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:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cell dysfunction or dysregulation in cancer generally results from complex gene interactions, numerous cellular events and environmental influences which modify gene expression or post-translational protein modifications. Genetic analysis in itself cannot always predict or diagnose multigenic diseases. The major technical difficulty is thus to detect, identify and measure simultaneously the expression of several genes and the post-translational modifications of their products. In order to progress to this direction, this paper describes a simple immunoblot method using several monoclonal anti-bodies to simultaneously analyze oncogene expression and cell cycle specific checkpoints in patient solid biopsies and transformed cell lines. One mg of normal human liver biopsy and HEPG2 (hepatoblastoma-derived cell line) protein samples have been separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were stained with amido black, scanned and tested separately with the nine monoclonal antibodies p53, c-myc, PCNA, MEK1, pan-ras, Cip1, Cdc2, Kip1, and TCTP. The nine antibodies of interest were then combined to form a mixture, and simultaneously used as the primary antibodies. This antibody mixture simultaneously detected the nine proteins of interest on both samples and it demonstrated the extensive expression changes and the presence of various isoforms most likely due to post-translational modifications of gene products.
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PMID:Simultaneous analysis of cyclin and oncogene expression using multiple monoclonal antibody immunoblots. 915 Sep 53

We have assessed five signal transduction pathways to determine the role each might play in the malignant transformation of mammary epithelium initiated by neu, heregulin/NDF, TGFalpha, v-Ha-ras and c-myc in transgenic mice. The study involves a molecular and pharmacologic assessment of Erk/MAP kinase, Jnk/SAP kinase, PI 3-kinase, protein kinase C, and the Src-related kinases Lck and Fyn. Our results indicate that oncogenes capable of transforming mammary gland epithelium activate and require specific signal transduction pathways. For example, mammary tumors initiated by neu, v-Ha-ras and c-myc have high levels of active Erk/MAP kinase and their anchorage independent growth is strongly inhibited by PD098059, an inhibitor of Mek/ MAP kinase kinase. By contrast, Erk/MAP kinase activity is weak in tumors initiated by TFGalpha and heregulin/NDF and the corresponding cell lines are not growth inhibited by PD098059. Similarly, PI 3-kinase is strongly activated in neu, TGFalpha and heregulin/NDF initiated tumor cell lines, but not in c-myc or v-Ha-ras initiated tumor cell lines. The anchorage independent growth of all these tumor cell lines are, however, inhibited by the specific PI 3-kinase inhibitor LY294001. Further illustrating this oncogene-based specificity, PP1, a specific inhibitor of the Src-like kinases, Lck and Fyn, blocks anchorage-independent cell growth only in the TGFalpha initiated mammary tumor cell line. Taken together with additional observations, we conclude that certain oncogenes reliably require the recruitment/activation of specific signal transduction pathways. Such specific relationships between the initiating oncogene and a required pathway may reflect a direct activating effect or the parallel activation of a pathway that is a necessary oncogenic collaborator for transformation in the mammary gland. The work points to a molecular basis for targeting therapy when an initiating oncogene can be implicated; for example, because of amplification, increased expression, genetic alteration, or heritable characteristics.
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PMID:Signal transduction pathways activated and required for mammary carcinogenesis in response to specific oncogenes. 948 37

Members of the erbB family of receptor tyrosine kinases are commonly overexpressed in human breast cancer. However, the relative contribution of particular signalling pathways activated downstream of these receptors to the mitogenic response of transformed breast epithelial cells remains poorly characterized. Administration of heregulin-beta2 (HRG), a ligand for erbB3 and erbB4, to growth arrested T-47D human breast cancer cells leads to activation of both the PI3-kinase and MAP kinase signalling pathways and potent stimulation of cell cycle progression. Specific inhibitors were used to assess the role of these pathways in HRG-induced mitogenesis and to identify underlying mechanisms in terms of regulation of gene expression. Treatment with the MEK inhibitor PD98059 led to a complete block of HRG-induced entry into S-phase, whilst administration of the PI3-kinase inhibitor wortmannin resulted in only modest inhibition. In addition, administration of PD98059 8 h after HRG was equipotent with simultaneous administration in inhibiting entry into S-phase. However, delaying addition for 14-16 h after HRG, when the cells were entering S-phase, was without effect. HRG stimulation led to sequential induction of c-myc, cyclin D1, cyclin E and cyclin A gene expression and hyperphosphorylation of the retinoblastoma protein pRB. p21 (WAF1/CIP1/SDI1) gene expression was rapidly induced by HRG, but significant changes in p27 (KIP1) protein levels were not detected. Preincubation with PD98059 blocked the HRG-dependent induction of cyclin D1 mRNA, p21 and c-Myc protein and pRB phosphorylation. These findings demonstrate that MEK activation is critical to HRG-induced S-phase entry in these cells whilst PI3-kinase plays a minor role. Moreover, these data are compatible with HRG-induced activation of MEK being critical for a mid-G1 transition point and implicate c-myc and cyclin D1 as key targets of the MAP kinase pathway involved in this response.
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PMID:Inhibition of the MAP kinase cascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cells. 965 48

In response to oxidant stress, the cardiovascular system is known to express a number of genes, which could occur owing to the participation of mitogen-activated protein kinases such as MAPKs, ERK and JNK (SAPK) followed by stimulation of at least two well-defined transcription factors NF-KB and AP-1 (c-Fos and c-Jun). Oxidants activate cytosolic and membrane-bound PLA2 activities with the subsequent production of AA metabolites such as HETEs, which subsequently stimulate ERK and JNK (SAPK) activities leading to the activation of transcriptional factors and the ultimate stimulation of the transcription of several mitogen-stress-responsive genes. LacCer, a ceramide analogue present in atherosclerotic plaques, has been found to induce proliferation of aortic smooth muscle cells. LacCer is involved in Ras-GTP loading, activation of kinase cascades (MEK, Raf, p44 MAPK) and c-fos expression. TNF-alpha, on the other hand, induces c-fos, c-myc and c-jun expression. Recent investigations link ceramide and its analogues to the extracellular signal-regulated kinase (ERK) cascade, stress-activated protein kinase-c-Jun kinase (SAPK/JNK) cascade and apoptotic responses. These critical steps in the signalling pathways are sensitive to intracellular thiol-redox and protease(s)-antiprotease(s) status, both of which can be modified by oxidants. Because mobilisation of intracellular Ca2+ caused by a variety of signals also plays a role in the activation of the signalling pathways, an important aspect of future work will be to ascertain the roles of oxidants and Ca2+ individually and in combination in the activation of the signalling pathways. The following two important questions also deserve future attention: (1) How does NF-kB shield cells from apoptotic death? and (2) By what mechanisms does the activated NF-kB cause cellular transformation? Furthermore, the role of AP-1 acting as transcriptional activator seems clear, but the target genes remain to be defined.
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PMID:Oxidant-mediated activation of mitogen-activated protein kinases and nuclear transcription factors in the cardiovascular system: a brief overview. 988 18

The mitogen-inducible gene c-myc is a key regulator of cell proliferation and transformation. Yet, the signaling pathway(s) that regulate its expression have remained largely unresolved. Using the mitogen-activated protein kinase kinase (MEK1/2) inhibitor PD98059 and dominant negative forms of Ras (N17) and ERK1 (K71R), we found that activation of Ras and extracellular signal-regulated kinase (ERK) is necessary for colony-stimulating factor-1 (CSF-1)-mediated c-Myc expression and DNA synthetic (S) phase entry. Quiescent NIH-3T3 cells expressing a partially defective CSF-1 receptor, CSF-1R (Y809F), exhibited impaired ERK1 activation and c-Myc expression and failed to enter the S phase of the cell division cycle in response to CSF-1 stimulation. Ectopic expression of a constitutively active form of MEK1 in cells expressing CSF-1R (Y809F) rescued c-Myc expression and S phase entry, but only in the presence of CSF-1-induced cooperating signals. Therefore, MEK1 participates in an obligate signaling pathway linking CSF-1R to c-Myc expression, but other signals from CSF-1R must cooperate with the MEK/ERK pathway to induce c-Myc expression and S phase entry in response to CSF-1 stimulation.
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PMID:Expression of c-Myc in response to colony-stimulating factor-1 requires mitogen-activated protein kinase kinase-1. 1003 49

The mitogenic response to insulin and epidermal growth factor (EGF) was studied in subconfluent and confluent cultures of primary rat hepatocytes. In subconfluent cultures, wortmannin, LY294002, and rapamycin reversed insulin- and EGF-induced [3H]thymidine incorporation into DNA. The mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) inhibitor PD98059 was without significant effect on either insulin- or EGF-induced [3H]thymidine incorporation. Insulin treatment did not alter levels of messenger RNAs (mRNAs) for c-fos, c-jun, and c-myc. EGF induced an increase in c-myc, but not c-fos or c-jun, mRNA levels in subconfluent hepatocyte cultures. This increase in c-myc mRNA was abolished by PD98059. In confluent cells that could not be induced to synthesize DNA, EGF treatment also promoted an increase in c-myc mRNA to levels seen in subconfluent cultures. This increase was also abrogated by PD98059. These data indicate that in primary rat hepatocyte cultures, 1) the phosphoinositol 3-kinase pathway, perhaps through p70s6k activation, regulates DNA synthesis in response to insulin and EGF; 2) the MAPKpathway is not involved in insulin- and EGF-induced DNA synthesis; and 3) p44/42 MAPKs are involved the induction of c-myc mRNA levels, although this induction is not required for DNA synthesis. These studies define two distinct signal transduction pathways that independently mediate growth-related responses in a physiologically relevant, normal cell system.
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PMID:Epidermal growth factor and insulin-induced deoxyribonucleic acid synthesis in primary rat hepatocytes is phosphatidylinositol 3-kinase dependent and dissociated from protooncogene induction. 1057 26

Cooperation of myc and activated ras has been suggested to cause malignant cell transformation but the mechanism is still unknown. Here we isolated a transformed cell line in which activation of c-Myc and Ras are independently controllable, and show that after establishment of the transformed state by c-myc and activated ras, removal of activated Ras initiates apoptosis that is dependent on c-Myc activity. Apoptosis is also initiated by an inhibitor of MEK (MAPK/ERK kinase), a kinase downstream of Ras, and apoptosis is blocked by activated Mek1. These results suggest that one of the conditions required for establishment of the transformed state is a block of apoptosis involving MEK activity. We tested the effect of MEK inhibition on cells transformed by various oncogenes. Suppression of apoptosis by MEK is not critical in general, but in cells transformed by c-myc plus a gene that activates the MAPK cascade it is necessary to avoid cell death. Activated Ras/MEK did not suppress c-myc-dependent apoptosis due to serum-limitation. Overexpression of chicken bcl-xL suppressed apoptosis under serum-limiting conditions, but not apoptosis initiated by Ras/MEK inhibition in cells transformed by myc and activated ras. Altogether, these results suggest the existence of a novel regulatory mechanism for myc-dependent apoptosis in certain transformed cells.
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PMID:Ras/MEK signaling suppresses Myc-dependent apoptosis in cells transformed by c-myc and activated ras. 1064 86

Recent studies have shown that during apoptosis protein synthesis is inhibited and that this is in part due to the proteolytic cleavage of eukaryotic initiation factor 4G (eIF4G). Initiation of translation can occur either by a cap-dependent mechanism or by internal ribosome entry. The latter mechanism is dependent on a complex structural element located in the 5' untranslated region of the mRNA which is termed an internal ribosome entry segment (IRES). In general, IRES-mediated translation does not require eIF4E or full-length eIF4G. In order to investigate whether cap-dependent and cap-independent translation are reduced during apoptosis, we examined the expression of c-Myc during this process, since we have shown previously that the 5' untranslated region of the c-myc proto-oncogene contains an IRES. c-Myc expression was determined in HeLa cells during apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand. We have demonstrated that the c-Myc protein is still expressed when more than 90% of the cells are apoptotic. The presence of the protein in apoptotic cells does not result from either an increase in protein stability or an increase in expression of c-myc mRNA. Furthermore, we show that during apoptosis initiation of c-myc translation occurs by internal ribosome entry. We have investigated the signaling pathways that are involved in this response, and cotransfection with plasmids which harbor either wild-type or constitutively active MKK6, a specific immediate upstream activator of p38 mitogen-activated protein kinase (MAPK), increases IRES-mediated translation. In addition, the c-myc IRES is inhibited by SB203580, a specific inhibitor of p38 MAPK. Our data, therefore, strongly suggest that the initiation of translation via the c-myc IRES during apoptosis is mediated by the p38 MAPK pathway.
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PMID:c-Myc protein synthesis is initiated from the internal ribosome entry segment during apoptosis. 1064 1

In a previous work we have reported evidences on the mitogenic activity of urokinase-type and tissue-type plasminogen activator (u-PA, t-PA) on serum-deprived human dermal fibroblasts. In this work we have studied the transcription-dependent changes of some cell-cycle related genes associated with the biological activity of PAs, as well as the possible involvement of protein tyr kinases (PTK) and/or protein kinase C (PKC) in the mitogenic signal transduction. The data obtained demonstrate that the growth factor activity of PAs is associated with: - a rapid transient activation of early response genes, c-fos, c-jun and c-myc; - the subsequent coordinated down-regulation of p53 and p21CIP1; - the constant expression of the MEK1 mRNA in every phase of the cell cycle. Quiescent (G0) cells did not express c-fos, c-jun, c-myc and cyclin A, but upon stimulation with mitogens (fetal calf serum (FCS), u-PA, t-PA) the cyclin A mRNA expression was observed in concomitance with the activation of DNA synthesis. Therefore u-PA, t-PA and FCS similarly modulate the expression of c-fos, c-jun, c-myc, p53, p21CIP1 and cyclin A with only slight differences likely related to the time required for activation of DNA synthesis. The PAs mitogenic stimulation of serum-starved cells was associated with the internalization of their molecules, as revealed by immunostaining. The biological activity of u-PA, t-PA, as well as that of limiting concentration of FCS (1%), was mediated by PTK and PKC. Conversely, PTK, but not PKC, was involved in the activation of the proliferative response of basic fibroblast growth factor in the same experimental conditions. In conclusion, u-PA and t-PA can utilize two different pathways, one depending on PTK and the other on PKC in a way similar to the mitogenic activity induced by low concentration of FCS (1%).
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PMID:Gene response of human skin fibroblasts to urokinase- and tissue-type plasminogen activators. 1080 Oct 75

Exposure of islet beta-cells to elevated glucose concentrations (30 versus 3 mm) prompts enhanced preproinsulin (PPI) gene transcription and the trans-location to the nucleoplasm of pancreatic duodenum homeobox-1 (PDX-1; Rafiq, I., Kennedy, H., and Rutter, G. A. (1998) J. Biol. Chem. 273, 23241-23247). Here, we show that in MIN6 beta-cells, over-expression of p110.CAAX, a constitutively active form of phosphatidylinositol 3-kinase (PI3K) mimicked the activatory effects of glucose on PPI promoter activity, whereas Deltap85, a dominant negative form of the p85 subunit lacking the p110-binding domain, and the PI3K inhibitor LY 294002, blocked these effects. Similarly, glucose-stimulated nuclear trans-location of endogenous PDX-1 was blocked by Deltap85 expression, and wortmannin or LY 294002 blocked the trans-location from the nuclear membrane to the nucleoplasm of epitope-tagged PDX-1.c-myc. By contrast, SB 203580, an inhibitor of stress-activated protein kinase-2 (SAPK2)/p38 MAP kinase, had no effect on any of the above parameters, and PPI promoter activity and PDX-1.c-myc localization were unaffected by over-expression of the upstream kinase MKK6 (MAP kinase kinase-6) or wild-type p38/SAPK2, respectively. Furthermore, no change in the activity of extracted p38/SAPK2 could be detected after incubation of cells at either 3 or 30 mm glucose. These data suggest that stimulation of PI3K is necessary and sufficient for the effects of glucose on PPI gene transcription, acting via a downstream signaling pathway that does not involve p38/SAPK2.
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PMID:Glucose-stimulated preproinsulin gene expression and nuclear trans-location of pancreatic duodenum homeobox-1 require activation of phosphatidylinositol 3-kinase but not p38 MAPK/SAPK2. 1082 51


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