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)

Ras p21 in the GTP-bound form was shown to act as an upstream activator for mitogen-activated protein (MAP) kinase kinase (MAPKK) and MAP kinase, and Raf-1 was reported to act as a MAPKK kinase. Further, physical association between Ras and Raf-1 was demonstrated. Here we have shown that incubation of Xenopus immature oocyte extracts with Ras enhances the ability of endogenous Raf-1 to activate MAPKK. Moreover, a dominant negative form of Raf-1 blocked the Ras-induced activation of MAPKK and MAP kinase in the extracts, but not the cyclin A-dependent activation of MAP kinase. When the extracts were depleted of 45-kDa MAPKK with polyclonal anti-MAPKK antibody, no activation of MAP kinase occurred even after incubation with Ras. These results suggest that Ras can activate the MAPKK kinase activity of Raf-1 in the extracts and that MAPKK is indispensable for the Ras-induced MAP kinase activation. It is well known that Ras can induce oocyte maturation when injected into immature Xenopus oocytes. Co-injection of Ras with an anti-MAPKK antibody that inhibits the MAPKK activity prevented the Ras-induced germinal vesicle breakdown, suggesting that MAPKK mediates, at least, one of cellular functions of Ras.
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PMID:Analysis of the Ras p21/mitogen-activated protein kinase signaling in vitro and in Xenopus oocytes. 780 37

Raf-1, a serine/threonine kinase, is required for the mitogenic action of ras p21. It has been recently demonstrated that ras p21 directly associates with Raf-1. The C-terminal region of ras p21 is modified by farnesylation and carboxyl methylation. This modification is necessary for ras p21 function. To elucidate the role of post-translational modification of ras p21 in Raf-1 activation, we examined ras p21-dependent Raf-1 activity in baculovirus/Sf9 cells overexpressing Raf-1 and ras p21. Coexpression of Raf-1 with v-ras p21 in Sf9 cells stimulated the autophosphorylating activity of Raf-1. The activity of Raf-1, as assessed by its ability to activate extracellular signal-regulated kinase kinase (MEK) in vitro, was also increased when Raf-1 was coexpressed with v-ras p21. However, neither the autophosphorylating activity of Raf-1 nor its ability to activate MEK was stimulated by v-ras p21 mutants which are not post-translationally modified. Raf-1 formed a complex with v-ras p21 and the v-ras p21 mutants in Sf9 cells. These results indicate that the post-translational modification of ras p21 is necessary for Raf-1 activation but that the association of Raf-1 with ras p21 is not sufficient to activate Raf-1.
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PMID:The post-translational modification of ras p21 is important for Raf-1 activation. 805 Oct 91

We describe a novel Triton-disrupted mammalian cell system wherein the pathways for activation of mitogen-activated protein (MAP) kinases (MAPKs) are capable of direct biochemical manipulation in vitro. MAPKs p42mapk and p44mapk are activated in signal transduction cascade(s) initiated by occupancy of plasma membrane receptors for peptide growth factors, hormones, and neurotransmitters. One likely activation pathway for MAPKs consists of sequential activations of c-ras, c-raf-1, and a protein-tyrosine/threonine kinase, MAP kinase kinase. Triton-disrupted cells retained capacity for activation of the pathway by both peptide growth factors and by addition of GTP-loaded p21 rasVal12. Incubation of disrupted cells with an antibody that neutralized the function of c-ras (Y13-259) abolished receptor-mediated stimulation of MAPK as did acute addition of 200 microM azatyrosine. Activation of the pathway was reconstituted in a cell-free system using high-speed supernatants generated from Triton-disrupted cells together with purified plasma membranes from parental cells and as a heterogeneous system using purified plasma membranes from v-ras-transformed cells. These systems will allow biochemical dissection in vitro of the interaction(s) between c-ras and the MAPK pathway in mammalian cells.
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PMID:Activation of the mitogen-activated protein kinase pathway in Triton X-100 disrupted NIH-3T3 cells by p21 ras and in vitro by plasma membranes from NIH 3T3 cells. 833 4

To identify the direct target molecule of ras p21 in higher eukaryotes, we have recently developed the cell-free system in which ras p21 activates mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK). In this cell-free system, the guanosine 5'-[gamma-thio]triphosphate- bound form of Ki-ras p21, but not the GDP-bound form, activates endogenous Xenopus MAP kinase as well as recombinant ERK2 in the presence of the cytosol fraction of Xenopus oocytes. We separated two protein factors from the cytosol fraction of Xenopus oocytes by column chromatography: one was the inactive form of MAP kinase kinase and the other was a factor tentatively named ras p21-dependent ERK-kinase stimulator (REKS). The former and latter showed M(r) values of approximately 45,000 and 150,000-200,000, respectively, as estimated by gel filtration. Both factors were necessary for Ki-ras p21-dependent activation of MAP kinase/ERK2. These results indicate that an additional protein factor (REKS) is essential for Ki-ras p21 to activate MAP kinase through MAP kinase kinase.
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PMID:A protein factor for ras p21-dependent activation of mitogen-activated protein (MAP) kinase through MAP kinase kinase. 838 39

Desensitization of p21(ras) after stimulation of cells by growth factors and phorbol 12-myristate 13-acetate (PMA) correlates with hyperphosphorylation of the guanine nucleotide exchange factor Son-of-sevenless (Sos) and its dissociation from the adaptor protein Grb2 (Cherniack, A., Klarlund, J. K., Conway, B. R., and Czech, M. P. (1995) J. Biol. Chem. 270, 1485-1488). To test the role of the Raf/mitogen-activated protein (MAP) kinase pathway, we utilized cells expressing a chimera composed of the catalytic domain of p74Raf-1 and the hormone binding domain of the estradiol receptor (DeltaRaf-1:ER). Estradiol markedly stimulated DeltaRaf-1:ER and the downstream MEK and MAP kinases in these cells as well as Sos phosphorylation. However, the dissociation of Grb2 from Sos observed in response to PMA was not apparent upon DeltaRaf-1:ER activation. Furthermore, stimulation of DeltaRaf-1:ER did not impair GTP loading of p21(ras) in response to platelet-derived growth factor or epidermal growth factor. We conclude that activation of the Raf/MAP kinase pathway alone in these cells is insufficient to cause disassembly of Sos from Grb2 or to interrupt the ability of Sos to catalyze activation of p21(ras).
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PMID:Role of the Raf/mitogen-activated protein kinase pathway in p21ras desensitization. 866 95

The nerve growth factor (NGF)-mediated activation of the mitogen-activated protein (MAP) kinase cascade is an obligatory step in the morphological differentiation of PC12 cells. Signal transduction through the MAP kinase cascade is dependent upon activation of p21(ras) which binds directly to Raf family protein kinases, mediating their association with the membrane and activation. PC12 cells express two Raf isoforms, c-Raf and B-Raf. The activation of the MAP kinase cascade in response to NGF is due principally to the action of B-Raf. NGF treatment of PC12 cells resulted in the enhanced phosphorylation of B-Raf and c-Raf, and both exhibit reduced electrophoretic mobilities following stimulation of the cells. The NGF-stimulated phosphorylation of B-Raf was correlated with its enzymatic activation as measured by the phosphorylation of its substrate MEK. However, c-Raf does not exhibit significant levels of activity. B-Raf was present as a component of a high molecular mass complex, which included the molecular chaperone, heat shock protein 90 (HSP90). Importantly, c-Raf did not participate in the formation of such complexes. The B-Raf containing HSP90 complexes were normally present in PC12 cells, and their assembly was not dependent upon NGF stimulation. These data suggest that the ability of B-Raf to activate the MAP kinase cascade is due to its association with a large signaling complex, which is likely to impart signaling pathway specificity.
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PMID:Nerve growth factor-mediated activation of the mitogen-activated protein (MAP) kinase cascade involves a signaling complex containing B-Raf and HSP90. 879 78

Both mitogen-activated protein kinase kinase 1 (MAPKK1) and MAPKK2 function downstream of the proto-oncogene product Raf in signaling pathways that affect cell proliferation and differentiation. The isoforms were previously shown to be differentially regulated in two significant ways: (a) MAPKK1, but not MAPKK2, was phosphorylated and inactivated by the cyclin-dependent kinase p34cdc2; and (b) p21 Ras formed a ternary complex with Raf/MAPKK1 but not with Raf/MAPKK2. To further characterize the regulation and function of the two isoforms, we compared their mode of activation by v-Mos and examined the transcriptional and morphological responses that they mediate in cultured mammalian cells. v-Mos enhanced the enzymatic activity of both isoforms to the same extent, by about 600-fold. Constitutively active MAPKK2 mutants were generated by introducing the same deletion and amino acid substitutions that have been shown to activate MAPKK1, suggesting that the conformational changes that lead to their activation are analogous. These mutants potentiated transcription from a promoter containing AP1-responsive elements and induced morphological transformation when expressed in mammalian cells, matching outcomes observed with constitutively active MAPKK1. The specific activity of p42 MAPK in the transformed cells was 3-fold higher than in cells expressing wild-type MAPKK, thereby implicating p42 MAPK as a common effector in vivo, and suggesting that sustained activation of p42 MAPK may represent a critical factor that contributes to the development of the transformed state. Altogether, the results demonstrate that the two isoforms elicit similar responses in vivo despite differences in their regulation.
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PMID:Constitutively active mitogen-activated protein kinase kinase 1 (MAPKK1) and MAPKK2 mediate similar transcriptional and morphological responses. 882 8

The c-Jun amino-terminal kinases (JNKs)/stress-activated protein kinases (SAPKs) play a crucial role in stress responses in mammalian cells. The mechanism underlying this pathway in the hematopoietic system is unclear, but it is a key in understanding the molecular basis of blood cell differentiation. We have cloned a novel protein kinase, termed hematopoietic progenitor kinase 1 (HPK1), that is expressed predominantly in hematopoietic cells, including early progenitor cells. HPK1 is related distantly to the p21(Cdc42/Rac1)-activated kinase (PAK) and yeast STE20 implicated in the mitogen-activated protein kinase (MAPK) cascade. Expression of HPK1 activates JNK1 specifically, and it elevates strongly AP-1-mediated transcriptional activity in vivo. HPK1 binds and phosphorylates MEKK1 directly, whereas JNK1 activation by HPK1 is inhibited by a dominant-negative MEKK1 or MKK4/SEK mutant. Interestingly, unlike PAK65, HPK1 does not contain the small GTPase Rac1/Cdc42-binding domain and does not bind to either Rac1 or Cdc42, suggesting that HPK1. activation is Rac1/Cdc42-independent. These results indicate that HPK1 is a novel functional activator of the JNK/SAPK signaling pathway.
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PMID:Human HPK1, a novel human hematopoietic progenitor kinase that activates the JNK/SAPK kinase cascade. 882 85

The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras-->Raf-->MEK-->ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein alpha and beta gamma subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families of closely related proteins. The potential for more than one signal to be conveyed down a pathway simultaneously (multiplex signalling) is discussed. The net effect of a given stimulus on the cell is the result of a complex intracellular integration of the intensity and duration of activation of the individual pathways. The specific outcome depends on the particular signalling molecules expressed by the target cells and on the dynamic balance among the pathways.
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PMID:Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling. 883 13

p21(waf1/cip1) gene expression is induced by DNA damage in cells with wild-type p53 and contributes to the arrest of cell growth. It was demonstrated that under many experimental conditions, including oxidative stress, p21(waf1/cip1) expression can be induced through p53-independent pathways. Since most of these experimental conditions induce the phosphorylation of mitogen-activated protein kinase (MAPK) and thus its activation, we evaluated p21(waf1/cip1) mRNA levels in cells exposed to an oxidative stress, induced by diethylmaleate (Et2Mal), and in which the MAPK pathway was blocked. The expression of a dominant-negative mutant of MEK, the MAPK kinase that phosphorylates and activates MAPK, and of a dominant-negative [Asn17]Ras mutant prevented the Et2Mal-induced accumulation of p21(waf1/cip1) mRNA. Similarly, the expression of MEK- and of [Asn17]Ras mutants decreased the 12-O-tetradecanoyl-phorbol 13-acetate (TPA)-mediated p21(waf1/cip1) induction. Furthermore, TPA-induced and serum-induced p21(waf1/cip1) mRNA accumulation was blocked by pretreating the cells with the antioxidant compound N-acetylcysteine, suggesting that oxidative stress is involved in these responses. p21(waf1/cip1) mRNA levels reached a maximum within 2 h of adding Et2Mal or TPA; however, the rate of transcription from a p21(waf1/cip1)-promoter construct did not increase during this period. In contrast, cells treated with actinomycin D show an increase of p21(waf1/cip1) mRNA stability after Et2Mal treatment. This result suggests that the increase in p21(waf1/cip1) mRNA at early times results from post-transcriptional regulatory events. Longer exposure to TPA may activate p21(waf1/cip1) gene transcription through an Sp1-dependent mechanism, while Et2Mal treatment gradually inhibits p21(waf1/cip1) gene transcription through oxidative changes that affect Sp1 binding to DNA.
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PMID:Redox-mediated regulation of p21(waf1/cip1) expression involves a post-transcriptional mechanism and activation of the mitogen-activated protein kinase pathway. 918 12


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