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)

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

The CSF-1 receptor (CSF-1R) is expressed in >50% of human breast cancers. To investigate the consequence of CSF-1R expression, hormone-dependent human breast cancer cell lines, MCF-7 and T-47D, were transfected with CSF-1R. Unexpectedly, CSF-1 substantially inhibited estradiol (E2) and insulin-dependent proliferation of MCF-7 transfectants (MCF-7fms) and prevented cyclin E/cdk2 and cyclin A/cdk2 activation, consistent with a G1 arrest. In contrast, CSF-1 increased DNA synthesis in T-47D transfectants (T-47Dfms) alone and with E2 or insulin. In response to CSF-1, there was a marked and sustained upregulation of the cyclin-dependent kinase inhibitor, p21Waf1/Cip1, in MCF-7fms but not T-47Dfms. CSF-1 also markedly upregulated cyclin D1 in MCF-7fms. The coordinate increase in cyclin D1 and p21 had the effect of decreasing the specific but not absolute activity of cyclin D1/cdk4. p53 was not involved since CSF-1 induction of p21 was unaffected by dominant-negative p53 expression. ERK activation by CSF-1 was robust and sustained in MCF-7fms and to a much lesser extent in T-47Dfms. Using pharmacological and transient transfection approaches, we showed that ERK activation was necessary and sufficient for p21 induction in MCF-7fms. Moreover, activated MEK inhibited E2-stimulated cdk2 activity. Our findings indicate that the consequence of CSF-1R-mediated signals in human breast cancer cells is dependent on the genetic background of the particular tumor.
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PMID:CSF-1 activates MAPK-dependent and p53-independent pathways to induce growth arrest of hormone-dependent human breast cancer cells. 1060 7

The Raf/MEK/MAP kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using deltaMEK1:ER, a conditionally-active form of MEK1, we demonstrate the ability of this dual specificity protein kinase to abrogate the cytokine-dependency of the human and murine hematopoietic cells lines TF-1, FDC-P1 and FL5.12. Cytokine-independent cells were obtained from TF-1, FDC-P1 and FL5.12 cells at frequencies of 2.5 x 10(-3), 5 x 10(-5) and 10(-7) respectively, indicating that not all cells expressing deltaMEK1:ER were factor-independent. In general, cells that were converted to a cytokine-independent phenotype displayed a higher level of MAP kinase activity in response to deltaMEK1:ER activation than those that remained cytokine-dependent. deltaME-K1:ER-responsive cells could be maintained long-term in the presence of beta-estradiol as well as the estrogen-receptor antagonist 4-Hydroxy-Tamoxifen and the anti-estrogen ICI 164383. Removal of hormone led to the rapid cessation of cell growth in a manner similar to that observed when cytokine is withdrawn from the parental cells. Treatment of deltaMEKI:ER-responsive cells with a specific and selective inhibitor, PD98059, prevented growth in response to beta-estradiol. GM-CSF mRNA transcripts were detected in the MEK1-responsive cells indicating that the activated deltaMEK1:ER may induce a pathway leading to autocrine proliferation. Treatment of MEK1-responsive cells with an anti-GM-CSF antibody, but not a control antibody, suppressed cell growth. The cell lines described here will be useful for elaborating the ability of the MAP kinase pathway to regulate cell proliferation in hematopoietic cells.
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PMID:A conditionally-active form of MEK1 results in autocrine tranformation of human and mouse hematopoietic cells. 1069 22

The MEK1 oncoprotein plays a critical role in Ras/Raf/MEK/MAPK-mediated transmission of mitogenic signals from cell surface receptors to the nucleus. In order to examine this pathway's role in leukemic transformation, a conditionally active (beta-estradiol-inducible) form of the MEK1 protein was created by ligating a cDNA encoding an N-terminal truncated form of MEK1 to the hormone-binding domain of the estrogen receptor (ER). We introduced this chimeric deltaMEK1:ER oncoprotein into cytokine-dependent human TF-1 and murine FDC-P1 hematopoietic cell lines. Two different types of cells were recovered after drug selection in medium containing either cytokine or beta-estradiol: (1) cells that expressed the deltaMEK1:ER oncoprotein but remained cytokine-dependent and (2) MEK1-responsive cells that grew in response to deltaMEK1:ER activation. Cytokine-dependent cells were recovered 10(2) to 10(4) times more frequently than MEK1-responsive cells depending upon the particular cell line. To determine whether BCL2 overexpression could synergize with the deltaMEK1:ER oncoprotein in relieving cytokine dependence, the cytokine-dependent deltaMEK1:ER-expressing cells were infected with a BCL2-containing retrovirus, and the frequency of MEK1-responsive cells determined. BCL2 overexpression, by itself, did not relieve cytokine dependency of the parental cells, however, it did increase the frequency at which MEK1-responsive cells were recovered approximately 10-fold. DeltaMEK1:ER+BCL2 cells remained viable for at least 3 days after estradiol deprivation, whereas viability was readily lost upon withdrawal of beta-estradiol in the MEK1-responsive cells which lacked BCL2 overexpression. The MAP kinases, ERK1 and ERK2 were activated in response to deltaMEK1:ER stimulation in both deltaMEK1:ER and deltaMEK1:ER+BCL2 cells. As compared to the cytokine-dependent deltaMEK1:ER and BCL2 infected cells, MEK1-responsive BCL2 infected cells expressed higher levels of BCL2. While both MEK1-responsive deltaMEK1:ER and deltaMEK1:ER+BCL2 infected cells expressed cDNAs encoding the autocrine cytokine GM-CSF, more GM-CSF cDNAs and bioactivity were detected in the MEK1-responsive deltaMEK1:ER+BCL2 cells than in the MEK1-responsive cells lacking BCL2 or cytokine-dependent cells. These conditionally transformed cells will be useful in furthering our understanding of the roles MEK1 and BCL2 play in the prevention of apoptosis in hematopoietic cells.
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PMID:Combined effects of aberrant MEK1 activity and BCL2 overexpression on relieving the cytokine dependency of human and murine hematopoietic cells. 1086 74

Human GM-CSF (hGM-CSF) induces proliferation and sustains the viability of a mouse IL-3-dependent lymphoid cell line BA/F3 that expresses the functional hGM-CSF receptor (hGMR). To reveal an antiapoptotic mechanism of hGM-CSF, we analyzed various apoptotic markers of BA/F3 cells in various conditions. Within 24 hours of factor depletion, caspase 3-like, but not caspase 1-like, enzyme activity and DNA fragmentation were augmented. Analysis with the tyrosine kinase inhibitor (genistein) and an MEK1 inhibitor (PD98059) on antiapoptosis activity indicates that the activation of either the genistein-sensitive signaling pathway or the PD98059-sensitive signaling pathway of the betac subunit may be sufficient to suppress apoptosis through hGMR. Because hGMR mutants (which activate JAK2 but neither STAT5 nor the MAPK cascade) have antiapoptotic activity in BA/F3 cells, the involvement of JAK2, excluding the molecules mentioned earlier, for antiapoptosis activity seems likely. Because the JAK2 inhibitor AG-490 suppressed the antiapoptotic activity of hGM-CSF, the essential role for JAK2 activation to maintain the viability is considered. Interestingly, hGMR mutants, which lack MAPK cascade activation, require a higher dose of hGM-CSF than that for wild-type hGMR. Because the expression level and affinity to hGM-CSF among wild-type hGMR and mutant hGMR are the same, we speculated that biologic response is determined by a combination of strength of various signaling events.
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PMID:Analysis of antiapoptosis activity of human GM-CSF receptor. 1088 29

Colony-stimulating factor 1 (CSF-1) supports the proliferation, survival, and differentiation of bone marrow-derived cells of the monocytic lineage. In the myeloid progenitor 32D cell line expressing CSF-1 receptor (CSF-1R), CSF-1 activation of the extracellular signal-regulated kinase (ERK) pathway is both Ras and phosphatidylinositol 3-kinase (PI3-kinase) dependent. PI3-kinase inhibition did not influence events leading to Ras activation. Using the activity of the PI3-kinase effector, Akt, as readout, studies with dominant-negative and oncogenic Ras failed to place PI3-kinase downstream of Ras. Thus, PI3-kinase appears to act in parallel to Ras. PI3-kinase inhibitors enhanced CSF-1-stimulated A-Raf and c-Raf-1 activities, and dominant-negative A-Raf but not dominant-negative c-Raf-1 reduced CSF-1-provoked ERK activation, suggesting that A-Raf mediates a part of the stimulatory signal from Ras to MEK/ERK, acting in parallel to PI3-kinase. Unexpectedly, a CSF-1R lacking the PI3-kinase binding site (DeltaKI) remained capable of activating MEK/ERK in a PI3-kinase-dependent manner. To determine if Src family kinases (SFKs) are involved, we demonstrated that CSF-1 activated Fyn and Lyn in cells expressing wild-type (WT) or DeltaKI receptors. Moreover, CSF-1-induced Akt activity in cells expressing DeltaKI is SFK dependent since Akt activation was prevented by pharmacological or genetic inhibition of SFK activity. The docking protein Gab2 may link SFK to PI3-kinase. CSF-1 induced Gab2 tyrosyl phosphorylation and association with PI3-kinase in cells expressing WT or DeltaKI receptors. However, only in DeltaKI cells are these events prevented by PP1. Thus in myeloid progenitors, CSF-1 can activate the PI3-kinase/Akt pathway by at least two mechanisms, one involving direct receptor binding and one involving SFKs.
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PMID:Both src-dependent and -independent mechanisms mediate phosphatidylinositol 3-kinase regulation of colony-stimulating factor 1-activated mitogen-activated protein kinases in myeloid progenitors. 1095 75

1. The extent to which the p38 mitogen-activated protein (MAP) kinase and MAP kinase kinase (MKK)-1-signalling pathways regulate the expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) from LPS-stimulated human monocytes has been investigated and compared to the well studied cytokine tumour necrosis factor-alpha (TNF alpha). 2. Lipopolysaccharide (LPS) evoked a concentration-dependent generation of GM-CSF from human monocytes. Temporally, this effect was preceded by an increase in GM-CSF mRNA transcripts and abolished by actinomycin D and cycloheximide. 3. LPS-induced GM-CSF release and mRNA expression were associated with a rapid and time-dependent activation of p38 MAP kinase, ERK-1 and ERK-2. 4. The respective MKK-1 and p38 MAP kinase inhibitors, PD 098059 and SB 203580, maximally suppressed LPS-induced GM-CSF generation by >90%, indicating that both of these signalling cascades co-operate in the generation of this cytokine. 5. Electrophoretic mobility shift assays demonstrated that LPS increased nuclear factor kappa B (NF-kappa B) : DNA binding. SN50, an inhibitor of NF-kappa B translocation, abolished LPS-induced NF-kappaB : DNA binding and the elaboration of TNFalpha, a cytokine known to be regulated by NF-kappaB in monocytes. In contrast, SN50 failed to affect the release of GM-CSF from the same monocyte cultures. 6. Collectively, these results suggest that the generation of GM-CSF by LPS-stimulated human monocytes is regulated in a co-operative fashion by p38 MAP kinase- and MKK-1-dependent signalling pathways independently of the activation of NF-kappa B.
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PMID:p38 MAP kinase and MKK-1 co-operate in the generation of GM-CSF from LPS-stimulated human monocytes by an NF-kappa B-independent mechanism. 1108 22

Steel factor (SLF) plus GM-CSF induces proliferative synergy in myeloid progenitors and factor-dependent cell line MO7e. We previously reported that the protein level of cyclin-dependent kinase inhibitor p21(cip1/waf1) (p21) increased synergistically when MO7e cells were stimulated with SLF plus GM-CSF and that p21 induction was required for SLF synergistic responses. Here we show that this p21 induction is regulated at the transcriptional level. Based on use of a multiprobe RNase protection assay, the synergistic increase of p21 mRNA was unique among many cell cycle regulators. While STAT5A and 5B were activated after stimulation with GM-CSF alone or SLF plus GM-CSF, there was no difference in activation between the groups. p44/42 MAP kinase (ERK1/2) was synergistically activated by SLF plus GM-CSF, but SAPK/JNK and p38 MAP kinase were not. Synergistic induction of p21 was significantly decreased with a MEK1 inhibitor, suggesting that the ERK1/2 pathway is involved in the synergistic increase of p21 after GM-CSF plus SLF stimulation.
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PMID:Transcriptional and ERK1/2-dependent synergistic upregulation of p21(cip1/waf1) associated with steel factor synergy in MO7e. 1116 74

Steel factor (SLF) plus GM-CSF induces proliferative synergy in factor-dependent cell line MO7e and hematopoietic progenitor cells. We previously reported ERK1/2 involvement in this synergy, but its downstream signaling molecules are not defined. Here, we investigated activation of the 90-kDa ribosomal S6 kinase (RSK) proteins by measuring the phosphorylation status and in vitro kinase activity in MO7e cells. Both GM-CSF and SLF induced activation of RSK, and the combined stimulation with these two cytokines induced synergistic and persistent activation of RSK. RSK activity was reduced by PI3 kinase inhibitor LY294002 or MEK1 inhibitor PD98059, suggesting that the ERK as well as the PI3 kinase pathways are involved in regulation of RSK activity. Sensitivities of RSK activity to inhibitory drugs correlated well with those of c-fos gene induction. Taken together, synergistic activation of RSK may contribute, at least in part, to the synergistic induction of c-fos after combined stimulation with GM-CSF plus SLF.
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PMID:Synergistic activation of RSK correlates with c-fos induction in MO7e cells stimulated with GM-CSF plus Steel factor. 1123 44

Survivin, a member of the inhibitors-of-apoptosis gene family, is expressed in a cell-cycle-dependent manner in all the most common cancers but not in normal differentiated adult tissues. Survivin expression and regulation were examined in acute myeloid leukemia (AML). Survivin was detected by Western blot analysis in all myeloid leukemia cell lines and in 16 of 18 primary AML samples tested. In contrast, normal CD34(+) cells and normal peripheral blood mononuclear cells expressed no or very low levels of survivin. Cytokine stimulation increased survivin expression in leukemic cell lines and in primary AML samples. In cultured primary samples, single-cytokine stimulation substantially increased survivin expression in comparison with control cells, and the combination of G-CSF, GM-CSF, and SCF increased survivin levels even further. Conversely, all-trans retinoic acid significantly decreased survivin protein levels in HL-60, OCI-AML3, and NB-4 cells within 96 hours, parallel to the induction of myelomonocytic differentiation. Using selective pharmacologic inhibitors, the differential involvement of mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol-3 kinase (PI3K) pathways were demonstrated in the regulation of survivin expression. The MEK inhibitor PD98059 down-regulated survivin expression in both resting and GM-CSF-stimulated OCI-AML3 cells, whereas the PI3K inhibitor LY294002 inhibited survivin expression only on GM-CSF stimulation. In conclusion, these results demonstrate that survivin is highly expressed and cytokine-regulated in myeloid leukemias and suggest that hematopoietic cytokines exert their antiapoptotic and mitogenic effects, at least in part, by increasing survivin levels.
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PMID:Cytokine-regulated expression of survivin in myeloid leukemia. 1131 72


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