|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 31, 29115-29120, August 1, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Targeting Membrane-localized Focal Adhesion Kinase to Focal Adhesions
ROLES OF TYROSINE PHOSPHORYLATION AND SRC FAMILY KINASES*
¶ 
From the
Craniofacial Developmental Biology and
Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland
20892, the ||Departments of Anesthesiology, Cell
Biology, and Pediatrics, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21224, the Hematology
Institute, Tel Aviv Medical Center, Tel Aviv 64239, Israel, and the
Department of Molecular Cell Biology,
The Weizmann Institute of Science, Rehovot 76100, Israel
Received for publication, December 5, 2002 , and in revised form, May 4, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In the present study, we examined regulation of activated focal adhesion kinase localization in focal adhesions. By using focal adhesion kinase fused to an inert transmembrane anchor, we found that the focal contact targeting region within focal adhesion kinase was preserved in the membrane-targeted fusion protein. However, upon tyrosine phosphorylation, full-length focal adhesion kinase became excluded from focal adhesions. This negative regulation of localization could be abolished by mutating key amino acid residues of focal adhesion kinase shown previously to be involved in adhesion-mediated signal transduction. Hyper-phosphorylation of endogenous focal adhesion kinase induced by pervanadate resulted in a similar reduction of localization at focal adhesions. We also show here that Src family kinases are essential for the phosphorylation-dependent exclusion of focal adhesion kinase from focal adhesions. We propose here a molecular model for the tyrosine phosphorylation-dependent regulation of focal adhesion kinase organization involving Src kinases and an inhibitory phosphorylation of the C-terminal (Tyr-925) tyrosine residue.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Adhesion-mediated signaling triggers tyrosine phosphorylation of a multitude of proteins, including paxillin, tensin, and focal adhesion kinase (FAK)1 (1–4). FAK phosphorylation following adhesion depends on its association with Src family kinases, leading to the formation of multimolecular signaling complexes in which FAK serves as a scaffold (5–7). Following activation, residue Tyr-397 of FAK becomes auto-phosphorylated, resulting in the formation of an SH2-docking site (5). Src family kinases then associate with Tyr-397 and phosphorylate several other tyrosine residues in FAK, including Tyr-925 that resides within the focal adhesion targeting (FAT) sequence at the FAK C terminus (5). The adaptor protein Grb2 then binds phosphorylated Tyr-925 and forms a signaling complex that includes the nucleotide exchange factor Sos and the small GTP-binding protein Ras (5). This sequence of events contributes to activation of the ERK1/2 response (5). Two additional pathways lead to FAK-mediated activation of ERK1/2: one involves binding of the adaptor protein p130Cas to the prolinerich region of FAK via an SH3 domain interaction, and the second is the result of FAK association with the adaptor protein Shc (8–10).
The mechanisms that underlie FAK activation are not yet clear. Although FAK
lacks any myristoylation or farnesylation signals, membrane translocation of
FAK could be initiated by direct association with the 
1
integrin tail (11). Close
indirect interaction between FAK and integrin cytoplasmic tails may also be
mediated via talin or paxillin
(12,
13). Indeed, several studies
(11,
14) have demonstrated that a
membrane-translocated form of FAK (following fusion to an inert transmembrane
anchor) results in an adhesion-independent, constitutive phosphorylation of
the molecule. This phosphorylated state of FAK depends on its association with
Src and is accompanied by constitutive association with several signaling
molecules including Cas and Grb2
(11,
14). Integrin/FAK association
may be essential for adhesion-mediated signaling, which can protect cells
against apoptosis (11).
However, adhesion-mediated signaling responses are transient, pointing to the
activity of negative regulatory mechanisms
(15,
16). For example, the
adhesion-induced ERK1/2 response is rapid, reaching a peak by 15 min, followed
by a swift decay
(15–17).
Potential negative regulators of FAK activities include the phosphatases
Shp-2, PTP-PEST, and the dual specificity tumor suppressor phosphatase PTEN
(18–20).
In the present study, we examined whether membrane association of FAK affects its interaction with focal adhesions and whether such interaction is regulated by the phosphorylation state of FAK. We found that constitutively activated FAK, induced by membrane translocation, is excluded from focal adhesions of murine 3T3 and primary embryonic fibroblasts, as well as primary human fibroblasts. The exclusion of hyper-phosphorylated FAK from focal adhesions was prevented by mutating to arginine the Lys-454 residue (in the ATP-binding site, making this a kinase-crippled mutation after a Lys-to-Arg point mutation), Tyr-397 (the Src family kinase docking site), or Tyr-925 (the C-terminal Grb2 docking site). All of these residues are known to contribute to the FAK/Src family kinase signaling complex. Moreover, induction of hyper-phosphorylation by pervanadate resulted in a similar reduction of FAK at focal adhesions without affecting vinculin localization. We found that the localization of a wild-type FAK transmembrane chimera to focal adhesions improved when Src or a combination of Src, Yes, and Fyn were absent. We suggest that the hyper-phosphorylation of juxtamembrane FAK inhibits its localization to focal adhesions. Furthermore, this hyper-phosphorylation and subsequent interference with FAK localization and function is regulated by Src family kinases. This study demonstrates a novel regulatory mode of FAK subcellular localization controlled by specific tyrosine phosphorylation.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Construction of Chimeric Receptors—Chimeric transmembrane receptors were constructed using standard methods (21). The sequences of all constructs were confirmed by nucleotide sequencing, and protein expression from the constructs described in this study was confirmed following transfection by both immunofluorescence staining and Western immunoblotting. The general approach to generate a chimera with each type of cytoplasmic domain was to insert partial or full-length cDNA molecules into HindIII-XhoI or HindIII-XbaI restriction sites of the plasmid vector pCMV/IL-2R (22). This vector is driven by the CMV promoter, and it expresses the extracellular and transmembrane domains of the non-signaling
subunit of the interleukin-2 receptor (IL-2R) as
a fusion protein with any molecule of interest as the cytoplasmic domain, as
illustrated in Fig.
1A. Mouse FAK cDNA clone (pT7-7-FAK) was obtained from
the American Type Culture Collection (Manassas, VA). Full-length mouse FAK
cDNA was inserted into pCMV/IL-2R as follows. A PCR product encoding the first
500 bp of FAK cDNA was generated using pT7-7-FAK as template with a primer
containing a HindIII site followed by 15 nucleotides encoding the
first 5 amino acids of FAK (GenBankTM MUSFAK, residue 110):
5'-GGCTAGAAGCTTATGGCAGCTGCTTAT-3', and the antisense
primer 5'-GGTGCTTCTAGAGGTCTACTTGATCAGC-3' corresponding
to nucleotides 586–602 followed by an XbaI site. The PCR
product was ligated into CMV/IL-2R using its HindIII and
XbaI sites. An AccI-XbaI fragment was then removed
from the ligation product and replaced by an AccI-XbaI
fragment from pT7-7-FAK, creating a full-length IL-2R/FAK chimera that
included the FAK 3' non-translated region. The FAK454R mutant
was generated by converting codon 454 encoding lysine to an arginine residue,
and FAK397F and FAK925F were obtained by replacing the
tyrosine residue corresponding to amino acid position 397 or 925 with a
phenylalanine residue, using PCR-directed mutagenesis. Mutations were
confirmed by DNA sequencing. IL-2R/FRNK chimera was kindly provided by T.
Takino (CDBRB, NIDCR, National Institutes of Health, Bethesda).
|
Cells and Transfections—Human foreskin fibroblasts were kindly provided by Susan S. Yamada (NIDCR, National Institutes of Health, Bethesda). NIH 3T3 cells were kindly provided by J. Silvio Gutkind (NIDCR, National Institutes of Health, Bethesda). pp60c-Src null cells (src–/–), derived from mice homozygous for a disruption of the Src gene, as well as wild-type mouse embryo fibroblasts (wt), were kindly provided by P. Schwartzberg and H. Varmus (NHGRI, National Institutes of Health, Bethesda). Fibroblasts homozygous for disruption of the Src, Fyn, and Yes genes were generously provided by P. Soriano (Fred Hutchinson Cancer Center, Seattle). All cell lines were maintained in culture in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, glutamine, penicillin, streptomycin (Biological Industries, Beit Ha'emek, Israel), and 1 mM sodium pyruvate (Sigma). Electroporation of these cells with 30 µg of DNA for each chimeric IL-2R/cytoplasmic protein construct was performed as described previously at 170 V and 960 microfarads with a Bio-Rad Gene Pulser but without any thymidine block (22). Some transfections were performed using LipofectAMINE Plus (Invitrogen) utilizing standard protocols. Treatment of cells with vanadate was performed using activation to pervanadate as described (23). Briefly, vanadate was activated to pervanadate with hydrogen peroxide, and excess hydrogen peroxide was removed with catalase as described (23). The solution was added to regular culture medium at a final concentration of 0.1 mM pervanadate, and cells were fixed for immunofluorescence after incubation for 5–120 min. Controls were untreated or treated with an equal quantity of control hydrogen peroxide/catalase solution.
Immunological Reagents—Mouse monoclonal antibodies 7G7/B6
against the human IL-2 receptor subunit (Tac antigen) and
anti-phosphotyrosine 4G10 were from Upstate Biotechnology, Inc. (Lake Placid,
NY). FITC-conjugated rat anti-human IL-2 receptor antibody MCA350F was
purchased from Serotec (Raleigh, NC). Mouse anti-human vinculin monoclonal
antibody was kindly provided by V. Koteliansky (Biogen, Boston). Mouse
monoclonal antibody against FAK was purchased from Transduction Laboratories
(Lexington, KY).
Immunoprecipitation and Western Blotting—Murine 3T3 cells were transfected with the indicated plasmids, and 48 h later the cells were rinsed with PBS containing 1 mM sodium orthovanadate and lysed with RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM Tris, pH 8.0, 20 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin A, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate). Protein extracts were subjected to SDS-PAGE and Western blotting.
Indirect Immunofluorescence—Transfected cells were stained by indirect immunofluorescence as described previously (24). Cells were fixed with 4% formaldehyde in PBS for 20 min and permeabilized with 0.5% Triton X-100 in PBS for 5 min. The cells were then incubated with the indicated first (45 min) and second (45 min) antibodies in PBS. Anti-FAK, anti-vinculin or anti-phosphotyrosine antibodies were used at 10 µg/ml followed by staining with secondary Alexa- or Cy3-conjugated goat anti-mouse antiserum diluted 1:200. In some experiments, FITC-conjugated rat anti-human IL-2 receptor antibody was utilized at a 1:50 dilution.
Digital Fluorescence Image Analysis of Subcellular
Distribution— The system used for quantitative fluorescence
microscopy and image analysis is described in detail elsewhere
(25). Briefly, the specimens
were examined with a DeltaVision digital microscope system (Applied
Precision). Images of focal adhesions were segmented by the Water algorithm
following high pass filtration (subtracting from each pixel the average over 4
x 4 µm area around the particle). The parameters in Water were
adjusted to the typical dimensions of focal adhesions and were kept constant
for all the analyses. To analyze the relationships between fluorescent probes
(IL-2R or phosphotyrosine), focal adhesions were segmented separately, and
fluorescence intensities were measured for each probe in the segmented focal
adhesions. Ratio images were calculated and presented in a spectral, log
scale, color look-up table that ranged from blue for low label ratios
(
0.1) to red for high label ratios (
10). To utilize this 2
order-of-magnitude range optimally, and to compensate for the differences in
photon yields of different fluorescent labels, all the ratios were normalized
linearly by a constant that shifted their average toward a ratio value of 1.
In order to compare fluorescence intensity visually, spectrum scale
presentations of filtered fluorescence data were presented using a blue-to-red
linear spectrum scale.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Expression and Tyrosine Phosphorylation of Transmembrane FAK Chimeras—It has been established that FAK can be activated by membrane translocation to mimic its proximity to the plasma membrane in focal adhesions (11, 14). In order to examine whether the phosphorylation of specific tyrosine residues can regulate the subcellular localization of highly phosphorylated FAK, we generated a membrane-targeted form of FAK by fusing it to the non-signaling
subunit of the IL-2R as an inert transmembrane anchor
(Fig. 1A). The IL-2R
tac antigen ( subunit) was utilized to follow the specific location of
the transfected FAK molecules in cells. In addition, we constructed several FAK transmembrane chimeras mutated at residues previously shown to be involved in functions of the wild-type molecule (Fig. 1A). First, we examined the levels of FAK tyrosine phosphorylation following expression of the chimeras in murine 3T3 cells. Previous studies indicated that membrane translocation was sufficient to induce FAK tyrosine phosphorylation even when the cells were maintained in suspension (11, 14). In agreement with these studies, we found that membrane-translocated FAK becomes hyper-phosphorylated (Fig. 1B). As reported previously, mutations of residues K454R (kinase-mutated) and Y397F (inactive Src family SH2 docking site) significantly reduced FAK hyper-phosphorylation (Fig. 1B) (11, 14). Mutating residue Tyr-925 had no apparent effect on the overall level of FAK phosphorylation (Fig. 1B). In concurrence with previous studies, we observed trans-phosphorylation of endogenous cytoplasmic FAK in cells expressing highly phosphorylated wild type FAK or Y925F chimeras (Fig. 1B) (14).
Subcellular Localization of Hyper-phosphorylated FAK and Its
Mutants—Our previous studies indicated that transmembrane chimeras
containing integrin tails or cytoskeletal molecules (e.g.
vinculin) as their cytoplasmic fusions can localize to focal adhesions, in a
similar manner to their wild-type counterparts
(Fig. 2)
(22). We tested whether the
FAK C-terminal segment designated the focal adhesion
targeting (FAT) region is indeed functional when located close to
the plane of the membrane. Therefore, we fused the FAK C terminus
corresponding to the FRNK region to IL-2R
(Fig. 1A). When
expressed in human foreskin fibroblasts, this IL-2R/FRNK chimera localized to
focal adhesions (not shown).
|
However, as shown in Figs. 2 and 3, IL-2R/FAK that contains the full-length FAK molecule did not localize to focal adhesions of primary human fibroblasts. Lack of IL-2R/FAK localization was also observed in murine 3T3 cells and primary murine fibroblasts (not shown). In a similar manner, the IL-2R molecule without any cytoplasmic tail that served as a negative control was diffusely distributed on the cell membrane in agreement with previous studies (Fig. 2) (22). The inability of the FAK chimera to localize was not due to destruction of cell-matrix adhesion sites, as confirmed by double-labeling the transfected cells with anti-vinculin antibody, demonstrating that focal adhesions persist in the transfected cells (Fig. 3). The lack of hyper-phosphorylated FAK localization has been reported previously and may depend on the cell type being examined (11). One possibility was that FAK turnover within focal adhesions might decrease, thereby reducing the rate of incorporation of the chimeric form into already-established focal adhesions. To test this hypothesis, the transfected cells were detached and replated on fibronectin-coated coverslips. After 10, 30, 60, and 120 min, the cells were fixed, permeabilized, and double-stained for the IL-2R portion of the chimera and for vinculin. These double-localization analyses demonstrated that even though focal adhesions were formed in the transfected cells (as confirmed by vinculin staining), the chimera was not incorporated into these structures (not shown). This analysis indicates that the IL-2R/FAK chimera is specifically excluded from focal adhesions.
|
To test whether the localization of endogenous FAK is also sensitive to its tyrosine phosphorylation state, cells were treated with pervanadate for 5, 15, 30, 60, or 120 min; they were then fixed, permeabilized, and double-stained with anti-Tyr-397-phosphorylated FAK and anti-FAK or with anti-Tyr-397-phosphorylated FAK and anti-vinculin. At the earliest time point examined (5 min), a substantial increase in diffusely localized Tyr-397-phosphorylated FAK was observed concomitant with a loss of hyper-phosphorylated FAK from focal adhesions that became striking by 15–30 min (Fig. 4). Because phospho-FAK remaining in focal adhesions might theoretically have been masked by the large increases in general cytoplasmic phospho-FAK staining, we also compared the localization of total FAK protein after pervanadate treatment. Total FAK localized to focal adhesions was also substantially reduced, whereas double-staining with anti-vinculin confirmed that the focal adhesions remained intact (Fig. 4). These analyses of endogenous FAK confirm that the amount of FAK in focal adhesions is reduced upon its hyper-phosphorylation. In order to examine the regulation of FAK localization to focal adhesions by specific tyrosine phosphorylation, we transfected human foreskin fibroblasts with IL-2R chimeras containing FAK mutated at the K454R residue (a kinase active-site mutation), Y397F (the Src family kinase docking site), or Y925F (the Grb2 docking site). As shown in Figs. 2 and 3, the IL-2R/FAK Y397F, K454R, and Y925F mutant chimeras localized properly to focal adhesions of these cells. Identical focal adhesion localization patterns were detected with the FAK-mutated chimeras in murine 3T3 fibroblasts and murine embryonic fibroblasts (not shown).
|
Regulation of FAK Localization to Focal Adhesions by Src Family
Kinases—Src family kinases are important for generating
adhesion-mediated tyrosine phosphorylation of FAK
(5,
7). We hypothesized that the
presence of Src family kinases may be necessary for regulating FAK association
with focal adhesions. Therefore, we transfected the wild-type IL-2R/FAK
chimera into primary embryonic murine fibroblasts, which served as the control
for null fibroblasts. As demonstrated for primary human fibroblasts and murine
3T3 cells, the chimera did not localize to the focal adhesions of these cells
(not shown). However, when transfected into Src-deficient fibroblasts (Src
–/–) the wild type IL-2R/FAK chimera localized to focal adhesions
in 
100% of the transfected cells (not shown).
Since Src family kinases Fyn and Yes may also bind to and phosphorylate
FAK, we transfected the wild type IL-2R/FAK chimera into fibroblasts obtained
from triple Src family null embryos (SYF –/–), and we examined its
subcellular localization. In this experiment, the wild-type IL-2R/FAK chimera
(as well as FAK mutants chimeras) localized effectively to focal adhesions of
89% of the transfected cells (Fig.
5). FAK mutant chimeras (e.g. FAK K454R) also
demonstrated similar localization to focal adhesions of SYF –/–
cells (Fig. 5).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Appropriate adhesion-mediated signaling responses require tight regulatory mechanisms. Indeed, signaling responses mediated by focal adhesion kinase have been shown to be activated by protein tyrosine kinases (e.g. Src family kinases) or down-regulated by specific phosphatases (e.g. Shp-2 and PTEN) (5, 7, 18–20). However, adhesion-mediated signaling may be mediated by a variety of molecularly and structurally distinct cell-matrix adhesion sites that include focal adhesions, fibrillar adhesions, three-dimensional matrix adhesions, and podosomes (4, 27, 29). In fact, cell-matrix adhesion sites not only mediate physical association of cells with the extracellular matrix but may also function as specialized sites that initiate and regulate signaling responses (30–32). Although focal adhesion targeting regions have been identified in several cytoskeletal and signaling molecules, including FAK, the regulation of these regions still remains to a large extent unclear. One interesting exception is vinculin. It was shown that vinculin/talin and vinculin/actin associations are regulated by the intramolecular interactions of the vinculin N-terminal head domain with its C-terminal tail (33, 34). Phosphatidylinositol 4,5-bisphosphate may govern the vinculin head/tail association, thereby controlling its molecular associations within focal adhesions (33, 34). Mutations in the proline-rich region of vinculin, which links the head and neck domains, may affect the localization of the molecule (35).
In the present study, we evaluated for the first time the regulation of
focal adhesion targeting of FAK by specific tyrosine phosphorylation. We also
tested the contribution of Src family kinases to the regulation of FAK
targeting to focal adhesions. Following association with the phosphorylated
FAK Tyr-397 residue, Src family kinases phosphorylate additional tyrosine
residues on FAK (5). Tyr-925 is
one of several tyrosine residues reported to be phosphorylated by Src family
kinases, generating a docking site for the adaptor protein Grb2 and a
signaling complex, resulting in activation of the ERK1/2 response
(5). It is interesting to note
that Tyr-925 resides within the region defined as the FAT sequence of FAK
(Fig. 1)
(36). Recent studies have
indicated that in some cell types, FAK molecules that reside close to the
plasma membrane are phosphorylated at residue Tyr-397. These molecules,
however, are predominantly diffusely distributed, and only a minor fraction is
localized in specific domains
(37). In contrast, the
predominant in vivo state of residue Tyr-925 is apparently
unphosphorylated (26).
Although focal adhesions of cells in vitro contain high levels of
tyrosine-phosphorylated molecules, non-phosphorylated or partially
phosphorylated FAK molecules appear to be the predominant form of this
molecule associated with matrix adhesions in vivo
(29). Tyr-397-phosphorylated
FAK does not localize to "three-dimensional matrix adhesions" in
fibroblasts attaching to three-dimensional matrices in vitro,
although it can be found in small focal complexes. Similarly,
5 integrin co-localizes with FAK in mouse embryonic tissue
sections but not with Tyr-397-phosphorylated FAK
(29).
We speculate that hyper-phosphorylated FAK is excluded from focal adhesions for a variety of reasons. Phosphorylation that includes Tyr-925 may generate a constitutive association with Grb2, as shown in previous studies (11). The constitutive signaling complex might sterically block the focal adhesion targeting site, thereby inhibiting the association of activated FAK with focal adhesions (Fig. 6). Alternatively, the phosphorylation per se could have an inhibitory effect. The fact that the highly phosphorylated IL-2R/FAK Y925F mutant does localize to focal adhesions indicates that inhibition of FAK incorporation into focal adhesions does not stem merely from total tyrosine phosphorylation of the molecule but from the phosphorylation of specific residue(s). As depicted in Fig. 1A, Tyr-925 resides within the FAT segment of FAK. Therefore, the specific tyrosine phosphorylation of the Tyr-925 residue may be important for the regulation of FAK localization to focal adhesions. An additional mechanism that may explain the failure of highly phosphorylated FAK to localize to focal adhesions requires a higher affinity association of hyper-phosphorylated FAK with other molecular complexes, the FAK "scaffold." These complexes may be tied up elsewhere within the cytoplasm, and the off-rates of these interactions significantly altered such that availability of FAK for standard focal adhesion targeting (e.g. via paxillin and talin) is decreased. Alternatively, the constitutively hyper-phosphorylated FAK may have more restricted mobility in the plane of the membrane due to interactions (directly or indirectly via phosphatidylinositol phosphate) with membrane lipoproteins.
|
The common denominator of these putative mechanisms is the basic principle
that tyrosine residues on FAK may function as molecular binary switches
("transistors"), cycling between states of reactivity according to
their phosphorylation level. Tyr-397 is trans-phosphorylated by nearby FAK
molecules and thus is regulated by the local density of the molecules
localized at the plasma membrane (Fig.
6). Phosphorylation at this site determines whether FAK will bind
Src family kinases and possibly other molecules, e.g. phospholipase
C
(28). Src family
kinases expand the biochemical signal to additional tyrosine residues,
including Tyr-925 (Fig. 6).
Phosphorylation of Tyr-925 may require its molecular association with an Src
family kinase domain. Such association may be transient, but at high molecular
density of FAK and Src family molecules close to the inner plasma membrane
plane it may become more continuous. This specific state is achieved by use of
the chimeric FAK, which may represent the early stages of cell adhesion, where
FAK is recruited to the plasma membrane via direct and/or indirect
interactions with integrin tails. A previous study
(11) indicated that
anti-apoptotic signals, mediated by cell adhesion, may be inhibited by
peptides that inhibit direct interactions between FAK and integrin tails. This
inhibition was effective only during the early stages of cell adhesion
(11).
Therefore, several tyrosine residues of FAK may have a dual function as follows: 1) regulation of biochemical signaling responses, by directly controlling FAK enzymatic activity and its associations with signaling molecules; and 2) regulation of FAK localization by modulation of intermolecular and/or intramolecular interactions. This concept may apply to other molecules as well. The two regulatory modes may be linked to an integrated mode combining the subcellular localization of active signaling complexes. In the case of FAK, hyper-phosphorylated molecules on specific tyrosine residues may be selectively excluded from cell-matrix adhesion sites.
|
FOOTNOTES |
|---|
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** Recipient of National Institutes of Health Grant HL03299, a Michael
Visiting Professorship from the Weizmann Institute, and Fogarty Senior
International Fellowship F06TW02341.
The E. Neter Chair in Cell and Tumor Biology.
¶ To whom correspondence should be addressed: Hematology Institute, Tel Aviv Medical Center, 6 Weizman St., Tel Aviv, Israel. Tel.: 972-3-6973517; Fax: 972-3-6974452.
1 The abbreviations used are: FAK, focal adhesion kinase; ERK, extracellular
signal-regulated kinase; FAT, focal adhesion targeting; FITC, fluorescein
isothiocyanate; PBS, phosphate-buffered saline; IL-2, interleukin-2; IL-2R,
interleukin-2 receptor; SH, Src homology; CMV, cytomegalovirus.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
- Vuori, K. (1998) J. Membr. Biol. 165, 191–199[CrossRef][Medline] [Order article via Infotrieve]
- Hanks, S. K., Calalb, M. B., Harper, M. C., and Patel, S. K.
(1992) Proc. Natl. Acad. Sci. U. S. A.
89,
8487–8491
[Abstract/Free Full Text] - Burridge, K., Turner, C. E., and Romer, L. H. (1992)
J. Cell Biol. 119,
893–903
[Abstract/Free Full Text] - Zamir, E., and Geiger, B. (2001) J. Cell Sci. 114, 3583–3590[Medline] [Order article via Infotrieve]
- Schlaepfer, D. D., Hanks, S. K., Hunter, T., and van der Geer, P. (1994) Nature 372, 786–791[Medline] [Order article via Infotrieve]
- Schaller, M. D. (2001) Biochim. Biophys. Acta 1540, 1–21[Medline] [Order article via Infotrieve]
- Schlaepfer, D. D., Hauck, C. R., and Sieg, D. J. (1999) Prog. Biophys. Mol. Biol. 71, 435–478[CrossRef][Medline] [Order article via Infotrieve]
- Gu, J., Tamura, M., Pankov, R., Danen, E. H., Takino, T.,
Matsumoto, K., and Yamada, K. M. (1999) J. Cell
Biol. 146,
389–403
[Abstract/Free Full Text] - Schlaepfer, D. D., and Hunter, T. (1996) Mol. Cell. Biol. 16, 5623–5633[Abstract]
- Ruest, P. J., Shin, N. Y., Polte, T. R., Zhang, X., and Hanks, S.
K. (2001) Mol. Cell. Biol.
21,
7641–7652
[Abstract/Free Full Text] - Frisch, S. M., Vuori, K., Ruoslahti, E., and Chan-Hui, P. Y.
(1996) J. Cell Biol.
134,
793–799
[Abstract/Free Full Text] - Chen, H. C., Appeddu, P. A., Parsons, J. T., Hildebrand, J. D.,
Schaller, M. D., and Guan, J. L. (1995) J. Biol.
Chem. 270,
16995–16999
[Abstract/Free Full Text] - Hayashi, I., Vuori, K., and Liddington, R. C. (2002) Nat. Struct. Biol. 9, 101–106[CrossRef][Medline] [Order article via Infotrieve]
- Chan, P. Y., Kanner, S. B., Whitney, G., and Aruffo, A.
(1994) J. Biol. Chem.
269,
20567–20574
[Abstract/Free Full Text] - Howe, A. K., Aplin, A. E., and Juliano, R. L. (2002) Curr. Opin. Genet. & Dev. 12, 30–35[CrossRef][Medline] [Order article via Infotrieve]
- Assoian, R. K., and Schwartz, M. A. (2002) Curr. Opin. Genet. & Dev. 11, 48–53
- Miyamoto, S., Teramoto, H., Coso, O. A., Gutkind, J. S., Burbelo,
P. D., Akiyama, S. K., and Yamada, K. M. (1995). J.
Cell Biol. 131,
791–805
[Abstract/Free Full Text] - Miao, H., Burnett, E., Kinch, M., Simon, E., and Wang, B. (2000) Nat. Cell Biol. 2, 62–69[CrossRef][Medline] [Order article via Infotrieve]
- Davidson, D., and Veillette, A. (2001) EMBO J. 20, 3414–3426[CrossRef][Medline] [Order article via Infotrieve]
- Tamura, M., Gu, J., Matsumoto, K., Aota, S., Parsons, R., and
Yamada, K. M. (1998) Science
280,
1614–1617
[Abstract/Free Full Text] - Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
- LaFlamme, S. E., Akiyama, S. K., and Yamada, K. M.
(1992) J. Cell Biol.
117,
437–447
[Abstract/Free Full Text] - Huyer, G., Liu, S., Kelly, J., Moffat, J., Payette, P., Kennedy,
B., Tsaprailis, G., Gresser, M. J., and Ramachandran, C. (1997)
J. Biol. Chem. 272,
843–851
[Abstract/Free Full Text] - Volberg, T., Romer, L., Zamir, E., and Geiger, B. (2001) J. Cell Sci. 114, 2279–2289[Medline] [Order article via Infotrieve]
- Zamir, E., Katz, B.-Z., Aota, S.-I., Yamada, K. M., Geiger, B., and Kam, Z. (1999) J. Cell Sci. 112, 1655–1669[Abstract]
- Nakamura, K., Yano, H., Schaefer, E., and Sabe, H. (2001) Oncogene 20, 2626–2635[CrossRef][Medline] [Order article via Infotrieve]
- Geiger, B., Bershadsky, A., Pankov, R., and Yamada, K. M. (2001) Nat. Rev. Mol. Cell Biol. 2, 793–805[CrossRef][Medline] [Order article via Infotrieve]
- Zhang, X., Chattopadhyay, A., Ji, Q. S., Owen, J. D., Ruest, P. J.,
Carpenter, G., and Hanks, S. K. (1999) Proc. Natl.
Acad. Sci. U. S. A. 96,
9021–9026
[Abstract/Free Full Text] - Cukierman, E., Pankov, R., Stevens, D. R., and Yamada, K. M.
(2001) Science
294,
1708–1712
[Abstract/Free Full Text] - Almeida, E. A., Ilic, D., Han, Q., Hauck, C. R., Jin, F.,
Kawakatsu, H., Schlaepfer, D. D., and Damsky, C. H. (2000)
J. Cell Biol. 149,
741–754
[Abstract/Free Full Text] - Calderwood, D. A., Shattil, S. J., Ginsberg, M. H.
(2000) J. Biol. Chem.
275,
22607–22610
[Free Full Text] - Miyamoto, S., Katz, B.-Z., Lafrenie, R. M., and Yamada, K. M. (1998) Ann. N. Y. Acad. Sci. 857, 119–129[CrossRef][Medline] [Order article via Infotrieve]
- Johnson, R. P., and Craig, S. W. (1995) Nature 373, 261–264[CrossRef][Medline] [Order article via Infotrieve]
- Johnson, R. P., and Craig, S. W. (2000) J.
Biol. Chem. 275,
95–105
[Abstract/Free Full Text] - Craig, S. W., and Johnson, R. P. (1996) Curr. Opin. Cell Biol. 8, 74–85[CrossRef][Medline] [Order article via Infotrieve]
- Shen, Y., and Schaller, M. D. (1999) Mol.
Biol. Cell. 10,
2507–2518
[Abstract/Free Full Text] - Ilic, D., Genbacev, O., Jin, F., Caceres, E., Almeida, E. A.,
Bellingard-Dubouchaud, V., Schaefer, E. M., Damsky, C. H., and Fisher, S. J.
(2001) Am. J. Pathol.
159,
93–108
[Abstract/Free Full Text]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  J. A. Quinn, C. T. Graeber, A. R. Frackelton Jr., M. Kim, J. E. Schwarzbauer, and E. J. Filardo Coordinate Regulation of Estrogen-Mediated Fibronectin Matrix Assembly and Epidermal Growth Factor Receptor Transactivation by the G Protein-Coupled Receptor, GPR30 Mol. Endocrinol., July 1, 2009; 23(7): 1052 - 1064. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Waschbusch, S. Born, V. Niediek, N. Kirchgessner, I. Y. Tamboli, J. Walter, R. Merkel, and B. Hoffmann Presenilin 1 Affects Focal Adhesion Site Formation and Cell Force Generation via c-Src Transcriptional and Posttranslational Regulation J. Biol. Chem., April 10, 2009; 284(15): 10138 - 10149. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. C Burghardt, J. R Burghardt, J. D Taylor II, A. T Reeder, B. T Nguen, T. E Spencer, K. J Bayless, and G. A Johnson Enhanced focal adhesion assembly reflects increased mechanosensation and mechanotransduction at maternal-conceptus interface and uterine wall during ovine pregnancy Reproduction, March 1, 2009; 137(3): 567 - 582. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Hayashida, M.-H. Wu, A. Pierce, A.-C. Poncelet, J. Varga, and H. W. Schnaper MAP-kinase activity necessary for TGFbeta1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397 J. Cell Sci., December 1, 2007; 120(23): 4230 - 4240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Papp, M. P. Fadel, H. Kim, C. A. McCulloch, and M. Opas Calreticulin Affects Fibronectin-based Cell-Substratum Adhesion via the Regulation of c-Src Activity J. Biol. Chem., June 1, 2007; 282(22): 16585 - 16598. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Cetin, C. L. Leaphart, J. Li, I. Ischenko, M. Hayman, J. Upperman, R. Zamora, S. Watkins, H. R. Ford, J. Wang, et al. Nitric oxide inhibits enterocyte migration through activation of RhoA-GTPase in a SHP-2-dependent manner Am J Physiol Gastrointest Liver Physiol, May 1, 2007; 292(5): G1347 - G1358. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Lim, H. Park, J. Jeon, I. Han, J. Kim, E.-H. Jho, and E.-S. Oh Focal Adhesion Kinase Is Negatively Regulated by Phosphorylation at Tyrosine 407 J. Biol. Chem., April 6, 2007; 282(14): 10398 - 10404. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Vicente-Manzanares, J. Zareno, L. Whitmore, C. K. Choi, and A. F. Horwitz Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells J. Cell Biol., February 26, 2007; 176(5): 573 - 580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Boulter, D. Grall, S. Cagnol, and E. Van Obberghen-Schilling Regulation of cell-matrix adhesion dynamics and Rac-1 by integrin linked kinase FASEB J, July 1, 2006; 20(9): 1489 - 1491. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. H. Romer, K. G. Birukov, and J. G.N. Garcia Focal Adhesions: Paradigm for a Signaling Nexus Circ. Res., March 17, 2006; 98(5): 606 - 616. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ballestrem, N. Erez, J. Kirchner, Z. Kam, A. Bershadsky, and B. Geiger Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer J. Cell Sci., March 1, 2006; 119(5): 866 - 875. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. V. Usatyuk and V. Natarajan Regulation of reactive oxygen species-induced endothelial cell-cell and cell-matrix contacts by focal adhesion kinase and adherens junction proteins Am J Physiol Lung Cell Mol Physiol, December 1, 2005; 289(6): L999 - L1010. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Hamadi, M. Bouali, M. Dontenwill, H. Stoeckel, K. Takeda, and P. Ronde Regulation of focal adhesion dynamics and disassembly by phosphorylation of FAK at tyrosine 397 J. Cell Sci., October 1, 2005; 118(19): 4415 - 4425. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Kiely, M. Leahy, D. O'Gorman, and R. O'Connor RACK1-mediated Integration of Adhesion and Insulin-like Growth Factor I (IGF-I) Signaling and Cell Migration Are Defective in Cells Expressing an IGF-I Receptor Mutated at Tyrosines 1250 and 1251 J. Biol. Chem., March 4, 2005; 280(9): 7624 - 7633. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. M. Fonseca, R. Y. Inoue, C. B. Kobarg, D. P. Crosara-Alberto, J. Kobarg, and K. G. Franchini Targeting to C-Terminal Myosin Heavy Chain May Explain Mechanotransduction Involving Focal Adhesion Kinase in Cardiac Myocytes Circ. Res., January 7, 2005; 96(1): 73 - 81. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Zhang, G. Izaguirre, S.-Y. Lin, H. Y. Lee, E. Schaefer, and B. Haimovich The Phosphorylation of Vinculin on Tyrosine Residues 100 and 1065, Mediated by Src Kinases, Affects Cell Spreading Mol. Biol. Cell, September 1, 2004; 15(9): 4234 - 4247. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |