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)

Midkine is a heparin-binding growth factor that promotes the growth, survival, migration and differentiation of various target cells. So far, receptor-type protein tyrosine phosphatase zeta, low-density-lipoprotein-receptor-related protein and anaplastic lymphoma kinase have been identified as receptors for midkine. We found beta1 integrin in midkine-binding proteins from 13-day-old mouse embryos. beta1-Integrin bound to a midkine-agarose column and was eluted mostly with EDTA. Further study revealed that the alpha-subunits capable of binding to midkine were alpha4 and alpha6. Purified alpha4beta1- and alpha6beta1-integrins bound midkine. Anti-alpha4 antibody inhibited the midkine-dependent migration of osteoblastic cells, and anti-alpha6 antibody inhibited the midkine-dependent neurite outgrowth of embryonic neurons. After midkine treatment, tyrosine phosphorylation of paxillin, an integrin-associated molecule, was transiently increased in osteoblastic cells. Therefore, we concluded that alpha4beta1- and alpha6beta1-integrins are functional receptors for midkine. We observed that the low-density-lipoprotein-receptor-related-protein-6 ectodomain was immunoprecipitated with alpha6beta1-integrin and alpha4beta1-integrin. The low-density-lipoprotein-receptor-related-protein-6 ectodomain was also immunoprecipitated with receptor-type protein tyrosine phosphatase zeta. alpha4beta1- and alpha6beta1-Integrins are expected to co-operate with other midkine receptors, possibly in a multimolecular complex that contains other midkine receptors.
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PMID:alpha4beta1- and alpha6beta1-integrins are functional receptors for midkine, a heparin-binding growth factor. 1546 86

Activation of the hepatocyte growth factor (HGF) receptor in epithelial cells results in lamellipodia protrusion, spreading, migration, and tubule formation. We have previously reported that these morphogenic effects are dependent on MAPK activation at focal adhesions. In the present study we demonstrate that activated ERK phosphorylates paxillin on serine 83 and that mutation of this site eliminates HGF-stimulated increased association of paxillin and FAK in subconfluent cells. Failure to activate FAK at focal adhesions results in a loss of FAK-PI 3-kinase association and the marked reduction of Rac activation after HGF stimulation. Expression of paxillin mutants that disrupt ERK association or phosphorylation inhibits HGF-induced cell spreading, migration, and tubulogenesis. These data demonstrate that the paxillin-MAPK complex serves as a central regulator of HGF-stimulated FAK and Rac activation in the vicinity of focal adhesions, thus promoting the rapid focal adhesion turnover and lamellipodia extension that are required for migratory and tubulogenic responses.
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PMID:Paxillin serves as an ERK-regulated scaffold for coordinating FAK and Rac activation in epithelial morphogenesis. 1549 12

Mounting evidence suggests that the focal adhesion targeting (FAT) domain, an antiparallel four-helix bundle, exists in alternative conformations that may modulate phosphorylation, ligand binding, and the subcellular localization of focal adhesion kinase (FAK). In order to characterize the conformational dynamics of the FAT domain, we have developed a novel method for reconstructing the folding pathway of the FAT domain by using discrete molecular dynamics (DMD) simulations, with free energy constraints derived from NMR hydrogen exchange data. The DMD simulations detect a folding intermediate, in which a cooperative unfolding event causes helix 1 to lose helical character while separating from the helix bundle. The conformational dynamic features of helix 1 in the intermediate state of the FAT domain are likely to facilitate Y926 phosphorylation, yet interfere with paxillin binding. The presence of this intermediate state in vivo may promote FAK signaling via the ERK/MAPK pathway and by release of FAK from focal adhesions.
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PMID:New insights into FAK signaling and localization based on detection of a FAT domain folding intermediate. 1557 30

Focal adhesion kinase (FAK) is a 125 kDa protein tyrosine kinase (PTK) associated with focal adhesion in many cells, which plays a major role in the integrity of cytoskeletal structure. Reactive oxygen species produced during ischemia and reperfusion injury has been found to be an important mediator of signal transduction process. We found that low dose H2O2 induced increased FAK production in pulmonary microvascular endothelial cells, which could be blocked by cycloheximide (CHX), a protein synthesis inhibitor. Pulmonary endothelial cells were cultured on DMEM medium till 100% confluent. H2O2 was added at 100 uM for 30 min. The cells were collected and lysed, then immuno-blotted with anti-FAK antibody. After 30 min treatment, we found a 30%+/-6% (N=5) increase of FAK in H2O2 treated endothelial cells. This increase could be blocked by pretreatment of cells with CHX at 5 ug/ml for 60 min. In both groups, increased phosphorylation of ERK was observed. Immuno-fluorescence revealed increased staining of FAK in the peri-nuclear region of the H2O2 treated endothelial cells. These findings suggest that H2O2 activated MAP kinase pathway leading to increased FAK production at the protein level. FAK is a 125 kDa PTK associated with focal adhesion in many cells, and it plays a major role in the integrity of cytoskeletal structure. FAK is discretely localized to focal adhesions via its C-terminal focal adhesion-targeting (FAT) sequence. FAK is regulated by integrin-dependent cell adhesion and can control tyrosine phosphorylation of downstream substrates, like paxillin. The reactive oxygen species produced during ischemia and reperfusion injury has been found to be an important mediator of the signal transduction process. Although the signaling pathways leading to hydrogen peroxide induced endothelial monolayer permeability remain ambiguous, cytoskeletal proteins are known to be essential for maintaining endothelial integrity and regulating solute flux through the monolayer. Recent evidence has shown that H2O2 stimulates cytoskeleton reorganization, cell growth/proliferation, and DNA synthesis in various cells. In our previous study, we found a significantly increased amount of FAK in endothelial cells treated with low doses of H2O2. Mitogen-activated protein (MAP) kinases are a group of 30- to 110-kDa serine/threonine kinases. MAPKs belong to the group of kinases that are rapidly activated in response to growth factor stimulation. This family of MAPKs includes ERK, and ERK2. The activated MAPK can translocate to the nucleus where it can regulate transcription factors. Activation of p44 and p42 extracellular signal-regulated protein kinases (ERK1 and ERK2) is an important step in the cascade leading to cell growth and proliferation. In order to determine the mechanism of increased FAK production, we investigated the relationship of FAK production and ERK activation.
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PMID:Reactive oxygen species increased focal adhesion kinase production in pulmonary microvascular endothelial cells. 1563 12

Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. Anti-phosphotyrosine and anti-EGFr affinity chromatography, isotope-coded muLC-MS/MS, and immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein. Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection, comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located despite the complexity of the peptide mixtures. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK, and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. Pharmacological inhibition of EGF receptor kinase activity by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cgamma (PLCgamma), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated by transient EGF stimulation suggesting a complex balance between growth factor induced kinase and phosphatase activities in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, lysophosphatidic acid (LPA) signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal S6.
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PMID:Phosphotyrosine signaling networks in epidermal growth factor receptor overexpressing squamous carcinoma cells. 1565 67

Here, we examined the in vitro and in vivo anti-angiogenesis and anti-tumor activities of PE, a new marine-derived compound. Inhibition of angiogenesis was assessed in vitro using proliferation, migration, adhesion, tube-formation and apoptosis assays in PE-treated HMECs and HUVECs. In vivo, CAM assays were used to assess inhibition effect of PE on physiological angiogenesis, and immunofluorescent microscopy was used to examine tumor microvessel density and apoptosis in PE-treated mouse tumor models. Finally, Western blotting analyses were performed to examine the effect of PE on VEGF signaling in HMECs. The results showed that PE inhibited proliferation of HMECs and HUVECs with IC50 values of 2.22 +/- 0.31 microM and 1.98 +/- 0.32 microM, induced endothelial cell apoptosis at concentrations <2 microM, induced dose-dependent suppression of cell migration, cell adhesion and tube formation in HMECs and HUVECs, and showed anti-proliferative activities against several tumor cell lines (IC50 values of approximately 4 microM). In vivo, PE (5 nM/egg) suppressed spontaneous angiogenesis in our CAM assay, and induced marked growth inhibition in mouse sarcoma 180 and hepatoma 22 models. Specifically, PE treatment reduced mouse sarcoma 180 tumor volume by triggering apoptosis of both tumor and tumor-associated endothelial cells, preferentially targeting on endothelial cells comparable with tumor cells. Finally, PE treatment suppressed the active (phosphorylated) forms of VEGFR2, Akt, ERK, FAK and paxillin, which are involved in endothelial cell survival, proliferation, adhesion and migration. Our results indicate that PE exerts an anti-angiogenic activity associated with inhibition of VEGFR2 signaling, and an anti-tumor activity associated with decreased proliferation of tumor cells and increased apoptosis of both endothelial cells and tumor cells.
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PMID:PE, a new sulfated saponin from sea cucumber, exhibits anti-angiogenic and anti-tumor activities in vitro and in vivo. 1610 48

OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3' kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non-receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell leukemia.
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PMID:Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930. 1609 34

Integrin-mediated cell adhesion induces activation of the EGF receptor tyrosine kinase independently of the soluble growth factor ligand. EGFR activation is instrumental for subsequent activation of additional signaling pathways in adherent cells, including the Ras-MAP kinase pathway and the phosphatidylinositol 3-kinase/Akt pathway. We demonstrate here that integrin-dependent EGFR activation is also essential for adhesion-induced formation of actin stress fibers, focal adhesion localization and tyrosine phosphorylation of the adapter protein paxillin, as well as transcriptional activation of the serum response factor. All these events are known to be mediated by the small GTPase RhoA. EGFR activity was not found to regulate the activity status of RhoA, however. Instead, we found that EGFR activity is required for integrin-induced phosphorylation of cofilin. Cofilin is an actin-binding protein, which, when unphosphorylated, stimulates depolymerization and severing of actin filaments. Thus, in the absence of the kinase activity of the EGFR, cofilin remains dephosphorylated and depolymerizes actin filaments, rendering cells unable to respond to RhoA signaling. These studies demonstrate adhesion-dependent regulation of cofilin phosphorylation, and identify a novel role for EGFR in integrin signaling.
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PMID:EGF receptor activity is essential for adhesion-induced stress fiber formation and cofilin phosphorylation. 1612 57

Upon cell adhesion to extracellular matrix proteins, focal adhesion kinase (FAK) rapidly undergoes autophosphorylation on its Tyr-397 which consequently serves as a binding site for the Src homology 2 domains of the Src family protein kinases and several other intracellular signaling molecules. In this study, we have attempted to examine the effect of the FAK Y397F mutant on v-Src-stimulated cell transformation by establishing an inducible expression of the Y397F mutant in v-Src-transformed FAK-null (FAK(-/-)) mouse embryo fibroblasts. We found that the FAK Y397F mutant had both positive and negative effects on v-Src-stimulated cell transformation; it promoted v-Src-stimulated invasion, but on the other hand it inhibited the v-Src-stimulated anchorage-independent cell growth in vitro and tumor formation in vivo . The positive effect of the Y397F mutant on v-Src-stimulated invasion was correlated with an increased expression of matrix metalloproteinase-2, both of which were inhibited by the specific phosphatidylinositol 3-kinase inhibitor wortmannin or a dominant negative mutant of AKT, suggesting a critical role for the phosphatidylinositol 3-kinase/AKT pathway in both events. However, the expression of the Y397F mutant rendered v-Src-transformed FAK(-/-) cells susceptible to anoikis, correlated with suppression on v-Src-stimulated activation of ERK and AKT. In addition, under anoikis stress, the induction of the Y397F mutant in v-Src-transformed FAK(-/-) cells selectively led to a decrease in the level of p130(Cas), but not other focal adhesion proteins such as talin, vinculin, and paxillin. These results suggest that FAK may increase the susceptibility of v-Src-transformed cells to anoikis by modulating the level of p130(Cas).
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PMID:Differential effect of the focal adhesion kinase Y397F mutant on v-Src-stimulated cell invasion and tumor growth. 1613 10

Interleukin-1beta (IL-1beta) mediates destruction of matrix collagens in diverse inflammatory diseases including arthritis, periodontitis, and pulmonary fibrosis by activating fibroblasts, cells that interact with matrix proteins through integrin-based adhesions. In vitro, IL-1beta signaling is modulated by focal adhesions, supramolecular protein complexes that are enriched with tyrosine kinases and phosphatases. We assessed the importance of tyrosine phosphatases in regulating cell-matrix interactions and IL-1beta signaling. In human gingival fibroblasts plated on fibronectin, IL-1beta enhanced the maturation of focal adhesions as defined by morphology and enrichment with paxillin and alpha-actinin. IL-1beta also induced activation of ERK and recruitment of phospho-ERK to focal complexes/adhesions. Treatment with the potent tyrosine phosphatase inhibitor pervanadate, in the absence of IL-1beta, recapitulated many of these responses indicating the importance of tyrosine phosphatases. Immunoblotting of collagen bead-associated complexes revealed that the tyrosine phosphatase, SHP-2, was also enriched in focal complexes/adhesions. Depletion of SHP-2 by siRNA or by homologous recombination markedly altered IL-1beta-induced ERK activation and maturation of focal adhesions. IL-1beta-induced tyrosine phosphorylation of SHP-2 on residue Y542 promoted focal adhesion maturation. Association of Gab1 with SHP-2 in focal adhesions correlated temporally with activation of ERK and was abrogated in cells expressing mutant (Y542F) SHP-2. We conclude that IL-1beta mediated maturation of focal adhesions is dependent on tyrosine phosphorylation of SHP-2 at Y542, leading to recruitment of Gab1, a process that may influence the downstream activation of ERK.
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PMID:Tyrosine phosphatase SHP-2 regulates IL-1 signaling in fibroblasts through focal adhesions. 1625 12


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