EC:2.7.11.24 - FACTA Search

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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The activation of mitogen-activated protein kinase (MAP kinase) in macrophages and the involvement of protein kinase C (PKC) in MAP kinase activation was investigated in macrophages exposed to agents that have previously been shown to activate the 85-kDa cytosolic phospholipase A2 (PLA2) and induce arachidonic acid release. Phorbol 12-myristate 13-acetate (PMA) and zymosan maximally stimulated MAP kinase activity by 5 and 15 min, respectively, whereas the response to okadaic acid was maximal by 60-90 min. MAP kinase activation correlated with tyrosine phosphorylation of p44 MAP kinase in PMA-stimulated cells and p44 and p42 MAP kinases in zymosan- and okadaic acid-stimulated cells. MAP kinase activity was not elevated in A23187-stimulated macrophages. Inhibition of PKC with the inhibitor, bisindolylmaleimide (GF109203X), or by prolonged exposure to PMA suppressed both arachidonic acid release and MAP kinase activation in PMA- and zymosan-stimulated macrophages but not in okadaic acid or A23187-treated cells. However, prolonged exposure to PMA did not suppress the increased cytosolic PLA2 activity in agonist-treated macrophages. This approach was complicated since initial exposure to PMA to down-regulate PKC increased cytosolic PLA2 activity which remained elevated for 16 h. In contrast, GF109203X treatment suppressed the increase in cytosolic PLA2 activity in response to zymosan and PMA but not to okadaic acid or A23187. The results demonstrate that PMA and zymosan trigger PKC activation that leads to the activation of MAP kinase and PLA2, whereas these responses are PKC independent in okadaic acid-treated cells. In addition, the results are consistent with a role for MAP kinase activation in regulating the activation of the 85-kDa PLA2 and arachidonic acid release in PMA-, zymosan-, and okadaic acid-stimulated cells, whereas these responses in A23187-treated cells are MAP kinase-and PKC-independent.
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PMID:Protein kinase C-dependent and -independent pathways of mitogen-activated protein kinase activation in macrophages by stimuli that activate phospholipase A2. 803 17

Previous studies have provided suggestive evidence for an interaction between ras activation and signalling pathways involved in agonist-stimulated arachidonic acid release in a variety of cell systems. In order to clarify this interaction, we have measured epidermal growth factor (EGF)-stimulated arachidonic acid release in rat-1 fibroblasts transfected with the N-17 dominant negative mutation of ras. Cells transfected with the N-17 ras mutant, display a markedly attenuated arachidonic acid-release response to EGF, compared to sham-transfected and non-transfected cells. In contrast, the response to phorbol myristate acetate (PMA) was not attenuated in the N-17-mutant expressing cells. No differences were detected between sham-transfected and N-17 mutant expressing cells in levels of immunodetectable EGF receptor, cytosolic phospholipase A2 or mitogen-activated protein (MAP) kinase. Attenuation of EGF-stimulated arachidonic acid release in the N-17 mutant expressing cells, was accompanied by a marked diminution in EGF-stimulated tyrosine phosphorylation of MAP kinase. We conclude that the signalling pathway involved in epidermal growth factor-stimulated arachidonic acid release is similar to the signalling pathway for mitogenic responses to epidermal growth factor and requires ras activation, likely followed by a downstream cascade of kinases eventuating in MAP kinase activation.
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PMID:RAS is required for epidermal growth factor-stimulated arachidonic acid release in rat-1 fibroblasts. 824 28

Arachidonic acid release in cells highly over expressing cytosolic phospholipase A2 has been attributed to mitogen-activated protein kinase phosphorylation of cytosolic phospholipase A2 on serine-505. To investigate the role of cytosolic phospholipase A2 in cellular physiology, we attempted to inhibit cytosolic phospholipase A2 in the intact cell employing an antisense RNA strategy. Swiss 3T3 cells were stably transfected with an antisense cytosolic phospholipase A2 expression vector. A clone of cells with reduced immunodetectable cytosolic phospholipase A2, compared to a vector transfected cell line, was identified by Western blotting and a corresponding decrease in phospholipase A2 activity was confirmed by enzymatic assay in cell free extracts. However, arachidonic acid release from intact cells in response to agonists was not different between antisense and control cell lines. Thus, arachidonic acid release in intact cells with decreased cytosolic phospholipase A2 activity is likely to be modulated by rate limiting factors that are extrinsic to cytosolic phospholipase A2.
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PMID:Reduced phospholipase A2 activity is not accompanied by reduced arachidonic acid release. 829 24

We compared the regulation of cytosolic phospholipase A2 (cPLA2) activity in undifferentiated and neutrophil-like HL60 cells. Although Ca(2+)-mobilizing P2-purinergic receptors are expressed in both cell types, arachidonic acid (AA) release stimulated by P2-purinergic agonists was 5-7-fold higher in the differentiated cells. Similarly, the stimulation of AA release by AlF4- in intact cells or by ATP and guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in electropermeabilized cells was significantly higher in the differentiated cells. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced A23187-stimulated AA release in intact HL60 granulocytes with minimal effects in the undifferentiated cells. Immunoblotting experiments showed similar levels of cPLA2 and of agonist-mediated activation of mitogen-activated protein kinase in both cell types. Experiments measuring stimulation of AA release by either melittin, using endogenously labeled intact cells, or Ca2+, using homogenates and exogenous substrate, indicated that undifferentiated cells do not lack an activatable PLA2. The stimulatory effects of GTP gamma S and Ca2+ on AA release in homogenates from endogenously labeled cells suggested that undifferentiated cells display G protein-cPLA2 coupling. Basal and PMA-stimulated phosphorylation of cPLA2 was detected in differentiated, but not in undifferentiated cells. However, the two cell types displayed only subtle differences in the time courses of phosphorylation of mitogen-activated protein kinase triggered by agonists and PMA. The observed defect in cPLA2 phosphorylation may represent the alteration preventing agonist-mediated stimulation of AA release in undifferentiated HL60 cells.
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PMID:Regulation of phospholipase A2 activity in undifferentiated and neutrophil-like HL60 cells. Linkage between impaired responses to agonists and absence of protein kinase C-dependent phosphorylation of cytosolic phospholipase A2. 830 Jun 48

Treatment of cells with agents that stimulate the release of arachidonic acid causes increased serine phosphorylation and activation of cytosolic phospholipase A2 (cPLA2). Here we report that cPLA2 is a substrate for mitogen-activated protein (MAP) kinase. Moreover, phosphorylation by MAP kinase increases the enzymatic activity of cPLA2. The site of cPLA2 phosphorylation by MAP kinase, Ser-505, is identical to the major site of cPLA2 phosphorylation observed in phorbol ester-treated cells. Replacement of Ser-505 with Ala resulted in a mutant cPLA2 that is not a substrate for MAP kinase and causes little or no enhanced agonist-stimulated arachidonate release from intact cells. Taken together, these data indicate that MAP kinase mediates, at least in part, the agonist-induced activation of cPLA2.
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PMID:cPLA2 is phosphorylated and activated by MAP kinase. 838 Oct 49

The 85 kDa cytosolic phospholipase A2 (cPLA2) preferentially catalyses the hydrolysis of arachidonic acid from the sn-2 position of phospholipids. cPLA2 can be activated by extracellular stimuli such as thrombin, platelet-derived growth factor and epidermal growth factor (EGF): A full activation of cPLA2 requires an increase of intracellular Ca2+ concentration and phosphorylation on Ser-505 by mitogen-activated protein (MAP) kinase. Because EGF can provoke an increase in intracellular [Ca2+] ([Ca2+]i) and activation of MAP kinase, we investigated the role of these pathways in EGF-induced activation of cPLA2. Characterization of two cell lines expressing different numbers of EGF receptors (HERc13 and HER14) revealed that both were activating MAP kinase in response to EGF, but only HER14 responded with an increase in [Ca2+]i. In this study we used both cell lines as a tool to clarify the role of each pathway in cPLA2 activation. We show that EGF stimulates cPLA2 activity in both cell lines in vitro as measured in cytosolic fractions, but only in HER14 in vivo as measured by 3H release from cells prelabelled with [3H]arachidonic acid. This latter activation can be restored in HERc13 cells by the addition of the ionophore A23187. Interestingly, this effect is only observed when EGF stimulation precedes A23187 addition. The phosphorylation of MAP kinase, however, was identical under identical conditions. We conclude that a maximal cPLA2 activation by EGF requires both, and in this order: MAP kinase activation followed by a rise in [Ca2+]i concentration.
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PMID:Maximal epidermal growth-factor-induced cytosolic phospholipase A2 activation in vivo requires phosphorylation followed by an increased intracellular calcium concentration. 854 15

The abilities of platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF-I) to regulate cAMP metabolism and mitogen-activated protein kinase (MAP kinase) activity were compared in human arterial smooth muscle cells (hSMC). PDGF-BB stimulated cAMP accumulation up to 150-fold in a concentration-dependent manner (EC50 approximately 0.7 nM). The peak of cAMP formation and cAMP-dependent protein kinase (PKA) activity occurred approximately 5 min after the addition of PDGF and rapidly declined thereafter. Incubating cells with PDGF and 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) enhanced the accumulation of cAMP and PKA activity by an additional 2.5-3-fold, whereas IBMX alone was essentially without effect. The PDGF-stimulated increase in cAMP was prevented by addition of the cyclooxygenase inhibitor indomethacin, consistent with release of prostaglandins stimulating cAMP. PDGF, but not IGF-I, stimulated MAPK activity, cytosolic phospholipase A2 (cPLA2) phosphorylation, and cAMP synthesis which indicated a key role for MAP kinase in the activation of cPLA2. Further, PDGF stimulated the rapid release of arachidonic acid and synthesis of prostaglandin E2 (PGE2) which could be inhibited by a cPLA2 inhibitor (AACOCF3). Calcium mobilization was required for PDGF-induced arachidonic acid release and PGE2 synthesis but not for MAPK activation, whereas PKC was required for PGE2-mediated activation of PKA. In summary, these results demonstrated that PDGF increases cAMP formation and PKA activity through a MAP kinase-mediated activation of cPLA2, arachidonic acid release, and PGE2 synthesis in human arterial smooth muscle cells.
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PMID:Platelet-derived growth factor stimulates protein kinase A through a mitogen-activated protein kinase-dependent pathway in human arterial smooth muscle cells. 855 Jun 11

The phosphorylation sites on the human, 85-kDa cytosolic phospholipase A2 (cPLA2) were identified using recombinant cPLA2 expressed in Spodoptera frugiperda (Sf9) cells. Analysis by high performance liquid chromatography of tryptic digests of 32P-labeled recombinant cPLA2 showed four major peaks of radiolabeled phosphopeptides. The phosphorylated residues were identified as Ser-437, Ser-454, Ser-505, and Ser-727 using mass spectrometry and automated Edman sequencing. Sf9 cells infected with recombinant virus expressing cPLA2 exhibited a time-dependent release of arachidonic acid in response to the calcium ionophore A23187 or the protein phosphatase inhibitor okadaic acid, which was not observed in Sf9 cells infected with wild-type virus. Stimulation of Sf9 cells with A23187 and okadaic acid also increased the level of phosphorylation of cPLA2. Okadaic acid, but not A23187, induced a gel shift of cPLA2 and increased the level of phosphorylation of Ser-727 by 4.5-fold, whereas the level of phosphorylation of the other sites increased by 60% or less in response to both agonists. To determine whether the same sites on cPLA2 were phosphorylated in mammalian cells, human monocytes were studied. Okadaic acid stimulation of monocytes induced a gel shift of cPLA2, increased the release of arachidonic acid, and increased the level of phosphorylation of cPLA2 on serine residues. Comparison of two-dimensional peptide maps of tryptic digests of 32P-labeled recombinant cPLA2 and human monocyte cPLA2 demonstrated that the same peptides on cPLA2 were phosphorylated in mammalian cells as in insect cells. These results show that the Sf9-baculovirus expression system is useful for investigation of the phosphorylation sites on cPLA2. The results also suggest that phosphorylation of the cPLA2 by protein kinases other than mitogen-activated protein kinase may be important for the regulation of arachidonic acid release.
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PMID:Identification of phosphorylation sites of human 85-kDa cytosolic phospholipase A2 expressed in insect cells and present in human monocytes. 863 28

Cell adhesion to the extracellular matrix triggers a cascade of intracellular biochemical signals regulated by the integrin family of receptors. Recent evidence suggests that integrin engagement may activate a mitogen-activated protein (MAP) kinase cascade that may cooperate with more clearly defined mitogenic signaling pathways to regulate cell proliferation, adhesion, and migration. Here we report that the adhesion-dependent activation of the MAP kinase Erk2 (extracellular signal-regulated kinase 2) occurs in serum-starved NIH3T3 cells, and that this activation of Erk2 is preceded by the activation of the small GTP-binding protein Ras in fibronectin-adherent cells. Inhibition of Ras signaling by expression of a dominant-inhibitory mutant of Ras (N17Ras) in NIH3T3 cells blocked adhesion-dependent activation of Erk2, although the focal adhesion kinase (FAK) was still activated in these cells. Furthermore, activation of this Ras-MAP kinase pathway activated cytosolic phospholipase A2, leading to the release of arachidonic acid metabolites, and N17Ras also inhibited these events. However, N17Ras expression does not inhibit cell adhesion, spreading, or focal contact and stress fiber formation. These results suggest that, while integrin-dependent activation of this MAP kinase pathway is Ras-dependent, the integrin-dependent activation of FAK and several morphological events are Ras-independent. Thus, integrin-mediated signals involved in regulating cell morphology appear to diverge from those regulating MAP kinase activation at a level upstream of Ras activation.
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PMID:Ras activation is necessary for integrin-mediated activation of extracellular signal-regulated kinase 2 and cytosolic phospholipase A2 but not for cytoskeletal organization. 866 48

The newly defined eicosatetraenoates (ETEs), 5-oxoETE and 5-oxo-15(OH)-ETE, share structural motifs, synthetic origins, and bioactions with leukotriene B4 (LTB4). All three eicosanoids stimulate Ca2+ transients and chemotaxis in human neutrophils (PMN). However, unlike LTB4, 5-oxoETE and 5-oxo-15(OH)-ETE alone cause little degranulation and no superoxide anion production. However, we show herein that, in PMN pretreated with granulocyte-macrophage or granulocyte colony-stimulating factor (GM-CSF or G-CSF), the oxoETEs become potent activators of the last responses. The oxoETEs also induce translocation of secretory vesicles from the cytosol to the plasmalemma, an effect not requiring cytokine priming. To study the mechanism of PMN activation in response to the eicosanoids, we examined the activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 (cPLA2). PMN expressed three proteins (40, 42, and 44 kDa) that reacted with anti-MAPK antibodies. The oxoETEs, LTB4, GM-CSF, and G-CSF all stimulated PMN to activate the MAPKs and cPLA2, as defined by shifts in these proteins' electrophoretic mobility and tyrosine phosphorylation of the MAPKs. However, the speed and duration of the MAPK response varied markedly depending on the stimulus. 5-OxoETE caused a very rapid and transient activation of MAPK. In contrast, the response to the cytokines was rather slow and persistent. PMN pretreated with GM-CSF demonstrated a dramatic increase in the extent of MAPK tyrosine phosphorylation and electrophoretic mobility shift in response to 5-oxoETE. Similarly, 5-oxoETE induced PMN to release some preincorporated [14C]arachidonic acid, while GM-CSF greatly enhanced the extent of this release. Thus, the synergism exhibited by these agents is prominent at the level of MAPK stimulation and phospholipid deacylation. Pertussis toxin, but not Ca2+ depletion, inhibited MAPK responses to 5-oxoETE and LTB4, indicating that responses to both agents are coupled through G proteins but not dependent upon Ca2+ transients. 15-OxoETE and 15(OH)-ETE were inactive while 5-oxo-15(OH)-ETE and 5(OH)-ETE had 3- and 10-fold less potency than 5-oxoETE, indicating a rather strict structural specificity for the 5-keto group. LY 255283, a LTB4 antagonist, blocked the responses to LTB4 but not to 5-oxoETE. Therefore, the oxoETEs do not appear to operate through the LTB4 receptor. In summary, the oxoETEs are potent activators of PMN that share some but not all activities with LTB4. The response to the oxoETEs is greatly enhanced by pretreatment with cytokines, indicating that combinations of these mediators may be very important in the pathogenesis of inflammation.
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PMID:5-Oxo-eicosanoids and hematopoietic cytokines cooperate in stimulating neutrophil function and the mitogen-activated protein kinase pathway. 866 32

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