共查询到20条相似文献,搜索用时 15 毫秒
1.
Background
Efficient cell movement requires the dynamic regulation of focal adhesion (FA) formation and turnover. FAs are integrin-associated sites of cell attachment and establish linkages to the cellular actin cytoskeleton. Cells without focal adhesion kinase (FAK), an integrin-activated tyrosine kinase, exhibit defects in FA turnover and cell motility. Cortactin is an actin binding adaptor protein that can influence FA dynamics. FAK and cortactin interact, but the cellular role of this complex remains unclear.Principal Findings
Using FAK-null fibroblasts stably reconstituted with green fluorescent protein (GFP) tagged FAK constructs, we find that FAK activity and FAK C-terminal proline-rich region 2 (PRR2) and PRR3 are required for FA turnover and cell motility. Cortactin binds directly to FAK PRR2 and PRR3 sites via its SH3 domain and cortactin expression is important in promoting FA turnover and GFP-FAK release from FAs. FAK-cortactin binding is negatively-regulated by FAK activity and associated with cortactin tyrosine phosphorylation. FAK directly phosphorylates cortactin at Y421 and Y466 and over-expression of cortactin Y421, Y466, and Y482 mutated to phenylalanine (3YF) prevented FAK-enhanced FA turnover and cell motility. However, phospho-mimetic cortactin mutated to glutamic acid (3YE) did not affect FA dynamics and did not rescue FA turnover defects in cells with inhibited FAK activity or with PRR2-mutated FAK that does not bind cortactin.Conclusions
Our results support a model whereby FAK-mediated FA remodeling may occur through the formation of a FAK-cortactin signaling complex. This involves a cycle of cortactin binding to FAK, cortactin tyrosine phosphorylation, and subsequent cortactin-FAK dissociation accompanied by FA turnover and cell movement. 相似文献2.
Background
Tumor cell motility and invasion is governed by dynamic regulation of the cortical actin cytoskeleton. The actin-binding protein cortactin is commonly upregulated in multiple cancer types and is associated with increased cell migration. Cortactin regulates actin nucleation through the actin related protein (Arp)2/3 complex and stabilizes the cortical actin cytoskeleton. Cortactin is regulated by multiple phosphorylation events, including phosphorylation of S405 and S418 by extracellular regulated kinases (ERK)1/2. ERK1/2 phosphorylation of cortactin has emerged as an important positive regulatory modification, enabling cortactin to bind and activate the Arp2/3 regulator neuronal Wiskott-Aldrich syndrome protein (N-WASp), promoting actin polymerization and enhancing tumor cell movement.Methodology/Principal Findings
In this report we have developed phosphorylation-specific antibodies against phosphorylated cortactin S405 and S418 to analyze the subcellular localization of this cortactin form in tumor cells and patient samples by microscopy. We evaluated the interplay between cortactin S405 and S418 phosphorylation with cortactin tyrosine phosphorylation in regulating cortactin conformational forms by Western blotting. Cortactin is simultaneously phosphorylated at S405/418 and Y421 in tumor cells, and through the use of point mutant constructs we determined that serine and tyrosine phosphorylation events lack any co-dependency. Expression of S405/418 phosphorylation-null constructs impaired carcinoma motility and adhesion, and also inhibited lamellipodia persistence monitored by live cell imaging.Conclusions/Significance
Cortactin phosphorylated at S405/418 is localized to sites of dynamic actin assembly in tumor cells. Concurrent phosphorylation of cortactin by ERK1/2 and tyrosine kinases enables cells with the ability to regulate actin dynamics through N-WASp and other effector proteins by synchronizing upstream regulatory pathways, confirming cortactin as an important integration point in actin-based signal transduction. Reduced lamellipodia persistence in cells with S405/418A expression identifies an essential motility-based process reliant on ERK1/2 signaling, providing additional understanding as to how this pathway impacts tumor cell migration. 相似文献3.
Background
Postsynaptic enrichment of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction (NMJ) depends on the activation of the muscle receptor tyrosine MuSK by neural agrin. Agrin-stimulation of MuSK is known to initiate an intracellular signaling cascade that leads to the clustering of AChRs in an actin polymerization-dependent manner, but the molecular steps which link MuSK activation to AChR aggregation remain incompletely defined.Methodology/Principal Findings
In this study we used biochemical, cell biological and molecular assays to investigate a possible role in AChR clustering of cortactin, a protein which is a tyrosine kinase substrate and a regulator of F-actin assembly and which has also been previously localized at AChR clustering sites. We report that cortactin was co-enriched at AChR clusters in situ with its target the Arp2/3 complex, which is a key stimulator of actin polymerization in cells. Cortactin was further preferentially tyrosine phosphorylated at AChR clustering sites and treatment of myotubes with agrin significantly enhanced the tyrosine phosphorylation of cortactin. Importantly, forced expression in myotubes of a tyrosine phosphorylation-defective cortactin mutant (but not wild-type cortactin) suppressed agrin-dependent AChR clustering, as did the reduction of endogenous cortactin levels using RNA interference, and introduction of the mutant cortactin into muscle cells potently inhibited synaptic AChR aggregation in response to innervation.Conclusion
Our results suggest a novel function of phosphorylation-dependent cortactin signaling downstream from agrin/MuSK in facilitating AChR clustering at the developing NMJ. 相似文献4.
Background
Epidermal growth factor receptor (EGFR) internalization following ligand binding controls EGFR downstream pathway signaling activity. Internalized EGFR is poly-ubiquitinated by Cbl to promote lysosome-mediated degradation and signal downregulation. ACK1 is a non-receptor tyrosine kinase that interacts with ubiquitinated EGFR to facilitate EGFR degradation. Dynamic reorganization of the cortical actin cytoskeleton controlled by the actin related protein (Arp)2/3 complex is important in regulating EGFR endocytosis and vesicle trafficking. How ACK1-mediated EGFR internalization cooperates with Arp2/3-based actin dynamics during EGFR downregulation is unclear.Methodology/Principal Findings
Here we show that ACK1 directly binds and phosphorylates the Arp2/3 regulatory protein cortactin, potentially providing a direct link to Arp2/3-based actin dynamics during EGFR degradation. Co-immunoprecipitation analysis indicates that the cortactin SH3 domain is responsible for binding to ACK1. In vitro kinase assays demonstrate that ACK1 phosphorylates cortactin on key tyrosine residues that create docking sites for adaptor proteins responsible for enhancing Arp2/3 nucleation. Analysis with phosphorylation-specific antibodies determined that EGFR-induced cortactin tyrosine phosphorylation is diminished coincident with EGFR degradation, whereas ERK1/2 cortactin phosphorylation utilized in promoting activation of the Arp2/3 regulator N-WASp is sustained during EGFR downregulation. Cortactin and ACK1 localize to internalized vesicles containing EGF bound to EGFR visualized by confocal microscopy. RNA interference and rescue studies indicate that ACK1 and the cortactin SH3 domain are essential for ligand-mediated EGFR internalization.Conclusions/Significance
Cortactin is a direct binding partner and novel substrate of ACK1. Tyrosine phosphorylation of cortactin by ACK1 creates an additional means to amplify Arp2/3 dynamics through N-WASp activation, potentially contributing to the overall necessary tensile and/or propulsive forces utilized during EGFR endocytic internalization and trafficking involved in receptor degradation. 相似文献5.
Head JA Jiang D Li M Zorn LJ Schaefer EM Parsons JT Weed SA 《Molecular biology of the cell》2003,14(8):3216-3229
Cortactin is an F-actin binding protein that activates actin-related protein 2/3 complex and is localized within lamellipodia. Cortactin is a substrate for Src and other protein tyrosine kinases involved in cell motility, where its phosphorylation on tyrosines 421, 466, and 482 in the carboxy terminus is required for cell movement and metastasis. In spite of the importance of cortactin tyrosine phosphorylation in cell motility, little is known regarding the structural, spatial, or signaling requirements regulating cortactin tyrosine phosphorylation. Herein, we report that phosphorylation of cortactin tyrosine residues in the carboxy terminus requires the aminoterminal domain and Rac1-mediated localization to the cell periphery. Phosphorylation-specific antibodies directed against tyrosine 421 and 466 were produced to study the regulation and localization of tyrosine phosphorylated cortactin. Phosphorylation of cortactin tyrosine 421 and 466 was elevated in response to Src, epidermal growth factor receptor and Rac1 activation, and tyrosine 421 phosphorylated cortactin localized with F-actin in lamellipodia and podosomes. Cortactin tyrosine phosphorylation is progressive, with tyrosine 421 phosphorylation required for phosphorylation of tyrosine 466. These results indicate that cortactin tyrosine phosphorylation requires Rac1-induced cortactin targeting to cortical actin networks, where it is tyrosine phosphorylated in hierarchical manner that is closely coordinated with its ability to regulate actin dynamics. 相似文献
6.
Background
Dystroglycan is a ubiquitously expressed cell adhesion receptor best understood in its role as part of the dystrophin glycoprotein complex of mature skeletal muscle. Less is known of the role of dystroglycan in more fundamental aspects of cell adhesion in other cell types, nor of its role in myoblast cell adhesion.Principal Findings
We have examined the role of dystroglycan in the early stages of myoblast adhesion and spreading and found that dystroglycan initially associates with other adhesion proteins in large puncta morphologically similar to podosomes. Using a human SH3 domain phage display library we identified Tks5, a key regulator of podosomes, as interacting with β-dystroglycan. We verified the interaction by immunoprecipitation, GST-pulldown and immunfluorescence localisation. Both proteins localise to puncta during early phases of spreading, but importantly following stimulation with phorbol ester, also localise to structures indistinguishable from podosomes. Dystroglycan overexpression inhibited podosome formation by sequestering Tks5 and Src. Mutation of dystroglycan tyrosine 890, previously identified as a Src substrate, restored podosome formation.Conclusions
We propose therefore, that Src-dependent phosphorylation of β-dystroglycan results in the formation of a Src/dystroglycan complex that drives the SH3-mediated association between dystroglycan and Tks5 which together regulate podosome formation in myoblasts. 相似文献7.
Background
The Focal Adhesion Kinase is a well studied tyrosine kinase involved in a wide number of cellular processes including cell adhesion and migration. It has also been shown to play important roles during embryonic development and targeted disruption of the FAK gene in mice results in embryonic lethality by day 8.5.Principal Findings
Here we examined the pattern of phosphorylation of FAK during Xenopus development and found that FAK is phosphorylated on all major tyrosine residues examined from early blastula stages well before any morphogenetic movements take place. We go on to show that FRNK fails to act as a dominant negative in the context of the early embryo and that the FERM domain has a major role in determining FAK’s localization at the plasma membrane. Finally, we show that autonomous expression of the FERM domain leads to the activation of endogenous FAK in a tyrosine 397 dependent fashion.Conclusions
Overall, our data suggest an important role for the FERM domain in the activation of FAK and indicate that integrin signalling plays a limited role in the in vivo activation of FAK at least during the early stages of development. 相似文献8.
Background
Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simple-type epithelia. Although K19 is known to be phosphorylated on tyrosine residue(s), conclusive site-specific characterization of these residue(s) and identification potential kinases that may be involved has not been reported.Methodology/Principal Findings
In this study, biochemical, molecular and immunological approaches were undertaken in order to identify and characterize K19 tyrosine phosphorylation. Upon treatment with pervanadate, a tyrosine phosphatase inhibitor, human K19 (hK19) was phosphorylated on tyrosine 391, located in the ‘tail’ domain of the protein. K19 Y391 phosphorylation was confirmed using site-directed mutagenesis and cell transfection coupled with the generation of a K19 phospho (p)-Y391-specific rabbit antibody. The antibody also recognized mouse phospho-K19 (K19 pY394). This tyrosine residue is not phosphorylated under basal conditions, but becomes phosphorylated in the presence of Src kinase in vitro and in cells expressing constitutively-active Src. Pervanadate treatment in vivo resulted in phosphorylation of K19 Y394 and Y391 in colonic epithelial cells of non-transgenic mice and hK19-overexpressing mice, respectively.Conclusions/Significance
Human K19 tyrosine 391 is phosphorylated, potentially by Src kinase, and is the first well-defined tyrosine phosphorylation site of any keratin protein. The lack of detection of K19 pY391 in the absence of tyrosine phosphatase inhibition suggests that its phosphorylation is highly dynamic. 相似文献9.
Elvira Nieto-Pelegrin Narcisa Martinez-Quiles 《Cell communication and signaling : CCS》2009,7(1):1-14
Background
Cortactin activates the actin-related 2/3 (Arp2/3) complex promoting actin polymerization to remodel cell architecture in multiple processes (e.g. cell migration, membrane trafficking, invadopodia formation etc.). Moreover, it was called the Achilles' heel of the actin cytoskeleton because many pathogens hijack signals that converge on this oncogenic scaffolding protein. Cortactin is able to modulate N-WASP activation in vitro in a phosphorylation-dependent fashion. Thus Erk-phosphorylated cortactin is efficient in activating N-WASP through its SH3 domain, while Src-phosphorylated cortactin is not. This could represent a switch on/off mechanism controlling the coordinated action of both nucleator promoting factors (NPFs). Pedestal formation by enteropathogenic Escherichia coli (EPEC) requires N-WASP activation. N-WASP is recruited by the cell adapter Nck which binds a major tyrosine-phosphorylated site of a bacterial injected effector, Tir (translocated intimin receptor). Tir-Nck-N-WASP axis defines the current major pathway to actin polymerization on pedestals. In addition, it was recently reported that EPEC induces tyrosine phosphorylation of cortactin.Results
Here we demonstrate that cortactin phosphorylation is absent on N-WASP deficient cells, but is recovered by re-expression of N-WASP. We used purified recombinant cortactin and Tir proteins to demonstrate a direct interaction of both that promoted Arp2/3 complex-mediated actin polymerization in vitro, independently of cortactin phosphorylation.Conclusion
We propose that cortactin binds Tir through its N-terminal part in a tyrosine and serine phosphorylation independent manner while SH3 domain binding and activation of N-WASP is regulated by tyrosine and serine mediated phosphorylation of cortactin. Therefore cortactin could act on Tir-Nck-N-WASP pathway and control a possible cycling activity of N-WASP underlying pedestal formation. 相似文献10.
Willian F. Zambuzzi Estevam A. Bonfante Ryo Jimbo Mariko Hayashi Martin Andersson Gutemberg Alves Esther R. Takamori Paulo J. Beltr?o Paulo G. Coelho José M. Granjeiro 《PloS one》2014,9(7)
Background
It is known that physico/chemical alterations on biomaterial surfaces have the capability to modulate cellular behavior, affecting early tissue repair. Such surface modifications are aimed to improve early healing response and, clinically, offer the possibility to shorten the time from implant placement to functional loading. Since FAK and Src are intracellular proteins able to predict the quality of osteoblast adhesion, this study evaluated the osteoblast behavior in response to nanometer scale titanium surface texturing by monitoring FAK and Src phosphorylations.Methodology
Four engineered titanium surfaces were used for the study: machined (M), dual acid-etched (DAA), resorbable media microblasted and acid-etched (MBAA), and acid-etch microblasted (AAMB). Surfaces were characterized by scanning electron microscopy, interferometry, atomic force microscopy, x-ray photoelectron spectroscopy and energy dispersive X-ray spectroscopy. Thereafter, those 4 samples were used to evaluate their cytotoxicity and interference on FAK and Src phosphorylations. Both Src and FAK were investigated by using specific antibody against specific phosphorylation sites.Principal Findings
The results showed that both FAK and Src activations were differently modulated as a function of titanium surfaces physico/chemical configuration and protein adsorption.Conclusions
It can be suggested that signaling pathways involving both FAK and Src could provide biomarkers to predict osteoblast adhesion onto different surfaces. 相似文献11.
Background
Despite effective radiotherapy for the initial stages of cancer, several studies have reported the recurrence of various cancers, including medulloblastoma. Here, we attempt to capitalize on the radiation-induced aggressive behavior of medulloblastoma cells by comparing the extracellular protease activity and the expression pattern of molecules, known to be involved in cell adhesion, migration and invasion, between non-irradiated and irradiated cells.Methodology/Principal Findings
We identified an increase in invasion and migration of irradiated compared to non-irradiated medulloblastoma cells. RT-PCR analysis confirmed increased expression of uPA, uPAR, focal adhesion kinase (FAK), N-Cadherin and integrin subunits (e.g., α3, α5 and β1) in irradiated cells. Furthermore, we noticed a ∼2-fold increase in tyrosine phosphorylation of FAK in irradiated cells. Immunoprecipitation studies confirmed increased interaction of integrin β1 and FAK in irradiated cells. In addition, our results show that overexpression of uPAR in cancer cells can mimic radiation-induced activation of FAK signaling. Moreover, by inhibiting FAK phosphorylation, we were able to reduce the radiation-induced invasiveness of the cancer cells. In this vein, we studied the effect of siRNA-mediated knockdown of uPAR on cell migration and adhesion in irradiated and non-irradiated medulloblastoma cells. Downregulation of uPAR reduced the radiation-induced adhesion, migration and invasion of the irradiated cells, primarily by inhibiting phosphorylation of FAK, Paxillin and Rac-1/Cdc42. As observed from the immunoprecipitation studies, uPAR knockdown reduced interaction among the focal adhesion molecules, such as FAK, Paxillin and p130Cas, which are known to play key roles in cancer metastasis. Pretreatment with uPAR shRNA expressing construct reduced uPAR and phospho FAK expression levels in pre-established medulloblastoma in nude mice.Conclusion/Significance
Taken together, our results show that radiation enhances uPAR-mediated FAK signaling and by targeting uPAR we can inhibit radiation-activated cell adhesion and migration both in vitro and in vivo. 相似文献12.
Erk/Src phosphorylation of cortactin acts as a switch on-switch off mechanism that controls its ability to activate N-WASP 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Martinez-Quiles N Ho HY Kirschner MW Ramesh N Geha RS 《Molecular and cellular biology》2004,24(12):5269-5280
The Arp2/3 complex can be independently activated to initiate actin polymerization by the VCA domain of WASP family members and by the acidic N-terminal and F-actin-binding repeat region of cortactin, which possesses a C-terminal SH3 domain. Cortactin is a target for phosphorylation by Src tyrosine kinases and by serine/threonine kinases that include Erk. Here we demonstrate that cortactin binds N-WASP and WASP via its SH3 domain, induces in vitro N-WASP-mediated actin polymerization, and colocalizes with N-WASP and WASP at sites of active actin polymerization. Erk phosphorylation and a mimicking S405,418D double mutation enhanced cortactin binding and activation of N-WASP. In contrast, Src phosphorylation inhibited the ability of cortactin previously phosphorylated by Erk, and that of S405,418D double mutant cortactin, to bind and activate N-WASP. Furthermore, Y-->D mutation of three tyrosine residues targeted by Src (Y421, Y466, and Y482) inhibited the ability of S405,418D cortactin to activate N-WASP. We propose that Erk phosphorylation liberates the SH3 domain of cortactin from intramolecular interactions with proline-rich regions, causing it to synergize with WASP and N-WASP in activating the Arp2/3 complex, and that Src phosphorylation terminates cortactin activation of N-WASP and WASP. 相似文献
13.
Effects of tyrosine phosphorylation of cortactin on podosome formation in A7r5 vascular smooth muscle cells 总被引:2,自引:0,他引:2
Cortactin, a predominant substrate of Src family kinases, plays an important role in Arp2/3-dependent actin polymerization in lamellipodia and membrane ruffles and was recently shown to be enriched in podosomes induced by either c-Src or phorbol ester. However, the mechanisms by which cortactin regulates podosome formation have not been determined. In this study, we showed that cortactin is required for podosome formation, using siRNA knockdown of cortactin expression in smooth muscle A7r5 cells. Treatment with phorbol ester or expression of constitutively active c-Src induced genesis of cortactin-containing podosomes as well as increase in phosphorylation of cortactin at Y421 and Y466, the Src phosphorylation sites on cortactin. The Src kinase inhibitor SU-6656 significantly inhibited formation of podosomes induced by phorbol ester and phosphorylation of cortactin, whereas PKC inhibitor did not affect podosome formation in c-Src-transfected cells. Unexpectedly, expression of cortactin mutants containing Y421F, Y421D, Y466F, or Y466D mutated sites did not affect podosome formation or cortactin translocation to podosomes, although endogenous tyrosine-phosphorylated cortactin at Y421 and Y466 was present in podosomes. Our data indicate that 1) PKC acts upstream of Src in phosphorylation of cortactin and podosome formation in smooth muscle cells; 2) expression of cortactin is essential for genesis of podosomes; 3) phosphorylation at Y421 and Y466 is not required for translocation of cortactin to podosomes, although phosphorylation at these sites appears to be enriched in podosomes; and 4) tyrosine phosphorylation of cortactin may be involved in regulation of stability and turnover of podosomes, rather than targeting this protein to the site of podosome formation. actin cytoskeleton; Src; protein kinase C 相似文献
14.
Aims
Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase of the focal adhesion kinase (FAK) family, is up-regulated in more than 60% of the tumors of hepatocellular carcinoma (HCC) patients. Forced overexpression of Pyk2 can promote the proliferation and invasion of HCC cells. In this study, we aimed to explore the underlying molecular mechanism of Pyk2-mediated cell migration of HCC cells.Methodology/Principal Findings
We demonstrated that Pyk2 transformed the epithelial HCC cell line Hep3B into a mesenchymal phenotype via the induction of epithelial to mesenchymal transition (EMT), signified by the up-regulation of membrane ruffle formation, activation of Rac/Rho GTPases, down-regulation of epithelial genes E-cadherin and cytokeratin as well as promotion of cell motility in presence of lysophosphatidic acid (LPA). Suppression of Pyk2 by overexpression of dominant negative PRNK domain in the metastatic HCC cell line MHCC97L transformed its fibroblastoid phenotype to an epithelial phenotype with up-regulation of epithelial genes, down-regulation of mesenchymal genes N-cadherin and STAT5b, and reduction of LPA-induced membrane ruffle formation and cell motility. Moreover, overexpression of Pyk2 in Hep3B cells promoted the phosphorylation and localization of mesenchymal gene Hic-5 onto cell membrane while suppression of Pyk2 in MHCC97L cells attenuated its phosphorylation and localization.Conclusion
These data provided new evidence of the underlying mechanism of Pyk2 in controlling cell motility of HCC cells through regulation of genes associated with EMT. 相似文献15.
Mizuno S Yasuo M Bogaard HJ Kraskauskas D Alhussaini A Gomez-Arroyo J Farkas D Farkas L Voelkel NF 《PloS one》2012,7(1):e30678
Background
Copper is an important regulator of hypoxia inducible factor 1 alpha (HIF-1α) dependent vascular endothelial growth factor (VEGF) expression, and is also required for the activity of lysyl oxidase (LOX) to effect matrix protein cross-linking. Cell detachment from the extracellular matrix can induce apoptosis (anoikis) via inactivation of focal adhesion kinase (FAK).Methodology
To examine the molecular mechanisms whereby copper depletion causes the destruction of the normal alveolar architecture via anoikis, Male Sprague-Dawley rats were fed a copper deficient diet for 6 weeks while being treated with the copper chelator, tetrathiomolybdate. Other groups of rats were treated with the inhibitor of auto-phosphorylation of FAK, 1,2,4,5-benzenetetraamine tetrahydrochloride (1,2,4,5-BT) or FAK small interfering RNA (siRNA).Principal Findings
Copper depletion caused emphysematous changes, decreased HIF-1α activity, and downregulated VEGF expression in the rat lungs. Cleaved caspase-3, caspase-8 and Bcl-2 interacting mediator of cell death (Bim) expression was increased, and the phosphorylation of FAK was decreased in copper depleted rat lungs. Administration of 1,2,4,5-BT and FAK siRNA caused emphysematous lung destruction associated with increased expression of cleaved capase-3, caspase-8 and Bim.Conclusions
These data indicate that copper-dependent mechanisms contribute to the pathogenesis of emphysema, which may be associated with decreased HIF-1α and FAK activity in the lung. 相似文献16.
Background
Despite its first discovery by in silico cloning of novel endothelial cell-specific genes a decade ago, the biological functions of endothelial cell-specific molecule 2 (ECSM2) have only recently begun to be understood. Limited data suggest its involvement in cell migration and apoptosis. However, the underlying signaling mechanisms and novel functions of ECSM2 remain to be explored.Methodology/Principal Findings
A rabbit anti-ECSM2 monoclonal antibody (RabMAb) was generated and used to characterize the endogenous ECSM2 protein. Immunoblotting, immunoprecipitation, deglycosylation, immunostaining and confocal microscopy validated that endogenous ECSM2 is a plasma membrane glycoprotein preferentially expressed in vascular endothelial cells (ECs). Expression patterns of heterologously expressed and endogenous ECSM2 identified that ECSM2 was particularly concentrated at cell-cell contacts. Cell aggregation and transwell assays showed that ECSM2 promoted cell-cell adhesion and attenuated basic fibroblast growth factor (bFGF)-driven EC migration. Gain or loss of function assays by overexpression or knockdown of ECSM2 in ECs demonstrated that ECSM2 modulated bFGF-directed EC motility via the FGF receptor (FGFR)-extracellular regulated kinase (ERK)-focal adhesion kinase (FAK) pathway. The counterbalance between FAK tyrosine phosphorylation (activation) and ERK-dependent serine phosphorylation of FAK was critically involved. A model of how ECSM2 signals to impact bFGF/FGFR-driven EC migration was proposed.Conclusions/Significance
ECSM2 is likely a novel EC junctional protein. It can promote cell-cell adhesion and inhibit bFGF-mediated cell migration. Mechanistically, ECSM2 attenuates EC motility through the FGFR-ERK-FAK pathway. The findings suggest that ECSM2 could be a key player in coordinating receptor tyrosine kinase (RTK)-, integrin-, and EC junctional component-mediated signaling and may have important implications in disorders related to endothelial dysfunction and impaired EC junction signaling. 相似文献17.
Background
Non-receptor tyrosine kinases (NTKs) regulate physiological processes such as cell migration, differentiation, proliferation, and survival by interacting with and phosphorylating a large number of substrates simultaneously. This makes it difficult to attribute a particular biological effect to the phosphorylation of a particular substrate. We developed the Functional Interaction Trap (FIT) method to phosphorylate specifically a single substrate of choice in living cells, thereby allowing the biological effect(s) of that phosphorylation to be assessed. In this study we have used FIT to investigate the effects of specific phosphorylation of p130Cas, a protein implicated in cell migration. We have also used this approach to address a controversy regarding whether it is Src family kinases or focal adhesion kinase (FAK) that phosphorylates p130Cas in the trimolecular Src-FAK-p130Cas complex. 相似文献18.
Background
One of the earliest activation events following stimulation of the T cell receptor (TCR) is the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD3-associated complex by the Src family kinase Lck. There is accumulating evidence that a large pool of Lck is constitutively active in T cells but how the TCR is connected to Lck and to the downstream signaling cascade remains elusive.Methodology/Principal Findings
We have analyzed the phosphorylation state of Lck and Fyn and TCR signaling in human naïve CD4+ T cells and in the transformed T cell line, Hut-78. The latter has been shown to be similar to primary T cells in TCR-inducible phosphorylations and can be highly knocked down by RNA interference. In both T cell types, basal phosphorylation of Lck and Fyn on their activatory tyrosine was observed, although this was much less pronounced in Hut-78 cells. TCR stimulation led to the co-precipitation of Lck with the transmembrane adaptor protein LAT (linker for activation of T cells), Erk-mediated phosphorylation of Lck and no detectable dephosphorylation of Lck inhibitory tyrosine. Strikingly, upon LAT knockdown in Hut-78 cells, we found that LAT promoted TCR-induced phosphorylation of Lck and Fyn activatory tyrosines, TCRζ chain phosphorylation and Zap-70 activation. Notably, LAT regulated these events at low strength of TCR engagement.Conclusions/Significance
Our results indicate for the first time that LAT promotes TCR signal initiation and suggest that this adaptor may contribute to maintain active Lck in proximity of their substrates. 相似文献19.
Tsang SM Liu L Teh MT Wheeler A Grose R Hart IR Garrod DR Fortune F Wan H 《PloS one》2010,5(12):e14211
Background
Desmoglein 3 (Dsg3), a desmosomal adhesion protein, is expressed in basal and immediate suprabasal layers of skin and across the entire stratified squamous epithelium of oral mucosa. However, increasing evidence suggests that the role of Dsg3 may involve more than just cell-cell adhesion.Methodology/Principal Findings
To determine possible additional roles of Dsg3 during epithelial cell adhesion we used overexpression of full-length human Dsg3 cDNA, and RNAi-mediated knockdown of this molecule in various epithelial cell types. Overexpression of Dsg3 resulted in a reduced level of E-cadherin but a colocalisation with the E-cadherin-catenin complex of the adherens junctions. Concomitantly these transfected cells exhibited marked migratory capacity and the formation of filopodial protrusions. These latter events are consistent with Src activation and, indeed, Src-specific inhibition reversed these phenotypes. Moreover Dsg3 knockdown, which also reversed the decreased level of E-cadherin, partially blocked Src phosphorylation.Conclusions/Significance
Our data are consistent with the possibility that Dsg3, as an up-stream regulator of Src activity, helps regulate adherens junction formation. 相似文献20.
Lee M. Wheldon Bryan P. Haines Rajit Rajappa Ivor Mason Peter W. Rigby John K. Heath 《PloS one》2010,5(4)