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)

Colony-stimulating factors (CSFs) promote the proliferation, differentiation, commitment, and survival of myeloid progenitors, whereas cyclic AMP (cAMP)-mediated signals frequently induce their growth arrest and apoptosis. The ERK/mitogen-activated protein kinase (MAPK) pathway is a target for both CSFs and cAMP. We investigated how costimulation by cAMP and colony-stimulating factor-1 (CSF-1) or interleukin-3 (IL-3) modulates MAPK in the myeloid progenitor cell line, 32D. cAMP dramatically increased ERK activity in the presence of CSF-1 or IL-3. IL-3 also synergized with cAMP to activate ERK in another myeloid cell line, FDC-P1. The increase in ERK activity was transmitted to a downstream target, p90(rsk). cAMP treatment of 32D cells transfected with oncogenic Ras was found to recapitulate the superactivation of ERK seen with cAMP and CSF-1 or IL-3. ERK activation in the presence of cAMP did not appear to involve any of the Raf isoforms and was blocked by expression of dominant-negative MEK1 or treatment with a MEK inhibitor, PD98059. Although cAMP had an overall inhibitory effect on CSF-1-mediated proliferation and survival, the inhibition was markedly increased if ERK activation was blocked by PD98059. These findings suggest that upregulation of the ERK pathway is one mechanism induced by CSF-1 and IL-3 to protect myeloid progenitors from the growth-suppressive and apoptosis-inducing effects of cAMP elevations.
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PMID:Synergistic activation of mitogen-activated protein kinase by cyclic AMP and myeloid growth factors opposes cyclic AMP's growth-inhibitory effects. 988 15

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

In this study, the abilities of constitutive and conditional forms of the three Raf kinases to abrogate the cytokine dependency of FDC-P1 cells were examined. The constitutively active forms (delta) of all three Raf kinases were fused to the hormone-binding domain of the estrogen receptor (ER), rendering their activities conditionally dependent upon exogenous beta-estradiol. The vast majority of deltaRaf:ER-infected FDC-P1 cells remained cytokine-dependent; however, cells were obtained at low frequency in which expression of deltaRaf:ER abrogated cytokine dependency. Isoform specific differences between the Raf kinases were observed as cytokine-independent cells were obtained more frequently from deltaA-Raf:ER than either deltaRaf-1:ER or deltaB-Raf:ER infected cells. To determine whether the regulatory phosphorylation sites in the Raf proteins were necessary for abrogation of cytokine dependency, they were changed by site-directed mutagenesis. Substitution with phenylalanine eliminated the transforming ability of the deltaB-Raf:ER and deltaRaf-1:ER kinases. However, a similar substitution in A-Raf did not extinguish its transforming activity. The activated Raf proteins induced essential downstream MEK1 activity as treatment with the MEK1 inhibitor, PD98059, suppressed Raf-mediated growth. Activated MAP kinases (ERK1 and ERK2) were detected in deltaRaf:ER-transformed cells, and their presence was dependent upon a functional MEK1 protein. The cytokine-independent phenotype required the continued activity of the deltaRaf:ER proteins as removal of beta-estradiol caused the cells to stop growing and undergo apoptosis. The Raf-responsive cells were found to express autocrine growth factors, which promoted their growth. Constitutive activation of the Raf-1 oncogene resulted in malignant transformation as cytokine-independent FDC-P1 cells infected with a retrovirus encoding an activated Raf-1 protein formed tumors upon injection of immunocompromised mice. In summary, Raf kinases can abrogate cytokine dependency, prevent apoptosis and induce the tumorigenicity of a certain subpopulation of FDC-P1 cells by a MEK1-dependent mechanism.
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PMID:Differential abilities of the Raf family of protein kinases to abrogate cytokine dependency and prevent apoptosis in murine hematopoietic cells by a MEK1-dependent mechanism. 1076 50

The Raf oncoprotein plays critical roles in the transmission of mitogenic signals from cytokine receptors to the nucleus. There are three Raf family members: A-Raf, B-Raf and Raf-1. Conditionally active forms of the Raf proteins were created by ligating N-terminal truncated activated forms to the estrogen-receptor (ER) hormone-binding domain resulting in beta-estradiol-inducible constructs. We introduced these chimeric deltaRaf:ER oncoproteins into the murine FDC-P1 hematopoietic cell line. Two different types of cells were recovered after drug selection in medium containing either cytokine or beta-estradiol: (1) cytokine-dependent cells that expressed the deltaRaf:ER oncoproteins; and (2) Raf-responsive cells that grew in response to the deltaRaf:ER oncoprotein. Depending upon the particular deltaRaf:ER oncoprotein, cytokine-dependent cells were recovered 10(3) to 10(5) times more frequently than Raf-responsive cells. To determine whether BCL2 could synergize with the deltaRaf:ER oncoproteins and increase the frequency of cytokine-independent cells, cytokine-dependent deltaRaf:ER-expressing cells were infected with either a BCL2 containing retrovirus or an empty retroviral vector. BCL2 overexpression, by itself, did not relieve cytokine dependency of the parental cell line. However, BCL2 overexpression increased the frequency of Raf-responsive cells approximately five- to 100-fold. Cytokine-dependent deltaRaf:ER-infected cells entered the G1 phase of the cell cycle after cytokine withdrawal and entered S phase only after cytokine addition. Raf-responsive deltaRaf:ER cells entered the G1 phase of the cell cycle after estrogen deprivation and re-entered the cell cycle after addition of either IL-3 or the estrogen receptor antagonist tamoxifen which activates the deltaRaf:ER constructs. Expression of the BCL2 oncoprotein often delayed the exit from the S and G2/M phases demonstrating the protective effects BCL2 provided to these Raf and BCL2 infected cells. The deltaRaf:ER cells expressed the deltaRaf:ER proteins and downstream MEK and ERK activities after beta-estradiol treatment. Raf-responsive cells that were also infected with BCL2 expressed higher levels of BCL2 than the cells that were not infected with BCL2. Thus BCL2 can synergize with the activated Raf in the abrogation of cytokine dependency of certain hematopoietic cells. These cells will be useful in furthering our understanding of the roles of the Raf and BCL2 oncoproteins in hematopoietic cell growth, cell cycle progression and prevention of apoptosis.
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PMID:Synergy between Raf and BCL2 in abrogating the cytokine dependency of hematopoietic cells. 1086 73

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

The PI3K/Akt and Raf/MEK/ERK signal transduction cascades are pivotal in transmitting signals from membrane receptors to downstream targets that regulate apoptosis, gene expression, and cell growth. The abilities of activated PI3K, Akt, Raf, and MEK proteins to abrogate the cytokine dependence of three different hematopoietic cell lines were determined. Activated PI3K or Akt expression by themselves did not efficiently annul cytokine dependence. Raf and MEK could abrogate the cytokine dependence of murine FDC-PI and human TF-1 cells; however, the frequency of transformation was dependent on the particular oncogene examined, as more factor-independent cells were isolated after infection with activated retroviruses encoding A-Raf or Raf-1 than were with MEK1 or B-Raf. Cytokine-independent deltaRaf-1-infected cells formed tumors on injection into immunocompromised mice, whereas cytokine-dependent cell lines did not, demonstrating the oncogenic effects of activation of the Raf/MEK/ERK pathway. Overexpression of the antiapoptotic Bcl-2 protein synergized with activation of the Raf/MEK/ERK cascade and increased the efficiency of transformation of FDC-PI and TF-1 cells. In contrast to the results observed with FDC-P1 and TF-I cells, the activated Raf genes did not relieve the cytokine dependence of murine FL5.12 cells. The abilities of the Raf and PI3K pathways to interact and annul the cytokine dependence of FL5.12 cells were determined. The combination of Raf and either PI3K or Akt expression relieved cytokine dependence of some FL5.12 cells, and the efficiency of transformation could be enhanced further by Bcl-2 or Bcl-XL overexpression. Thus, the antiapoptotic PI3K/Akt and Bcl-2/Bcl-XL proteins can interact with the growth-promoting Raf/MEK/ERK pathway and annul the cytokine dependence of certain hematopoietic cells.
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PMID:Interactions between the PI3K and Raf signaling pathways can result in the transformation of hematopoietic cells. 1153 Oct 15

The Raf/MEK/ERK kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using DeltaMEK1:ER, a conditionally active form of MEK1 which responds to either beta-estradiol or the estrogen receptor antagonist 4 hydroxy-tamoxifen (4HT), we previously documented the ability of this dual specificity protein kinase to abrogate the cytokine-dependency of human (TF-1) and murine (FDC-P1 and FL5.12) hematopoietic cells lines. Here we demonstrate the ability of DeltaMEK1:ER to activate the phosphatidylinositol 3-kinase (PI3K)/Akt/p70 ribosomal S6 kinase (p70(S6K)) pathway and the importance of this pathway in MEK1-mediated prevention of apoptosis. MEK1-responsive cells can be maintained long term in the presence of beta-estradiol, 4HT or IL-3. Removal of hormone led to the rapid cessation of cell proliferation and the induction of apoptosis in a manner similar to cytokine deprivation of the parental cells. Stimulation of DeltaMEK1:ER by 4HT resulted in ERK, PI3K, Akt and p70(S6K) activation. Treatment with PI3K, Akt and p70(S6K) inhibitors prevented MEK-responsive growth. Furthermore, the apoptotic effects of PI3K/Akt/p70(S6K) inhibitors could be enhanced by cotreatment with MEK inhibitors. Use of a PI3K inhibitor and a constitutively active form of Akt, [DeltaAkt(Myr(+))], indicated that activation of PI3K was necessary for MEK1-responsive growth and survival as activation of Akt alone was unable to compensate for the loss of PI3K activity. Cells transduced by MEK or MEK+Akt displayed different sensitivities to signal transduction inhibitors, which targeted these pathways. These results indicate a requirement for the activation of the PI3K pathway during MEK-mediated transformation of certain hematopoietic cells. These experiments provide important clues as to why the identification of mutant signaling pathways may be the Achilles heel of leukemic cell growth. Leukemia treatment targeting multiple signal transduction pathways may be more efficacious than therapy aimed at inhibiting a single pathway.
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PMID:Requirement for the PI3K/Akt pathway in MEK1-mediated growth and prevention of apoptosis: identification of an Achilles heel in leukemia. 1276 69

The Raf/MEK/ERK and PI3K/Akt pathways regulate proliferation and prevent apoptosis, and their altered expression is commonly observed in human cancer due to the high mutation frequency of upstream regulators. In this study, the effects of Raf, MEK, and PI3K inhibitors on conditionally transformed hematopoietic cells were examined to determine if they would display cytotoxic differences between cytokine- and oncogene-mediated proliferation, and whether inhibition of both pathways was a more effective means to induce apoptosis. In the hematopoietic model system employed, proliferation was conditional and occurred when either interleukin-3 (IL-3) or the estrogen receptor antagonist 4-hydroxytamoxifen (4HT), which activates the conditional oncoprotein (DeltaRaf:ER), were provided. Thus, upon the addition of the signal transduction inhibitors and either IL-3 or 4HT, the effects of these drugs were examined in the same cell under 'cytokine-' and 'oncoprotein' -mediated growth conditions avoiding genetic and differentiation stage heterogeneity. At drug concentrations around the reported IC(50) for the Raf inhibitor L-779,450, it suppressed DNA synthesis and induced apoptosis in hematopoietic FDC-P1 cells transformed to grow in response to either Raf-1 or A-Raf (FD/DeltaRaf-1:ER and FD/DeltaA-Raf:ER), but it displayed less effects on DNA synthesis and apoptosis when the cells were cultured in IL-3. This Raf inhibitor was less effective on B-Raf- or MEK1-responsive cells, demonstrating the specificity of this drug. MEK inhibitors also suppressed DNA synthesis and induced apoptosis in Raf-responsive cells and the effects were more significant on Raf-responsive compared to cytokine-mediated growth. The PI3K inhibitor LY294002 suppressed Raf-mediated growth, indicating that part of the long-term proliferative effects mediated by Raf are PI3K dependent. Simultaneous inhibition of both Raf/MEK/ERK and PI3K/Akt pathways proved a more efficient means to suppress DNA synthesis and induce apoptosis at lower drug concentrations.
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PMID:Differential effects of kinase cascade inhibitors on neoplastic and cytokine-mediated cell proliferation. 1297 Jul 75

FDC-P1 hematopoietic cells were conditionally transformed to grow in response to (delta)B Raf:ER, (delta)Raf-1:ER or DA-Raf:ER in which the hormone binding domain of the estrogen receptor (ER) was linked to the N-terminal truncated (delta) Raf genes. When these cells were deprived of IL-3 or beta-estradiol for 24 hrs, they exited the cell cycle and underwent apoptosis. FD/(delta)Raf-1:ER and FD/(delta)A-Raf:ER, but not FD/(delta)B-Raf:ER cells, were readily induced to re-enter the cell cycle after addition of beta-estradiol or IL-3. Deprived FD/(delta)Raf-1:ER, but not FD/(delta)B-Raf:ER cells, expressed activated forms of MEK1 and ERK after beta-estradiol or IL-3 stimulation. Insulin or beta-estradiol alone did not induce FD/(delta)B-Raf:ER cells to re-enter the cell cycle, whereas cell cycle entry was observed upon their co-addition. Apoptosis was prevented in FD/(delta)B-Raf:ER cells when they were cultured in the presence of IL-3 or beta-estradiol, whereas they underwent apoptosis in their absence. Insulin by itself did not prevent apoptosis, however, upon DB-Raf:ER or DRaf-1:ER activation and addition of insulin, more than an additive effect was observed in both lines indicating that these path- ways synergized to prevent apoptosis. Raf isoforms differ in their abilities to control apoptosis and cell cycle progression and B-Raf requires insulin-activated pathways for full antiapoptotic and proliferative activity.
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PMID:B-raf and insulin synergistically prevent apoptosis and induce cell cycle progression in hematopoietic cells. 1471 85

The insulin like growth factor-1 (IGF-1) receptor (R) induced PI3K/Akt signal transduction cascade has critical roles in prevention of apoptosis and regulation of cell cycle progression. Here, we discuss the effects of IGF-1R-mediated signal transduction on hematopoietic cells which normally require interleukin-3 (IL-3) for growth and prevention of apoptosis. Cytokine-dependent FDC-P1 hematopoietic cells were conditionally transformed to grow in response to overexpression of IGF-1R in the presence of IGF-1. When these cells were deprived of IL-3 or IGF-1 for 24 hrs, they exited the cell cycle, activated caspase 3 and underwent apoptosis. The effects of inhibitors which targeted the PI3K/Akt and Raf/MEK/ERK pathways were determined. When the cells were cultured with IGF-1 and either PI3K or MEK inhibitors, cell cycle progression and DNA synthesis were inhibited and caspase 3 activity and apoptosis were induced. Coinhibition of both pathways synergized to prevent cell cycle progression, inhibit DNA synthesis and induce apoptosis. These inhibitors had more apoptotic inducing effects when the cells were grown in response to IGF-1 than IL-3, indicating that IL-3 can induce additional anti-apoptotic pathways. These results demonstrate that the PI3K/Akt and Raf/MEK/ERK pathways are intimately involved in IGF-1R-mediated cell cycle progression and prevention of apoptosis in hematopoietic cells.
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PMID:Synergy between PI3K/Akt and Raf/MEK/ERK pathways in IGF-1R mediated cell cycle progression and prevention of apoptosis in hematopoietic cells. 1472 97


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