allowed cells to proceed through mitosis into the next G1 phase, suggesting that activation of the p38 alpha/beta 2 pathway can promote a G2 cell cycle arrest.
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PMID:Delta MEKK3:ER* activation induces a p38 alpha/beta 2-dependent cell cycle arrest at the G2 checkpoint. 1244 45

mRNA stabilization plays an important role in the changes in protein expression initiated by inducers of inflammation or direct cell stress such as UV light. This study provides evidence that stabilization in response to UV light differs from that induced by proinflammatory stimuli such as bacterial lipopolysaccharide or interleukin (IL)-1. Firstly, UV-induced stabilization is independent of the p38 MAP kinase pathway, which has previously been shown to mediate stabilization induced by IL-1 or lipopolysaccharide. UV-induced mRNA stabilization was insensitive to the dominant negative forms of p38 MAP kinase and its substrate MAP kinase-activated protein kinase 2 (MK2), or to the p38 MAP kinase inhibitor SB 203580, demonstrating that it occurs through a different signaling mechanism. Secondly, UV-induced stabilization exhibits a different transcript selectivity. Activation of the p38 MAP kinase pathway, by expressing active MAP kinase kinase 6, induced stabilization only of transcripts containing AU-rich elements. UV light also induced stabilization of transcripts lacking AU-rich elements. This effect could not be mimicked by expressing MEKK1, an upstream activator of the p38, JNK, ERK and NF-kappaB pathways. UV light also stabilized endogenous histone mRNA, which lacks AU-rich elements and a poly(A) tail. This effect was not mimicked by active MAP kinase kinase 6 and not sensitive to a p38 MAP kinase inhibitor. This suggests that UV light induces stabilization through a mechanism that is independent of p38 MAP kinase and affects a broad spectrum of mRNAs.
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PMID:Evidence for general stabilization of mRNAs in response to UV light. 1244 71

Oxygen-dependent regulation of HIF-1 activity occurs at multiple levels in vivo. The mechanisms regulating HIF-1alpha protein expression have been most extensively analyzed but the ones modulating HIF-1 transcriptional activity remain unclear. Changes in the phosphorylation and/or redox status of HIF-1alpha certainly play a role. Here, we show that ionomycin could activate HIF-1 transcriptional activity in a way that was additive to the effect of hypoxia without affecting HIF-1alpha protein level. In addition, a calmodulin dominant negative mutant and W7, a calmodulin antagonist, as well as BAPTA, an intracellular calcium chelator, inhibited the hypoxia-induced HIF-1 activation. These results indicate that elevated calcium in hypoxia could participate in HIF-1 activation. Furthermore, ERK but not JNK phosphorylation was evidenced in both conditions, ionomycin and hypoxia. PD98059, an inhibitor of the ERK pathway as well as a ERK1 dominant negative mutant also blocked HIF-1 activation by hypoxia and by ionomycin. A MEKK1 (a kinase upstream of JNK) dominant negative mutant had no effect. In addition, BAPTA, calmidazolium, a calmodulin antagonist and PD98059 inhibited VEGF secretion by hypoxic HepG2. All together, these results suggest that calcium and calmodulin would act upstream of ERK in the hypoxia signal transduction pathway.
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PMID:Role of ERK and calcium in the hypoxia-induced activation of HIF-1. 1244 87

Mixed lineage kinases (MLKs) are MAPKKK members that activate JNK and reportedly lead to cell death. However, the agonist(s) that regulate MLK activity remain unknown. Here, we demonstrate ceramide as the activator of Drosophila MLK (dMLK) and identify ceramide and TNF-alpha as agonists of mammalian MLK3. dMLK and MLK3 are activated by a ceramide analog and bacterial sphingomyelinase in vivo, whereas a low nanomolar concentration of natural ceramide activates them in vitro. Specific inhibition of dMLK and MLK3 significantly attenuates activation of JNK by ceramide in vivo without affecting ceramide-induced p38 or ERK activation. In addition, TNF-alpha also activates MLK3 and evidently leads to JNK activation in vivo. Thus, the ceramide serves as a common agonist of dMLK and MLK3, and MLK3 contributes to JNK activation induced by TNF-alpha.
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PMID:Activation of the Drosophila MLK by ceramide reveals TNF-alpha and ceramide as agonists of mammalian MLK3. 1250 27

Leukocyte infiltration of the cortico-interstitium is characteristic of many forms of progressive renal disease. The principal adhesion molecule expressed on resident interstitial cells and recognized by leukocytes is intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is an inducible transmembrane receptor, which forms the counter-receptor for the leukocyte beta 2 integrins. ICAM-1-dependent binding induces the synthesis of the chemokine RANTES and of ICAM-1 itself. This study examines some of the signaling pathways involved in this induction. After ICAM-1 cross-linking on fibroblasts, the mRNA and protein for both RANTES and ICAM-1 were induced. This induction was calcium-dependent and inhibited by BAPTA-AM. The p38, ERK1, and ERK2 MAP kinases were activated in a [Ca2+]i-dependent manner, with a maximum phosphorylation at approximately 3 min after cross-linking. Through the use of selective inhibitors of p38 MAP kinase (SB203580) or MEKK (PD98059), p38 but not ERK activation was shown to be essential for the induction of ICAM-1. Neither was involved in RANTES activation, however. These mechanisms differed from those initiated by TNF-alpha, which were not [Ca2+]i-dependent. Electrophoretic mobility shift analysis demonstrated a time-dependent induction of both AP-1 and NF-kappaB binding activity in nuclear extracts, maximal at approximately 15 min after ICAM-1 cross-linking. Only AP-1 activation, however, was calcium-dependent, suggesting the central involvement of this transcription factor in ICAM-1 and RANTES induction after the ligation of ICAM-1. This study suggests an independent mechanism of inflammatory amplification, which may be characteristic of a persistent leukocytic involvement in areas of chronic inflammation rather than in cytokine-induced acute inflammation.
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PMID:Selective regulation of ICAM-1 and RANTES gene expression after ICAM-1 ligation on human renal fibroblasts. 1250 44

MAP kinase pathways comprise a group of parallel protein phosphorylation cascades, which are involved in signaling triggered by a variety of stimuli. Previous findings suggested that the ERK and the JNK pathways have opposing roles in regulating proliferation and survival or apoptosis and that apoptosis can be promoted by inhibiting the ERK pathway or by activation of the JNK pathway. In order to test this hypothesis and explore whether it can be exploited as a strategy for killing human cancer cells, we used gene transfer experiments with a range of cancer cell lines. We expressed the catalytic fragment of human MEKK1 to activate JNK and the Ras-binding domain (RBD) of Raf-1 to inhibit the Ras-ERK pathway. In addition, we designed several RBD-MEKK1 fusion proteins aiming to simultaneously activate the JNK and block the ERK pathway. We found that the MEKK1 proteins as well as the RBD alone could reduce colony formation in all cell lines. The survival time of MEKK1-expressing cells depended on the cell line. In HeLa cells, survival could be prolonged by inhibition of caspases but not by coexpression of the anti-apoptotic protein Bcl-2. Due to a lower kinase activity the RBD-MEKK1 fusion proteins were less effective in apoptosis induction than the MEKK1 kinase domain alone. Using mutant forms of Ras and Raf-1 we could show that the reduced kinase activity of RBD-MEKK1 fusion proteins was caused by binding to the Ras protein. The expression of lethal doses of MEKK1 resulted in a strong activation of all three major MAP kinase families JNK, ERK, and p38. Blocking these pathways either by coexpressing a dominant negative form of MKK4 or with inhibitors of MEK or p38 failed to inhibit apoptosis. This suggests that MEKK1 induces apoptosis by causing a general deregulation of MAP kinase signaling rather than by the activation of a single pathway.
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PMID:The kinase domain of MEKK1 induces apoptosis by dysregulation of MAP kinase pathways. 1256 21

In skin, the profibrotic protein connective tissue growth factor (CTGF) is not normally expressed. However, when skin cells are exposed to transforming growth factor-beta (TGF-beta), CTGF is induced in fibroblasts but not in epithelial cells. We have begun to investigate the requirements for the fibroblast-selective induction of CTGF by TGF-beta. Previously we found that this response was Smad-dependent. Now we show that protein kinase C and Ras/MEK/ERK are necessary for the TGF-beta induction of the CTGF promoter but not of a generic Smad-responsive promoter (SBE-lux). Induction of the CTGF promoter is antagonized by c-Jun or by MEKK1, suggesting that a proper balance between the Ras/MEK/ERK and JNK MAPK cascades is necessary for TGF-beta induction of CTGF. We identify the minimal CTGF promoter element necessary and sufficient to confer TGF-beta responsiveness to a heterologous promoter and show that a tandem repeat of a consensus transcription enhancer factor binding element, 5'-GAGGAATGG-3', is necessary for this induction. This element has not been previously shown to play a role in TGF-beta induction of gene expression in fibroblasts. Gel shift analysis shows that this sequence binds nuclear factors that are greatly enriched in fibroblasts relative to epithelial cells. Thus Smads, Ras/MEK/ERK, protein kinase C, and fibroblast-enriched factors that bind GAGGAATGG act together to drive the TGF-beta-mediated induction of CTGF in fibroblasts.
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PMID:Connective tissue growth factor gene regulation. Requirements for its induction by transforming growth factor-beta 2 in fibroblasts. 1257 Dec 53

The abnormal accumulation of methylglyoxal (MG), a physiological glucose metabolite, is strongly related to the development of diabetic complications by affecting the metabolism and functions of organs and tissues. These disturbances could modify the cell response to hormones and growth factors, including insulin-like growth factor-1 (IGF-I). In this study, we investigated the effect of MG on IGF-I-induced cell proliferation and the mechanism of the effect in two cell lines, a human embryonic kidney cell line (HEK293), and a mouse fibroblast cell line (NIH3T3). MG rendered these cells resistant to the mitogenic action of IGF-I, and this was associated with stronger and prolonged activation of ERK and over-expression of P21(Waf1/Cip1). The synergistic effect of MG with IGF-I in activation of ERK was completely abolished by PD98059 but not by a specific PI3K inhibitor, LY294002, or a specific PKC inhibitor, bisindolylmaleimide. Blocking of Raf-1 activity by expression of a dominant negative form of Raf-1 did not reduce the enhancing effect of MG on IGF-I-induced activation of ERK. However, transfection of a catalytically inactive form of MEKK1 resulted in inactivation of the MG-induced activation of ERK and partial inhibition of the enhanced activation of ERK and over-expression of p21(Waf1/Cip1) induced by co-stimulation of MG and IGF-I. These results suggested that the alteration of intracellular milieu induced by MG through a MEKK1-mediated and PI3K/PKC/Raf-1-independent pathway resulted in the modification of cell response to IGF-I for p21(Waf1/Cip1)-mediated growth arrest, which may be one of the crucial mechanisms for MG to promote the development of chronic clinical complications in diabetes.
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PMID:Involvement of MEKK1/ERK/P21Waf1/Cip1 signal transduction pathway in inhibition of IGF-I-mediated cell growth response by methylglyoxal. 1264 5


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