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Query: UNIPROT:P51812 (mitogen-activated protein)
 10,636 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The mitogen-activated protein (MAP) kinase homologue FUS3 mediates both transcription and G1 arrest in a pheromone-induced signal transduction cascade in Saccharomyces cerevisiae. We report an in vitro kinase assay for FUS3 and its use in identifying candidate substrates. The assay requires catalytically active FUS3 and pheromone induction. STE7, a MAP kinase kinase homologue, is needed for maximal activity. At least seven proteins that specifically associate with FUS3 are phosphorylated in the assay. Many of these substrates are physiologically relevant and are affected by in vivo levels of numerous signal transduction components. One substrate is likely to be the transcription factor STE12. A second is likely to be FAR1, a protein required for G1 arrest. FAR1 was isolated as a multicopy suppressor of a nonarresting fus3 mutant and interacts with FUS3 in a two hybrid system. Consistent with this FAR1 is a good substrate in vitro and generates a FUS3-associated substrate of expected size. These data support a model in which FUS3 mediates transcription and G1 arrest by direct activation of STE12 and FAR1 and phosphorylates many other proteins involved in the response to pheromone.
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PMID:FUS3 phosphorylates multiple components of the mating signal transduction cascade: evidence for STE12 and FAR1. 833 5

Xenopus 45-kDa mitogen-activated protein (MAP) kinase kinase (MAPKK) is a serine/threonine/tyrosine kinase, which activates MAP kinase (MAPK) by phosphorylating its threonine and tyrosine residues. MAPKK is active only when its threonine and/or serine residues are phosphorylated. We have identified from Xenopus eggs two protein kinases responsible for phosphorylation of MAPKK. The two kinases are separated by Sephacryl S-300 gel filtration chromatography. The higher molecular weight kinase phosphorylates MAPKK previously dephosphorylated and inactivated by phosphatase 2A treatment on mainly serine and slightly threonine residues, and reactivates the MAPKK, and is thus assumed to work as MAPKK kinase (MAPKKK) in vivo. The lower molecular weight kinase, identified as MAPK, phosphorylates the dephosphorylated MAPKK on mainly threonine and faintly serine residues, but does not reactivate the MAPKK activity. As Xenopus MAPKK contains a single phosphorylation consensus sequence (PXT388P) for MAPK in the C-terminal region, this T388 residue may be a major phosphorylation site catalyzed by MAPK. Thus, Xenopus MAPKK is phosphorylated in mature oocytes by not only an upstream kinase, MAPKKK, but also a downstream kinase, MAPK.
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PMID:Phosphorylation of Xenopus mitogen-activated protein (MAP) kinase kinase by MAP kinase kinase kinase and MAP kinase. 838 23

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

Several protein kinases, including Mos, maturation-promoting factor (MPF), mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MAPKK), are activated when Xenopus oocytes enter meiosis. De novo synthesis of the Mos protein is required for progesterone-induced meiotic maturation. Recently, bacterially synthesized maltose-binding protein (MBP)-Mos fusion protein was shown to be sufficient to initiate meiosis I and MPF activation in fully grown oocytes in the absence of protein synthesis. Here we show that MAP kinase is rapidly phosphorylated and activated following injection of wild-type, but not kinase-inactive mutant, MBP-Mos into fully grown oocytes. MAP kinase activation by MBP-Mos occurs within 20 min, much more rapidly than in progesterone-treated oocytes. The MBP-Mos fusion protein also activates MPF, but MPF activation does not occur until approximately 2 h after injection. Extracts from oocytes injected with wild-type but not kinase-inactive MBP-Mos contain an activity that can phosphorylate MAP kinase, suggesting that Mos directly or indirectly activates a MAPKK. Furthermore, activated MBP-Mos fusion protein is able to phosphorylate and activate a purified, phosphatase-treated, rabbit muscle MAPKK in vitro. Thus, in oocytes, Mos is an upstream activator of MAP kinase which may function through direct phosphorylation of MAPKK.
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PMID:Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro. 838 11

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.
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PMID:MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C. 838 20

Virtually all mitogens lead to the rapid activation of one or more mitogen-activated protein (MAP) kinases. In almost all cases, mitogen-activated surface signaling complexes transmit an essential signal via ras on to a protein kinase cascade that involves the serine/threonine kinase raf. Raf appears to be a MAP kinase kinase kinase, activating MAP kinase kinase which, in turn, activates MAP kinase. Among the targets of MAP kinase are other kinases, nuclear transcription factors and other proteins with roles in cell cycle activation. Both G0-arrested somatic cells and G2-arrested oocytes use many of the same signaling mechanisms to break cell cycle arrest; this is a useful concept in light of newly developed cell-free systems from quiescent oocytes that can be used to study signal transduction in vitro.
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PMID:MAP kinase and the activation of quiescent cells. 838 66

In Xenopus oocytes, mitogen-activated protein (MAP) kinase can be activated by progesterone treatment or by microinjection of cyclin A, both of which lead to activation of the cdc2 protein kinase. The tyrosine kinase pp60v-src has previously been shown to accelerate progesterone-induced oocyte maturation and to increase the phosphorylation of ribosomal protein S6 by pp90rsk, most likely by activating MAP kinase. In extracts of resting oocytes, MAP kinase kinase and MAP kinase were activated by addition of pp60v-src or cyclin A. Activation by pp60v-src was blocked by a dominant-negative p21ras protein (RAST), but activation by cyclin A/cdc2 was unaffected. Thus these two pathways that converge at MAP kinase kinase but are clearly divergent upstream of a p21ras-dependent step can be studied in a cell-free system.
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PMID:Reconstitution of p21ras-dependent and -independent mitogen-activated protein kinase activation in a cell-free system. 839 92

p74raf-1, a serine/threonine kinase, is structurally related to the protein kinase C (PKC) family and contains a cysteine motif in its N-terminal domain, which is essential for its regulation. It has been shown that p74raf-1 functions upstream of mitogen-activated protein (MAP) kinase kinase. We have constructed a p74raf-1 mutant (N delta raf) that only contains the N-terminal regulatory domain. When transiently expressed in COS-M6 cells, N delta raf efficiently blocked the activation of the MAP extracellular signal regulated kinase (ERK2), induced by either epidermal growth factor, phorbol ester, serum, or oncogenic p21ras. Similar constructs with the cysteine motifs from either PKC-alpha or diacylglycerol kinase did not inhibit activation of ERK2. Overexpression of full-length p74raf-1 rescued the inhibition of ERK2 by N delta raf in a stimulus dependent manner, indicating that N delta raf acts as a competitive inhibitor of wild-type p74raf-1. In contrast, overexpression of either PKC-alpha, -epsilon, or -zeta in N delta raf-containing cells could not rescue the inhibition of ERK2. We conclude that p74raf-1 is an essential mediator of epidermal growth factor- and phorbol ester-induced ERK2 activation and that the MAP kinase kinase activity of p74raf-1 cannot be substituted with either PKC-alpha, -epsilon or -zeta.
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PMID:A dominant-negative mutant of raf blocks mitogen-activated protein kinase activation by growth factors and oncogenic p21ras. 839 1

Activation of tyrosine kinase receptors causes mitogen-activated protein (MAP) kinase stimulation via a pathway involving p21ras, p74raf-1 (acting as a MAP kinase kinase kinase), and MAP kinase kinases; however, the pathway by which heterotrimeric G-protein-coupled receptors activate MAP kinases is undefined. Since there are several MAP kinase kinase kinases it has been suggested that p74raf-1 may only couple tyrosine kinase receptors to MAP kinase activation. We therefore investigated the requirement for p21ras and p74raf-1 in G-protein receptor-mediated MAP kinase activation. Lysophosphatidic acid stimulates MAP kinase via a pertussis toxin-sensitive pathway, which is blocked by dominant negative Ras. Lysophosphatidic acid-stimulated MAP kinase activation is potentiated by overexpression of p74raf-1 and blocked by expression of a dominant negative Raf protein comprising the N-terminal 259 amino acids. We conclude that lysophosphatidic acid activates MAP kinases by a G-protein-coupled pathway that requires both p21ras and p74raf-1.
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PMID:Lysophosphatidic acid stimulates mitogen-activated protein kinase activation via a G-protein-coupled pathway requiring p21ras and p74raf-1. 840 93


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