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)

Previous work has demonstrated that sequence-selective DNA-binding drugs can inhibit transcription factors from binding to their target sites on gene promoters. In this study, the potency and effectiveness of DNA-binding drugs to inhibit transcription were assessed using the c-fos promoter's serum response element (SRE) as a target. The drugs chosen for analysis included the minor groove binding agents chromomycin A(3) and Hoechst 33342, which bind to G/C-rich and A/T-rich regions, respectively, and the intercalating agent nogalamycin, which binds G/C-rich sequences in the major groove. The transcription factors targeted, Elk-1 and serum response factor (SRF), form a ternary complex (TC) on the SRE that is necessary and sufficient for induction of c-fos by serum. The drugs' abilities to prevent TC formation on the SRE in vitro were nogalamycin > Hoechst 33342 > chromomycin. Their potencies in inhibiting cell-free transcription and endogenous c-fos expression in NIH3T3 cells, however, were chromomycin > nogalamycin > Hoechst 33342. The latter order of potency was also obtained for the drugs' cytotoxicity and inhibition of general transcription as measured by [(3)H]uridine incorporation. These systematic analyses provide insight into how drug and transcription factor binding characteristics are related to drugs' effectiveness in inhibiting gene expression.
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PMID:Evaluation of the effectiveness of DNA-binding drugs to inhibit transcription using the c-fos serum response element as a target. 1101 5

The biological functions of Rit (Ras-like protein in tissues) and Rin (Ras-like protein in neurons), members of a novel branch of Ras-related GTP-binding proteins that are approximately 50% identical to Ras, have not been characterized. Therefore, we assessed their activity in growth control, transformation and signaling. NIH cells stably expressing a constitutively activated mutant of Rit [Rit(79L)] (analogous to the oncogenic mutant H-Ras(61L)) demonstrated strong growth transformation, proliferating rapidly in low serum and forming colonies in soft agar and tumors in nude mice. Although Rit(79L) alone did not promote morphologically transformed foci, it cooperated with both Raf and Rho A to form Rac/Rho-like foci. Rin [Rin(78L)] cooperated only with Raf. Rit(79L) but not Rin(78L) stimulated transcription from luciferase reporter constructs regulated by SRF, NF-kappaB, Elk-1 and Jun. However, neither activated ERK, JNK or p38, or PI3-K/Akt kinases in immune complex kinase assays. Interestingly, although Rit lacks any known recognition signal for C-terminal lipidation, Rit-transformed cell growth and survival in low serum is dependent on a farnesylated protein, as treatment with farnesyltransferase inhibitors caused apoptosis. Rin cooperated with Raf in focus assays but did not otherwise function in these assays, perhaps due to a lack of appropriate effector pathways in NIH3T3 fibroblasts for this neural-specific Ras family member. In summary, although Rit shares most core effector domain residues with Ras, our results suggest that Rit uses novel effector pathways to regulate proliferation and transformation.
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PMID:Rit, a non-lipid-modified Ras-related protein, transforms NIH3T3 cells without activating the ERK, JNK, p38 MAPK or PI3K/Akt pathways. 1103 18

The ternary complex factor Elk-1, a major nuclear target of extracellular signal-regulated kinases, is a strong transactivator of serum-responsive element (SRE) driven gene expression. We report here that mature brain neurons and nerve growth factor (NGF)-differentiated PC12 cells also express a second, smaller isoform of Elk-1, short Elk-1 (sElk-1). sElk-1 arises from an internal translation start site in the Elk-1 sequence, which generates a protein lacking the first 54 amino acids of the DNA-binding domain. This deletion severely compromises the ability of sElk-1 to form complexes with serum response factor on the SRE in vitro and to activate SRE reporter genes in the presence of activated Ras. Instead, sElk, but not a mutant that cannot be phosphorylated, inhibits transactivation driven by Elk-1. More pertinent to the neuronal-specific expression of sElk-1, we show it plays an opposite role to Elk-1 in potentiating NGF-driven PC12 neuronal differentiation. Overexpression of sElk-1 but not Elk-1 increases neurite extension, an effect critically linked to its phosphorylation. Interestingly, in the presence of sElk-1, Elk-1 loses its strictly nuclear localization to resemble the nuclear/cytoplasm pattern observed in the mature brain. This is blocked by mutating a normally cryptic nuclear export signal in Elk-1. These data provide new insights into molecular events underlying neuronal differentiation of PC12 cells mediated by the NGF-ERK signaling cascade.
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PMID:Opposing roles of Elk-1 and its brain-specific isoform, short Elk-1, in nerve growth factor-induced PC12 differentiation. 1105 86

Proliferative signals lead to the rapid and transient induction of the c-fos proto-oncogene by targeting the ternary complex assembled on the serum response element (SRE). Transactivation by both components of this complex, serum response factor (SRF) and the ternary complex factor Elk-1, can be potentiated by the coactivator CREB-binding protein (CBP). We report a novel interaction between the bromodomain of CBP, amino acids 1100-1286, and Elk-1. DNA binding and glutathione S-transferase pull-down assays demonstrate that binding requires Elk-1(1-212) but not the C-terminal transactivation domain. Competition and antibody controls show that the bromocomplex involves both SRF and Elk-1 on the c-fos SRE and uniquely Elk-1 on the E74 Ets binding site. Interestingly, methylation interference and DNA footprinting analyses show almost indistinguishable patterns between ternary and bromocomplexes, suggesting that CBP-(1100-1286) interacts via Elk-1 and does not require specific DNA contacts. Functionally, the bromocomplex blocks activation, because cotransfection of CBP-(1100-1286) reduces RasV12-driven activation of SRE and E74 luciferase reporters. Repression is relieved moderately or strongly by linking the bromodomain to the N- or C-terminal transactivation domains of CBP, respectively. These results are consistent with a model in which CBP is constitutively bound to the SRE in a higher order complex that would facilitate the rapid transcriptional activation of c-fos by signaling-driven phosphorylation.
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PMID:Induction-independent recruitment of CREB-binding protein to the c-fos serum response element through interactions between the bromodomain and Elk-1. 1108 68

This report describes the development and application of a dual vector coexpression system for the overproduction of heteromeric cell cycle and transcriptional regulatory protein complexes in bacteria. To facilitate these studies we constructed a T7-based expression plasmid, pRM1 that contains an origin of replication derived from p15A, and a gene encoding kanamycin resistance. This expression vector is compatible with ColE1-derived plasmids found in the pET family of T7 expression vectors, which encode ampicillin resistance. It also has the same multiple cloning sites as the pET- derived pRSET vector, allowing easy shuttling between the two expression vectors. Cotransformation of the pRM1 and pET-derived expression vectors into an Escherichia coli strain such as BL21(DE3) results in a significant level of coexpression of heteromeric protein complexes. We demonstrate the applicability of combining the pRM1 and pET-derived vectors for the coexpression of cell cycle regulatory components, pRB/E7 and pRB/E1a, and the transcriptional regulatory complexes, SRF/SAP-1 and SRF/Elk-1. We further use the pRB/E1a complex to demonstrate that these coexpressed complexes can be purified to homogeneity for further studies. Use of the pRM1 vector in combination with the pET-derived vectors should be generally applicable for the large-scale coexpression and purification of a wide variety of heteromeric protein complexes for biochemical, biophysical, and structural studies.
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PMID:Coexpression of proteins in bacteria using T7-based expression plasmids: expression of heteromeric cell-cycle and transcriptional regulatory complexes. 1108 83

17beta-Estradiol (E2) induces c-fos protooncogene expression in MCF-7 human breast cancer cells, and deletion analysis of the c-fos promoter showed that the serum response element (SRE) at -325 to -296 was E2-responsive. The mechanism of ligand-activated estrogen receptor alpha (ERalpha)-dependent activation of gene expression through the SRE was determined by mutational analysis of the promoter, analysis of mitogen-activated protein kinase (MAPK) pathway activation by E2, and transforming growth factor alpha (TGF-alpha) as a positive control. In addition, ERalpha-negative MDA-MB-231 breast cancer and Chinese hamster ovary cells were used as reference cell lines. The results showed that transcriptional activation of the SRE by E2 was due to ERalpha activation of the MAPK pathway and increased binding of the serum response factor and Elk-1 to the SRE. Subsequent studies with dominant negative Elk-1, wild type, and variant GAL4-Elk-1 fusion proteins confirmed that phosphorylation of Elk-1 at serines 383 and 389 in the C-terminal region of Elk-1 is an important downstream target associated with activation of an SRE by E2. Both E2 (ERalpha-dependent) and growth factors (ERalpha-independent) activated the SRE in breast cancer cells via the Ras/MAPK pathway; however, in ER-negative CHO cells that do not express a receptor for TGF-alpha, only hormone-induced activation was observed in cells transfected with ERalpha.
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PMID:Estrogen receptor-mediated activation of the serum response element in MCF-7 cells through MAPK-dependent phosphorylation of Elk-1. 1114 55

The serum response element (SRE) of the c-fos promoter is a convergence point for mitogenic signaling pathways. Several transcription factors regulate SRE, including serum response factor (SRF), ternary complex factors, and CCAAT/enhancer-binding protein-beta (C/EBPbeta). C/EBPbeta can interact with both SRF and the ternary complex factor family member Elk-1, but only in response to activated Ras. Transactivation of the SRE by C/EBPbeta is also greatly stimulated by Ras. The Ras effectors that signal to C/EBPbeta are unknown. In this report, we demonstrate that a consensus MAPK site in C/EBPbeta is necessary for Ras stimulation of both C/EBPbeta-SRF interaction and transactivation of the SRE by C/EBPbeta. To dissect signaling pathways activated downstream of Ras, different Ras effector constructs were analyzed. We show that activated forms of Raf and phosphatidylinositol 3-kinase stimulate C/EBPbeta-SRF interaction. We also show a novel selectivity for the MAPK family member ERK2, where dominant-negative ERK2, but not dominant-negative ERK1, blocks Ras stimulation of C/EBPbeta-SRF interaction. In addition, recombinant C/EBPbeta protein is phosphorylated by ERK2, but not by ERK1, in vitro. Finally, we demonstrate a requirement for p90(Rsk2) in regulation of C/EBPbeta-SRF interaction. These data show that multiple Ras effectors are required to regulate C/EBPbeta and SRF association.
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PMID:ERK2- and p90(Rsk2)-dependent pathways regulate the CCAAT/enhancer-binding protein-beta interaction with serum response factor. 1150 Apr 90

Lipopolysaccharide (LPS) induces human monocytes to express many proinflammatory mediators, including the procoagulant molecule tissue factor (TF) and the cytokine tumor necrosis factor alpha (TNF-alpha). The TF and TNF-alpha genes are regulated by various transcription factors, including nuclear factor (NF)-kappaB/Rel proteins and Egr-1. In this study, the role of the MEK-ERK1/2 mitogen-activated protein kinase (MAPK) pathway in LPS induction of TF and TNF-alpha gene expression in human monocytic cells was investigated. The MAPK kinase (MEK)1 inhibitor PD98059 reduced LPS induction of TF and TNF-alpha expression in a dose-dependent manner. PD98059 did not affect LPS-induced nuclear translocation of NF-kappaB/Rel proteins and minimally affected LPS induction of kappaB-dependent transcription. In contrast, PD98059 and dominant-negative mutants of the Ras-Raf1-MEK-ERK (extacellular signal-regulated kinase) pathway strongly inhibited LPS induction of Egr-1 expression. In kinetic experiments LPS induction of Egr-1 expression preceded induction of TF expression. In addition, mutation of the Egr-1 sites in the TF and TNF-alpha promoters reduced expression of these proinflammatory genes. It was demonstrated that LPS induction of the Egr-1 promoter was mediated by 3 SRE sites, which bound an LPS-inducible complex containing serum response factor and Elk-1. LPS stimulation transiently induced phosphorylation of Elk-1 and increased the functional activity of a GAL4-Elk-1TA chimeric protein via the MEK-ERK1/2 pathway. The data indicate that LPS induction of Egr-1 gene expression is required for maximal induction of the TNF-alpha and TF genes in human monocytic cells.
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PMID:Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor alpha expression by inducing Elk-1 phosphorylation and Egr-1 expression. 1152 Jul 92

Replicative senescence is characterized by numerous phenotypic alterations including the loss of proliferative capacity in response to mitogens and numerous changes in gene expression including impaired serum inducibility of the immediate-early genes c-fos and erg-1. Transcription of c-fos in response to mitogens depends on the activation of a multiprotein complex formed on the c-fos serum response element (SRE), which includes the transcription factors SRF (serum response factor) and TCF (ternary complex factor). Our data indicate that at least two defects are responsible for the decreased c-fos transcription in senescent cells, one caused by diminished DNA binding activity of the SRF and another resulting from impaired activation of the TCF, Elk-1. In nuclei isolated from serum stimulated senescent cells the activating phosphorylation of p62(TCF)/Elk-1, which is catalyzed by the members of the extracellular-regulated kinase (ERK) family was strikingly diminished and correlated with a decrease in the abundance of activated ERK proteins. In contrast, in total cell lysates ERK phosphorylation and ERK activity (normalized to total protein) reached similar levels following stimulation of early- and late-passage cells. Interestingly, senescent cells consistently exhibited higher ERK protein abundance. Thus, the proportion of phosphorylated (active) ERK molecules in stimulated senescent cells was lower than in early passage cells. The accumulation of unphosphorylated ERK molecules in senescent cells correlated with the diminished abundance of phosphorylated (active) MEK. These data indicate that in senescent cells there is a general dysregulation in the ERK signaling pathway, which results in the accumulation of inactive ERK molecules, decreased abundance of active ERK in the nucleus of senescent cells, and subsequent lack of activation of the transcription factor TCF(Elk-1). These impairments, together with the impaired DNA binding activity of SRF, could potentially account for the lack of c-fos expression in senescent cells and for multiple other molecular changes dependent upon this pathway.
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PMID:Lack of Elk-1 phosphorylation and dysregulation of the extracellular regulated kinase signaling pathway in senescent human fibroblast. 1157 Aug 21

Heparan sulfate (HS) is one of the components of extracellular matrix and a potent anti-growth factor in various cells. Heparin has a similar structure to HS and is demonstrated to inhibit myocardial cell hypertrophy. We examined the intracellular signal mechanisms linking to the inhibitory effects of heparin and HS on endothelin-1 (ET-1)-induced hypertrophy in cultured rat neonatal myocardial cells (MCs). Heparin inhibited ET-1-induced c-fos mRNA expression. Heparin and HS inhibited ET-1-induced activation of c-fos promoter/enhancer in MCs. Although heparin and HS inhibited ET-1-induced activation of the wild-type c-fos serum response element (SRE), the activation of a mutated c-fos SRE that contains an intact binding site for the serum response factor (SRF) but lacks the ternary complex factor (TCF) binding site, was not inhibited. In addition, heparin and HS inhibited the activation of TPA response element (TRE). However, heparin did not inhibit the activation of cyclic AMP response element (CRE). Furthermore, heparin and HS inhibited ET-1-induced activation of extracellular signal-regulated kinase (ERK) and phosphorylation of Elk-1, which is one of the TCFs. These results indicate that heparin and HS inhibited ET-1-induced ERK activation, resulting in suppression of Elk-1 phosphorylation, and lead to inhibition of c-fos gene expression through SRF-independent manner. Moreover, heparin and HS inhibited ET-1-induced [3H] leucine incorporation. These results suggest that heparin and HS inhibit ET-1 induced myocardial cell hypertrophy through the inhibition of gene expression and protein synthesis.
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PMID:Heparin and heparan sulfate inhibit extracellular signal-regulated kinase activation and myocardial cell hypertrophy induced by endothelin-1. 1159 24


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