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 pp125FAK-dependent tyrosine phosphorylation of paxillin creates a high-affinity binding site for Crk.

1995 ◽  
Vol 15 (5) ◽  
pp. 2635-2645 ◽  
Author(s):  
M D Schaller ◽  
J T Parsons
Keyword(s):  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Protein Tyrosine Kinase ◽  
Focal Adhesions ◽  
Sh2 Domain ◽  
Binding Motif ◽  
Adapter Protein ◽  
Tyrosine Residues ◽  
Sh2 Domains ◽  
High Affinity Binding Site

Paxillin, a focal-adhesion-associated protein, becomes phosphorylated in response to a number of stimuli which also induce the tyrosine phosphorylation of the focal-adhesion-associated protein tyrosine kinase pp125FAK. On the basis of their colocalization and coordinate phosphorylation, paxillin is a candidate for a substrate of pp125FAK. We describe here conditions under which the phosphorylation of paxillin on tyrosine is pp125FAK dependent, supporting the hypothesis that paxillin phosphorylation is regulated by pp125FAK. pp125FAK must localize to focal adhesions and become autophosphorylated to induce paxillin phosphorylation. Phosphorylation of paxillin on tyrosine creates binding sites for the SH2 domains of Crk, Csk, and Src. We identify two sites of phosphorylation as tyrosine residues 31 and 118, each of which conforms to the Crk SH2 domain binding motif, (P)YXXP. These observations suggest that paxillin serves as an adapter protein, similar to insulin receptor substrate 1, and that pp125FAK may regulate the formation of signaling complexes by directing the phosphorylation of paxillin on tyrosine.

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 Tyrosine phosphorylation of focal adhesion kinase (p125FAK): regulation by cAMP and thrombin in mesangial cells.

1996 ◽  
Vol 7 (3) ◽  
pp. 415-423
Author(s):  
D A Troyer ◽  
A Bouton ◽  
R Bedolla ◽  
R Padilla
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Actin Filaments ◽  
Mesangial Cells ◽  
Close Contact ◽  
Focal Adhesions ◽  
Cytochalasin D ◽  
Fiber Formation ◽  
Stress Fibers

Stress fibers, composed of actin filaments, converge upon and associate with a number of proteins, including focal adhesion kinase (p125FAK), and integrin receptors to form areas of close contact between cells and the extracellular matrix referred to as focal adhesions. Treatment of mesangial cells with cAMP-elevating agents causes a loss of focal adhesions, fragmentation of stress fibers, and decreased tyrosine phosphorylation of p125FAK. Thrombin reverses these effects of cAMP, and this model can be used to address some of the cellular mechanisms involved in regulating the loss and formation of focal adhesions. This study reports the effects of cAMP and thrombin on mesangial cell shape, distribution of actin, formation of stress fibers, and tyrosine phosphorylation of p125FAK. cAMP-treated cells display a condensed cell body with slender processes that traverse the area formerly covered by the cell. Addition of thrombin to these cells restores actin filaments (stress fibers) and increases tyrosine phosphorylation of p125FAK, and the cells resume a flattened morphology, even in the continued presence of cAMP-elevating agents. Peptides that mimic the tethered ligand portion of the thrombin receptor have the same effects on cell morphology and stress fiber formation as thrombin. In selected experiments, agents that disrupt either stress fibers (cytochalasin D) or microtubules (nocodazole; Sigma Chemical, St. Louis, MO) were used to examine the role of these cytoskeletal elements in thrombin-induced restoration of focal adhesions. Cytochalasin D blocked the ability of thrombin to restore focal adhesions and phosphorylate p125FAK. The effects of nocodazole, an agent that destabilizes microtubules (but which has no known receptor), are very similar to those of thrombin. The findings discussed in this study indicate that thrombin can modulate the formation of focal adhesions. The organization of stress fibers and microtubules is apparently intimately related to the phosphorylation of p125FAK and can be modulated by soluble receptor agonists such as thrombin or via altered polymerization of microtubules.

scholarly journals Identification of integrin-stimulated sites of serine phosphorylation in FRNK, the separately expressed C-terminal domain of focal adhesion kinase: a potential role for protein kinase A

1997 ◽  
Vol 324 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Alan RICHARDSON ◽  
John D. SHANNON ◽  
Reid B. ADAMS ◽  
Michael D. SCHALLER ◽  
J. Thomas PARSONS
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Serine Phosphorylation ◽  
Early Event ◽  
Kinase A

Focal adhesion kinase (pp125FAK) is a protein tyrosine kinase that is localized to focal adhesions in many cell types and which undergoes tyrosine phosphorylation after integrin binding to extracellular matrix. In some cells the C-terminal non-catalytic domain of pp125FAK is expressed as a separate protein referred to as FRNK (FAK-related, non-kinase). We have previously shown that overexpression of FRNK inhibits tyrosine phosphorylation of pp125FAK and its substrates as well as inhibiting cell spreading on fibronectin. In this report we identify Ser148 and Ser151 as residues in FRNK that are phosphorylated after tyrosine phosphorylation of pp125FAK and in response to integrin binding to fibronectin. Tyrosine phosphorylation of pp125FAK appears to be an early event after integrin occupancy, and serine phosphorylation of FRNK occurs significantly later. Treatment of fibroblasts with a series of protein kinase A inhibitors delayed serine phosphorylation of FRNK as well as cell spreading on fibronectin and tyrosine phosphorylation of pp125FAK. However, these PKA inhibitors are unlikely to delay cell spreading simply by preventing serine phosphorylation of FRNK, as overexpression of FRNK containing mutations of Ser148 and Ser151 either singly or jointly to either alanine or glutamate residues did not significantly alter the ability of FRNK to act as an inhibitor of pp125FAK.

scholarly journals Human enhancer of filamentation 1, a novel p130cas-like docking protein, associates with focal adhesion kinase and induces pseudohyphal growth in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (7) ◽  
pp. 3327-3337 ◽  
Author(s):  
S F Law ◽  
J Estojak ◽  
B Wang ◽  
T Mysliwiec ◽  
G Kruh ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Tyrosine Kinases ◽  
Focal Adhesions ◽  
Sh3 Domain ◽  
Physical Interaction ◽  
Cell Periphery ◽  
Isolation And Characterization ◽  
Docking Protein

Budding in Saccharomyces cerevisiae follows a genetically programmed pattern of cell division which can be regulated by external signals. On the basis of the known functional conservation between a number of mammalian oncogenes and antioncogenes with genes in the yeast budding pathway, we used enhancement of pseudohyphal budding in S. cerevisiae by human proteins expressed from a HeLa cDNA library as a morphological screen to identify candidate genes that coordinate cellular signaling and morphology. In this report, we describe the isolation and characterization of human enhancer of filamentation 1 (HEF1), an SH3-domain-containing protein that is similar in structure to pl30cas, a recently identified docking protein that is a substrate for phosphorylation by a number of oncogenic tyrosine kinases. In contrast to p130cas, the expression of HEF1 appears to be tissue specific. Further, whereas p130cas is localized predominantly at focal adhesions, immunofluorescence indicates that HEF1 localizes to both the cell periphery and the cell nucleus and is differently localized in fibroblasts and epithelial cells, suggesting a more complex role in cell signalling. Through immunoprecipitation and two-hybrid analysis, we demonstrate a direct physical interaction between HEF1 and p130cas, as well as an interaction of the SH3 domain of HEF1 with two discrete proline-rich regions of focal adhesion kinase. Finally, we demonstrate that as with p130cas, transformation with the oncogene v-abl results in an increase in tyrosine phosphorylation on HEF1, mediated by a direct association between HEF1 and v-Abl. We anticipate that HEF1 may prove to be an important linking element between extracellular signalling and regulation of the cytoskeleton.

scholarly journals Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src

1994 ◽  
Vol 14 (3) ◽  
pp. 1680-1688
Author(s):  
M D Schaller ◽  
J D Hildebrand ◽  
J D Shannon ◽  
J W Fox ◽  
R R Vines ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Tyrosine Kinases ◽  
Cellular Protein ◽  
Sh2 Domain ◽  
Major Site ◽  
Sh2 Domains ◽  
High Affinity Binding Site

The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.

scholarly journals Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase.

1995 ◽  
Vol 6 (6) ◽  
pp. 637-647 ◽  
Author(s):  
J D Hildebrand ◽  
M D Schaller ◽  
J T Parsons
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Tyrosine Kinases ◽  
Focal Adhesions ◽  
Stable Complex ◽  
Suspension Cells ◽  
Terminal Domain ◽  
Carboxy Terminal

Focal adhesion kinase (pp125FAK or FAK) and paxillin colocalize with integrins in structures called focal adhesions. pp125FAK plays an important role in the transmission of integrin-induced cytoplasmic signals. Paxillin has also been implicated in cell signaling by virtue of its association with the protein tyrosine kinases pp60src and Csk (C-terminal Src kinase) as well as with the adapter/oncoprotein p47gag-crk. In this report we show that endogenous pp125FAK and paxillin form a stable complex both in vivo and in vitro and that this interaction is direct, requiring only pp125FAK and paxillin. The paxillin binding site on pp125FAK has been localized to the carboxy-terminal 148 residues of pp125FAK, but appears to be distinct from the previously identified focal adhesion-targeting sequence also present in the carboxy-terminal domain of pp125FAK. The interaction of paxillin and pp125FAK is independent of the adhesion of cells to the extracellular matrix, as the association can be detected in suspension cells as well as those attached to fibronectin.

scholarly journals Focal Adhesion Targeting: The Critical Determinant of FAK Regulation and Substrate Phosphorylation

1999 ◽  
Vol 10 (8) ◽  
pp. 2507-2518 ◽  
Author(s):  
Yu Shen ◽  
Michael D. Schaller
Keyword(s):  
Cell Adhesion ◽  
Subcellular Localization ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Wild Type ◽  
Critical Determinant ◽  
Single Region ◽  
Dependent Cell ◽  
Substrate Phosphorylation

The focal adhesion kinase (FAK) is discretely localized to focal adhesions via its C-terminal focal adhesion–targeting (FAT) sequence. FAK is regulated by integrin-dependent cell adhesion and can regulate tyrosine phosphorylation of downstream substrates, like paxillin. By the use of a mutational strategy, the regions of FAK that are required for cell adhesion–dependent regulation and for inducing tyrosine phosphorylation of paxillin were determined. The results show that the FAT sequence was the single region of FAK that was required for each function. Furthermore, the FAT sequence of FAK was replaced with a focal adhesion–targeting sequence from vinculin, and the resulting chimera exhibited cell adhesion–dependent tyrosine phosphorylation and could induce paxillin phosphorylation like wild-type FAK. These results suggest that subcellular localization is the major determinant of FAK function.

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