scholarly journals Phosphatidylinositol 3,4,5-Trisphosphate Directs Association of Src Homology 2-containing Signaling Proteins with Gelsolin

2001 ◽  
Vol 276 (50) ◽  
pp. 47434-47444 ◽  
Author(s):  
Meenakshi A. Chellaiah ◽  
Rajat S. Biswas ◽  
David Yuen ◽  
Ulises M. Alvarez ◽  
Keith A. Hruska
Keyword(s):  
Bone Resorption ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Protein Interactions ◽  
Lipid Extraction ◽  
Focal Adhesions ◽  
Amino Acid Sequences ◽  
Signaling Proteins ◽  
Src Homology 2 ◽  
Src Homology

Podosomes are adhesion structures in osteoclasts and are structurally related to focal adhesions mediating cell motility during bone resorption. Here we show that gelsolin coprecipitates some of the focal adhesion-associated proteins such as c-Src, phosphoinositide 3-kinase (PI3K), p130Cas, focal adhesion kinase, integrin αvβ3, vinculin, talin, and paxillin. These proteins were inducibly tyrosine-phosphorylated in response to integrin activation by osteopontin. Previous studies have defined unique biochemical properties of gelsolin related to phosphatidylinositol 3,4,5-trisphosphate in osteoclast podosomes, and here we demonstrate phosphatidylinositol 3,4,5-trisphosphate/gelsolin function in mediating organization of the podosome signaling complex. Overlay and GST pull-down assays demonstrated strong phosphatidylinositol 3,4,5-trisphosphate-PI3K interactions based on the Src homology 2 domains of PI3K. Furthermore, lipid extraction of lysates from activated osteoclasts eliminated interaction between gelsolin, c-Src, PI3K, and focal adhesion kinase despite equal amounts of gelsolin in both the lipid-extracted and unextracted experiment. The cytoplasmic protein tyrosine phosphatase (PTP)-proline-glutamic acid-serine-threonine amino acid sequences (PEST) was also found to be associated with gelsolin in osteoclast podosomes and with stimulation of αvβ3-regulated phosphorylation of PTP-PEST. We conclude that gelsolin plays a key role in recruitment of signaling proteins to the plasma membrane through phospholipid-protein interactions and by regulation of their phosphorylation status through its association with PTP-PEST. Because both gelsolin deficiency and PI3K inhibition impair bone resorption, we conclude that phosphatidylinositol 3,4,5-trisphosphate-based protein interactions are critical for osteoclast function.

scholarly journals In Situ Photoactivation of a Caged Phosphotyrosine Peptide Derived from Focal Adhesion Kinase Temporarily Halts Lamellar Extension of Single Migrating Tumor Cells

2005 ◽  
Vol 280 (23) ◽  
pp. 22091-22101 ◽  
Author(s):  
David Humphrey ◽  
Zenon Rajfur ◽  
M. Eugenio Vazquez ◽  
Danielle Scheswohl ◽  
Michael D. Schaller ◽  
...  
Keyword(s):  
Cell Migration ◽  
Focal Adhesion Kinase ◽  
Cell Body ◽  
Focal Adhesion ◽  
Tumor Cell Line ◽  
Leading Edge ◽  
Src Homology 2 ◽  
Src Homology ◽  
Phosphoinositide 3 Kinase

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, mediates integrin-based cell signaling by transferring signals regulating cell migration, adhesion, and survival from the extracellular matrix to the cytoplasm. Following autophosphorylation at tyrosine 397, FAK binds the Src homology 2 domains of Src and phosphoinositide 3-kinase, among several other possible binding partners. To further investigate the role of phosphorylated FAK in cell migration in situ, peptides comprising residues 391–406 of human FAK with caged phosphotyrosine 397 were synthesized. Although the caged phosphopeptides were stable to phosphatase activity, the free phosphopeptides showed a half-life of ∼10–15 min in cell lysates. Migrating NBT-II cells (a rat bladder tumor cell line) were microinjected with the caged FAK peptide and locally photoactivated using a focused laser beam. The photoactivation of caged FAK peptide in 8-μm diameter spots over the cell body led to the temporary arrest of the leading edge migration within ∼1 min of irradiation. In contrast, cell body migration was not inhibited. Microinjection of a non-caged phosphorylated tyrosine 397 FAK peptide into migrating NBT-II cells also led to lamellar arrest; however, this approach lacks the temporal control afforded by the caged phosphopeptides. Photoactivation of related phosphotyrosine peptides with altered sequences did not result in transient lamellar arrest. We hypothesize that the phosphorylated FAK peptide competes with the endogenous FAK for binding to FAK effectors including, but not limited to, Src and phosphoinositide 3-kinase, causing spatiotemporal misregulation and subsequent lamellar arrest.

scholarly journals Complex Formation with Focal Adhesion Kinase: A Mechanism to Regulate Activity and Subcellular Localization of Src Kinases

1999 ◽  
Vol 10 (10) ◽  
pp. 3489-3505 ◽  
Author(s):  
Michael D. Schaller ◽  
Jeffrey D. Hildebrand ◽  
J. Thomas Parsons
Keyword(s):  
Subcellular Localization ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Tyrosine Kinases ◽  
Focal Adhesions ◽  
Src Kinases ◽  
High Affinity Binding Site ◽  
Src Homology ◽  
Src Homology 2 Domain

Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60c-srcor p59fynresults in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60c-srcor p59fynto induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60c-srcis hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60c-srcfrom a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60c-srcto focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.

scholarly journals PLC-γ directly binds activated c-Src, which is necessary for carbachol-mediated inhibition of NHE3 activity in Caco-2/BBe cells

2013 ◽  
Vol 305 (3) ◽  
pp. C266-C275 ◽  
Author(s):  
Nicholas C. Zachos ◽  
Luke J. Lee ◽  
Olga Kovbasnjuk ◽  
Xuhang Li ◽  
Mark Donowitz
Keyword(s):  
Sh2 Domain ◽  
Signaling Proteins ◽  
Multiprotein Complexes ◽  
Sh2 Domains ◽  
Src Homology 2 ◽  
Src Inhibitor ◽  
Intracellular Ca ◽  
Direct Binding ◽  
Src Homology

Elevated levels of intracellular Ca2+([Ca2+]i) inhibit Na+/H+exchanger 3 (NHE3) activity in the intact intestine. We previously demonstrated that PLC-γ directly binds NHE3, an interaction that is necessary for [Ca2+]iinhibition of NHE3 activity, and that PLC-γ Src homology 2 (SH2) domains may scaffold Ca2+signaling proteins necessary for regulation of NHE3 activity. [Ca2+]iregulation of NHE3 activity is also c-Src dependent; however, the mechanism by which c-Src is involved is undetermined. We hypothesized that the SH2 domains of PLC-γ might link c-Src to NHE3-containing complexes to mediate [Ca2+]iinhibition of NHE3 activity. In Caco-2/BBe cells, carbachol (CCh) decreased NHE3 activity by ∼40%, an effect abolished with the c-Src inhibitor PP2. CCh treatment increased the amount of active c-Src as early as 1 min through increased Y416phosphorylation. Coimmunoprecipitation demonstrated that c-Src associated with PLC-γ, but not NHE3, under basal conditions, an interaction that increased rapidly after CCh treatment and occurred before the dissociation of PLC-γ and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC-γ SH2 domains, an interaction that was prevented by blocking the PLC-γ SH2 domain. This study demonstrated that c-Src 1) activity is necessary for [Ca2+]iinhibition of NHE3 activity, 2) activation occurs rapidly (∼1 min) after CCh treatment, 3) directly binds PLC-γ SH2 domains and associates dynamically with PLC-γ under elevated [Ca2+]iconditions, and 4) does not directly bind NHE3. Under elevated [Ca2+]iconditions, PLC-γ scaffolds c-Src into NHE3-containing multiprotein complexes before dissociation of PLC-γ from NHE3 and subsequent endocytosis of NHE3.

scholarly journals Src Homology 2 (SH2)-Containing 5′-Inositol Phosphatase Localizes to Podosomes, and the SH2 Domain Is Implicated in the Attenuation of Bone Resorption in Osteoclasts

Endocrinology ◽  
2006 ◽  
Vol 147 (7) ◽  
pp. 3307-3317 ◽  
Author(s):  
Keiichiro Yogo ◽  
Megumi Mizutamari ◽  
Kazuta Mishima ◽  
Hiromi Takenouchi ◽  
Norihiro Ishida-Kitagawa ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Nuclear Factor Κb ◽  
Sh2 Domain ◽  
Terminal Region ◽  
Spectrometric Analysis ◽  
Inositol Phosphatase ◽  
Amino Terminal ◽  
Novel Proteins ◽  
Src Homology 2 ◽  
Src Homology

c-Src plays an important role in bone resorption by osteoclasts. Here, we show using wild-type and ship−/− osteoclasts that Src homology 2 (SH2)-containing 5′-inositol phosphatase (SHIP) appeared to negatively regulate bone resorption activated by c-Src. SHIP was found to localize to podosomes under the influence of c-Src, and the presence of either the amino-terminal region comprising the SH2 domain or the carboxyl-terminal region was sufficient for its localization. Although SHIP lacking a functional SH2 domain was still found in podosomes, it could not rescue the hyper-bone resorbing activity and hypersensitivity to receptor activator of nuclear factor-κB ligand in ship−/− osteoclasts, suggesting that the localization of SHIP to podosomes per se was not sufficient and the SH2 domain was indispensable for its function. Cas and c-Cbl, known to function in podosomes of osteoclasts, were identified as novel proteins binding to the SHIP SH2 domain by mass spectrometric analysis, and this interaction appeared to be dependent on the Src kinase activity. These results demonstrate that c-Src enhances the translocation of SHIP to podosomes and regulates its function there through the SH2 domain, leading to an attenuation of bone resorption.

Possible involvement of focal adhesion kinase, p125FAK, in osteoclastic bone resorption

1995 ◽  
Vol 58 (4) ◽  
pp. 424-435 ◽  
Author(s):  
Sakae Tanaka ◽  
Naoyuki Takahashi ◽  
Nobuyuki Udagawa ◽  
Hiroshi Murakami ◽  
Ichiro Nakamura ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Osteoclastic Bone Resorption

scholarly journals Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration

2009 ◽  
Vol 296 (3) ◽  
pp. H627-H638 ◽  
Author(s):  
Ana Maria Manso ◽  
Seok-Min Kang ◽  
Sergey V. Plotnikov ◽  
Ingo Thievessen ◽  
Jaewon Oh ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Adult Mouse ◽  
Cardiac Fibroblasts ◽  
Focal Adhesions ◽  
Protein Levels ◽  
Tyrosine Kinase 2 ◽  
A Cell ◽  
And Migration ◽  
Proliferation And Migration

Migration and proliferation of cardiac fibroblasts (CFs) play an important role in the myocardial remodeling process. While many factors have been identified that regulate CF growth and migration, less is known about the signaling mechanisms involved in these processes. Here, we utilized Cre-LoxP technology to obtain focal adhesion kinase (FAK)-deficient adult mouse CFs and studied how FAK functioned in modulating cell adhesion, proliferation, and migration of these cells. Treatment of FAKflox/flox CFs with Ad/Cre virus caused over 70% reduction of FAK protein levels within a cell population. FAK-deficient CFs showed no changes in focal adhesions, cell morphology, or protein expression levels of vinculin, talin, or paxillin; proline-rich tyrosine kinase 2 (Pyk2) expression and activity were increased. Knockdown of FAK protein in CFs increased PDGF-BB-induced proliferation, while it reduced PDGF-BB-induced migration. Adhesion to fibronectin was not altered. To distinguish between the function of FAK and Pyk2, FAK function was inhibited via adenoviral-mediated overexpression of the natural FAK inhibitor FAK-related nonkinase (FRNK). Ad/FRNK had no effect on Pyk2 expression, inhibited the PDGF-BB-induced migration, but did not change the PDGF-BB-induced proliferation. FAK deficiency had only modest effects on increasing PDGF-BB activation of p38 and JNK MAPKs, with no alteration in the ERK response vs. control cells. These results demonstrate that FAK is required for the PDGF-BB-induced migratory response of adult mouse CFs and suggest that FAK could play an essential role in the wound-healing response that occurs in numerous cardiac pathologies.

scholarly journals Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin-containing focal adhesions

2010 ◽  
pp. n/a-n/a ◽  
Author(s):  
David W. Dumbauld ◽  
Heungsoo Shin ◽  
Nathan D. Gallant ◽  
Kristin E. Michael ◽  
Harish Radhakrishna ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Focal Adhesions

scholarly journals Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms

2000 ◽  
Vol 348 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Madeleine TOUTANT ◽  
Jeanne-Marie STUDLER ◽  
Ferran BURGAYA ◽  
Alicia COSTA ◽  
Pascal EZAN ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Src Family Kinases ◽  
Critical Residue ◽  
Green Fluorescent ◽  
And Function

In brain, focal adhesion kinase (FAK) is regulated by neurotransmitters and has a higher molecular mass than in other tissues, due to alternative splicing. Two exons code for additional peptides of six and seven residues (‘boxes’ 6 and 7), located on either side of Tyr397, which increase its autophosphorylation. Using in situ hybridization and a monoclonal antibody (Mab77) which does not recognize FAK containing box 7, we show that, although mRNAs coding for boxes 6 and 7 have different patterns of expression in brain, FAK+6,7 is the main isoform in forebrain neurons. The various FAK isoforms fused to green fluorescent protein were all targeted to focal adhesions in non-neuronal cells. Phosphorylation-state-specific antibodies were used to study in detail the phosphorylation of Tyr397, a critical residue for the activation and function of FAK. The presence of boxes 6 and 7 increased autophosphorylation of Tyr397 independently and additively, whereas they had a weak effect on FAK kinase activity towards poly(Glu,Tyr). Src-family kinases were also able to phosphorylate Tyr397 in cells, but this phosphorylation was decreased in the presence of box 6 or 7, and abolished in the presence of both. Thus the additional exons characteristic of neuronal isoforms of FAK do not alter its targeting, but change dramatically the phosphorylation of Tyr397. They increase its autophosphorylation in vitro and in transfected COS-7 cells, whereas they prevent its phosphorylation when co-transfected with Src-family kinases.

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