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)

Axin is a multidomain protein that plays a critical role in Wnt signaling, serving as a scaffold for down-regulation of beta-catenin. It also activates the JNK mitogen-activated protein kinase by binding to MEKK1. However, it is intriguing that Axin requires several additional elements for JNK activation, including a requirement for homodimerization, sumoylation at the extreme C-terminal sites, and a region in the protein phosphatase 2A-binding domain. In our present study, we have shown that another MEKK family member, MEKK4, also binds to Axin in vivo and mediates Axin-induced JNK activation. Surprisingly MEKK4 binds to a region distinct from the MEKK1-binding site. Dominant negative mutant of MEKK4 attenuates the JNK activation by Axin. Activation of JNK by Axin in MEKK1-/- mouse embryonic fibroblast cells supports the idea that another MEKK can mediate Axin-induced JNK activation. Expression of specific small interfering RNA against MEKK4 effectively attenuates JNK activation by the MEKK1 binding-defective Axin mutant in 293T cells and inhibits JNK activation by wild-type Axin in MEKK1-/- cells, confirming that MEKK4 is indeed another mitogen-activated protein kinase kinase kinase that is specifically involved in Axin-mediated JNK activation independently of MEKK1. We have also identified an additional domain between MEKK1- and MEKK4-binding sites as being required for JNK activation by Axin. MEKK1 and MEKK4 compete for Axin binding even though they bind to sites far apart, suggesting that Axin may selectively bind to MEKK1 or MEKK4 depending on distinct signals or cellular context. Our findings will provide new insights into how scaffold proteins mediate ultimate activation of different mitogen-activated protein kinase kinase kinases.
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PMID:Axin utilizes distinct regions for competitive MEKK1 and MEKK4 binding and JNK activation. 1287 10

The BRAF gene, encoding a mitogen-activated protein kinase kinase kinase, is mutated in several human cancers, with the highest incidence occurring in cutaneous melanoma. The activating V599E mutation accounted for 80% of all mutations detected in cutaneous melanoma cell lines. Reconstitution experiments have shown that this mutation increases ectopically expressed B-Raf kinase activity and induces NIH3T3 cell transformation. Here we used tumor-derived cell lines to characterize the activity of endogenous mutated B-Raf protein and assess its specific role in transformation. We show that three cell lines (OCM-1, MKT-BR, and SP-6.5) derived from human choroidal melanoma, the most frequent primary ocular neoplasm in humans, express B-Raf containing the V599E mutation. These melanoma cells showed a 10-fold increase in endogenous B-RafV599E kinase activity and a constitutive activation of the MEK/ERK pathway that is independent of Ras. This, as well as melanoma cell proliferation, was strongly diminished by siRNA-mediated depletion of the mutant B-Raf protein. Moreover, blocking B-RafV599E-induced ERK activation by different experimental approaches significantly reduced cell proliferation and anchorage-independent growth of melanoma cells. Finally, quantitative immunoblot analysis allowed us to identify signaling and cell cycle proteins that are differentially expressed between normal melanocytes and melanoma cells. Although the expression of signaling molecules was not sensitive to U0126 in melanoma cells, the expression of a cluster of cell cycle proteins remained regulated by the B-RafV599E/MEK/ERK pathway. Our results pinpoint this pathway as an important component in choroidal melanoma cell lines.
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PMID:Mutation of B-Raf in human choroidal melanoma cells mediates cell proliferation and transformation through the MEK/ERK pathway. 1291 19

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase family member that plays a central role in cytokine- and stress-induced apoptosis by activating c-Jun N-terminal kinase and p38 signaling cascades. ASK1-induced apoptotic activity is up-regulated by two cellular factors, Daxx and TRAF2, through direct protein-protein interactions. Daxx and TRAF2 are death receptor-associated proteins in Fas and tumor necrosis factor-alpha pathways, respectively. Recent studies suggest that calcium signaling may regulate ASK1 pathway. Here we report that human D53L1, a member of the tumor protein D52 family involved in cell proliferation and calcium signaling, up-regulates the ASK1-induced apoptosis. The human D53L1 physically interacts with the C-terminal regulatory domain of ASK1 and promotes ASK1-induced apoptotic activity by activating caspase signaling in mammalian cells. In luciferase reporter assays, hD53L1 activates c-Jun N-terminal kinase-mediated transactivation in the presence of ASK1. Expression of hD53L1 enhances autophosphorylation and kinase activity of ASK1 but has no effect on ASK1 oligomerization that is necessary for kinase activity and on binding of ASK1 to MKK6, a downstream factor of ASK1. Taken together, these results suggest that activation of ASK1 by hD53L1 may provide a novel mechanism for ASK1 regulation.
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PMID:Positive regulation of apoptosis signal-regulating kinase 1 by hD53L1. 1476 63

Cellular and genetic approaches were used to investigate the requirements for activation during spermatogenesis of the extracellular signal-regulated protein kinases (ERKs), more commonly known as the mitogen-activated protein kinases (MAPKs). The MAPKS and their activating kinases, the MEKs, are expressed in specific developmental patterns. The MAPKs and MEK2 are expressed in all premeiotic germ cells and spermatocytes, while MEK1 is not expressed abundantly in pachytene spermatocytes. Phosphorylated (active) variants of these kinases are diminished in pachytene spermatocytes. Treatment of pachytene spermatocytes with okadaic acid (OA), to induce transition from meiotic prophase to metaphase I (G2/MI), resulted in phosphorylation and enzymatic activation of ERK1/2. However, U0126, an inhibitor of the ERK-activating kinases, MEK1/2, did not inhibit OA-induced MAPK activation or chromosome condensation. Analysis of spermatocytes lacking MOS, a mitogen-activated protein kinase kinase kinase responsible for MEK and MAPK activation, revealed that MOS is not required for OA-induced activation of the MAPKs. OA-induced MAPK activation was inhibited by butyrolactone I, an inhibitor of cyclin-dependent kinases 1 and 2 (CDK1, CDK2); thus, these kinases may regulate MAPK activity. Additionally, spermatocytes lacking CDC25C condensed bivalent chromosomes and activated both MPF and MAPKs in response to OA treatment; therefore, there is a CDC25C-independent pathway for MPF and MAPK activation. These studies reveal that spermatocytes do not require either MOS or CDC25C for onset of the meiotic division phase or for activation of MPF and the MAPKs, thus implicating a novel pathway for activation of the ERK1/2 MAPKs in spermatocytes.
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PMID:Mitogen-activated protein kinase dynamics during the meiotic G2/MI transition of mouse spermatocytes. 1508 80

Cancer osaka thyroid (COT) is a member of the mitogen-activated protein kinase kinase kinase family of enzymes and plays a pivotal role in tumor necrosis factor-alpha production in macrophages. Consequently, COT is considered to be a promising target for antiinflammatory drug discovery. We describe here the development of in vitro COT assays in several formats and the advantages and disadvantages of each. A cascade assay requires very small amounts of enzyme and can provide a useful tool for high-throughput screening, but it is not desirable for compound mechanistic studies due to complicated kinetics. Direct assays are superior to cascade assays and are suitable for both compound screening and mechanistic studies. Among the direct assays, the homogeneous time-resolved fluorescence (HTRF) format is preferred over the radiometric format due to the robustness, throughput, and ease of use of the HTRF format. When the physiological protein substrate MEK1 (MAP/Erk kinase 1) was used to determine inhibitor potencies, false positives were observed due to compound interference by binding to MEK1. Using a MEK1 peptide substrate, these false positives were eliminated. In addition, we describe a simple method to study the ATP competitiveness of compounds. The knowledge gained through our studies with COT, and the methods described for our assays and compound mechanistic studies, can be readily applied to other kinase targets.
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PMID:Comparative analysis of various in vitro COT kinase assay formats and their applications in inhibitor identification and characterization. 1635 59

MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IkappaB kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3-dependent activation of an NF-kappaB reporter gene as well as ERK, JNK, and p38 MAP kinases correlated with a requirement for serine at position 526. Constitutively active mutants of MEKK3, consisting of S526D and S526E, were capable of activating a NF-kappaB luciferase reporter gene as well as ERK and MEK, suggesting that a negative charge at Ser526 was necessary for MEKK3 activity and implicating Ser526 as a phosphorylation site. An antibody was developed that specifically recognized phospho-Ser526 of MEKK3 but did not recognize the S526A point mutant. The catalytically inactive (K391M) mutant of MEKK3 was not phosphorylated at Ser526, indicating that phosphorylation of Ser526 occurs via autophosphorylation. Endogenous MEKK3 was phosphorylated on Ser526 in response to osmotic stress. In addition, phosphorylation of Ser526 was required for MKK6 phosphorylation in vitro, whereas dephosphorylation of Ser526 was mediated by protein phosphatase 2A and sensitive to okadaic acid and sodium fluoride. Finally, the association between MEKK3 and 14-3-3 was dependent on Ser526 and prevented dephosphorylation of Ser526. In summary, Ser526 of MEKK3 is an autophosphorylation site within the T-loop that is regulated by PP2A and 14-3-3 proteins.
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PMID:Phosphorylation of serine 526 is required for MEKK3 activity, and association with 14-3-3 blocks dephosphorylation. 1640 1

The small guanosine triphosphatase KRAS and the protein kinases BRAF, which is a mitogen-activated protein kinase kinase kinase (MAPKKK), and mitogen-activated protein kinase kinase 1 and 2 (MAPKK1/2, also known as MKK1/2 or MEK1/2) are signaling partners in the MAPK signal transduction pathway. They are involved in many biological processes and play crucial roles during embryonic development. When inappropriately expressed, KRAS, BRAF, and MEK1/2 are also frequently implicated in tumor progression. Hence, it might reasonably have been predicted that either loss- or gain-of-function germline mutations in the genes that encode them would cause embryonic death. However, in a surprising development, two articles report that germline mutations in the KRAS, BRAF, and MEK1/2 genes are associated with cardio-facio-cutaneous (CFC) syndrome. This unexpected discovery demonstrates that mutations in KRAS, BRAF, and MEK can pass through the germline to cause specific developmental syndromes. This finding will undoubtedly stimulate further research into the function of these proteins in development and in both inherited and sporadic cancers.
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PMID:BRAF and MEK mutations make a late entrance. 1656 17

Apoptosis signal-regulating kinase (ASK) 1 is a mitogen-activated protein kinase kinase kinase (MAP3K) in the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways that play multiple important roles in cytokine and stress responses. Here we show that ASK2, a highly related serine/threonine kinase to ASK1, also functions as a MAP3K only in a heteromeric complex with ASK1. We found that endogenous ASK2 was constitutively degraded in ASK1-deficient cells, suggesting that ASK1 is required for the stability of ASK2. ASK2 in a heteromeric complex with a kinase-negative mutant of ASK1 (ASK1-KN) effectively activated MAP2K and was more competent to respond to oxidative stress than ASK2 alone. Knockdown of ASK2 revealed that ASK2 was required for oxidative stress-induced JNK activation. These results suggest that ASK2 forms a functional MAP3K complex with ASK1, in which ASK1 supports the stability and the active configuration of ASK2. Moreover, ASK2 was found to activate ASK1 by direct phosphorylation, suggesting that ASK1 and ASK2 in a heteromeric complex facilitate their activities to each other by distinct mechanisms. Such a formation of functional heteromeric complex between different MAP3Ks may be advantageous for cells to cope with a wide variety of stimuli by fine regulation of cellular responses.
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PMID:Apoptosis signal-regulating kinase (ASK) 2 functions as a mitogen-activated protein kinase kinase kinase in a heteromeric complex with ASK1. 1721 May 79

Fusarium graminearum produces trichothecenes in aerial hyphae, a process which is markedly suppressed by NaCl without a significant effect on fungal growth. Here we report on the involvement of kinases of the two-component osmotic signal transduction pathway in the regulation of secondary metabolism in F. graminearum. While a deletion null mutant of FgOs1 (encoding the osmosensor histidine kinase) (deltaFgOs1) produced a reduced amount of the red pigment aurofusarin and was unaltered in its ability to produce trichothecenes, deletion null mutants of FgOs4 (encoding mitogen-activated protein kinase kinase kinase; MAPKKK), FgOs5 (MAPKK), and FgOs2 (MAPK) showed markedly enhanced pigmentation and failed to produce trichothecenes in aerial hyphae. Also, the transcript levels of PKS12 and GIP2 (aurofusarin biosynthetic pathway and regulatory genes, respectively) were significantly enhanced in the deltaFgOs4, deltaFgOs5, and deltaFgOs2 mutants and were reduced in the deltaFgOs1 mutant. In addition, expression of Tri4 and Tri6 (trichothecene biosynthetic pathway and regulatory genes) and production of trichothecenes in rice medium were markedly reduced in the former three protein kinase mutants. This is the first report demonstrating the involvement of a MAPK in the regulation of secondary metabolism.
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PMID:Involvement of the osmosensor histidine kinase and osmotic stress-activated protein kinases in the regulation of secondary metabolism in Fusarium graminearum. 1789 20

Parathyroid hormone-related peptide (PTHrP) and the parathyroid hormone-PTHrP receptor increase chondrocyte proliferation and delay chondrocyte maturation in endochondral bone development at least partly through cyclic AMP (cAMP)-dependent signaling pathways. Because data suggest that the ability of cAMP to stimulate cell proliferation involves the mitogen-activated protein kinase kinase kinase B-Raf, we hypothesized that B-Raf might mediate the proliferative action of PTHrP in chondrocytes. Though B-Raf is expressed in proliferative chondrocytes, its conditional removal from cartilage did not affect chondrocyte proliferation and maturation or PTHrP-induced chondrocyte proliferation and PTHrP-delayed maturation. Similar results were obtained by conditionally removing B-Raf from osteoblasts. Because A-raf and B-raf are expressed similarly in cartilage, we speculated that they may fulfill redundant functions in this tissue. Surprisingly, mice with chondrocytes deficient in both A-Raf and B-Raf exhibited normal endochondral bone development. Activated extracellular signal-regulated kinase (ERK) was detected primarily in hypertrophic chondrocytes, where C-raf is expressed, and the suppression of ERK activation in these cells by PTHrP or a MEK inhibitor coincided with a delay in chondrocyte maturation. Taken together, these results demonstrate that B-Raf and A-Raf are dispensable for endochondral bone development and they indicate that the main role of ERK in cartilage is to stimulate not cell proliferation, but rather chondrocyte maturation.
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PMID:A-raf and B-raf are dispensable for normal endochondral bone development, and parathyroid hormone-related peptide suppresses extracellular signal-regulated kinase activation in hypertrophic chondrocytes. 1796 76


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