scholarly journals Identification of Tyr-397 as the primary site of tyrosine phosphorylation and pp60src association in the focal adhesion kinase, pp125FAK.

1995 ◽  
Vol 15 (5) ◽  
pp. 2819-2827 ◽  
Author(s):  
B L Eide ◽  
C W Turck ◽  
J A Escobedo
Keyword(s):  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Consensus Sequence ◽  
Sh2 Domain ◽  
Protein Tyrosine ◽  
Adhesion Plaques

A number of cellular processes, such as proliferation, differentiation, and transformation, are regulated by cell-extracellular matrix interactions. Previous studies have identified a novel tyrosine kinase, the focal adhesion kinase p125FAK, as a component of cell adhesion plaques. p125FAK was identified as a 125-kDa tyrosine-phosphorylated protein in cells transformed by the v-src oncogene. p125FAK is an intracellular protein composed of three domains: a central domain with homology to protein tyrosine kinases, flanked by two noncatalytic domains of 400 amino acids which bear no significant homology to previously cloned proteins. p125FAK is believed to play an important regulatory role in cell adhesion because it localizes to cell adhesion plaques and because its phosphorylation on tyrosine residues is regulated by binding of cell surface integrins to the extracellular matrix. Recent studies have shown that Src, through its SH2 domain, stably associates with pp125FAK and that this association prevents dephosphorylation of pp125FAK in vitro by protein tyrosine phosphatases. In this report, we identify Tyr-397 as the primary in vivo and in vitro site of p125FAK tyrosine phosphorylation and association with Src. Substituting phenylalanine for tyrosine at position 397 significantly reduces p125FAK tyrosine phosphorylation and association with Src but does not abolish p125FAK kinase activity. In addition, p125FAK kinase is able to trans-phosphorylate Tyr-397 in vitro in a kinase-deficient p125FAK variant. Phosphorylation of Tyr-397 provides a site [Y(P)AEI] that fits the consensus sequence for the binding of Src.

Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: identification of a vinculin and pp125Fak-binding region

1994 ◽  
Vol 107 (6) ◽  
pp. 1583-1591 ◽  
Author(s):  
C.E. Turner ◽  
J.T. Miller
Keyword(s):  
Cell Adhesion ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Growth Control ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Cytoskeletal Protein ◽  
Binding Motifs ◽  
Amino Terminal

Paxillin is a cytoskeletal protein involved in actin-membrane attachment at sites of cell adhesion to the extracellular matrix. Extensive tyrosine phosphorylation of this protein occurs during integrin-mediated cell adhesion, embryonic development, fibroblast transformation and following stimulation of cells by mitogens that operate through the family of seven membrane-spanning G-protein-coupled receptors. Paxillin binds in vitro to the focal adhesion protein vinculin as well as to the SH3 domain of c-src and, when tyrosine phosphorylated, to the SH2 domain of v-crk. Here, we report the complementary DNA, and derived amino acid sequence, that codes for approximately 90% of the paxillin protein. We have identified a region in the amino-terminal half of the protein that supports the binding of both vinculin and the focal adhesion tyrosine kinase, pp125Fak. Although there is no significant overall homology with other identified proteins, the carboxyl third of paxillin contains one LIM domain and three LIM-like sequences. The LIM motif is common to a number of transcription factors and to two other focal adhesion proteins, zyxin and cysteine-rich protein. In addition to several potential tyrosine phosphorylation sites there are five tyrosine-containing sequences that conform to SH2-binding motifs. The protein also contains a short proline-rich region indicative of a SH3-binding domain. Taken together, these data suggest that paxillin is a unique cytoskeletal protein capable of interaction with a variety of intracellular signalling, and structural, molecules important in growth control and the regulation of cytoskeletal organization.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Candida albicans Expresses a Focal Adhesion Kinase-Like Protein That Undergoes Increased Tyrosine Phosphorylation upon Yeast Cell Adhesion to Vitronectin and the EA.hy 926 Human Endothelial Cell Line

2002 ◽  
Vol 70 (7) ◽  
pp. 3804-3815 ◽  
Author(s):  
Giorgio Santoni ◽  
Roberta Lucciarini ◽  
Consuelo Amantini ◽  
Jordan Jacobelli ◽  
Elisabetta Spreghini ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Candida Albicans ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Yeast Cell ◽  
Focal Adhesion Kinase ◽  
Cell Line ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Human Endothelial Cell Line

ABSTRACT The signaling pathways triggered by adherence of Candida albicans to the host cells or extracellular matrix are poorly understood. We provide here evidence in C. albicans yeasts of a p105 focal adhesion kinase (Fak)-like protein (that we termed CaFak), antigenically related to the vertebrate p125Fak, and its involvement in integrin-like-mediated fungus adhesion to vitronectin (VN) and EA.hy 926 human endothelial cell line. Biochemical analysis with different anti-chicken Fak antibodies identified CaFak as a 105-kDa protein and immunofluorescence and cytofluorimetric analysis on permeabilized cells specifically stain C. albicans yeasts; moreover, confocal microscopy evidences CaFak as a cytosolic protein that colocalizes on the membrane with the integrin-like VN receptors upon yeast adhesion to VN. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A strongly inhibited C. albicans yeast adhesion to VN and EA.hy 926 endothelial cells. Moreover, engagement of αvβ3 and αvβ5 integrin-like on C. albicans either by specific monoclonal antibodies or upon adhesion to VN or EA.hy 926 endothelial cells stimulates CaFak tyrosine phosphorylation that is blocked by PTK inhibitor. A role for CaFak in C. albicans yeast adhesion was also supported by the failure of VN to stimulate its tyrosine phosphorylation in a C. albicans mutant showing normal levels of CaFak and VNR-like integrins but displaying reduced adhesiveness to VN and EA.hy 926 endothelial cells. Our results suggest that C. albicans Fak-like protein is involved in the control of yeast cell adhesion to VN and endothelial cells.

scholarly journals Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains.

1995 ◽  
Vol 130 (5) ◽  
pp. 1181-1187 ◽  
Author(s):  
M D Schaller ◽  
C A Otey ◽  
J D Hildebrand ◽  
J T Parsons
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Cytoplasmic Domain ◽  
Embryo Cell ◽  
Vitro Assay ◽  
Protein Tyrosine ◽  
Signaling Complex

The integrins have recently been implicated in signal transduction. A likely mediator of integrin signaling is focal adhesion kinase (pp125FAK or FAK), a structurally distinct protein tyrosine kinase that becomes enzymatically activated upon engagement of integrins with their ligands. A second candidate signaling molecule is paxillin, a focal adhesion associated, cytoskeletal protein that coordinately becomes phosphorylated on tyrosine upon activation of pp125FAK. Paxillin physically complexes with two protein tyrosine kinases, pp60src and Csk (COOH-terminal src kinase), and the oncoprotein p47gag-crk, each of which could function as part of a paxillin signaling complex. Using an in vitro assay we have established that the cytoplasmic domain of the beta 1 integrin can bind to paxillin and pp125FAK from chicken embryo cell lysates. The NH2-terminal, noncatalytic domain of pp125FAK can bind directly to the cytoplasmic tail of beta 1 and recognizes integrin sequences distinct from those involved in binding to alpha-actinin. Paxillin binding is independent of pp125FAK binding despite the fact that both bind to the same region of beta 1. These results demonstrate that the cytoplasmic domain of the beta subunits of integrins contain binding sites for both signaling molecules and structural proteins suggesting that integrins can coordinate the generation of cytoplasmic signals in addition to their role in anchoring components of the cytoskeleton.

scholarly journals Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

1997 ◽  
Vol 17 (3) ◽  
pp. 1702-1713 ◽  
Author(s):  
D D Schlaepfer ◽  
M A Broome ◽  
T Hunter
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Intracellular Signaling ◽  
Protein Tyrosine Kinase ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Sh2 Domain ◽  
Mitogen Activated Protein Kinase

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.

scholarly journals Minimal features of paxillin that are required for the tyrosine phosphorylation of focal adhesion kinase

2005 ◽  
Vol 393 (2) ◽  
pp. 565-573 ◽  
Author(s):  
Ramon Wade ◽  
Scott Vande Pol
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Embryonic Stem ◽  
Adaptor Protein ◽  
Structural Features ◽  
Lim Domains

Tyrosine phosphorylation of FAK (focal adhesion kinase) regulates signalling that results from the interaction of integrins with extracellular matrix and growth factor receptors. A critical step in this process is the phosphorylation of Tyr397 of FAK, which creates a binding site for Src family kinases, PI3K (phosphoinositide 3-kinase) and Shc (Src homology and collagen homology). An intact Tyr397 site is required for FAK-mediated regulation of cell migration, survival signals and full responsiveness to soluble growth factors. We showed previously that the adaptor protein paxillin is required for the overall tyrosine phosphorylation of FAK in embryonic stem cells [Wade, Bohl and Vande Pol (2002) Oncogene 21, 96–107]. In the present paper, we identify the minimal structural features of paxillin that are required to support overall FAK tyrosine phosphorylation and Tyr397 phosphorylation. Paxillin contains N-terminal leucine-rich LD motifs that bind directly to FAK and four LIM (Lin-11, Isl-1 and Mec-3) domains in the C-terminus. We show that paxillin LIM domains 1, 2 and 3 are each required for FAK tyrosine phosphorylation, while LIM4 is dispensable. In addition to paxillin LIM domains 1, 2 and 3, a single LD motif on paxillin is required to support FAK tyrosine phosphorylation in embryonic stem cells. Both sequence and spatial requirements exist for LD motifs to support FAK tyrosine phosphorylation. Interestingly, synthetic LD motifs that fail to bind FAK in vitro are able to fully support FAK tyrosine phosphorylation, indicating that minimal interactions of LD motifs with FAK suffice. Our results demonstrate at least four distinct structural domains of paxillin support at least three distinct functions that are each required for FAK tyrosine phosphorylation.

scholarly journals Cloning and Characterization of Cell Adhesion Kinase β, a Novel Protein-tyrosine Kinase of the Focal Adhesion Kinase Subfamily

1995 ◽  
Vol 270 (36) ◽  
pp. 21206-21219 ◽  
Author(s):  
Hiroko Sasaki ◽  
Kazuko Nagura ◽  
Masaho Ishino ◽  
Hirotoshi Tobioka ◽  
Kiyoshi Kotani ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Tyrosine Kinase ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Protein Tyrosine Kinase ◽  
Protein Tyrosine ◽  
Kinase Subfamily ◽  
Novel Protein

scholarly journals Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

1994 ◽  
Vol 5 (4) ◽  
pp. 413-421 ◽  
Author(s):  
Z Xing ◽  
H C Chen ◽  
J K Nowlen ◽  
S J Taylor ◽  
D Shalloway ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Intracellular Signaling ◽  
Direct Interaction ◽  
Sh2 Domain ◽  
Sh2 Domains ◽  
Intracellular Signaling Molecules

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.

scholarly journals Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation.

1994 ◽  
Vol 14 (8) ◽  
pp. 5249-5258 ◽  
Author(s):  
C Couture ◽  
G Baier ◽  
C Oetken ◽  
S Williams ◽  
D Telford ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Molecular Mechanisms ◽  
Sh2 Domain ◽  
Lymphoid Cells ◽  
Intact Cells ◽  
Protein Tyrosine

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.

scholarly journals Characterization of an activated mutant of focal adhesion kinase: ‘SuperFAK’

2002 ◽  
Vol 365 (3) ◽  
pp. 591-603 ◽  
Author(s):  
Veronica GABARRA-NIECKO ◽  
Patricia J. KEELY ◽  
Michael D. SCHALLER
Keyword(s):  
Catalytic Activity ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Human Cancer ◽  
Point Mutations ◽  
Enzymic Activity ◽  
Wild Type

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in normal cellular processes such as adhesion, spreading, migration, proliferation and survival. In addition, FAK is overexpressed in a variety of cancer cells and tumours and may play a role in the development of human cancer. As a prelude to modelling the role of aberrant FAK signalling in the initiation of cancer, the goal of the present study was to engineer point mutations in FAK that would enhance enzymic activity. A number of substitutions that were reported as activating mutations in other tyrosine kinases were introduced into FAK. Glutamic acid substitutions for two lysine residues in the activation loop of FAK, based upon the K650E (Lys650→Glu) mutant of fibroblast-growth-factor receptor 3, were made to create ‘SuperFAK'. Two brain-specific exons were engineered into avian FAK to create FAK6.7. SuperFAK and, to a lesser extent, FAK6.7, exhibited increased catalytic activity in vitro compared with wild-type FAK. The expression of SuperFAK and FAK6.7 in fibroblasts led to hyperphosphorylation of FAK substrates. Although the catalytic activity of SuperFAK and FAK6.7 was largely independent of cell adhesion, tyrosine phosphorylation of downstream substrates was adhesion-dependent. Further, since SuperFAK exhibited the same ability as wild-type FAK to recruit Src family kinases, tyrosine phosphorylation of substrates was likely due to direct phosphorylation by FAK. In addition to enhanced biochemical signalling, SuperFAK also increased the motility of epithelial cells. SuperFAK and FAK6.7 may be valuable molecular tools to investigate the potential role of aberrant FAK signalling in human disease.

Sign in / Sign up

Export Citation Format

Share Document