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)

Stimulation of T cells via the T cell receptor (TCR) activates a number of signaling pathways that are potentially involved in the elicitation of physiological responses, such as the production of cytokines. The extracellular signal-regulated kinases (ERK) are a group of molecules activated in response to TCR ligation, whose role in T cell cytokine production is controversial. In this study, we have asked whether ERK activation is coupled to the production of a number of T cell-derived cytokines, and whether particular cytokines are differentially affected by ERK activation. To address these questions, we have utilized a constitutively active version of the immediate upstream activator of both ERK1 and ERK2, mitogen-activated/extracellular signal-regulated kinase 1 (MEK1), to activate ERK signaling selectively in the absence of other TCR-activated signaling pathways. The effect of constitutive MEK/ERK activation on T cell cytokine production was measured by transiently co-transfecting newly activated mouse T cells with DNA encoding constitutively active MEK1 (CA-MEK1) and the human interleukin-2 (IL-2) receptor alpha chain (hCD25), purifying hCD25+ transfectants by flow-cytometric cell sorting, and measuring the production of IL-3, IL-4, interferon (IFN)-gamma and granulocyte/macrophage-colony-stimulating factor (GM-CSF) either in the presence or absence of ionomycin stimulation. Newly activated T cells were used in these experiments as they more closely resemble T cells activated in vivo than do transformed T cells or long-term established T cell clones. CA-MEK1 expression led to constitutive ERK activation, which acted synergystically with ionomycin treatment to stimulate cytokine production. Furthermore, these experiments revealed a hierarchy of cytokine responsiveness to MEK/ERK activation, such that the production of IL-3 was most affected, followed by GM-CSF, IFN-gamma, and IL-4.
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PMID:Differential activation of T cell cytokine production by the extracellular signal-regulated kinase (ERK) signaling pathway. 889 34

Activation of p38 MAP kinase (p38) as well as JNK/SAPK has been described as being induced by a variety of environmental stresses such as osmotic shock, ultraviolet radiation, and heat shock, or the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1 (IL-1). We found that the hematopoietic cytokines erythropoietin (Epo) and IL-3, which regulate growth and differentiation of erythroids and hematopoietic progenitors, respectively, also activate a p38 cascade. Immunoblot analyses and in vitro kinase assay clearly showed that Epo and IL-3 rapidly and transiently phosphorylated and activated p38 in Epo- or IL-3-dependent mouse hematopoietic progenitor cells. p38 can generally be activated by the upstream kinase MKK3 or MKK6. However, in vitro kinase assays in the immunoprecipitates with anti-MKK6 antibody and anti-phosphorylated MKK3/MKK6 antibody showed that activation of neither MKK3 nor MKK6 was detected after Epo or IL-3 stimulation, while osmotic shock clearly induced activation of both MKK3/MKK6 and p38. Together with previous observations, these results suggest that both p38 and JNK cascades play an important role not only in stress and proinflammatory cytokine responses but also in hematopoietic cytokine actions.
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PMID:Activation of p38 MAP kinase pathway by erythropoietin and interleukin-3. 924 20

Productive T cell activation leading to cytokine secretion requires the cooperation of multiple signaling pathways coupled to the TCR and to costimulatory molecules such as CD28. Here, we utilized two pharmacophores, PD98059 and FK506, that inhibit, respectively, mitogen-activated protein (MAP) kinase kinase 1 (MEK 1) and calcineurin, to determine the relative role of the signaling pathways controlled by these enzymes in T cell activation. Although the two compounds had distinctive effects on CD69 induction, they both suppressed T cell proliferation induced by anti-CD3 mAb, in a manner reversible by exogenous IL-2, suggesting that PD98059, like FK506, affects the production of, rather than the responsiveness to growth-promoting cytokines. Accordingly, IL-2 production by T cells stimulated with anti-CD3 mAb in conjunction with PMA or with anti-CD28 mAb was inhibited by both compounds. However, these compounds differentially affected the production of other cytokines, depending on the mode of activation. PD98059 inhibited TNF-alpha, IL-3, granulocyte-macrophage (GM)-CSF, IFN-gamma, and to a lesser extent IL-6 and IL-10 production but enhanced IL-4, IL-5, and IL-13 production induced by CD3/PMA or CD3/CD28. FK506 suppressed CD3/PMA-induced production of all cytokines examined here but to a lesser extent IL-13. FK506 also reduced CD3/CD28-induced production of IL-3, IL-4, IL-10, TNF-alpha, and IL-6 but augmented that of GM-CSF, IL-5, IFN-gamma, and IL-13. Therefore, the biochemical targets of PD98059 and FK506 contribute differently to the production of various cytokines by T cells, which may have implications for the therapeutic manipulation of this production.
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PMID:Inhibition of T cell activation by pharmacologic disruption of the MEK1/ERK MAP kinase or calcineurin signaling pathways results in differential modulation of cytokine production. 951 Jan 55

The extracellular signal-regulated kinase (ERK) signaling pathway is strongly activated in response to TCR stimulation in normal T cells. However, the extent to which activation of the ERK pathway is necessary for TCR-stimulated cytokine production is not clear. We have addressed this question by use of two separate methods to interfere with TCR activation of the ERK cascade. The first approach utilized transient expression of a catalytically inactive form of mitogen-activated/ERK 1 (CI-MEK1), while the second involved using the MEK1- and MEK2-specific inhibitor PD98059 to block ERK activation by the TCR. In order to assess the requirement for ERK activation in T cell cytokine production, we have measured the effect of ERK inhibition upon the production of six cytokines, IL-3, IL-4, IL-5, IL-10, granulocyte macrophage colony stimulating factor (GM-CSF) and IFN-gamma, by newly activated normal mouse T cells in response to TCR stimulation. The results of experiments using both methods to block ERK activation have revealed a requirement for intact ERK signaling for the full elicitation of TCR-stimulated cytokine production. Dose-response analyses using the MEK inhibitor PD98059 showed that the TCR-stimulated production of all cytokines measured was affected by this treatment. However, the production of IL-3 and IL-4 was only partially dependent upon ERK activation, whereas IL-5, IL-10, IFN-gamma and GM-CSF production was severely affected by diminished ERK activation. We conclude that the ERK pathway is differentially involved in the activation of different cytokine genes in normal T cells.
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PMID:Activation of the extracellular signal-regulated kinase pathway is differentially required for TCR-stimulated production of six cytokines in primary T lymphocytes. 953 50

While normal hematopoietic progenitor cells are dependent on colony-stimulating factors for in vitro proliferation, myeloid leukemic cells are frequently factor-independent. In this study we investigated several signalling intermediates of the Ras-Er1,2 pathway which may be involved in the development of growth factor independence. In the growth factor independent cell line KG1, an extremely short activation pattern of Erk1,2 with a maximum at 30 s was observed in response to FBS. In contrast, stimulation of the IL-3 receptor in AML193 cells resulted in a transient Erk activation peaking at 5 min and returning to base levels after 15 min. Although the Erk activation in KG1 cells is short-lived, using the MEK inhibitor PD98059, we demonstrated that Erk phosphorylation is essential for proliferation of these cell lines. We also detected major differences in Shc phosphorylation between factor-dependent and -independent cells. These data suggest that Erk activation is essential for proliferation of growth factor-dependent and -independent leukemic cells. The minimal Erk activation observed in KG1 cells in response to serum is sufficient for mitogenesis of these cells.
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PMID:Unconventional rapid Erk1,2 activation is indispensable for proliferation of the growth factor-independent myeloid leukemic cell line KG1. 959 67

Flt3 ligand (FL) is an early-acting potent co-stimulatory cytokine that regulates proliferation and differentiation of a number of blood cell lineages. Its receptor Flt3/Flk2 belongs to class III receptor tyrosine kinases that also include the receptors for colony-stimulating factor 1, Steel factor, and platelet-derived growth factor. Using CSF-1 receptor/Flt3 chimeras, two groups have characterized some of the post-receptor signaling events and substrate specificity of murine Flt3 receptor. However, there are few studies on the signaling pathway through human Flt3. We examined human Flt3 signaling pathways in a murine IL-3-dependent hematopoietic cell line Baf3, which stably expresses full-length human Flt3 receptor. This subline proliferates in response to human FL. Like the chimeric murine Flt3, human Flt3 undergoes autophosphorylation, associates with Grb2, and leads to tyrosine phosphorylation of Shc on ligand binding. We found that SHP-2, but not SHP-1, is tyrosine-phosphorylated by FL stimulation. SHP-2 does not associate with Flt3, but binds directly to Grb2. SHIP is also tyrosine-phosphorylated and associates with Shc after FL simulation. We further examined the downstream signaling pathway. FL transiently activates MAP kinase. This activation could be blocked by PD98059, a specific MEK inhibitor. PD98059 also blocked cell proliferation in response to FL. These results demonstrate that SHP-2 and SHIP are important components in the human Flt3 signaling pathway and suggest that SHP-2 and SHIP, by forming complexes with adapter proteins Grb2 and Shc, may modulate MAP kinase activation, which may be necessary for the mitogenic signaling of Flt3.
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PMID:Flt3 signaling involves tyrosyl-phosphorylation of SHP-2 and SHIP and their association with Grb2 and Shc in Baf3/Flt3 cells. 1008 May 42

In FDCP2 myeloid cells, IL-4 activated cyclic nucleotide phosphodiesterases PDE3 and PDE4, whereas IL-3, granulocyte-macrophage CSF (GM-CSF), and phorbol ester (PMA) selectively activated PDE4. IL-4 (not IL-3 or GM-CSF) induced tyrosine phosphorylation of insulin-receptor substrate-2 (IRS-2) and its association with phosphatidylinositol 3-kinase (PI3-K). TNF-alpha, AG-490 (Janus kinase inhibitor), and wortmannin (PI3-K inhibitor) inhibited activation of PDE3 and PDE4 by IL-4. TNF-alpha also blocked IL-4-induced tyrosine phosphorylation of IRS-2, but not of STAT6. AG-490 and wortmannin, not TNF-alpha, inhibited activation of PDE4 by IL-3. These results suggested that IL-4-induced activation of PDE3 and PDE4 was downstream of IRS-2/PI3-K, not STAT6, and that inhibition of tyrosine phosphorylation of IRS molecules might be one mechnism whereby TNF-alpha could selectively regulate activities of cytokines that utilized IRS proteins as signal transducers. RO31-7549 (protein kinase C (PKC) inhibitor) inhibited activation of PDE4 by PMA. IL-4, IL-3, and GM-CSF activated mitogen-activated protein (MAP) kinase and protein kinase B via PI3-K signals; PMA activated only MAP kinase via PKC signals. The MAP kinase kinase (MEK-1) inhibitor PD98059 inhibited IL-4-, IL-3-, and PMA-induced activation of MAP kinase and PDE4, but not IL-4-induced activation of PDE3. In FDCP2 cells transfected with constitutively activated MEK, MAP kinase and PDE4, not PDE3, were activated. Thus, in FDCP2 cells, PDE4 can be activated by overlapping MAP kinase-dependent pathways involving PI3-K (IL-4, IL-3, GM-CSF) or PKC (PMA), but selective activation of PDE3 by IL-4 is MAP kinase independent (but perhaps IRS-2/PI3-K dependent).
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PMID:IL-3 and IL-4 activate cyclic nucleotide phosphodiesterases 3 (PDE3) and 4 (PDE4) by different mechanisms in FDCP2 myeloid cells. 1020 31

ERYTHROPOIETIN (EPO): Erythropoietin (EPO) is a hormone that promotes the proliferation and differentiation of erythroid progenitor cells and regulates the number of erythrocytes in peripheral blood. EPO is produced mainly by the kidneys, and transcription of the EPO gene is promoted by a reduction in the oxygen concentration in the blood. The existence of EPO was suggested near the end of the 19th century by the discovery that hypoxia increases the production of red blood cells. EPO was identified as a serum factor in the 1950s, and in 1970 Miyake and coworkers succeeded in purifying it by using the urine of patients with aplastic anemia as a starting material. The human EPO gene was cloned in 1985 using a partial amino acid sequence from this purified EPO, and it is well known that recombinant EPO is currently used as a drug to treat anemia associated with chronic renal failure and other illnesses. ACTION OF EPO: When human bone marrow cells are cultured in a semisolid medium containing EPO, they form small erythroblast colonies in five to seven days, and by day 10 large erythroblast colonies appear that resemble fireworks ("burst" colonies). The original cells in the former colonies are called colony forming units-erythroid (CFU-E) or late-stage erythroblast progenitor cells and in the latter colonies they are called burst forming units-erythroid (BFU-E) or early-stage erythroblast progenitor cells. As shown in Figure 1, red blood cells are produced through differentiation from stem cells to BFU-E, CFU-E, and erythroblasts. Although EPO acts on both BFU-E and CFU-E cells, CFU-E cells show greater sensitivity to EPO, and other factors such as stem cell factor (SCF), interleukin (IL)-3, IL-4, and granulocyte macrophage colony-stimulating factor (GM-CSF) must be present together with EPO for BFU-E cell proliferation. In erythroblasts beyond the CFU-E stage, sensitivity to EPO decreases as the cells mature. THE EPO RECEPTOR AND THE CYTOKINE RECEPTOR FAMILY: The EPO receptor gene was cloned by D'Andrea and coworkers in 1989 from murine erythroleukemia cells [1]. It became clear that the EPO receptor belongs to the cytokine receptor family that comprises receptors for the various interleukins, GM-CSF, granulocyte colony-stimulating factor (G-CSF), growth hormone and prolactin. The special characteristic of this family of receptors is that they are switched on (i.e., the receptor is activated) and transduce signals to the interior of the cell by the formation of homo- or hetero-oligomers (dimers or trimers). Moreover, hetero-oligomers of these receptors share a common receptor subunit. As shown in Figure 2, the IL-3, IL-5 and GM-CSF receptors have a common &bgr; subunit, and their ligand specificity is determined by the &agr; subunit. In the same manner, the IL-6, LIF and oncostatin M (OSM) receptors all share gp130, which is the &bgr; subunit of the IL-6 receptor. The IL-2, IL-4 and IL-7 receptors all share the &ggr; subunit of the IL-2 receptor. All the above receptors are activated by the formation of hetero-oligomers, but the G-CSF receptor, EPO receptor, and growth hormone receptor are activated by the formation of homodimers of the same types of molecules [2]. We can see that groups of cytokines such as the interleukins that affect a relatively wide range of cells and have redundant biological activity create this redundancy through the common use of a single receptor subunit. On the other hand, EPO and G-CSF act with high specificity on a relatively limited range of cells, so it was probably unnecessary for their receptors to share one of the subunits. EPO RECEPTOR AND JAK2 KINASE: The signal for cellular proliferation and differentiation into erythroblasts is thought to originate at the EPO receptor. The cytoplasmic domain of the EPO receptor can be divided into two major regions. Roughly half of the cytoplasmic domain, the part lying nearest the plasma membrane, is required for generating the signals for proliferation and differentiation such as the induction of globin synthesis [3, 4]. The remaining half is not required for this signaling, and, conversely, it acts to dampen the signals. It is known that a tyrosine kinase called JAK2 associates with the region near the plasma membrane, undergoes autophosphorylation, and phosphorylates the EPO receptor, and a transcription factor called a STAT [5]. It is thought that JAK2 plays an important role in promoting cellular proliferation. The STAT is activated by the phosphorylation, and it then translocates to the nucleus, recognizes a specific base sequence in the promoter region of its target gene, and initiates transcription. At present, we know that the STAT whose activation is mediated by the EPO receptor is STAT5, and the target genes are CIS [6], which has an SH2 domain (a molecular structure that recognizes a phosphorylated tyrosine) and OSM [7], which is a pleiotropic cytokine. However, activation of STAT5 and activation of the target genes are not unique to the EPO receptor, and they also occur with the IL-2 and IL-3 receptors. Moreover, the JAK2 substrate that is directly linked to cellular proliferation is still unknown. At present, studies are under way to determine the transcription factors specific to EPO and their target genes, as well as the substrates of JAK2. RECEPTOR PHOSPHORYLATION AND CESSATION OF THE SIGNAL: On the other hand, tyrosine phosphorylation of the receptor is necessary at the cytoplasmic tail region far from the plasma membrane, and the signal transduction pathway that originates with this phosphorylated tyrosine and is mediated by proteins with SH2 domains becomes activated. First, a GTP/GDP exchange factor called SOS, which is mediated by Shc and Grb2, migrates to the plasma membrane and converts a ras protein to its GTP form. The activated ras protein then activates the Raf-MAP kinase kinase-MAP kinase cascade, and ultimately initiates the transcription of oncogenes such as c-fos and c-jun. An enzyme called PI3 kinase binds to the tyrosine phosphorylation site of the receptor and a second messenger is born. It is known that this pathway is a requirement for DNA synthesis in certain types of fibroblasts. However, these signal transduction pathways are not unique to the EPO receptor, and they are also activated by most growth factor receptors, so they are not necessarily required for EPO-induced proliferation. Conversely, the tyrosine phosphatase SH-PTP1 (also called HCP) that has an SH2 domain and is specific to blood cells associates with the tyrosine phosphorylation site of the receptor and promotes the dephosphorylation of JAK2. In other words, the role of SH-PTP1 is to stop generation of the signal [8]. Therefore, in mutations lacking this cytoplasmic tail region of the receptor far from the plasma membrane, the receptors do not undergo tyrosine phosphorylation, JAK2 activation continues for a longer period of time, and thus the signal is generated more efficiently. In fact, in one patient with a mild case of familial erythrocytosis a mutation was discovered in which the C-terminus of the EPO receptor was missing 70 amino acids [9]. This was a dominant genetic trait, and the patient's erythroblasts showed an increased sensitivity to EPO. In this family the impairment was not severe enough to be called an illness, and in fact it is said that this patient was proficient enough athletically to compete for a gold medal at the Olympics. More specifically, the reason that athletes undergo training at high altitudes is to boost EPO production because of the lower oxygen partial pressure, and this brings about the desired effect of sustained athletic capability due to a resultant increase in red blood cells. However, the same effect has occurred naturally in this athlete thanks to accelerated receptor capability.
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PMID:Physician Education: The Erythropoietin Receptor and Signal Transduction. 1038 12

Bcl2 phosphorylation at Ser-70 may be required for the full and potent suppression of apoptosis in IL-3-dependent myeloid cells and can result from agonist activation of mitochondrial protein kinase C (PKC). Paradoxically, expression of exogenous Bcl2 can protect parental cells from apoptosis induced by the potent PKC inhibitor, staurosporine (stauro). High concentrations of stauro of up to 1 microM only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKC-mediated Bcl2 phosphorylation in vitro. These data indicate a role for a stauro-resistant Bcl2 kinase (SRK). We show that aurintricarboxylic acid (ATA), a nonpeptide activator of cellular MEK/mitogen-activated protein kinase (MAPK) kinase, can induce Ser-70 phosphorylation of Bcl2 and support survival of cells expressing wild-type but not the phosphorylation-incompetent S70A mutant Bcl2. A role for a MEK/MAPK as a responsible SRK was implicated because the highly specific MEK/MAPK inhibitor, PD98059, also can only partially inhibit IL-3-induced Bcl2 phosphorylation, whereas the combination of PD98059 and stauro completely blocks phosphorylation and synergistically enhances apoptosis. p44MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 are activated by IL-3, colocalize with mitochondrial Bcl2, and can directly phosphorylate Bcl2 on Ser-70 in a stauro-resistant manner both in vitro and in vivo. These findings suggest a role for the ERK1/2 kinases as SRKs. Thus, the SRKs can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways and, in a novel capacity, may explain how Bcl2 can suppress stauro-induced apoptosis. In addition, although the mechanism of regulation of Bcl2 by phosphorylation is not yet clear, our results indicate that phosphorylation may functionally stabilize the Bcl2-Bax heterodimerization.
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PMID:Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases. 1067 2

Cytokine-dependent activation of distinct signaling pathways is a common scheme thought to be required for the subsequent programmation into cell proliferation and survival. The PI 3-kinase/Akt, Ras/MAP kinase, Ras/NFIL3 and JAK/STAT pathways have been shown to participate in cytokine mediated suppression of apoptosis in various cell types. However the relative importance of these signaling pathways seems to depend on the cellular context. In several cases, individual inhibition of each pathway is not sufficient to completely abrogate cytokine mediated cell survival suggesting that cooperation between these pathways is required. Here we showed that individual inhibition of STAT5, PI 3-kinase or MEK activities did not or weakly affected the IL-3 dependent survival of the bone marrow derived Ba/F3 cell line. However, the simultaneous inhibition of STAT5 and PI 3-kinase activities but not that of STAT5 and MEK reduced the IL-3 dependent survival of Ba/F3. Analysis of the expression of the Bcl-2 members indicated that phosphorylation of Bad and Bcl-x expression which are respectively regulated by the PI 3-kinase/Akt pathway and STAT5 probably explain this cooperation. Furthermore, we showed by co-immunoprecipitation studies and pull down experiments with fusion proteins encoding the GST-SH2 domains of p85 that STAT5 in its phosphorylated form interacts with the p85 subunit of the PI 3-kinase. These results indicate that the activations of STAT5 and the PI 3-kinase by IL-3 in Ba/F3 cells are tightly connected and cooperate to mediate IL-3-dependent suppression of apoptosis by modulating Bad phosphorylation and Bcl-x expression.
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PMID:Cooperation between STAT5 and phosphatidylinositol 3-kinase in the IL-3-dependent survival of a bone marrow derived cell line. 1071 4


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