scholarly journals Integrin and cytoskeletal regulation of growth factor signaling to the MAP kinase pathway

1999 ◽  
Vol 112 (5) ◽  
pp. 695-706 ◽  
Author(s):  
A.E. Aplin ◽  
R.L. Juliano
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Actin Stress Fiber ◽  
Growth Factor Signaling ◽  
Cortical Actin ◽  
Suspension Cells ◽  
Egf Signaling

Integrin-mediated anchorage of NIH3T3 fibroblasts to the extracellular matrix component fibronectin permits efficient growth factor signaling to the p42 and p44 forms of mitogen-activated protein kinase (MAPK). Since integrins bridge the extracellular matrix to focal adhesion sites and to the actin cytoskeleton, we analyzed the role of these integrin-associated structures in efficient growth factor activation of p42 and p44-MAPKs. Use of specific reagents that disrupt actin stress fiber and focal adhesion formation demonstrated that upon readhesion of NIH3T3 cells to fibronectin, cells that were poorly spread and lacked prominent focal adhesions but that formed cortical actin structures, efficiently signaled to p42 and p44-MAPKs upon EGF stimulation. In contrast, failure to form the cortical actin structures, despite attachment to fibronectin, precluded effective EGF signaling to p42 and p44-MAPKs. Actin cytoskeletal changes induced by expression of dominant-negative and constitutively active forms of Rho GTPases did not alter EGF activation of MAPK in adherent cells. However, active Cdc42, but not active Rac1 or RhoA, partially rescued EGF signaling to p44-MAPK in cells maintained in suspension. These data indicate that a limited degree of adhesion-mediated cytoskeletal organization and focal adhesion complex formation are required for efficient EGF activation of p42 and p44-MAPKs. Our studies exclude a major role for the GTPases RhoA and Rac1 in the formation of cytoskeletal structures relevant for signaling, but indicate that structures regulated by Cdc42 enhance the ability of suspension cells to activate MAPK in response to growth factors.

Focal Adhesion Kinase Regulates Critical Functions in Hematopoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1407-1407
Author(s):  
Sasidhar Vemula ◽  
Benjamin P. Abratigue ◽  
Premchand Gandra ◽  
John T. O’Malley ◽  
Ayek-Nati N. Ahyi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Growth Factor ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Egf Receptor ◽  
Bone Marrow Cells ◽  
Focal Adhesions ◽  
Growth Factor Receptors ◽  
Fold Reduction

Abstract Focal adhesion kinase (FAK) initially identified as a unique cytoplasmic tyrosine kinase involved in focal adhesions, has been studied extensively in fibroblasts. In these cells, FAK has been shown to play an essential role in bridging signals between integrin and growth factor receptors such as the PDGF and the EGF receptor. In fibroblasts, FAK localizes to regions of the cell that attach to the extracellular matrix and coordinates signals from integrins, cytokines, growth factor receptors, and oncogenes. In some tumors, FAK is over-expressed or constitutively activated, which correlates with increased motility, invasiveness, and proliferation. More recently, expression of FAK in acute myeloid leukemia was associated with enhanced blast migration, increased cellularity, and poor prognosis. However, virtually nothing is known about FAKs role in normal hematopoiesis. FAK is expressed in blood cells, including in bone marrow derived KIT+, Gr-1+, Mac-1+, CD4+, CD8+ and B220+ cells. To determine how loss of FAK affects hematopoiesis, we have generated a mouse model with hematopoietic restricted deletion of FAK. We deleted FAK in bone marrow cells by crossing the FAK-flox mice to Mx.Cre+ expressing mice and by treating Mx.cre+FAK+/+ and Mx.cre+FAKflox/flox mice with poly (I)-poly(C) and then analyzing mice 1 month after the last injection. After one month of poly(I)-poly(C) induction, the progeny failed to express detectable levels of FAK in bone marrow, spleen as well as in bone marrow derived macrophages as determined by PCR and western blotting. Evaluation of peripheral blood counts in control and FAK deleted mice revealed modest but significant differences in different lineages (WBC k/μl: FAK; 14 vs. FAK−/−; 10.3, n=7, *p<0.05, LY k/μl: FAK; 10.48 vs. FAK−/−; 7.26, n=7, *p<0.005, RBC k/μl: FAK; 9.76 X106 vs. FAK−/−;8.58 X106 n=7 *p<0.003, PLT k/μl: FAK; 644 vs. FAK−/−; 434 n=7 *p<0.007). Since macrophages express abundant levels of FAK and are rapidly recruited in large numbers to sites of infection, we initially examined the role of FAK in macrophages by creating a well studied model of aseptic thioglycolate-induced peritonitis. Our results demonstrate a ∼1.5 fold reduction in the migration of macrophages to the peritoneal cavity of FAK−/− mice compared to controls (n=5, FAK; 1.8 X 106 vs. FAK−/−; 1.213 X106, *p<0.03). The reduction in recruitment of FAK−/− macrophages was observed in spite of comparable levels of F4/80 expression (WT; 92.98% vs. FAK−/−; 94.55%) as well as integrin (α4β1 & α5β1) expression (WT; 68% & 83.79% vs. FAK−/−; 60.39% & 83.17%, respectively) between WT and FAK−/− macrophages. Further analysis of FAK−/− macrophages revealed a significant decrease in extracellular matrix/integrin directed migration of these cells in response to M-CSF on fibronectin (40% reduction), laminin (55% reduction) and collagen (60% reduction) (n=3, *p<0.004) coated plates as well as a decrease in migration in a wound healing assay (n=3, *p<0.003). The reduction in migration of FAK−/− macrophages was associated with a significant decrease in adhesion on fibronectin (63%), laminin (52%) and collagen (56%) as well as spreading (n=3, *p<0.03). Taken together, our results provide a critical physiologic role for FAK in regulating several adhesive and migratory functions in cells of myeloid lineage. Additional functions of FAK in other hematopoietic lineages will be discussed.

scholarly journals Focal adhesion kinase signaling is decreased in polyamine-depleted IEC-6 cells

2001 ◽  
Vol 281 (2) ◽  
pp. C475-C485 ◽  
Author(s):  
Ramesh M. Ray ◽  
Mary Jane Viar ◽  
Shirley A. McCormack ◽  
Leonard R. Johnson
Keyword(s):  
Extracellular Matrix ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Integrin Signaling ◽  
Actin Stress Fiber ◽  
Stress Fiber Formation ◽  
And Migration

Polyamines are essential to the migration of epithelial cells in the intestinal mucosa. Cells depleted of polyamines do not attach as rapidly to the extracellular matrix and do not form the actin stress fibers essential for migration. Because both attachment and stress fiber formation depend on integrin signaling and the formation of focal adhesions, we examined these and related processes in polyamine-depleted IEC-6 cells. There was general decreased tyrosine phosphorylation of focal adhesion kinase (FAK), and, specifically, decreased phosphorylation of Tyr-925, the paxillin binding site. In control cells, FAK phosphorylation was rapid after attachment to the extracellular matrix, while attached cells depleted of polyamines had significantly delayed phosphorylation. FAK activity was also significantly inhibited in polyamine-depleted cells as was the phosphorylation of paxillin. Polyamine-depleted cells failed to spread normally after attachment, and immunocytochemistry showed little colocalization of FAK and actin compared with controls. Focal adhesion complex formation was greatly reduced in the absence of polyamines. These data suggest that defective integrin signaling may, at least in part, account for the decreased rates of attachment, actin stress fiber formation, spreading, and migration observed in polyamine-depleted cells.

Smooth muscle cell rigidity and extracellular matrix organization influence endothelial cell spreading and adhesion formation in coculture

2007 ◽  
Vol 293 (3) ◽  
pp. H1978-H1986 ◽  
Author(s):  
Charles S. Wallace ◽  
Sophie A. Strike ◽  
George A. Truskey
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Endothelial Cell ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Tissue Engineered Blood Vessels ◽  
Focal Adhesion Formation ◽  
Fibrillar Adhesion

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the α5β1-integrin complex, whereas ECs used either α5β1- or αvβ3-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC α5β1-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.

Syndecan-4 and integrins: combinatorial signaling in cell adhesion

1999 ◽  
Vol 112 (20) ◽  
pp. 3415-3420 ◽  
Author(s):  
J.R. Couchman ◽  
A. Woods
Keyword(s):  
Extracellular Matrix ◽  
Heparan Sulfate ◽  
Focal Adhesion ◽  
Core Protein ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Dominant Negative ◽  
Focal Adhesion Formation ◽  
Dominant Negative Inhibitor ◽  
Phosphatidylinositol 4

It is now becoming clear that additional transmembrane components can modify integrin-mediated adhesion. Syndecan-4 is a transmembrane heparan sulfate proteoglycan whose external glycosaminoglycan chains can bind extracellular matrix ligands and whose core protein cytoplasmic domain can signal during adhesion. Two papers in this issue of JCS demonstrate, through transfection studies, that syndecan-4 plays roles in the formation of focal adhesions and stress fibers. Overexpression of syndecan-4 increases focal adhesion formation, whereas a partially truncated core protein that lacks the binding site for protein kinase C(α) and phosphatidylinositol 4, 5-bisphosphate acts as a dominant negative inhibitor of focal adhesion formation. Focal adhesion induction does not require interaction between heparan sulfate glycosaminoglycan and ligand but can occur when non-glycanated core protein is overexpressed; this suggests that oligomerization of syndecan-4 plays a major role in signaling from the extracellular matrix in adhesion.

EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLCgamma signaling pathway

1998 ◽  
Vol 111 (5) ◽  
pp. 615-624 ◽  
Author(s):  
H. Xie ◽  
M.A. Pallero ◽  
K. Gupta ◽  
P. Chang ◽  
M.F. Ware ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Motility ◽  
Map Kinase ◽  
Signaling Pathway ◽  
Focal Adhesion ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Focal Adhesions ◽  
Short Term ◽  
Egfr Kinase

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these biophysical events are actuated by biochemical signaling pathways, we investigate here whether epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. We find that EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFR) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30% within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility, being noted at 0.1 nM EGF. EGFR kinase activity is required as cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. We further examine here known motility-associated pathways to determine whether these contribute to EGF-induced effects. We have previously demonstrated that phospholipase C(gamma) (PLCgamma) activation and mobilization of gelsolin from a plasma membrane-bound state are required for EGFR-mediated cell motility. In contrast, we find here that short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973) which fails to activate PLCgamma or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLCgamma. Because both WT and c'973 EGFR activate the erk MAP kinase pathway, we additionally explore here this signaling pathway, not previously associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, we characterize for the first time the ability of EGFR kinase activity to directly stimulate focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration. Furthermore, we propose a model of EGF-induced motogenic cell responses in which the PLCgamma pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion.

scholarly journals Localization of Phospholipase D1 to Caveolin-enriched Membrane via Palmitoylation: Implications for Epidermal Growth Factor Signaling

2002 ◽  
Vol 13 (11) ◽  
pp. 3976-3988 ◽  
Author(s):  
Jung Min Han ◽  
Yong Kim ◽  
Jun Sung Lee ◽  
Chang Sup Lee ◽  
Byoung Dae Lee ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Point Mutations ◽  
Dominant Negative ◽  
Growth Factor Signaling ◽  
Wild Type ◽  
Caveolin 1 ◽  
Epidermal Growth ◽  
Egf Signaling

Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCα mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.

Indomethacin Delays Gastric Restitution: Association with the Inhibition of Focal Adhesion Kinase and Tensin Phosphorylation and Reduced Actin Stress Fibers

2002 ◽  
Vol 227 (6) ◽  
pp. 412-424 ◽  
Author(s):  
Imre L. Szabó ◽  
Rama Pai ◽  
Michael K. Jones ◽  
George R. Ehring ◽  
Hirofumi Kawanaka ◽  
...  
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Actin Stress Fiber ◽  
Stress Fiber Formation ◽  
Actin Stress Fiber Formation

Repair of superficial gastric mucosal injury is accomplished by the process of restitution—migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.

scholarly journals Mapping the Mechanical Cross-Talk between Epidermal Growth Factor Receptor and Focal Adhesion Formation

2018 ◽  
Vol 114 (3) ◽  
pp. 516a
Author(s):  
Tejeshwar C. Rao ◽  
Tara M. Urner ◽  
Victor Pui-Yan Ma ◽  
Khalid Salaita ◽  
Alexa L. Mattheyses
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Focal Adhesion ◽  
Cross Talk ◽  
Adhesion Formation ◽  
Growth Factor Receptor ◽  
Focal Adhesion Formation ◽  
Epidermal Growth

scholarly journals Src-mediated coupling of focal adhesion kinase to integrin αvβ5 in vascular endothelial growth factor signaling

2002 ◽  
Vol 157 (1) ◽  
pp. 149-160 ◽  
Author(s):  
Brian P. Eliceiri ◽  
Xose S. Puente ◽  
John D. Hood ◽  
Dwayne G. Stupack ◽  
David D. Schlaepfer ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Critical Event ◽  
Growth Factor Signaling ◽  
Vascular Endothelial ◽  
Signaling Complex ◽  
Specific Growth Factor ◽  
Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) promotes vascular permeability (VP) and neovascularization, and is required for development. We find that VEGF-stimulated Src activity in chick embryo blood vessels induces the coupling of focal adhesion kinase (FAK) to integrin αvβ5, a critical event in VEGF-mediated signaling and biological responsiveness. In contrast, FAK is constitutively associated with β1 and β3 integrins in the presence or absence of growth factors. In cultured endothelial cells, VEGF, but not basic fibroblast growth factor, promotes the Src-mediated phosphorylation of FAK on tyrosine 861, which contributes to the formation of a FAK/αvβ5 signaling complex. Moreover, formation of this FAK/αvβ5 complex is significantly reduced in pp60c-src-deficient mice. Supporting these results, mice deficient in either pp60c-src or integrin β5, but not integrin β3, have a reduced VP response to VEGF. This FAK/αvβ5 complex was also detected in epidermal growth factor-stimulated epithelial cells, suggesting a function for this complex outside the endothelium. Our findings indicate that Src can coordinate specific growth factor and extracellular matrix inputs by recruiting integrin αvβ5 into a FAK-containing signaling complex during growth factor–mediated biological responses.

Gingival epithelial cell signalling and cytoskeletal responses to Porphyromonas gingivalis invasion

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2417-2426 ◽  
Author(s):  
Özlem Yilmaz ◽  
Patrick A. Young ◽  
Richard J. Lamont ◽  
George E. Kenny
Keyword(s):  
Porphyromonas Gingivalis ◽  
Focal Adhesion ◽  
Cell Signalling ◽  
Focal Adhesions ◽  
Wild Type ◽  
Cortical Actin ◽  
Membrane Ruffling ◽  
Invasion Mechanism ◽  
Signalling Molecules ◽  
Gingival Epithelial Cell

Porphyromonas gingivalis, an oral pathogen, can internalize within primary gingival epithelial cells (GECs) through an invasion mechanism mediated by interactions between P. gingivalis fimbriae and integrins on the surface of the GECs. Fimbriae–integrin-based signalling events were studied by fluorescence microscopy, and the subcellular localization of integrin-associated signalling molecules paxillin and focal adhesion kinase (FAK), and the architecture of the actin and microtubule cytoskeleton were examined. GECs infected with P. gingivalis for 30 min demonstrated significant redistribution of paxillin and FAK from the cytosol to cell peripheries and assembly into focal adhesion complexes. In contrast, a fimbriae-deficient mutant of P. gingivalis did not contribute substantially to activation of paxillin or FAK. After 24 h, the majority of paxillin and FAK had returned to the cytoplasm with significant co-localization with P. gingivalis in the perinuclear region. Wild-type P. gingivalis induced nucleation of actin filaments forming microspike-like protrusions and long stable microfilaments distributed throughout the cells. Fimbriae mutants promoted a rich cortical actin meshwork accompanied by membrane ruffling dispersed along the cell membrane. Remarkable disassembly and nucleation of the actin and microtubule filamentous network was observed following 24 h infection with either wild-type or fimbriae-deficient mutants of P. gingivalis. The results show that fimbriated P. gingivalis cells induce formation of integrin-associated focal adhesions with subsequent remodelling of the actin and tubulin cytoskeleton.

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