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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cyclin-dependent kinase (CDK)-activating kinase (CAK) phosphorylates a conserved threonine residue on CDKs and activates them. Two known classes of CAKs are represented by monomeric Cak1p in budding yeast Saccharomyces cerevisiae and by heterotrimeric CDK7-cyclin H-Mat1 in human and other metazoa. We report here the identification of p42, a novel CAK activity in human cells. p42 has sequence homology to both Cak1p and CDK7 groups of CAKs. p42 is essential for the phosphorylation of Thr-160 and activation of CDK2. A dominant-negative p42 mutant, T161A, and posttranscriptional gene silencing of p42 with RNA(i)-impaired Thr-160 phosphorylation and activity of CDK2. Purified p42 phosphorylated glutathione S-transferase-CDK2 at Thr-160 within the T-loop and activated its histone H1 kinase activity. Finally, p42 is indispensable for cell growth. Cells lacking p42 were incapable of growing and forming colonies whereas cells with a reduced level of p42 grew at significantly slower rates than control cells. Our findings suggest that p42 represents a novel CAK activity in mammalian cells.
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PMID:p42, a novel cyclin-dependent kinase-activating kinase in mammalian cells. 1459 12

Previous studies have shown that the adaptor protein Shb is involved in receptor tyrosine kinase signaling. In this study, we demonstrate that Shb is phosphorylated in an Src-dependent manner upon vascular endothelial growth factor (VEGF) stimulation using porcine aortic endothelial cells expressing the human VEGF receptor 2 (VEGFR-2) (KDR). In co-immunoprecipitation experiments, we could detect an interaction between Shb and the VEGFR-2 in human telomerase-immortalized microvascular endothelial cells. Furthermore, in a glutathione S-transferase pull-down assay, the Src homology 2 domain of Shb was shown to interact with phosphorylated tyrosine 1175 in the C-terminal tail of VEGFR-2. VEGF-induced Shb phosphorylation was lost in porcine aortic endothelial cells expressing a chimeric murine VEGFR-2 (Flk-1) with a mutation at the corresponding position. Shb expression was specifically decreased by 80%, in a transient manner, by using the short interfering RNA technique. Reduced Shb expression led to a loss of stimulation of phosphatidylinositol 3-kinase, phosphorylation of focal adhesion kinase at tyrosine 576, the generation of focal adhesions, and stress fiber formation in response to VEGF. Furthermore, we show that VEGF-induced migration is inhibited in Shb short interfering RNA-treated cells. Our data demonstrate that Shb is important for VEGF signaling in endothelial cells. This is achieved by Shb binding to tyrosine 1175 in the VEGFR-2, which regulates VEGF-induced formation of focal adhesions and cell migration, of which the latter occurs in a phosphatidylinositol 3-kinase-dependent manner.
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PMID:The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration. 1502 17

We have used structure-based design techniques to introduce the drug O(2)-[2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino) phenyl] 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), which is efficiently metabolized to potentially cytolytic nitric oxide by the pi isoform of glutathione S-transferase, an enzyme expressed at high levels in many tumors. We have used mouse embryo fibroblasts (MEFs) null for GSTpi (GSTpi(-/-)) to show that the absence of GSTpi results in a decreased sensitivity to PABA/NO. Cytotoxicity of PABA/NO was also examined in a mouse skin fibroblast (NIH3T3) cell line that was stably transfected with GSTpi and/or various combinations of gamma-glutamyl cysteine synthetase and the ATP-binding cassette transporter MRP1. Overexpression of MRP1 conferred the most significant degree of resistance, and in vitro transport studies confirmed that a GSTpi-activated metabolite of PABA/NO was effluxed by MRP1 in a GSH-dependent manner. Additional studies showed that in the absence of MRP1, PABA/NO activated the extracellular-regulated and stress-activated protein kinases ERK, c-Jun NH(2)-terminal kinase (JNK), and p38. Selective inhibition studies showed that the activation of JNK and p38 were critical to the cytotoxic effects of PABA/NO. Finally, PABA/NO produced antitumor effects in a human ovarian cancer model grown in SCID mice.
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PMID:Tumor cell responses to a novel glutathione S-transferase-activated nitric oxide-releasing prodrug. 1510 35

Discoidin domain receptors 1 and 2 (DDR1 and DDR2) are tyrosine kinase receptors activated by triple-helical collagens. Aberrant expression and signaling of these receptors have been implicated in several human diseases linked to accelerated matrix degradation and remodeling including tumor invasion, atherosclerosis and liver fibrosis. The objective of this study is to characterize the collagen-binding sites in the discoidin domains of DDR1 and DDR2 at a molecular level. We expressed glutathione S-transferase fusion proteins containing the discoidin and extracellular domains of DDR1 and DDR2 in insect cells and subjected them to a solid-phase collagen-binding assay. We found high affinity binding of the DDR extracellular domains to immobilized type I collagen and confirmed the discoidin-collagen interaction with an enzyme-linked immunosorbent assay-based read-out. Furthermore, we created a three-dimensional model of the DDR1 discoidin domain based on the related domains of blood coagulation factors V and VIII. This model predicts the presence of four neighboring, surface-exposed loops that are topologically equivalent to a major phospholipid-binding site in factors V and VIII. To test the involvement of these loops in collagen binding, we mutated individual amino acid residues to alanine or deleted short sequence stretches within these loops. We found that several residues within loop 1 (Ser-52-Thr-57) and loop 3 (Arg-105-Lys-112) as well as Ser-175 in loop 4 are critically involved in collagen binding. Our structure-function analysis of the DDR discoidin domains provides new insights into this non-integrin-mediated collagen-signaling mechanism and may ultimately lead to the design of small molecule inhibitors that interfere with aberrant DDR function.
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PMID:Exploring the collagen-binding site of the DDR1 tyrosine kinase receptor. 1513 80

In mammalian systems, detoxification enzymes of the GST (glutathione S-transferase) family regulate JNK (c-Jun N-terminal kinase) signal transduction by interaction with JNK itself or other proteins upstream in the JNK pathway. In the present study, we have studied GSTs and their interaction with components of the JNK pathway from Diptera. We have evaluated the effects of four Delta class Anopheles dirus GSTs, GSTD1-1, GSTD2-2, GSTD3-3 and GSTD4-4, on the activity of full-length recombinant Drosophila HEP (mitogen-activated protein kinase kinase 7; where HEP stands for hemipterous) and the Drosophila JNK, as well as the reciprocal effect of these kinases on GST activity. Interestingly, even though these four GSTs are alternatively spliced products of the same gene and share >60% identity, they exerted different effects on JNK activity. GSTD1-1 inhibited JNK activity, whereas the other three GST isoforms activated JNK. GSTD2-2, GSTD3-3 and GSTD4-4 were inhibited 50-80% by HEP or JNK but GSTD1-1 was not inhibited by JNK. However, there were some similarities in the actions of HEP and JNK on these GSTs. For example, binding constants for HEP or JNK inhibiting a GST were similar (20-70 nM). Furthermore, after incubation of the GSTs with JNK, both JNK and the GSTs changed catalytic properties. The substrate specificities of both GSTs and JNK were also altered after their co-incubation. In addition, glutathione modulated the effects of JNK on GST activity. These results emphasize that different GST spliceforms possess different properties, both in their catalytic function and in their regulation of signalling through the JNK pathway.
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PMID:Reciprocal regulation of glutathione S-transferase spliceforms and the Drosophila c-Jun N-terminal kinase pathway components. 1525 Aug 26

Sphingosine 1-phosphate (S1P) is a lipid agonist that regulates smooth muscle cell (SMC) and endothelial cell functions by activating several members of the S1P subfamily of G-protein-coupled Edg receptors. We have shown previously that SMC differentiation is regulated by RhoA-dependent activation of serum response factor (SRF). Because S1P is a strong activator of RhoA, we hypothesized that S1P would stimulate SMC differentiation. Treatment of primary rat aortic SMC cells with S1P activated RhoA as measured by precipitation with a glutathione S-transferase-rhotekin fusion protein. In SMC and 10T1/2 cells, S1P treatment up-regulated the activities of several transiently transfected SMC-specific promoters, and these effects were inhibited by the Rho-kinase inhibitor, Y-27632. S1P also increased smooth muscle alpha-actin protein levels in SMC but had no effect on SRF binding to the smooth muscle alpha-actin CArG B element. Quantitative reverse transcriptase-PCR showed that S1P treatment of SMC or 10T1/2 cells did not increase the mRNA level of either of the recently identified SRF co-factors, myocardin or myocardin-related transcription factor-A (MRTF-A). MRTF-A protein was expressed highly in SMC and 10T1/2 cultures, and importantly the effects of S1P were inhibited by a dominant negative form of MRTF-A indicating that S1P may regulate the transcriptional activity of MRTF-A. Indeed, S1P treatment increased the nuclear localization of FLAG-MRTF-A, and the effect of MRTF-A overexpression on smooth muscle alpha-actin promoter activity was inhibited by dominant negative RhoA. S1P also stimulated SMC growth by activating the early growth response gene, c-fos. This effect was not attenuated by Y-27632 but could be inhibited by the MEK inhibitor, UO126. S1P enhanced SMC growth through ERK-mediated phosphorylation of the SRF co-factor, Elk-1, as measured by gel shift and Elk-1 activation assays. Taken together these results demonstrate that S1P activates multiple signaling pathways in SMC and regulates proliferation by ERK-dependent activation of Elk-1 and differentiation by RhoA-dependent activation of MRTF-A.
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PMID:Sphingosine 1-phosphate stimulates smooth muscle cell differentiation and proliferation by activating separate serum response factor co-factors. 1529 66

Insulin-like growth factor-1 (IGF-1) is an angiogenic and oncogenic factor that activates signal transduction pathways involved in the expression of transcriptional regulators of tumorigenesis. RUNX2, a member of the Ig-loop family of transcription factors is expressed in vascular endothelial cells (EC) and regulates EC migration, invasion, and proliferation. Here we show that IGF-1 and its receptor regulate post-translational changes in RUNX2 to activate DNA binding in proliferating EC. The phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, reduced both basal and IGF-1-stimulated RUNX2 DNA binding activity in the absence of changes in RUNX2 protein as did the overexpression of the phosphatidylinositol 3-phosphate phosphatase, confirming that PI3K signaling mediates RUNX2 activation. IGF-1 increased ERK1/2 activation, which was abrogated by the inhibition of PI3K, thus linking these two pathways in EC. Treatment with U0126, which inhibits ERK1/2 activation, reduced IGF-1-stimulated RUNX2 DNA binding without affecting RUNX2 protein levels. Overexpression of constitutively active MKK1 increased RUNX2 DNA binding and phosphorylation. No additive effects of PI3K or ERK inhibitors on DNA binding were evident. Surprisingly, these IGF-1-mediated effects on RUNX2 were not regulated by Akt phosphorylation, a common downstream target of PI3K, as determined by pharmacological or genetic inhibition. However, an inhibitor of the p21-activated protein kinase-1, glutathione S-transferase-Pak1-(83-149), inhibited both basal and IGF-1-stimulated RUNX2 DNA binding, suggesting that Pak1 mediates IGF-1 signaling to increase RUNX2 activity. These results indicate that the angiogenic growth factor, IGF-1, can regulate RUNX2 DNA binding through sequential activation of the PI3K/Pak1 and ERK1/2 signaling cascade.
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PMID:Insulin-like growth factor-1 regulates endogenous RUNX2 activity in endothelial cells through a phosphatidylinositol 3-kinase/ERK-dependent and Akt-independent signaling pathway. 1530 89

A widely expressed protein containing UBA (ubiquitin-associated) and UBX (ubiquitin-like) domains was identified as a substrate of SAPKs (stress-activated protein kinases). Termed SAKS1 (SAPK substrate-1), it was phosphorylated efficiently at Ser200 in vitro by SAPK3/p38gamma, SAPK4/p38delta and JNK (c-Jun N-terminal kinase), but weakly by SAPK2a/p38alpha, SAPK2b/p38beta2 or ERK (extracellular-signal-regulated kinase) 2. Ser200, situated immediately N-terminal to the UBX domain, became phosphorylated in HEK-293 (human embryonic kidney) cells in response to stressors. Phosphorylation was not prevented by SB 203580 (an inhibitor of SAPK2a/p38alpha and SAPK2b/p38beta2) and/or PD 184352 (which inhibits the activation of ERK1 and ERK2), and was similar in fibroblasts lacking both SAPK3/p38gamma and SAPK4/p38delta or JNK1 and JNK2. SAKS1 bound ubiquitin tetramers and VCP (valosin-containing protein) in vitro via the UBA and UBX domains respectively. The amount of VCP in cell extracts that bound to immobilized GST (glutathione S-transferase)-SAKS1 was enhanced by elevating the level of polyubiquitinated proteins, while SAKS1 and VCP in extracts were coimmunoprecipitated with an antibody raised against S5a, a component of the 19 S proteasomal subunit that binds polyubiquitinated proteins. PNGase (peptide N-glycanase) formed a 1:1 complex with VCP and, for this reason, also bound to immobilized GST-SAKS1. We suggest that SAKS1 may be an adaptor that directs VCP to polyubiquitinated proteins, and PNGase to misfolded glycoproteins, facilitating their destruction by the proteasome.
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PMID:A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs. 1536 74

The c-Jun/AP-1 transcription complex is associated with diverse cellular processes such as differentiation, proliferation, transformation, and apoptosis. These different biological endpoints are likely achieved by the regulation of specific target gene expression. We describe the identification of Ras guanine nucleotide exchange factor 1, Ras-GRF1, by microarray analysis as a c-Jun/AP-1 regulated gene essential for anchorage-independent growth of immortalized rat fibroblasts. Increased Ras-GRF1 expression, in response to inducible c-Jun expression in Rat1a fibroblasts, was confirmed by both real-time PCR and Northern blot analysis. We show that c-Jun/AP-1 can bind and activate the Ras-GRF1 promoter in vivo. A 75-kDa c-Jun/AP-1-inducible protein, p75-Ras-GRF1, was detected, and the inhibition of its expression with antisense oligomers significantly blocked c-Jun-regulated anchorage-independent cell growth. p75-Ras-GRF1 expression occurred with a concomitant increase in activated Ras (GTP bound), and the activation of Ras was significantly inhibited by antisense Ras-GRF1 oligomers. Moreover, p75-Ras-GRF1 could be coprecipitated with a Ras dominant-negative glutathione S-transferase (GST) construct, GST-Ras15A, demonstrating an interaction between p75-Ras-GRF1 and Ras. A downstream target of Ras activation, Elk-1, had increased transcriptional activity in c-Jun-expressing cells, and this activation was inhibited by dominant-negative Ras. In addition, c-Jun overexpression resulted in an increase in phospho-AKT while phosphorylation of ERK1/2 remained largely unaffected. The inhibition of phosphatidylinositol 3-kinase (PI3K)-AKT signal transduction by Ly294002 and wortmannin significantly blocked c-Jun-regulated morphological transformation, while inhibition of basal MEK-ERK activity with PD98059 and U0126 had little effect. We conclude that c-Jun/AP-1 regulates endogenous p75-Ras-GRF1 expression and that c-Jun/AP-1-regulated anchorage-independent cell growth requires activation of Ras-PI3K-AKT signal transduction.
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PMID:p75-Ras-GRF1 is a c-Jun/AP-1 target protein: its up regulation results in increased Ras activity and is necessary for c-Jun-induced nonadherent growth of Rat1a cells. 1579 16

Mitogen-activated protein kinase (MAPK) kinases (MKKs, also called MAPK/extracellular signal-regulated kinase [ERK] kinase [MEK]) are constituents of numerous signal transduction pathways involved in growth, differentiation, and stress response. One of its members, MKK4, directly phosphorylates and activates the c-Jun terminal kinases (also called stress-activated protein kinase [SAPK]) in response to stress and pro-inflammatory cytokines. Recent evidence suggest that control of MKK4 activity may provide a novel approach for the treatment of cancer or as anti-inflammatory therapy. To screen for novel low-molecular-weight inhibitors of MKK4, we established a quantitative, non-radioactive in vitro kinase assay. Human MKK4 was expressed as fusion protein with glutathione S-transferase (GST) in Escherichia coli. Co-expression of a constitutive active fragment of the MAPK/ERK kinase kinase-1 yielded active GST-MKK4 using GST-SAPK alpha-kinase-negative (KN) mutant as substrate. We determined the kinetic constants for ATP and GST-SAPK alpha-KN. The apparent Km value for GST-SAPKalpha-KN was 3.7 microM, while the apparent Km value for ATP was 0.17 microM. Staurosporine inhibited GST-MKK4 with an IC50 of 70 nM. The kinase assay was adapted to a 384-well non-radioactive format. After the kinase reaction the phosphorylated product was captured onto a streptavidin-coated microtiter plate, and phosphorylation was detected with a europium-labeled anti-phosphotyrosine antibody, which allowed time-resolved fluorescence measurement.
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PMID:Development of a non-radioactive, 384-well format assay to detect inhibitors of the mitogen-activated protein kinase kinase 4. 1579 97


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