scholarly journals Paxillin-Kinase-Linker Tyrosine Phosphorylation Regulates Directional Cell Migration

2009 ◽  
Vol 20 (22) ◽  
pp. 4706-4719 ◽  
Author(s):  
Jianxin A. Yu ◽  
Nicholas O. Deakin ◽  
Christopher E. Turner
Keyword(s):  
Cell Migration ◽  
Cell Adhesion ◽  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Wound Repair ◽  
Cell Polarization ◽  
Dependent Manner ◽  
Directed Cell Migration ◽  
Focal Adhesion Protein ◽  
Directional Cell Migration

Directed cell migration requires the coordination of growth factor and cell adhesion signaling and is of fundamental importance during embryonic development, wound repair, and pathological conditions such as tumor metastasis. Herein, we demonstrate that the ArfGAP, paxillin-kinase-linker (PKL/GIT2), is tyrosine phosphorylated in response to platelet-derived growth factor (PDGF) stimulation, in an adhesion dependent manner and is necessary for directed cell migration. Using a combination of pharmacological inhibitors, knockout cells and kinase mutants, FAK, and Src family kinases were shown to mediate PDGF-dependent PKL tyrosine phosphorylation. In fibroblasts, expression of a PKL mutant lacking the principal tyrosine phosphorylation sites resulted in loss of wound-induced cell polarization as well as directional migration. PKL phosphorylation was necessary for PDGF-stimulated PKL binding to the focal adhesion protein paxillin and expression of paxillin or PKL mutants defective in their respective binding motifs recapitulated the polarization defects. RNA interference or expression of phosphorylation mutants of PKL resulted in disregulation of PDGF-stimulated Rac1 and PAK activities, reduction of Cdc42 and Erk signaling, as well as mislocalization of βPIX. Together these studies position PKL as an integral component of growth factor and cell adhesion cross-talk signaling, controlling the development of front–rear cell polarity and directional cell migration.

scholarly journals Nerve Growth Factor Stimulates Multisite Tyrosine Phosphorylation and Activation of the Atypical Protein Kinase C's via a src Kinase Pathway

2001 ◽  
Vol 21 (24) ◽  
pp. 8414-8427 ◽  
Author(s):  
Marie W. Wooten ◽  
Michel L. Vandenplas ◽  
M. Lamar Seibenhener ◽  
Thangiah Geetha ◽  
Maria T. Diaz-Meco
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Pc12 Cells ◽  
Catalytic Domain ◽  
Src Kinase ◽  
Dependent Manner ◽  
Nerve Growth ◽  
Atypical Protein Kinase

ABSTRACT Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated differentiation of PC12 cells. In the present study, we report that PKC-ι becomes tyrosine phosphorylated in the membrane coincident with activation posttreatment with nerve growth factor. Tyrosine phosphorylation and activation of PKC-ι were inhibited in a dose-dependent manner by both PP2 and K252a, src and TrkA kinase inhibitors. Purified src was observed to phosphorylate and activate PKC-ι in vitro. In PC12 cells deficient in src kinase activity, both NGF-induced tyrosine phosphorylation and activation of PKC-ι were also diminished. Furthermore, we demonstrate activation of src by NGF along with formation of a signal complex including the TrkA receptor, src, and PKC-ι. Recruitment of PKC-ι into the complex was dependent on the tyrosine phosphorylation state of PKC-ι. The association of src and PKC-ι was constitutive but was enhanced by NGF treatment, with the src homology 3 domain interacting with a PXXP sequence within the regulatory domain of PKC-ι (amino acids 98 to 114). Altogether, these findings support a role for src in regulation of PKC-ι. Tyrosine 256, 271, and 325 were identified as major sites phosphorylated by src in the catalytic domain. Y256F and Y271F mutations did not alter src-induced activation of PKC-ι, whereas the Y325F mutation significantly reduced src-induced activation of PKC-ι. The functional relevance of these mutations was tested by determining the ability of each mutant to support TRAF6 activation of NF-κB, with significant impairment by the Y325F PKC-ι mutant. Moreover, when the Y352F mutant was expressed in PC12 cells, NGF's ability to promote survival in serum-free media was reduced. In summary, we have identified a novel mechanism for NGF-induced activation of atypical PKC involving tyrosine phosphorylation by c-Src.

Kinetics and Regulation of Tyrosine Phosphorylation of the Platelet Derived Growth Factor Receptor

1989 ◽  
Vol 75 (4) ◽  
pp. 362-366
Author(s):  
Lilia Alberghina ◽  
Renata Zippel ◽  
Enzo Martegani ◽  
Emmapaola Sturani
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Tyrosine Phosphorylation ◽  
Growth Factor Receptor ◽  
Growth Conditions ◽  
Platelet Derived Growth Factor ◽  
Dependent Manner ◽  
Pdgf Receptor ◽  
Receptor Complexes ◽  
Phosphorylated Form

Platelet derived growth factor (PDGF) interaction with the cells induces rapid tyrosine phosphorylation of the PDGF receptor in a dose dependent manner. At 37 °C phosphorylation of the receptor is followed by its dephosphorylation and internalization. It is observed that the higher the ligand concentration, the more transient is the response, and the observed kinetics are explained by a simple kinetic model. At 4 °C the phosphorylated form of the receptor is more stable; however, if PDGF is dissociated from the cell surface-associated ligand-receptor complexes, the receptors are rapidly dephosphorylated, indicating that phosphatases specific for phosphotyrosine groups are very active within the cells. In fact, addition of orthovanadate stabilizes the phosphorylated form of the receptor and helps in recognizing possible physiological substrates of the PDGF receptor kinase. The expression of PDGF receptors on the cell surface has been investigated under different growth conditions: a positive correlation exists between the amount of PDGF receptors and the duplication times of exponentially growing cultures. Moreover, during exponential growth the PDGF receptors are scarcely expressed, and their number increases reaching a maximal value when the population enters the stationary phase.

scholarly journals Scribble, Lgl1, and myosin II form a complex in vivo to promote directed cell migration

2020 ◽  
Vol 31 (20) ◽  
pp. 2234-2248
Author(s):  
Maha Abedrabbo ◽  
Shoshana Ravid
Keyword(s):  
Cell Migration ◽  
Cell Adhesion ◽  
Myosin Ii ◽  
Leading Edge ◽  
Directed Cell Migration ◽  
Lethal Giant Larvae ◽  
And Migration ◽  
Migrating Cells

Here we show that Scribble (Scrib), Lethal giant larvae 1 (Lgl1), and myosin II form a complex in vivo and colocalize at the cell leading edge of migrating cells, and this colocalization is interdependent. Scrib and Lgl1 are required for proper cell adhesion, polarity, and migration.

scholarly journals The Inhibitory Effects of Gold Nanoparticles on VEGF-A-Induced Cell Migration in Choroid-Retina Endothelial Cells

2019 ◽  
Vol 21 (1) ◽  
pp. 109 ◽  
Author(s):  
Chi-Ming Chan ◽  
Chien-Yu Hsiao ◽  
Hsin-Ju Li ◽  
Jia-You Fang ◽  
Der-Chen Chang ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Cell Adhesion ◽  
Age Related Macular Degeneration ◽  
Adhesion Assay ◽  
Dependent Manner ◽  
Inhibitory Effects ◽  
Viability Assay ◽  
Age Related

Background: Vascular endothelial growth factor (VEGF) is upregulated by hypoxia and is a crucial stimulator for choroidal neovascularization (CNV) in age-related macular degeneration and pathologic myopia, as well as retinal neovascularization in proliferative diabetic retinopathy. Retinal and choroidal endothelial cells play key roles in the development of retinal and CNV, and subsequent fibrosis. At present, the effects of gold nanoparticles (AuNPs) on the VEGF-induced choroid-retina endothelial (RF/6A) cells are still unknown. In our study, we investigated the effects of AuNPs on RF/6A cell viabilities and cell adhesion to fibronectin, a major ECM protein of fibrovascular membrane. Furthermore, the inhibitory effects of AuNPs on RF/6A cell migration induced by VEGF and its signaling were studied. Methods: The cell viability assay was used to determine the viability of cells treated with AuNPs. The migration of RF/6A cells was assessed by the Transwell migration assay. The cell adhesion to fibronectin was examined by an adhesion assay. The VEGF-induced signaling pathways were determined by western blotting. Results: The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay revealed no cytotoxicity of AuNPs on RF/6A cells. AuNPs inhibited VEGF-induced RF/6A cell migration in a concentration-dependent manner but showed no significant effects on RF/6A cell adhesion to fibronectin. Inhibitory effects of AuNPs on VEGF-induced Akt/eNOS were found. Conclusions: These results suggest that AuNPs are an effective inhibitor of VEGF-induced RF/6A cell migration through the Akt/eNOS pathways, but they have no effects on their cell viabilities and cell adhesion to fibronectin.

scholarly journals N-Cadherin and Keratinocyte Growth Factor Receptor Mediate the Functional Interplay between Ki-RASG12V and p53V143A in Promoting Pancreatic Cell Migration, Invasion, and Tissue Architecture Disruption

2006 ◽  
Vol 26 (11) ◽  
pp. 4185-4200 ◽  
Author(s):  
Therese B. Deramaudt ◽  
Munenori Takaoka ◽  
Rabi Upadhyay ◽  
Mark J. Bowser ◽  
Jess Porter ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Adhesion ◽  
Growth Factor ◽  
Tumor Suppressor ◽  
Keratinocyte Growth Factor ◽  
Growth Factor Receptor ◽  
Genetic Alterations ◽  
Pancreatic Cell ◽  
Ductal Cells ◽  
Tumor Suppressor Gene Inactivation

ABSTRACT The genetic basis of pancreatic ductal adenocarcinoma, which constitutes the most common type of pancreatic malignancy, involves the sequential activation of oncogenes and inactivation of tumor suppressor genes. Among the pivotal genetic alterations are Ki-RAS oncogene activation and p53 tumor suppressor gene inactivation. We explain that the combination of these genetic events facilitates pancreatic carcinogenesis as revealed in novel three-dimensional cell (spheroid cyst) culture and in vivo subcutaneous and orthotopic xenotransplantation models. N-cadherin, a member of the classic cadherins important in the regulation of cell-cell adhesion, is induced in the presence of Ki-RAS mutation but subsequently downregulated with the acquisition of p53 mutation as revealed by gene microarrays and corroborated by reverse transcription-PCR and Western blotting. N-cadherin modulates the capacity of pancreatic ductal cells to migrate and invade, in part via complex formation with keratinocyte growth factor receptor and neural cell adhesion molecule and in part via interaction with p120-catenin. However, modulation of these complexes by Ki-RAS and p53 leads to enhanced cell migration and invasion. This preferentially induces the downstream effector AKT over mitogen-activated protein kinase to execute changes in cellular behavior. Thus, we are able to define molecules that in part are directly affected by Ki-RAS and p53 during pancreatic ductal carcinogenesis, and this provides a platform for potential new molecularly based therapeutic interventions.

scholarly journals Rapid Up-Regulation of α4 Integrin-mediated Leukocyte Adhesion by Transforming Growth Factor-β1

2003 ◽  
Vol 14 (1) ◽  
pp. 54-66 ◽  
Author(s):  
Rubén A. Bartolomé ◽  
Francisco Sanz-Rodrı́guez ◽  
Mar M. Robledo ◽  
Andrés Hidalgo ◽  
Joaquin Teixidó
Keyword(s):  
Cell Migration ◽  
Cell Adhesion ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Human Peripheral Blood ◽  
Transforming Growth Factor Β1 ◽  
Small Gtpase ◽  
Mitogen Activated Protein Kinase ◽  
Intracellular Camp ◽  
Tgf Β1

The α4 integrins (α4β1 and α4β7) are cell surface heterodimers expressed mostly on leukocytes that mediate cell-cell and cell-extracellular matrix adhesion. A characteristic feature of α4 integrins is that their adhesive activity can be subjected to rapid modulation during the process of cell migration. Herein, we show that transforming growth factor-β1 (TGF-β1) rapidly (0.5–5 min) and transiently up-regulated α4 integrin-dependent adhesion of different human leukocyte cell lines and human peripheral blood lymphocytes (PBLs) to their ligands vascular cell adhesion molecule-1 (VCAM-1) and connecting segment-1/fibronectin. In addition, TGF-β1 enhanced the α4 integrin-mediated adhesion of PBLs to tumor necrosis factor-α–treated human umbilical vein endothelial cells, indicating the stimulation of α4β1/VCAM-1 interaction. Although TGF-β1 rapidly activated the small GTPase RhoA and the p38 mitogen-activated protein kinase, enhanced adhesion did not require activation of both signaling molecules. Instead, polymerization of actin cytoskeleton triggered by TGF-β1 was necessary for α4 integrin-dependent up-regulated adhesion, and elevation of intracellular cAMP opposed this up-regulation. Moreover, TGF-β1 further increased cell adhesion mediated by α4 integrins in response to the chemokine stromal cell-derived factor-1α. These data suggest that TGF-β1 can potentially contribute to cell migration by dynamically regulating cell adhesion mediated by α4 integrins.

scholarly journals The Molecular Basis of Phospholipase D2-Induced Chemotaxis: Elucidation of Differential Pathways in Macrophages and Fibroblasts

2010 ◽  
Vol 30 (18) ◽  
pp. 4492-4506 ◽  
Author(s):  
Katie Knapek ◽  
Kathleen Frondorf ◽  
Jennalee Post ◽  
Stephen Short ◽  
Dianne Cox ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Migration ◽  
Growth Factor ◽  
Cell Line ◽  
Protein Interactions ◽  
Wound Repair ◽  
Molecular Mechanisms ◽  
Growth Factor Receptor ◽  
Cell Transfection ◽  
Phospholipase D1

ABSTRACT We report the molecular mechanisms that underlie chemotaxis of macrophages and cell migration of fibroblasts, cells that are essential during the body's innate immune response and during wound repair, respectively. Silencing of phospholipase D1 (PLD1) and PLD2 reduced cell migration (both chemokinesis and chemotaxis) by ∼60% and >80%, respectively; this migration was restored by cell transfection with PLD2 constructs refractory to small interfering RNA (siRNA). Cells overexpressing active phospholipase D1 (PLD1) but, mostly, active PLD2 exhibited cell migration capabilities that were elevated over those elicited by chemoattractants alone. The mechanism for this enhancement is complex. It involves two pathways: one that is dependent on the activity of the lipase (and signals through its product, phosphatidic acid [PA]) and another that involves protein-protein interactions. The first is evidenced by partial abrogation of chemotaxis with lipase activity-defective constructs (PLD2-K758R) and by n-butanol treatment of cells. The second is evidenced by PLD association with the growth factor receptor-bound protein 2 (Grb2) through residue Y169, located within a Src homology 2 (SH2) consensus site. The association Grb2-PLD2 could be visualized by fluorescence microscopy in RAW/LR5 macrophages concentrated in actin-rich membrane ruffles, making possible that Grb2 serves as a docking or intermediary protein. The Grb2/PLD2-mediated chemotaxis process also depends on Grb2's ability to recognize other motility proteins, like the Wiskott-Aldrich syndrome protein (WASP). Cell transfection with WASP, PLD2, and Grb2 constructs yields the highest levels of cell migration response, particularly in a macrophage cell line (RAW/LR5) and only modestly in the fibroblast cell line COS-7. Further, RAW/LR5 macrophages utilize for cell migration an additional pathway that involves S6 kinase (S6K) through PLD2-Y296, known to be phosphorylated by epidermal growth factor receptor (EGFR) kinase. Thus, both fibroblasts and macrophages use activity-dependent and activity-independent signaling mechanisms. However, highly mobile cells like macrophages use all signaling machinery available to them to accomplish their required function in rapid immune response, which sets them apart from fibroblasts, cells normally nonmobile that are only briefly involved in wound healing.

scholarly journals αvβ3Integrin Mediates the Cell-adhesive Capacity and Biological Activity of Basic Fibroblast Growth Factor (FGF-2) in Cultured Endothelial Cells

1997 ◽  
Vol 8 (12) ◽  
pp. 2449-2461 ◽  
Author(s):  
Marco Rusnati ◽  
Elena Tanghetti ◽  
Patrizia Dell’Era ◽  
Anna Gualandris ◽  
Marco Presta
Keyword(s):  
Tissue Culture ◽  
Endothelial Cells ◽  
Cell Adhesion ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Amino Acid Sequences ◽  
Dependent Manner ◽  
Fibroblast Growth ◽  
Tissue Culture Plastic ◽  
Cell Adhesive

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38–61 and 82–101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-αvβ3antibodies prevent cell adhesion to FGF-2. Also, purified human αvβ3binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-αvβ3monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with αvβ3integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.

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