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)

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

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

32D cells expressing v-Ha-Ras fail to show a transformed phenotype. Since Ras requires an active IGF-1R for transformation of fibroblasts, we asked whether expression of IRS-1 or Shc (two of the major substrates of the IGF-1R) could co-operate with oncogenic Ras in transforming 32D cells. We find that IRS-1, but not Shc, in combination with v-Ha-Ras generates a fully transformed phenotype in 32D cells. 32D cells expressing both IRS-1 and v-Ha-Ras (32D/IRS1/Ras) survive and proliferate in the absence of IL-3, do not undergo granulocytic differentiation in the presence of G-CSF and form tumors in nu/nu and syngeneic mice. In contrast, 32D cells expressing singly IRS-1 or v-Ha-Ras exhibit only a block in differentiation capacity. Over-expression of Shc proteins, by itself, promotes differentiation of 32D cells. Concomitant expression of IRS-1 and v-Ha-Ras synergistically phosphorylates ERK-1 and ERK-2 whereas a MEK inhibitor rapidly induces death of 32D/IRS1/Ras transformed cells. Furthermore, transformed 32D/IRS1/Ras cells display high levels of PI3-K activation and undergo rapid apoptosis when exposed to PI3-K inhibitors. The data indicate that: (1) a fully transformed phenotype in 32D cells is generated when a block in differentiation (v-Ha-Ras) is coupled with another differentiation block (IRS-1); (2) PI3-K and MAPK activity are required for the survival of transformed cells; (3) the signals generated by IRS-1 and oncogenic Ras converge on ERK and PI3-K resulting in high levels of activation.
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PMID:Cooperative transformation of 32D cells by the combined expression of IRS-1 and V-Ha-Ras. 1091 81

Erythropoietin (EPO) and its receptor (EPOR) are required for development of erythrocytes. It has been shown that the ectopic expression of EPOR confers EPO-dependent proliferation on an interleukin 3 (IL3)-dependent cell line, Ba/F3, whereas the IL2-dependent T cell line, CTLL-2 expressing the EPOR (T-ER), fails to proliferate in response to EPO. However, the molecular basis of the EPO unresponsiveness in CTLL-2 has not been clarified. We found that the expression level of JAK2 in T-ER cells was much lower than that in Ba/F3 cells. Therefore, we examined the effects of forced expression of JAK2 in T-ER cells. In T-ER transformants expressing JAK2 (T-JER), EPO induced tyrosine phosphorylation of the EPOR, JAK2, and STAT5, and consequently STAT5-responsive genes including bcl-X and cis1 were normally induced. Furthermore, T-JER cells were resistant to apoptosis until at least 72 h after switching from IL2 to EPO. Although T-JER cells could not continuously proliferate in the presence of EPO, additional expression of JAK2 in T-JER (T-JJER) to a level similar to that in Ba/F3 cells supported long term proliferation in response to EPO. JAK2 was equally co-immunoprecipitated with the EPOR among T-JER, T-JJER, and Ba/F3 cells expressing the EPOR (BF-ER). However, EPO-dependent mitogen-activated protein (MAP) kinase activation was observed in T-JJER and BF-ER cells but not in T-JER cells. EPO-dependent long term proliferation of T-JER cells was conferred by expression of the constitutively activated form of MEK1. Our results suggest that MAP kinase activation is, at least in part, an important component for mitotic signal from the EPOR, and CTLL-2 cells probably lack signaling molecule(s) in JAK2 and the Ras-MAP kinase pathway.
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PMID:Mitogen-activated protein kinase plays an essential role in the erythropoietin-dependent proliferation of CTLL-2 cells. 1096 Apr 79

We have investigated heat shock stimulation of MAPK cascades in an interleukin 3-dependent cell line, BaF3. Following exposure to 42 degrees C, the stress-activated JNK MAPKs were phosphorylated and activated, but p38 MAPKs remained unaffected. Surprisingly, heat shock also activated ERK MAPKs in a potent (>60-fold), delayed (>30 min), and sustained (>/=120 min) manner. These characteristics suggested a novel mechanism of ERK MAPK activation and became the focus of this study. A MEK-specific inhibitor, PD98059, inhibited heat shock ERK MAPK activation by >75%. Surprisingly, a role for Ras in the heat shock response was eliminated by the failure of a dominant-negative Ras(Asn-17) mutant to inhibit ERK MAPK activation and the failure to observe increases in Ras.GTP. Heat shock also failed to stimulate activation of A-, B-, and c-Raf. Instead, a serine/threonine phosphatase inhibitor, okadaic acid, activated ERK MAPK in a similar manner to heat shock. Furthermore, pretreatment with suramin, generally recognized as a broad range inhibitor of growth factor receptors, inhibited both okadaic acid-stimulated and heat shock-stimulated ERK MAPK activity by >40%. Inhibiting ERK MAPK activation during heat shock with PD98059 enhanced losses in cell viability. These results demonstrate Ras- and Raf-independent ERK MAPK activation maintains cell viability following heat shock.
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PMID:The mechanism of heat shock activation of ERK mitogen-activated protein kinases in the interleukin 3-dependent ProB cell line BaF3. 1100 8

We investigated neurotrophic effects of interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on cultured sympathetic neurons obtained from mouse superior cervical ganglia. After 1 day of culture with physiological concentrations of mouse recombinant IL-3 and GM-CSF, the numbers of process-bearing neurons were increased. Maximum responses were elicited by 10 U/ml IL-3 and 1 U/ml GM-CSF, which were equivalent to the action of a submaximal dose (5 ng/ml) of nerve growth factor (NGF). The effects of IL-3 and GM-CSF were completely blocked by their corresponding antibodies, but not by anti-NGF, indicting their action is specific and completely independent of NGF. IL-3 and, to a lesser extent, GM-CSF were also able to protect NGF-differentiated neurons from apoptotic cell death caused by NGF withdrawal. The mitogen-activated protein (MAP) kinase signal transduction pathway is known to be involved in action of IL-3 and GM-CSF on hemopoietic cells, and thus we examined the participation of this pathway in the neurotrophic activities of IL-3 and GM-CSF. IL-3 and GM-CSF stimulation of the differentiated neurons was found to result in a rapid elevation of MAP kinase activity, and PD98059, an inhibitor of MAP kinase kinase activity, blocked both the neuritogenic and neuroprotective effects of IL-3 and GM-CSF. Immunocytochemical studies showed that IL-3 and GM-CSF receptors were present on the differentiated neurons. Thus, IL-3 and GM-CSF appear to be able to stimulate sympathetic nerve growth, via specific cytokine receptors on neurons, which lead to activation of the MAP kinase pathway that then mediates the observed neurotrophic effects.
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PMID:Neurotrophic action of interleukin 3 and granulocyte-macrophage colony-stimulating factor on murine sympathetic neurons. 1112 79

Although considered tightly linked, the linkage effectors for proliferation and antiapoptotic signaling pathways are not clear. Phosphorylation of Bcl2 at serine 70 is required for suppression of apoptosis in interleukin 3 (IL-3)-dependent myeloid cells deprived of IL-3 or treated with antileukemic drugs and can result from agonist activation of mitochondrial protein kinase C alpha (PKCalpha). However, we have recently found that high concentrations of staurosporine up to 1 microM: can only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKCalpha-mediated Bcl2 phosphorylation in vitro, indicating the existence of a non-PKC, staurosporine-resistant Bcl2 kinase (SRK). Although the RAF-1MEK-1-mitogen-activated protein kinase (MAPK) cascade is required for factor-dependent mitogenic signaling, a direct role in antiapoptosis signaling is not clear. In particular, the role of phosphorylation in the regulation of death substrates is not yet clear. Our findings indicate a potential role for the MEK/MAPK pathway in addition to PKC in antiapoptosis signaling, involving Bcl2 phosphorylation that features a role for extracellular signal-regulated kinase (ERK)1 and 2 as SRKs. These findings indicate a novel role for ERK1 and 2 as molecular links between proliferative and survival signaling and may, at least in part, explain the apparent paradox by which Bcl2 may suppress staurosporine-induced apoptosis. Although the effect of phosphorylation on Bcl2 function is not clear, effector molecules that regulate Bcl2 phosphorylation may have clinical significance in patients with acute myelogenous leukemia (AML) who express detectable levels of Bcl2. Preliminary findings suggest that expression of PKCalpha, ERK2, and Bax in leukemic blast cells from patients with AML, although individually not prognostic, appears to have potential clinical value in predicting chemoresistance and survival outcomes.
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PMID:Regulation of Bcl2 phosphorylation and potential significance for leukemic cell chemoresistance. 1115 4

Two Ras effector pathways leading to the activation of Raf-1 and phosphatidylinositol 3-kinase (PI3K) have been implicated in the survival signaling by the interleukin 3 (IL-3) receptor. Analysis of apoptosis suppression by Raf-1 demonstrated the requirement for mitochondrial translocation of the kinase in this process. This could be achieved either by overexpression of the antiapoptotic protein Bcl-2 or by targeting Raf-1 to the mitochondria via fusion to the mitochondrial protein Mas p70. Mitochondrially active Raf-1 is unable to activate extracellular signal-related kinase 1 (ERK1) and ERK2 but suppresses cell death by inactivating the proapoptotic Bcl-2 family member BAD. However, genetic and biochemical data also have suggested a role for the Raf-1 effector module MEK-ERK in apoptosis suppression. We thus tested for MEK requirement in cell survival signaling using the interleukin 3 (IL-3)-dependent cell line 32D. MEK is essential for survival and growth in the presence of IL-3. Upon growth factor withdrawal the expression of constitutively active MEK1 mutants significantly delays the onset of apoptosis, whereas the presence of a dominant negative mutant accelerates cell death. Survival signaling by MEK most likely results from the activation of ERKs since expression of a constitutively active form of ERK2 was as effective in protecting NIH 3T3 fibroblasts against doxorubicin-induced cell death as oncogenic MEK. The survival effect of activated MEK in 32D cells is achieved by both MEK- and PI3K-dependent mechanisms and results in the activation of PI3K and in the phosphorylation of AKT. MEK and PI3K dependence is also observed in 32D cells protected from apoptosis by oncogenic Raf-1. Additionally, we also could extend these findings to the IL-3-dependent pro-B-cell line BaF3, suggesting that recruitment of MEK is a common mechanism for survival signaling by activated Raf. Requirement for the PI3K effector AKT in this process is further demonstrated by the inhibitory effect of a dominant negative AKT mutant on Raf-1-induced cell survival. Moreover, a constitutively active form of AKT synergizes with Raf-1 in apoptosis suppression. In summary these data strongly suggest a Raf effector pathway for cell survival that is mediated by MEK and AKT.
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PMID:Apoptosis suppression by Raf-1 and MEK1 requires MEK- and phosphatidylinositol 3-kinase-dependent signals. 1125 82

Interleukin (IL)-3-induced Bcl2 phosphorylation at Ser(70) may be required for its full and potent antiapoptotic activity. However, in the absence of IL-3, increased expression of Bcl2 can also prolong cell survival. To determine how Bcl2 may be functionally phosphorylated following IL-3 withdrawal, a stress-activated Bcl2 kinase (SAK) was sought. Results indicate that anisomycin, a potent activator of the stress kinase JNK/SAPK, can induce Bcl2 phosphorylation at Ser(70) and that JNK1 can be latently activated following IL-3 withdrawal to mediate Bcl2 phosphorylation. JNK1 directly phosphorylates Bcl2 in vitro, co-localizes with Bcl2, and collaborates with Bcl-2 to mediate prolonged cell survival in the absence of IL-3 or following various stress applications. Dominant-negative (DN)-JNK1 can block both anisomycin and latent IL-3 withdrawal-induced Bcl2 phosphorylation (>90%) and potently enhances cell death. Furthermore, low dose okadaic acid (OA), a potent protein phosphatase 1 and 2A inhibitor, can activate the mitogen-activated protein kinases JNK1 and ERK1/2, but not p38 kinase, to induce Bcl2 phosphorylation and prolong cell survival in factor-deprived cells. Since PD98059, a specific MEK inhibitor, can only partially inhibit OA-induced Bcl2 phosphorylation but completely blocks OA-induced Bcl2 phosphorylation in cells expressing DN-JNK1, this supports the conclusion that OA may stimulate Bcl2 phosphorylation via a mechanism involving both JNK1 and ERK1/2. Collectively, these findings indicate a novel role for JNK1 as a SAK and may explain, at least in part, how functional phosphorylation of Bc12 can occur in the absence of growth factor.
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PMID:Novel role for JNK as a stress-activated Bcl2 kinase. 1132 15

IL-3 removal was reported to induce membrane association of the apoptotic effector Bax. This report demonstrates that IL-3-dependent cells from Bax-null mice failed to activate caspases after IL-3 removal and survived in an 10-fold lower concentration of IL-3. As IL-3 removal also down-regulates expression of Bcl-X, we examined the relationship between Bcl-X decrease and Bax membrane association. IL-3 removal from BAF-3 cells, followed by sorting caspase-active and caspase-inactive populations, showed that both expressed similar levels of Bcl-X. Inhibition of IL-3 signalling via PI-3 kinase and MEK1/2 resulted in cells with minimal Bcl-X, which remained viable with soluble Bax. However BAF-3-derived cells, which maintained Bcl-X expression without IL-3, also remained viable with soluble Bax on IL-3 removal. Therefore a decrease in Bcl-X is necessary, though not sufficient, for Bax membrane association on IL-3 removal. In contrast, treatment of BAF-3 cells with hydroxyurea induced apoptosis in the absence of a Bcl-X decrease. Furthermore, IL-3-dependent cells from Bax-null mice activated caspases after hydroxyurea treatment and show the same sensitivity to a variety of cytotoxic drugs. Thus, apoptosis after IL-3 removal requires a decrease in Bcl-X and Bax membrane association, whereas that induced by cytotoxic drugs does not.
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PMID:Role of Bax in apoptosis of IL-3-dependent cells. 1149 43


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