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)

Endothelial cell migration, a key process in angiogenesis, requires the coordinated integration of motogenic signals elicited by the adhesion of endothelial cells to extracellular matrices and by angiogenic cytokines such as the vascular endothelial growth factor (VEGF). In this study, we found that addition of VEGF to human umbilical vein endothelial cells cultivated on vitronectin triggers a synergistic interaction between the VEGF receptor VEGFR2 and the clustered integrin receptor alphavbeta3. The interaction between VEGFR2 and alphavbeta3 is required for full phosphorylation of VEGFR2 and to drive the activation of motogenic pathways involving focal adhesion kinase (FAK) and stress-activated protein kinase-2/p38 (SAPK2/p38). The signal emanating from the VEGFR2 and alphavbeta3 interaction and leading to SAPK2/p38 activation proceeds directly from VEGFR2. The chaperone Hsp90 is found in a complex that coprecipitates with inactivated VEGFR2, and the association is increased by VEGF and decreased by geldanamycin, a specific inhibitor of Hsp90-mediated events. Geldanamycin also impairs the phosphorylation of FAK that results from the interaction between VEGFR2 and alphavbeta3, and this is accompanied by an inhibition of the recruitment of vinculin to VEGFR2. We conclude that a necessary cross talk should occur between VEGFR2 and the integrin alphavbeta3, to transduce the VEGF signals to SAPK2/p38 and FAK and that Hsp90 is instrumental in the building up of focal adhesions by allowing the phosphorylation of FAK and the recruitment of vinculin to VEGFR2.
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PMID:Integrin alphavbeta3, requirement for VEGFR2-mediated activation of SAPK2/p38 and for Hsp90-dependent phosphorylation of focal adhesion kinase in endothelial cells activated by VEGF. 1282 Jun 53

Up to now, most of the studies addressing the critical roles played by protrusive and contractile cell-matrix contacts in cell adhesion, guidance, migration, matrix assembly, and activation of signaling molecules have been performed on two-dimensional surfaces. Here, we analysed the organization of chondrosarcoma cell contacts in a new three-dimensional environment made of titanium beads. Surface charges were modified by deposition of polyelectrolyte multilayer films built up by alternated polycations poly-(L-lysine) or poly(allylamine hydrochloride) and polyanions poly-(L-glutamic acid) or poly(sodium 4-styrenesulfonate). Negatively charged 3-D titanium surfaces amplified the occurrence and length of cell protrusions. These protrusions had pseudopod characteristics extended to 200 microm in length, growing off the substratum to distant beads. Pseudopod formation is inhibited by the exocytosis inhibitor concanamycin A and is triggered by a secreted factor. Chondrosarcoma cells adhering on uncoated or on negatively charged surfaces contained discrete focal spots of vinculin and actin cables. In cells plated onto these surfaces, phosphorylation of p44/42 MAPK/ERK was twofold increased. In contrast, no cytoskeletal vinculin and actin organization was observed when the surface was positively charged. These data suggest that chondrosarcoma cells adapt a more stable adhesion on uncoated or negatively charged surfaces. This point may be critical in tissue engineering strategies designed for cartilage repair.
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PMID:3-D surface charges modulate protrusive and contractile contacts of chondrosarcoma cells. 1456 95

We quantitatively compared vinculin-related adhesion parameters in osteoblastic cells submitted to two opposing mechanical stresses: low deformation and frequency strain regimens (stretch conditions) and microgravity exposure (relaxed conditions). In both ROS 17/2.8 cells and rat primary osteoblastic cells, 1% cyclic deformations at 0.05 Hz for 10 min per day for seven days stimulated cell growth compared to static culture conditions, while relaxed ROS cells proliferated in a similar way to static cultures (BC). We studied the short-term (up to 24 h) adaptation of focal contact reorganization under these two conditions. Cyclic deformation induced a biphasic response comprising the formation of new focal contacts followed by clustering of these focal contacts in both ROS cells and primary osteoblasts. Microgravity exposure induced a reduction in focal contact number and clustering in ROS cells. To evaluate whether the proliferation (stretch) or survival (relaxed) status of ROS cells influences focal contact organization, we inhibited the ERK proliferative-dependent pathway. Inhibition of proliferation by PD98059 was partially reversed, but not fully restored by stretch. Stretch-induced clustering of vinculin-positive contacts also persisted in the presence of PD98059, whereas the increase in focal contact number was abolished. In conclusion, we show that focal contacts are mechanoeffectors, and we suggest that their morphologic organization might serve as a discriminant functional parameter between survival and proliferation status in ROS 17/2.8 osteoblastic cells.
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PMID:Focal contact clustering in osteoblastic cells under mechanical stresses: microgravity and cyclic deformation. 1468 Oct 58

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

Clinicians have observed that keloids preferentially form in body areas subject to increased skin tension. We hypothesized a difference exists in the transcriptional response of keloid fibroblasts to mechanical strain compared with normal fibroblasts. Normal and keloid fibroblasts were seeded in a device calibrated to deliver a known level of equibiaxial strain. We examined the transcriptional response of TGF-beta isoforms and collagen Ialpha, genes differentially expressed in keloids. Keloid fibroblasts produced more mRNA for TGF-beta1, TGF-beta2, and collagen Ialpha after mechanical strain compared to normals, and this was correlated with protein production. Inhibiting the major mechanical signal transduction pathway with the ERK inhibitor, U0126, blocked upregulation of gene expression. In addition, keloid fibroblasts formed more focal adhesion complexes as measured by immunofluorescence for focal adhesion kinase, integrin beta1, and vinculin. Finally, there is increased activation of focal adhesion kinase when we detected the phosphorylated form of focal adhesion kinase with immunofluorescence and immunoblotting. In summary, keloid fibroblasts have an exaggerated response to mechanical strain compared to normal fibroblasts leading to increased production of pro-fibrotic growth factors. This may be one molecular mechanism for the development of keloids.
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PMID:Increased transcriptional response to mechanical strain in keloid fibroblasts due to increased focal adhesion complex formation. 1615 10

Keratinocytes migrate directionally into the wound bed to initiate re-epithelialization, necessary for wound closure and restoration of barrier function. They solely express the beta2-adrenergic receptor (beta2-AR) subtype of beta-ARs and can also synthesize beta-AR agonists generating a hormonal mediator network in the skin. Emerging studies from our laboratory demonstrate that beta-AR agonists decrease keratinocyte migration via a protein phosphatase (PP) 2A-dependent mechanism. Here we have extended our investigations to observe the effects of beta2-AR activation on keratinocyte polarization, migration, and ERK phosphorylation at the wound edge, cytoskeletal organization, phospho-ERK intracellular localization, proliferation, human skin wound re-epithelialization, wound-induced ERK phosphorylation, and murine skin wound healing. We demonstrate that in keratinocytes, beta2-AR activation is anti-motogenic and anti-mitogenic with both mechanisms being PP2A dependent. beta2-AR activation dramatically alters the organization of the actin cytoskeleton and prevents localization of phospho-ERK to the lamellipodial edge and its colocalization with vinculin. Finally, we demonstrate a beta2-AR-mediated delay in re-epithelialization and decrease in wound-induced epidermal ERK phosphorylation in human skin wounds and a delay in re-epithelialization in murine tail-clip wounds. Our work uncovers novel keratinocyte biology and a previously unrecognized role for the adrenergic hormonal mediator network in the wound repair process.
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PMID:Beta2-adrenergic receptor activation delays wound healing. 1639 70

Rac1 is an intracellular signal transducer regulating a variety of cell functions. Previous studies by overexpression of dominant-negative or constitutively active mutants of Rac1 in clonal cell lines have established that Rac1 plays a key role in actin lamellipodia induction, cell-matrix adhesion, and cell anoikis. In the present studies, we have examined the cellular behaviors of Rac1 gene-targeted primary mouse embryonic fibroblasts (MEFs) after Cre recombinase-mediated deletion of Rac1 gene. Rac1-null MEFs became contracted and elongated in morphology and were defective in lamellipodia formation, cell spreading, cell-fibronectin adhesion, and focal contact formation in response to platelet-derived growth factor or serum. Unexpectedly, deletion of Rac1 also abolished actin stress fibers in the cells without detectable alteration of endogenous RhoA activity. Although the expression and/or activation status of focal adhesion complex components such as Src, FAK, and vinculin were not affected by Rac1 deletion, the number and size of adhesion plaques were significantly reduced, and the molecular complex between Src, FAK, and vinculin was dissembled in Rac1-null cells. Overexpression of an active RhoA mutant or ROK failed to rescue the stress fiber and adhesion plaque defects of the Rac1-null cells. Although Rac1 deletion caused a significant reduction in phospho-PAK1, -AKT, and -ERK under serum stimulation, reconstitution of active PAK1, but not AKT or MEK1, was able to rescue the actin cytoskeleton and adhesion phenotypes of the Rac1-deficient cells. Furthermore, Rac1 deletion led to a marked increase in spontaneous apoptosis that could be rescued by active PAK1, AKT, or MEK1 expression. Our results obtained from gene-targeted primary MEFs indicate that Rac1 is essential not only for lamellipodia induction but also for the RhoA-regulated actin stress fiber and focal adhesion complex formation and that Rac1 is involved in cell survival regulation through anoikis-dependent as well as -independent mechanisms.
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PMID:Genetic deletion of Rac1 GTPase reveals its critical role in actin stress fiber formation and focal adhesion complex assembly. 1669 90

The Src homology 2 phosphotyrosyl phosphatase (SHP2) is a nonreceptor-type phosphatase that acts as a positive transducer of receptor Tyr kinase (RTK) signaling, particularly the Ras-REK and PI3K-Akt pathways. Recently, we have demonstrated that SHP2 is required for cell transformation induced by the constitutively active fibroblast growth factor receptor 3 (K/E-FR3) (Oncogene, 22, 6909-6918). In that study, we had detected a phosphotyrosyl protein of approximately 100 KDa (p100) in cells expressing dominant-negative SHP2 (R/E-SHP2), but its identity and relevance in SHP2-meditaed transformation was not known. Here, we report the identification of p100 as alpha-catenin, a vinculin-related protein involved in adherens junction-mediated intercellular adhesion. We show that alpha-catenin becomes Tyr phosphorylated in intercellular adhesion-dependent manner and this event is counteracted by SHP2. Substrate trapping in intact cells and immunocomplex phosphatse assays confirmed that alpha-catenin is in deed an SHP2 substrate. Tyr phosphorylation of alpha-catenin enhances its translocation to the plasma membrane and its interaction with beta-catenin, leading to enhanced actin polymerization and stabilization of adherens junction-mediated intercellular adhesion, a phenomenon commensurate with loss of the transformation phenotype. Site-directed mutagenesis studies also suggested that Tyr phosphorylation of alpha-catenin enhances its inhibitory role on cell transformation. Based on our previous work and the current report, we demonstrate that mediation of cell transformation by SHP2 is a complex process that involves modulation of the Ras-ERK and PI3K-Akt signaling pathways, intercellular adhesion, focal adhesion and actin cytoskeletal reorganization. To our knowledge, this is the first report showing regulation of alpha-catenin function by Tyr phosphorylation and its inhibitory effect on cell transformation.
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PMID:Modulation of alpha-catenin Tyr phosphorylation by SHP2 positively effects cell transformation induced by the constitutively active FGFR3. 1676 62

Maintenance of bone structural integrity depends in part on the rate of apoptosis of bone-forming osteoblasts. Because substrate adhesion is an important regulator of apoptosis, we have investigated the role of focal adhesions in regulating bone cell apoptosis. To test this, we expressed a truncated form of alpha-actinin (ROD-GFP) that competitively displaces endogenous alpha-actinin from focal adhesions, thus disrupting focal adhesions. Immunofluorescence and morphometric analysis of vinculin and tyrosine phosphorylation revealed that ROD-GFP expression dramatically disrupted focal adhesion organization and reduced tyrosine phosphorylation at focal adhesions. In addition, Bcl-2 protein levels were reduced in ROD-GFP-expressing cells, but caspase 3 cleavage, poly(ADP-ribose) polymerase cleavage, histone H2A.X phosphorylation, and cytotoxicity were not increased due to ROD-GFP expression alone. Increases in both ERK and Akt phosphorylation were also observed in ROD-GFP-expressing cells, although inhibition of either ERK or Akt individually or together failed to induce apoptosis. However, we did find that ROD-GFP expression sensitized, whereas alpha-actinin-GFP expression protected, cells from TNF-alpha-induced apoptosis. Further investigation revealed that activation of TNF-alpha-induced survival signals, specifically Akt phosphorylation and NF-kappaB activation, was inhibited in ROD-GFP-expressing cells. The reduced expression of antiapoptotic Bcl-2 and inhibited survival signaling rendered ROD-GFP-expressing cells more susceptible to TNF-alpha-induced apoptosis. Thus we conclude that alpha-actinin plays a role in regulating cell survival through stabilization of focal adhesions and regulation of TNF-alpha-induced survival signaling.
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PMID:Disruption of alpha-actinin-integrin interactions at focal adhesions renders osteoblasts susceptible to apoptosis. 1680 2

Within the mammalian genome, there are many multimember gene families that encode membrane proteins with extracellular leucine rich repeats which are thought to act as cell adhesion or signalling molecules. We previously showed that the members of the NLRR gene family are expressed in a developmentally restricted manner in the mouse with NLRR-1 being expressed in the developing myotome. The FLRT gene family shows a similar genomic layout and predicted protein secondary structure to the NLRRs so we analysed expression of the three FLRT genes during mouse development. FLRTs are glycosylated membrane proteins expressed at the cell surface which localise in a homophilic manner to cell-cell contacts expressing the focal adhesion marker vinculin. Each member of the FLRT family has a distinct, highly regulated expression pattern, as was seen for the NLRR family. FLRT3 has a provocative expression pattern during somite development being expressed in regions of the somite where muscle precursor cells migrate from the dermomyotome and move into the myotome, and later in myotomal precursors destined to migrate towards their final destination, for example, those that form the ventral body wall. FLRT3 is also expressed at the midbrain/hindbrain boundary and in the apical ectodermal ridge, regions where FGF signalling is known to be important, suggesting that the role for FLRT3 in FGF signalling identified in Xenopus is conserved in mammals. FLRT1 is expressed at brain compartmental boundaries and FLRT2 is expressed in a subset of the sclerotome, adjacent to the region that forms the syndetome, suggesting that interaction with FGF signalling may be a general property of FLRT proteins. We confirmed this by showing that all FLRTs can interact with FGFR1 and FLRTs can be induced by the activation of FGF signalling by FGF-2. We conclude that FLRT proteins act as regulators of FGF signalling, being induced by the signal and then able to interact with the signalling receptor, in many tissues during mouse embryogenesis. This process may, in part, be dependent on homophilic intercellular interactions between FLRT molecules.
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PMID:Regulated expression of FLRT genes implies a functional role in the regulation of FGF signalling during mouse development. 1687 96


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