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)

Both MAP kinases and the protein kinase p74raf-1 are activated by many growth factors in a c-ras-dependent manner and by oncogenic p21ras. We were therefore interested in determining the relationship between MAP kinases and raf. The MAP kinase ERK2 is activated by expression of oncogenically activated raf, independently of cellular ras. Overexpressed p74raf-1 potentiates activation of ERK2 by EGF and TPA. MAP kinase kinase inactivated by phosphatase 2A treatment is phosphorylated and reactivated by incubation with p74raf-1 immunoprecipitated from phorbol ester-treated cells. We conclude that raf protein kinase is upstream of MAP kinases and is either a MAP kinase kinase kinase or a MAP kinase kinase kinase kinase.
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PMID:Activation of the MAP kinase pathway by the protein kinase raf. 133 Mar 21

Interferons (IFNs) exert antiproliferative effects on many types of cells. The underlying molecular mechanism, however, is unclear. One possibility is that IFNs block growth factor-induced mitogenic signaling, which involves activation of Ras/Raf-1/MEK/mitogen-activated protein kinase. We have tested this hypothesis by using HER14 cells (NIH 3T3 cell expressing both platelet-derived growth factor [PDGF] and epidermal growth factor [EGF] receptors) as a model system. Our studies showed that IFNs (alpha/beta and gamma) blocked PDGF-and phorbol ester- but not EGF-stimulated DNA synthesis and cell proliferation. While the ligand-stimulated receptor tyrosine phosphorylation and interaction with downstream signaling molecules, such as GRB2, were not affected, IFNs specifically blocked PDGF- and phorbol ester- but not EGF-stimulated activation of Raf-1, mitogen-activated protein kinases, and tyrosine phosphorylation of an unidentified 34-kDa protein. This inhibition could be detected as early as 5 min after IFN treatments and was insensitive to cycloheximide, indicating that de novo protein synthesis is not required. The IFN-induced inhibition acted upstream of Raf-1 kinase and downstream of diacyl glycerol/phorbol ester, suggesting that protein kinase C (PKC) is the potential primary target. Consistently, downregulation of PKC by chronic phorbol myristate acetate treatment or inhibition of PKC by H7 and staurosporine blocked PDGF- and phorbol myristate acetate- but not EGF-induced signaling and DNA synthesis. Moreover, incubating cells with antisense oligodeoxyribonucleotides of PKC delta eliminated production of PKC delta protein and specifically blocked PDGF- but not EGF-stimulated mitogenesis in these cells. Thus, these studies have elucidated a major difference in the early events of EGF-and PDGF-stimulated signal transduction and, more importantly, revealed a novel mechanism by which IFNs may execute their antiproliferative function.
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PMID:Interferons block protein kinase C-dependent but not-independent activation of Raf-1 and mitogen-activated protein kinases and mitogenesis in NIH 3T3 cells. 862 73

Src homology/collagen (SHC) proteins are thought to participate in signaling through both receptor tyrosine kinases, such as the insulin receptor and the EGF (epidermal growth factor) receptor, and cytoplasmic tyrosine kinases, such as v-src and v-fps. Here we approached the insulin-induced and the insulin-like-growth-factor-I-induced (IGF-I-induced) phosphorylation of SHC proteins, and the possible role of these proteins in insulin and IGF-I signaling. First, we showed that SHC proteins are phosphorylated on tyrosine residues upon insulin and IGF-I treatment of fibroblasts transfected with a SHC cDNA construct. More important, ligand-activated insulin and IGF-I receptors phosphorylate SHC proteins in vitro, indicating that SHC proteins could be direct substrates for insulin and IGF-I receptors. Further, insulin or IGF-I treatment of SHC-transfected fibroblasts leads to immunoprecipitation of SHC proteins with insulin-receptor substrate 1 (IRS-1). We next looked at the possible effect of SHC proteins on biological responses in SHC-transfected fibroblasts. We found that the expression of exogenous SHC proteins results in an increased basal MEK (MAPK/ERK-activating kinase) activity. Further, neither the basal nor the insulin-induced or IGF-I-induced PtdIns-3-kinase activity were modified by expression of exogenous SHC proteins. These results illustrate that SHC proteins are implicated in the MAP (mitogen-activated protein)-kinase pathway, but not in that of PtdIns-3-kinase. Finally, we show that SHC-transfected cells, unlike control cells, are able to advance into the early phases of the cell cycle, and are more sensitive to the growth-promoting effect of insulin. In conclusion, SHC proteins are substrates for insulin and IGF-I receptors, and would appear to function as early post-receptor signaling components.
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PMID:Involvement of Src-homology/collagen (SHC) proteins in signaling through the insulin receptor and the insulin-like-growth-factor-I-receptor. 803 92

We have studied in cultured rat astroglial cells MAP kinases, known for their role in intracellular signal transduction. The MAP kinase activity was stimulated by growth factors (FGFb, FGFa, EGF, PDGF, and IGF1), by a phorbol ester (TPA) activating-protein kinase C (PKC), by a neuropeptide (endothelin-1), and by a neuromediator (carbachol). Astrocytes pretreated for 18 h with TPA were still stimulated by growth factors and endothelin, suggesting that down-regulated isoforms of PKC are not involved in MAP kinase activation. In contrast, the small effect of carbachol was suppressed by TPA pretreatment. Astrocytes contained two proteins (p41 and p44) recognized by MAP kinase antibody. These proteins were phosphorylated on tyrosine residues in the cytosols of stimulated astrocytes. The kinetics of MAP kinase activation by FGFb and IGF1 were very different. FGFb promoted a rapid activation of MAP kinase (about 10 min) plus a prolonged phase that lasted at least 12 h. IGF1 produced only a rapid transient peak of activation at about 20 min. Hence, extracellular signals might generate different effects in astrocytes by differentially modulating the MAP kinase cascade. On a Mono Q column the growth factor-stimulated MAP kinase activity was separated into two peaks containing p41 and p44. Stimulation of astrocytes altered the elution pattern of p44 as a result of its phosphorylation. An ATP-dependent MAP kinase activator (MW = 40-45 kDa) was found in fractions of FGFb-stimulated cells which were not retained on Mono Q column, indicating the existence of a MAP kinase kinase (MEK) in astrocytes. C-Raf, identified in other cells as a MAP kinase kinase kinase, was also present in astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:MAP kinase cascade in astrocytes. 816 69

Mitogen activated protein kinases (MAP) or extracellular signal regulated protein kinases (ERK) are a family of protein serine/threonine kinases that are activated very rapidly in response to many extracellular stimuli. elk-1, an ets related gene codes for two transcriptional factors elk-1, which regulates c-fos transcription and delta elk-1, both of which are substrates for MAP kinases. A part of the C-terminal transcriptional activation domain (ETA-2) which is common to both the proteins was previously shown to function as an activator of MAP kinases. In this report, in an attempt to investigate the mechanism of activation of MAP kinases, purified preparations of recombinant elk-1 and P44mpk/ERK-1/ERK-2 proteins were used to show the association of elk-1 proteins with MAP kinases. The specific interactions of elk-1 proteins with MAP kinases were confirmed by co-immunoprecipitation studies. Thus elk-1 proteins appear to regulate the activity of MAP kinases by interacting with them ensuring a conformational change and stimulating their autophosphorylation and activation property. The activation was dependent on the presence of ATP and Mg2+. In vitro phosphorylation of elk-1 protein was not regulatory for autonomous DNA binding activity of elk-1 protein. Cells which were exposed to EGF showed a rapid stimulation of an elk-1 specific kinase activity, probably MAP kinase which phosphorylated MBP and was found to be associated with immobilized GST-elk-1. Furthermore, dephosphorylation studies indicate that elk-1 proteins can activate only tyrosine phosphorylated MAP kinase. These results demonstrate the presence of an alternative pathway/mechanism (other than MAP kinase kinase, MAPKK/Mek) for the activation of MAP kinases with tyrosine phosphorylation occurring before serine/threonine autophosphorylation and activation by elk-1 proteins.
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PMID:elk-1 proteins interact with MAP kinases. 820 31

The Drosophila argos gene encodes a secreted protein with an EGF motif which acts as an inhibitor of cellular differentiation in multiple developmental processes. To investigate the cellular pathways regulated by Argos, we screened for mutations which could modify the phenotype caused by overexpression of argos. We show that the effects of argos overexpression on the eye and wing vein development are suppressed by gain-of-function mutations of the MAPKK/D-MEK gene (Dsor1/D-mek) and the MAPK/ERK-A gene (rolled) and were enhanced by loss-of-function mutations of Star. Loss-of-function mutations in components of the Ras/MAPK signaling cascade act as dominant suppressors of the phenotype caused by the argos null mutations. A loss-of-function argos mutation enhanced the overproduction of R7 neurons caused by gain-of-function alleles of Son of sevenless and Dsor1. Conversely, overexpression of argos inhibited formation of the extra R7 cells that was caused by high-level MAPK/ERK-A activity. A phenotype of the sev; argos double mutants revealed that sev is epistatic to argos. These results provide evidence that Argos negatively regulates signal transduction events in the Ras/MAPK cascade.
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PMID:The Drosophila secreted protein Argos regulates signal transduction in the Ras/MAPK pathway. 881 5

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) caused a significant decrease in estradiol (E2) production when it was administered to human luteinized granulosa cells (hLGCs) in culture. We investigated the involvement of the epidermal growth factor receptor (EGFR) and protein tyrosine kinase (PTK) in this TCDD-induced toxicity. Upregulation in 125I-EGF binding to EGFR was measured after 24 h of TCDD treatment, while downregulation in EGFR binding was measured after 72 h of TCDD treatment. Upregulation of EGFR binding was associated with a significant decrease in postnuclear (7000 x g supernatant) PTK activity, but this activity was stimulated after 72 h of TCDD treatment. TCDD altered the level of tyrosine phosphorylation in proteins with molecular weights 35, 40, 43, 45, 60, and > 205 kDa. TCDD caused a significant increase in postnuclear cAMP-dependent protein kinase (PKA) after 24 h of treatment. The actions of TCDD on protein kinases were partially blocked by the protein synthesis inhibitor, cycloheximide. On the other hand, TCDD increased nuclear PTK and decreased nuclear PKA activity. E2 inhibited the postnuclear and nuclear activity of both PTK and PKA in control samples, but did not affect TCDD actions on either postnuclear or nuclear PTK activity. However, E2 abolished the stimulatory effect of TCDD on PKA activity in postnuclear protein. In the presence of insulin, TCDD did not induce any additional changes in postnuclear or nuclear PTK. Forskolin (FK) alone inhibited postnuclear PTK activity and stimulated its nuclear activity. The addition of TCDD 20 min after FK resulted in an increase in postnuclear PTK, but there was little change in nuclear PTK as compared to the effect of FK alone. The stimulatory effect of TCDD on postnuclear PKA activity was enhanced by insulin and TCDD reversed the negative effect of FK, but there was no effect of either insulin or FK on the inhibition by TCDD of nuclear PKA activity. TCDD decreased the activity of MAP2 kinase and reduced the binding activity of AP-1 DNA when given alone, and also blocked the E2 stimulation of MAP2K. These findings suggest that TCDD may interrupt the endocrine function of hLGCs through the blockage of the mitotic signal directly or indirectly through the interaction of PTK/MAP2K and PKA signaling.
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PMID:Mechanism of toxic action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in cultured human luteinized granulosa cells. 894 64

Tyrphostins are synthetic compounds that have been described as in vitro inhibitors of epidermal growth factor receptor (EGF-R) tyrosine kinase activity. The inhibitory effect of tyrphostins in intact cells has been shown only after prolonged treatment. However, these compounds appear to be readily incorporated, which suggests that tyrphostin acts indirectly on EGF-R. We studied the effects of a tyrphostin derivative, RG 50864, without preincubation in intact epithelial cells. We selected two human cell lines differing in degree of expression of the p185erbB2 protein, which is closely related to EGF-R. We showed that tyrphostin (RG 50864) had no effect on EGF-dependent EGF-R tyrosine phosphorylation in the parental cell line. On the contrary, it prolonged the EGF-dependent EGF-R and p185erbB2(V-E) tyrosine phosphorylation in p185erbB2(V-E)-expressing cells. Because tyrphostin has been shown to be an inhibitor of p185erbB2 and EGF-R in vitro, this finding indicates that the tyrphostin effect on p185erbB2(V-E) and EGF-R was the result of an indirect mechanism in transfected cells. Tyrphostin treatment alone led to the activation of mitogen-activated protein (MAP) kinase kinase or MAP kinase or extracellular signal-regulated kinase kinase (MEK), suggesting that one of the tyrphostin targets was upstream of MEK1. MAP kinase, however, was not activated after tyrphostin treatment. This finding indicates that tyrphostin had another target in intact cells because MEK1 activation by tyrphostin alone did not correlate with MAP kinase activation. In the two cell lines, tyrphostin modified the time course of EGF-dependent MEK and MAP kinase activation. We conclude that whereas tyrphostins were designed to inhibit EGF-R tyrosine kinase activity, under our conditions EGF-R is not a physiological target for tyrphostin, nor is one of its related protein tyrosine kinases, p185erbB2(V-E). On the contrary, our results show that tyrphostin targets are multiple, leading to complex effects on receptor signaling in these epithelial cells.
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PMID:Epidermal growth factor receptor signaling cascade as target for tyrphostin (RG 50864) in epithelial cells. Paradoxical effects on mitogen-activated protein kinase kinase and mitogen-activated protein kinase activities. 906 32

Binding of estrogen to its receptor (ER) activates early genes that drive responsive cells through the proliferative phase. Earlier studies to evaluate the expression of protooncogenes, growth factors, growth factor receptor and steroid hormone receptor gene activities in the rat uterine system indicated complex pathways that involve significant 'crosstalk' between ER-systems and signal transduction pathways (Bhattacharyya et al., 1994). To analyze the interactions between these factors, we examined two well characterized estrogen dependent (MCF-7) and estrogen independent (MDA-MB-231) human breast cancer cell lines. Antibodies to estrogen receptor, epidermal growth factor receptor, c-Fos, c-Jun, and Ras proteins, protein kinases involved in receptor tyrosine kinase signal transduction pathway, MEK1 and phosphotyrosine were utilized in immunocytochemical localization experiments to evaluate temporal expression of these factors in response to estrogen treatment. ER, which was diminished in MCF-7 cells grown in estrogen-stripped medium, increased 9-fold in estrogen-reconstituted medium by 120 min. Fos and Jun appeared at nuclear and perinuclear cytoplasmic sites within 60 min after estrogen treatment in MCF-7 cells. Fos/Jun proteins were prominent in MDA-MB-231 cells, especially in association with actin filaments. Immunolabeling studies revealed no EGF-r in MCF-7 cells, while MDA-MB-231 cells contained intense EGF-r labeling in the plasma membrane. Ras protein was prominent in the cytoplasm and at the cell surface within 60 min after treatment of MCF-7 cells with estrogen. Ras was intense in MDA cells. Similarly, MCF-7 and MDA cells contained high concentrations of MEK1 and phosphotyrosine (pTyr) containing proteins in their cytoplasm and immunolabeling remained high as long as MCF-7 cells were grown in medium containing estrogen. It is speculated that MEK1 (cytoplasmic) functioning through Fos/Jun or Myc/Max (nuclear) may regulate the activity of AP-1 transcription factor. In all cases however, MEK1 and pTyr protein labeling was more intense in the highly metastatic and hormone independent MDA-MB-231 breast cancer cells. Results revealed signal transduction pathway proteins in ER+ estrogen dependent cells suggesting possible crosstalk between both receptor pathways during the proliferative phase of MCF-7 cells.
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PMID:Estrogen receptor, growth factor receptor and protooncogene protein activities and possible signal transduction crosstalk in estrogen dependent and independent breast cancer cell lines. 906 37

SOS, the guanine nucleotide exchange factor for Ras, becomes phosphorylated on serine and threonine residues following stimulation of cells with growth factors. These phosphorylations may play a role in negative feedback of Ras stimulation and have been shown to be mediated in part by the MAP kinases Erk-1 and Erk-2. Here we show that in addition to MAP kinase, a major mitogen activated kinase for SOS is p90 Rsk-2, a downstream target of MAP kinase. p90 Rsk-2 phosphorylates SOS in an in gel assay and also in solution in vitro. The ability of p90 Rsk-2 to phosphorylate SOS increases greatly following EGF treatment of PC12 cells and is blocked by expression of N17 Ras or treatment with the MEK inhibitor PD98059. Phosphopeptide mapping revealed that the sites phosphorylated by p90 Rsk-2 in vitro were also phosphorylated in intact cells in response to EGF treatment. Several major sites of in vivo phosphorylation correlated with p90 Rsk-2 phosphorylation sites rather than MAP kinase sites. It is therefore likely that p90 Rsk-2 plays an important role in the down regulation of the Ras activation pathway through SOS.
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PMID:EGF induced SOS phosphorylation in PC12 cells involves P90 RSK-2. 924 73


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