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)

We have recently shown that the degradation products of hyaluronan of 3 to 10 disaccharides (o-HA), but not native high molecular weight hyaluronan, can induce angiogenesis in vivo and, as such, o-HA is an important regulator of the neovascularization process. As a continuation of this work, we have studied the cytoplasmic signal transduction pathways responsible for o-HA-activated endothelial cell proliferation. We show that the addition of o-HA (1 microg/ml) to bovine aortic endothelial cells induces tyrosine phosphorylation of multiple proteins within 1 minute and that the activity remains above basal levels for at least 24 hours. Increased phosphorylation of the CD44 receptor was also observed. Pretreatment of cells with an anti-CD44-receptor antibody (5 microg/ml) or the tyrosine kinase inhibitor genistein (10 microM) inhibited both o-HA-induced proliferation (p < 0.05) and protein tyrosine phosphorylation. In comparison, native hyaluronan had little effect on tyrosine phosphorylation across the same time period. Protein kinase C (PKC) activity was increased 2- to 3-fold in the membranes of cells treated with o-HA, and a pretreatment with phorbol 12,13-dibutyrate (PDBu) to down-regulate PKC significantly inhibited o-HA-induced cell proliferation (p < 0.05). Examination by Western blotting showed that only the betaI and epsilon isoforms remained translocated to the membrane for at least 24 hours. These isoforms seem to be involved in modulating the proliferative effects of o-HA, because the transient translocation of PKC isoforms by PDBu was not sufficient to induce mitogenesis. Furthermore, we show that PKC activation of the cytoplasmic kinase cascade (Raf-1 kinase, MAP kinase kinase [MEK-1], and extracellular signal-regulated kinase [ERK-1]) by o-HA culminated in the nuclear translocation of ERK-1. This pathway is essentially linear, as shown by the ability of specific enzyme inhibitors (PDBu and PD98059) to prevent both activation of ERK-1- and o-HA-induced proliferation. We conclude that phosphorylation of the CD44 receptor results in an increase in tyrosine phosphorylation, leading to the activation of a cytoplasmic cascade and cell proliferation; this concurs with previous work, which showed that o-HA-induced proliferation of endothelial cells is CD44-receptor-mediated and accompanied by early response gene activation.
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PMID:Angiogenic oligosaccharides of hyaluronan induce protein tyrosine kinase activity in endothelial cells and activate a cytoplasmic signal transduction pathway resulting in proliferation. 971 86

CD44 is a cell surface adhesion molecule for several extracellular matrix components. We previously showed that CD44 expressed in cancer cells is proteolytically cleaved at the ectodomain through membrane-anchored metalloproteases and that CD44 cleavage plays a critical role in cancer cell migration. Therefore, cellular signals that promote the migration and metastatic activity of cancer cells may regulate the CD44 ectodomain cleavage. Here, we demonstrate that the expression of the dominant active mutant of Ha-Ras (Ha-Ras(Val-12)) induces redistribution of CD44 to the newly generated membrane ruffling area and CD44 ectodomain cleavage. The migration assay revealed that the CD44 cleavage contributes to the Ha-Ras(Val-12)-induced migration of NIH3T3 cells on hyaluronate substrate. Treatment with LY294002, an inhibitor for phosphoinositide 3-OH kinase (PI3K), significantly inhibits Ha-Ras(Val-12)-induced CD44 cleavage, whereas that with PD98059, an inhibitor for MEK, does not. The active mutant p110 subunit of PI3K has also been shown to enhance the CD44 cleavage, suggesting that PI3K mediates the Ras-induced CD44 cleavage. Moreover, the expression of dominant negative mutants of Cdc42 and Rac1 inhibits the Ha-Ras(Val-12)-induced CD44 cleavage. These results suggest that Ras > PI3K > Cdc42/Rac1 pathway plays an important role in CD44 cleavage and may provide a novel molecular basis to explain how the activated Ras facilitates cancer cell migration.
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PMID:Ras oncoprotein induces CD44 cleavage through phosphoinositide 3-OH kinase and the rho family of small G proteins. 1089 35

CD44/hyaluronan interactions and epidermal growth factor (EGF) stimulation are both known to enhance tumour invasion in vitro. The frequent amplification of the EGF receptor (EGFR) in high-grade astrocytomas led to the examination of the hypothesis that CD44-dependent astrocytoma invasion is regulated by EGF. It has been shown that human astrocytoma cells express only the standard (haemopoietic) form of CD44 (CD44s) and that EGF up-regulates CD44 mRNA and protein in a time- and dose-dependent (10-100 ng/ml) manner. EGF stimulation did not result in induction of additional splice variants. No EGF-induced increase in CD44s was observed after treatment of cells with the wild-type EGFR tyrosine kinase inhibitor Tyrphostin AG1478 (30 nM). Up-regulation of CD44 by EGF is also prevented by the transcriptional inhibitor actinomycin D (5 microg/ml) and by blocking the MAP kinase (MAPK) pathway using the MEK inibitor U0126 (100 microM). CD44 up-regulation was associated with a 50% increase in invasion through hyaluronan-supplemented Matrigel(trade mark), which was abrogated by ligating CD44 with the specific antibody KM201. These results suggest that increased CD44 expression in response to EGF stimulation plays a significant role in astrocytoma invasion.
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PMID:Epidermal growth factor up-regulates CD44-dependent astrocytoma invasion in vitro. 1111 70

Signal transduction initiated by B cell Ag receptor (BCR) cross-linking plays an important role in the development and activation of B cells. Therefore, considerable effort has gone into determining the biochemical signaling events initiated by the BCR and delineating which events participate in specific biological responses to Ag. We used two inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 1 and MEK2, PD98059, and U0126, to assess the role the Ras-mitogen-activated protein kinase pathway plays in several BCR-induced responses. PD98059 or U0126 treatment substantially inhibited the BCR-induced activation of the extracellular signal-regulated kinase (ERK) forms of mitogen-activated protein kinase in the immature B cell line WEHI-231, in immature splenic B cells, and in mature splenic B cells. However, MEK-ERK inhibition did not block BCR-induced growth arrest or apoptosis of WEHI-231 cells or apoptosis of immature splenic B cells, indicating that the MEK-ERK pathway is not required for these events. In contrast, PD98059 and U0126 treatment did inhibit the up-regulation of specific BCR-induced proteins, including the transcription factor Egr-1 in WEHI-231 and mature splenic B cells, and the CD44 adhesion molecule and CD69 activation marker in mature splenic B cells. Moreover, both inhibitors suppressed BCR-induced proliferation of mature splenic B cells, in the absence and in the presence of IL-4. Therefore, activation of the MEK-ERK pathway is necessary for a subset of B cell responses to Ag.
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PMID:Inhibition of the MEK/ERK signaling pathway blocks a subset of B cell responses to antigen. 1123 29

We investigated the production of hyaluronan (HA) and its effect on cell motility in cells expressing the v-src mutants. Transformation of 3Y1 by v-src virtually activated HA secretion, whereas G2A v-src, a nonmyristoylated form of v-src defective in cell transformation, had no effect. In cells expressing the temperature-sensitive mutant of v-Src, HA secretion was temperature dependent. In addition, HA as small as 1 nM, on the other side, activated cell motility in a tumor-specific manner. HA treatment strongly activated the motility of v-Src-transformed 3Y1, whereas it showed no effect on 3Y1- and 3Y1-expressing G2A v-src. HA-dependent cell locomotion was strongly blocked by either expression of dominant-negative Ras or treatment with a Ras farnesyltransferase inhibitor. Similarly, both the MEK1 inhibitor and the kinase inhibitor clearly inhibited HA-dependent cell locomotion. In contrast, cells transformed with an active MEK1 did not respond to the HA. Finally, an anti-CD44-neutralizing antibody could block the activation of cell motility by HA as well as the HA-dependent phosphorylation of mitogen-activated protein kinase and Akt. Taken together, these results suggest that simultaneous activation of the Ras-mitogen-activated protein kinase pathway and the phosphoinositide 3-kinase pathway by the HA-CD44 interaction is required for the activation of HA-dependent cell locomotion in v-Src-transformed cells.
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PMID:Hyaluronan activates cell motility of v-Src-transformed cells via Ras-mitogen-activated protein kinase and phosphoinositide 3-kinase-Akt in a tumor-specific manner. 1140 91

Differential gene expression through alternative pre-mRNA splicing is crucial to various physiological and pathological conditions. Upon activation of B and T lymphocytes during an immune response, variant isoforms of the cell surface molecule CD44 are generated by alternative pre-mRNA splicing. We show here that in primary mouse T cells as well as in the murine LB-17 T-cell line upregulation of variant CD44 mRNA species upon T-cell activation requires activation of the MEK-ERK pathway. By employing mutant signaling molecules and a novel luciferase-based splice reporter system we demonstrate that the Ras-Raf-MEK-ERK signaling cascade, but not the p38 MAP-kinase pathway, activates a mechanism that retains variant CD44 exon v5 sequence in mature mRNA. The findings demonstrate that a highly conserved pleiotropic signaling pathway links extracellular cues to splice regulation, providing an avenue for tissue-specific, developmental or pathology-associated splicing decisions.
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PMID:Regulation of alternative pre-mRNA splicing by the ERK MAP-kinase pathway. 1148 22

To further our understanding of the functions of the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), and other myelin proteins, such as 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and myelin-associated glycoprotein (MAG), bovine brain myelin was extracted with Triton X-100, and protein complexes in the detergent-soluble fraction were isolated by coimmunoprecipitation and sucrose density gradient sedimentation. MBP, PLP, and the small isoform of MAG (S-MAG) were coimmunoprecipitated from the detergent-soluble fraction by anti-PLP, anti-MBP or anti-MAG monoclonal antibodies. Additionally, a 30 kDa phosphoserine-containing protein and two phosphotyrosine-containing proteins (M(r) 30 and 42 kDa) were found in the coimmunoprecipitates. The 42 kDa protein is probably p42MAPK, in that MAPK was shown also to be present in the immunoprecipitated complex. CNP, the small PLP isoform DM20, the large MAG isoform L-MAG, MOG, CD44, MEK, p44MAPK, and actin were not present in the immunoprecipitates, although they were present in the detergent-soluble fraction. Lipid analysis revealed that the PLP-MBP-S-MAG coimmunoprecipitated with some phospholipids and sulfatide but not cholesterol or galactosylceramide. However, the complex had a high density, indicating that the lipid/protein ratio is low, and it was retained on a Sepharose CL6B column, indicating that it is not a large membrane fragment. Given that MAG is localized mainly in the periaxonal region of myelin, where it interacts with axonal ligands, the PLP-MBP-S-MAG complex may come from these regions, where it could participate in dynamic functions in the myelin sheath and myelin-axonal interactions.
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PMID:Myelin proteolipid protein, basic protein, the small isoform of myelin-associated glycoprotein, and p42MAPK are associated in the Triton X-100 extract of central nervous system myelin. 1223 60

It has been established that fragmented hyaluronic acid (HA), but not native high molecular weight HA, can induce angiogenesis, cell proliferation and migration. We have studied the outside-in signal transduction pathways responsible for fragmented HA-mediated cancer cell invasion. In our study, we have studied the effects of CD44 stimulation by ligation with HA upon the expression of matrix metalloproteinases (MMPs)-2 and -9 as well as urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1) and the subsequent induction of invasion of human chondrosarcoma cell line HCS-2/8. Our study indicates that (i) CD44 stimulation by fragmented HA upregulates expression of uPA and uPAR mRNA and protein but does not affect MMPs secretion or PAI-1 mRNA expression; (ii) the effects of HA fragments are critically HA size dependent: high molecular weight HA is inactive, but lower molecular weight fragmented HA (Mr 3.5 kDa) is active; (iii) cells can bind avidly Mr 3.5 kDa fragmented HA through a CD44 molecule, whereas cells do not effectively bind higher Mr HA; (iv) a fragmented HA induces phosphorylation of MAP kinase proteins (MEK1/2, ERK1/2 and c-Jun) within 30 min; (v) CD44 is critical for the response (activation of MAP kinase and upregulation of uPA and uPAR expression); and (vi) cell invasion induced by CD44 stimulation with a fragmented HA is inhibited by anti-CD44 mAb, MAP kinase inhibitors, neutralizing anti-uPAR pAb, anti-catalytic anti-uPA mAb or amiloride. Therefore, our study represents the first report that CD44 stimulation induced by a fragmented HA results in activation of MAP kinase and, subsequently, enhances uPA and uPAR expression and facilitates invasion of human chondrosarcoma cells.
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PMID:CD44 stimulation by fragmented hyaluronic acid induces upregulation of urokinase-type plasminogen activator and its receptor and subsequently facilitates invasion of human chondrosarcoma cells. 1240 8

The tyrosine kinase receptor c-Met and its ligand HGF/SF, ezrin, and splice variants of CD44 have independently been identified as tumor metastasis-associated proteins. We now show that these proteins cooperate. A CD44 isoform containing variant exon v6 sequences is strictly required for c-Met activation by HGF/SF in rat and human carcinoma cells, in established cell lines as well as in primary keratinocytes. CD44v6-deficient tumor cells were unable to activate c-Met unless they were transfected with a CD44v6-bearing isoform. Antibodies to two v6-encoded epitopes inhibited autophosphorylation of c-Met by interfering with the formation of a complex formed by c-Met, CD44v6, and HGF/SF. In addition, signal transduction from activated c-Met to MEK and Erk required the presence of the cytoplasmic tail of CD44 including a binding motif for ERM proteins. This suggests a role for ERM proteins and possibly their link to the cortical actin cytoskeleton in signal transfer.
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PMID:CD44 is required for two consecutive steps in HGF/c-Met signaling. 1246 36

Elevated expression of matrix metalloproteinases (MMPs) is associated with increased metastatic potential in many tumor cells. As activation of the ERK pathway has been linked to the expression of MMP-9, we examined a possible correlation between ERK activation, MMP-9 expression, and invasive phenotype in human tumor cells. Activation state of the ERK pathway in tumor cells was well correlated with the invasive phenotype, which was determined by the ability of cells to invade through reconstituted extracellular matrix. Elevated expression of MMP-9 as well as of MMP-3, MMP-14, and CD44 was observed in tumor cells in which constitutive activation of the ERK pathway is detected. Blockade of the ERK pathway by treatment with PD184352, a specific and powerful inhibitor of mitogen-activated protein (MAP) kinase/ERK kinase (MEK), suppressed the expression of MMP-3, MMP-9, MMP-14, and CD44, and inhibited markedly the invasiveness of tumor cells. These results imply that, in addition to anti-proliferative effects, specific blockade of the ERK pathway is expected to result in anti-metastatic effects in tumor cells.
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PMID:Specific blockade of the ERK pathway inhibits the invasiveness of tumor cells: down-regulation of matrix metalloproteinase-3/-9/-14 and CD44. 1272 28


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