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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

In KB epidermoid cells, we previously showed that interleukin-1 alpha (IL-1) and various mitogens activate the mitogen-activated protein (MAP) kinases ERK1 and ERK2, which phosphorylate both myelin basic protein (MBP) and a peptide containing Thr669 of the epidermal growth factor receptor. In cell-free extracts made from gingival fibroblasts treated with platelet-derived growth factor or HepG2 hepatoma cells stimulated with phorbol myristate acetate, MBP and Thr669 kinase were both elevated 4-fold, and ERK1 and ERK2 were tyrosine-phosphorylated. In these cells IL-1 activated a kinase(s) that phosphorylated Thr669 peptide but not MBP and failed to cause tyrosine phosphorylation of ERK1/ERK2. Ceramide has been proposed as an intracellular mediator of IL-1 action, but C2-ceramide or sphingosine stimulated predominantly MBP-specific kinase activity in fibroblasts and had no effect in HepG2 cells. p54 MAP kinase (also called stress-activated protein kinase) is a c-Jun kinase first isolated from livers of cycloheximide-treated rats. After IL-1 stimulation, immunoprecipitates of lysates made from all three cell types with specific anti-p54 MAP kinase serum contained Thr669 and c-Jun phosphorylating activity, whereas precipitates from unstimulated cells contained no detectable p54 kinase activity. The major peak of IL-1-stimulated HepG2 Thr669 kinase activity co-chromatographed on Mono Q and phenyl-Superose with immunodetectable p54 MAP kinase. IL-1 did not cause p21ras activation in any cell type. Induction of Thr 669 kinase activity was not abrogated by elevation of cAMP levels, which has been shown to interfere with the activation of Raf-1. We could not detect MAP kinase kinase phosphorylating activity in unfractionated lysates made from IL-1-stimulated fibroblasts or HepG2 cells. KB cells contained a small amount of this activity, but it was not precipitated with an anti-Raf-1 antibody. We conclude that most of the IL-1-activated Thr669 kinase activity in fibroblasts and HepG2 cells, and a portion in KB cells, is due to p54 MAP kinase and that its activation is Ras-, Raf-, and MAP kinase kinase-independent.
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PMID:Interleukin-1 activates p54 mitogen-activated protein (MAP) kinase/stress-activated protein kinase by a pathway that is independent of p21ras, Raf-1, and MAP kinase kinase. 752 98

Protein phosphorylation has evolved as the most versatile posttranslational modification widely used by cells. Signal transduction pathways mediated by activation of MAP kinases and protein kinase C trigger the exit of cells from the quiscence (Go-->G1 transition). Indeed, binding of growth factors at the cell surface triggers their receptors, usually possessing a tyrosine kinase on the cytoplasmic side, to phosphorylate other molecules passing on the information sequentially to GRB2 protein, to p21ras, to c-Raf-1, to MAP kinase kinase, to MAP kinase, to p90rsk, to transcription factors. Activated PKC, MAP kinase, and pp90src can translocate to the nucleus where they phosphorylate a number of protein transcription regulators in a cell cycle-dependent manner or in response to cell stimulation for exit from quiescence. The cell cycle is mainly regulated by p34cdc2 or otherwise called cdc2 in association with cyclins B at G2/M and by Cdk2 in association with cyclins A, D1, and E at G1/S checkpoints; phosphorylation of histone H1 and lamins by cdc2 triggers chromosome assembly and nuclear envelope breakdown, respectively, as a prelude to mitosis. Cdc2 activities functioning as a G2/M regulator are controlled by its phosphorylation and dephosphorylation at Ser/Thr residues. MAP kinases might be the missing link in the chain connecting the Go to G1 transition with the cell cycle regulation, whereas phosphorylation of replication protein factors, retinoblastoma, and p53 might link the G1 to S transition with the control of DNA synthesis. A number of transcription factors are known to stimulate DNA replication, including p53, c-Myc, AP-1, Oct-1, T-antigen; the DNA binding activities of all these proteins and their interaction with other transcription factors are controlled by phosphorylation. The nuclear import of several proteins including NF kappa B, Dorsal, glucocorticoid receptor, ISGF3, rNFIL-6, T antigen, and the kinases PKC, MAP, and p90rsk, are dependent on their phosphorylation at specific sites. Histone phosphorylation stimulated at discrete stages of the cell cycle or in response to cAMP or other stimuli might induce profound changes in chromatin organization.
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PMID:Phosphorylation of transcription factors and control of the cell cycle. 754 80

A pyrazolo-quinoline compound, 6-methoxy-4-[2-[(2-hydroxyethoxyl)-ethyl]amino]-3-methyl-1M-pyrazo lo [3,4-b]quinoline (SCH 51344), was identified based on its ability to derepress human smooth muscle alpha-actin promoter activity in ras-transformed cells. In this study, we show that SCH 51344 reverts several key aspects of ras transformation, such as morphological changes, actin filament organization, and anchorage-independent growth, and also inhibits Val-12 Ras-induced maturation of Xenopus oocytes. SCH 51344 is also a potent inhibitor of the anchorage-independent growth of human tumor lines known to contain multiple genetic alterations in addition to activated ras genes. We have sought to determine whether SCH 51344 disrupts the signaling pathway that activates mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK) in normal and ras-transformed fibroblast cells. NIH 3T3 cells transformed by different oncogenes, which have products that participate at different steps of the Ras signaling pathway, were tested in a soft-agar colony formation assay to determine which step of the pathway is inhibited by SCH 51344. Our results indicate that SCH 51344 inhibits the ability of v-abl, v-mos, H-ras, v-raf, and mutant active MAP kinase kinase-transformed NIH 3T3 cells to grow in soft agar. Only v-fos-transformed cells were found to be resistant to the treatment of SCH 51344. SCH 51344 treatment had very little effect, if any, on the activation of MAP kinase kinase, MAP kinase, and p90RSK activity in response to growth factor stimulation. Treatment of ras-transformed cells with SCH 51344 led to stimulation of serum response factor DNA binding activity and activation of serum response element-dependent gene transcription, accounting for its ability to activate alpha-actin promoter activity in ras-transformed cells. Our results indicate that SCH 51344 inhibits ras transformation by a novel mechanism and acts at a point either downstream or parallel to extracellular signal-regulated kinase-dependent Ras signaling pathway.
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PMID:SCH 51344 inhibits ras transformation by a novel mechanism. 758 59

Insulin and epidermal growth factor receptors transmit signals for cell proliferation and gene regulation through formation of active GTP-bound p21ras mediated by the guanine nucleotide exchange factor Sos. Sos is constitutively bound to the adaptor protein Grb2 and growth factor stimulation induces association of the Grb2/Sos complex with Shc and movement of Sos to the plasma membrane location of p21ras. Insulin or epidermal growth factor stimulation induces a rapid increase in p21ras levels, but after several minutes levels decline toward basal despite ongoing hormone stimulation. Here we show that deactivation of p21ras correlates closely with phosphorylation of Sos and dissociation of Sos from Grb2, and that inhibition of mitogen-activated protein (MAP) kinase kinase (also known as extracellular signal-related kinase (ERK) kinase, or MEK) blocks both events, resulting in prolonged p21ras activation. These data suggest that a negative feedback loop exists whereby activation of the Raf/MEK/MAP kinase cascade by p21ras causes Sos phosphorylation and, therefore, Sos/Grb2 dissociation, limiting the duration of p21ras activation by growth factors. A serine/threonine kinase downstream of MEK (probably MAP kinase) mediates this desensitization feedback pathway.
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PMID:Negative feedback regulation and desensitization of insulin- and epidermal growth factor-stimulated p21ras activation. 759 90

We have shown that the interaction of interleukin (IL)-5 with the receptor activates Lyn tyrosine kinase within 1 min and Jak2 tyrosine kinase within 1-3 min. IL-5 also stimulates GTP binding to p21ras. The signal is subsequently propagated through the activation of Raf-1, MEK, and MAP kinases as shown by their increased autophosphorylation in vitro and phosphorylation in situ. Jak2 kinase has been shown to phosphorylate STAT nuclear proteins. The activation of STAT nuclear factors was studied by electrophoretic mobility shift assay using a gamma activation site (GAS) probe. We found that IL-5 induces two GAS-binding proteins in eosinophils, one of which is STAT1. We conclude that IL-5 induced signals are propagated through two distinct pathways: (1) Lyn-->Ras-->Raf-1-->MEK-->MAP kinase and (2) Jak2-->STAT1.
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PMID:The interleukin-5/receptor interaction activates Lyn and Jak2 tyrosine kinases and propagates signals via the Ras-Raf-1-MAP kinase and the Jak-STAT pathways in eosinophils. 761 38

Ras proteins function through the formation of specific complexes with Raf-1, B-raf, PI-3 kinase and RalGDS. These interactions all require Ras-GTP with an intact effector binding domain (Switch I region). We have examined the requirements of the Switch II region (amino acids 60-72) for the production of stable interactions between Ras and its downstream effectors. A point mutation at position 65 or 64 combined with additional mutations at either position 65 or 71 rendered nucleotide-free Ras protein unable to stably interact with Ras specific guanine nucleotide exchange factors. Ha-Ras containing point mutations at positions 65 and 71 possessed a twofold higher affinity for B-raf and consequently MEK1. The point mutation at 64, in combination with additional point mutations at either position 65 or 71, resulted in a protein which failed to interact with either PI-3 kinase or neurofibromin, though these Ras mutants effectively bound both Raf-1 and B-raf. An activated form of Ras, Q61L-Ras, associated with all effector proteins independent of the bound guanine nucleotide. Q61L-Ras-GDP was almost as effective as wild type Ras-GMPPNP in the in vitro activation of MEK1 and MAP kinase. Competitive studies with the catalytic domain if neurofibromin, NF1-GRD, demonstrated that its interaction with Ras-GMPPNP is mutually exclusive with both Raf-1 and B-raf. These data suggest that rasGAP and neurofibromin are unable to downregulate Ras-GTP complexed to Raf-1 or B-raf.
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PMID:Different structural requirements within the switch II region of the Ras protein for interactions with specific downstream targets. 763 Jun 28

The precise role of the protein tyrosine phosphatase Syp in insulin signaling is not well understood. We previously reported that expression of catalytically inactive Syp phosphatase blocked stimulation of mitogen-activated protein (MAP) kinase by insulin. In this study, we investigated the effect of dominant negative Syp on the intermediates in MAP kinase pathway. The expression of dominant negative Syp blocked the activation of MEK and raf-1 kinase in response to insulin and had no detectable effect on insulin-induced activation of p21ras. These data suggest that the target of the Syp phosphatase may reside in proteins immediately downstream of p21ras.
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PMID:Expression of a catalytically inert Syp blocks activation of MAP kinase pathway downstream of p21ras. 767 89

Ligation of membrane immunoglobulin M (mIgM) receptor in the Ramos B-cell line induced tyrosine phosphorylation of several intracellular substrates, including the adaptor protein. Shc. Phosphorylated Shc could be seen to associate with Grb2 in a complex which included hSOS. Inasmuch as hSOS is involved in p21ras activation, we also demonstrated that mIgM ligation activated a Ras-dependent kinase cascade in which sequential activation of Raf-1 and MEK-1 culminates in the activation of p42 mitogen-activated protein (MAP) kinase (ERK-2). The tumour promoter and protein kinase C agonist, phorbol 12-myristate 13-acetate (PMA), also activated Raf-1, MEK-1, and MAP kinase in Ramos cells, but did not induce tyrosine phosphorylation of Shc or Shc/Grb2 association. Okadaic acid, another tumour promoter and serine/threonine phosphatase inhibitor, activated p42 MAP kinase without activating Raf-1 or MEK-1, suggesting the existence of a serine/threonine phosphatase which directly regulates MAP kinase activity.
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PMID:The membrane immunoglobulin receptor utilizes a Shc/Grb2/hSOS complex for activation of the mitogen-activated protein kinase cascade in a B-cell line. 771 78

Activation of mitogen-activated protein kinase (MAP kinase) plays an important role in the cellular effects of nerve growth factor (NGF). Although the precise pathway by which NGF activates MAP kinase is not clear, several enzymes have been identified that may form a linear phosphorylation cascade, in which MAP kinase is activated by MAP kinase kinase (MEK). A key enzyme that links the ras-GTP complex to MEK is widely believed to be the raf kinase. However, immunoprecipitation experiments in PC-12 cells revealed that raf is not the major NGF-dependent MEK kinase [Zheng, Ohmichi, Saltiel and Guan (1994) Biochemistry 33, 5595-5599]. We have identified a protein kinase from PC-12 cells that catalyses both the phosphorylation and activation of MEK. This activity is stimulated 3-fold in cells treated with NGF. The partial purification on FPLC and characterization of this MEK kinase indicate that it is distinct from raf, MEK, MAP kinase and other previously described NGF-stimulated protein kinases. The activity of this enzyme is unaffected by direct addition to the assay of heparin, staurosporine, K252A and the heat-stable cyclic AMP-dependent kinase peptide inhibitor, but is slightly inhibited by NaF and calcium ions. Comparison of its behaviour on gel permeation and sucrose-density gradients indicates a molecular mass in the region of 50,000 Da. Moreover, isoelectric focusing of the enzyme revealed a pI of approx. 7.3. The kinase activity is specific for ATP as substrate with a Km of 11 microM, and requires Mg2+ as a cofactor. Analysis of the activation of this enzyme in PC-12 cells transfected with a dominant inhibitory mutant of p21ras suggests that this MEK kinase resides downstream of ras in the MAP kinase activation pathway. Moreover, site-directed mutation of the residues on MEK that are phosphorylated by raf does not completely abrogate phosphorylation by the MEK kinase, suggesting that this enzyme may share some phosphorylation sites with raf, but also phosphorylates MEK on other sites.
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PMID:Nerve growth factor stimulates a novel protein kinase in PC-12 cells that phosphorylates and activates mitogen-activated protein kinase kinase (MEK). 773 91


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