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Departments of Cell Biology [P. J. R., S. R., S. K. H.] Biochemistry [R. L. M.], and The Vanderbilt-Ingram Cancer Center [E. S., R. L. M., S. K. H], Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Abstract
Focal adhesion kinase (FAK) is a key signaling molecule regulating cellular responses to integrin-mediated adhesion. Integrin engagement promotes FAK phosphorylation at multiple sites to achieve full FAK activation. Phosphorylation of FAK Tyr-397 creates a binding site for Src-family kinases, and phosphorylation of FAK Tyr-576/Tyr-577 in the kinase domain activation loop enhances catalytic activity. Using novel phosphospecific antibody reagents, we show that FAK activation loop phosphorylation is significantly elevated in cells expressing activated Src and is an early event following cell adhesion to fibronectin. In both cases, this regulation is largely dependent on Tyr-397. We also show that the FAK activation loop tyrosines are required for maximal Tyr-397 phosphorylation. Finally, immunostaining analyses revealed that tyrosine-phosphorylated forms of FAK are present in both newly forming and mature focal adhesions. Our findings support a model for reciprocal activation of FAK and Src-family kinases and suggest that FAK/Src signaling may occur during both focal adhesion assembly and turnover.
Introduction
Cell adhesion to components of the
ECM3
triggers signaling events affecting diverse cellular traits and
activities including survival, proliferation, differentiation, and
migration. FAK has emerged as a key signaling molecule regulating
cellular responses to integrin-mediated adhesion. Integrin-ligand
engagement promotes FAK tyrosine phosphorylation at multiple sites,
which prompts FAK signaling activity (reviewed in Refs. 1
and 2
). Phosphorylation of FAK Tyr-397, the only apparent
autophosphorylation site, creates a high-affinity binding site for SH2
domains of Src-family kinases including c-Src and Fyn
(3, 4, 5)
. This interaction likely contributes both to the
recruitment of Src-family kinases to sites of cell adhesion and to
their catalytic activation through release of inhibitory intramolecular
interactions (6)
. Phosphorylated Tyr-397 also binds to SH2
domains from other effector molecules, including
phosphatidylinositol-3-kinase (7)
, phospholipase C-
(8)
, and Shc (9)
, indicating a
multifunctional signaling role for this site. Tyr-397 is not strictly
an autophosphorylation site but can also be phosphorylated by Src
(10)
. Other known FAK phosphoacceptor tyrosines are also
phosphorylated by Src but are not sites of autophosphorylation
(10)
. Among these Src-specific sites are FAK tyrosines 576
and 577, which lie within the FAK kinase domain activation loop
(10)
, a region recognized as a general target for
phosphorylation-mediated regulation of protein kinase activity.
Consistent with Tyr-576/577 phosphorylation being required for maximal
FAK kinase activity, phenylalanine mutations of these residues result
in a 2–3-fold reduction of kinase activity (10, 11, 12)
.
Evidence indicates that FAK signaling significantly influences cell behavior. FAK overexpression enhances migration of Chinese hamster ovary cells, requiring both FAK Tyr-397 (13) and a binding site for p130Cas (Crk-associated substrate; 14 ). A requirement for FAK Tyr-397 in a cell spreading response involving the substrate paxillin has been indicated (15) . The FAK activation loop tyrosines 576/577, in addition to Tyr-397, were found to be important for maximal cell spreading and migration responses in an inducible expression system (12) . FAK has been implicated in anchorage-dependent cell survival (16, 17, 18) , requiring both FAK kinase activity and Tyr-397 (16) in a signaling response that may suppress the p53-regulated cell death pathway (19) . Finally, FAK overexpression accelerates cell cycle progression through the G1 phase, and Tyr-397 phosphorylation has again been implicated in this response (20 , 21) .
In this report, we describe the production of phosphospecific polyclonal antibodies against the FAK activation loop and the use of these antibodies, together with another phosphospecific antibody against the FAK autophosphorylation site, to examine the mechanism of FAK activation and the localization of activated FAK within spreading and migrating fibroblasts. Our results support a model for signal amplification by reciprocal activation of FAK and Src-family kinases and indicate that FAK exists in its active signaling form at both nascent and mature focal adhesions.
Results
FAK Phosphospecific Antibody Characterization and Analysis of FAK
Phosphorylation in Cos-7 Cells Coexpressing Activated Src.
Polyclonal antibodies were prepared from rabbits immunized with
peptides, derived from the FAK activation loop, in which
phosphotyrosines were incorporated in place of either Tyr-576 alone or
both Tyr-576 and Tyr-577 (see "Materials and Methods"). To
determine the specificity of these pFAK576 and pFAK576/577 antibodies,
as well as a commercial polyclonal antibody generated against the FAK
phosphoTyr-397 site (pFAK397), immunoblot analyses were performed on
whole-cell lysates of Cos-7 cells expressing WT FAK with a
COOH-terminal HA epitope tag (WT-FAKHA) versus various FAKHA
mutants (F397-FAKHA, F576-FAKHA, F577-FAKHA, and
FF576/F577-FAKHA) in which phosphoacceptor tyrosines were
substituted with phenylalanine (Fig. 1)
. The FAKHA variants were coexpressed with a "kinase active" form
of c-Src in which the COOH-terminal regulatory tyrosine was changed to
phenylalanine (KA Src; Fig. 1E
) to achieve high levels of
total FAK phosphotyrosine, as assessed using the 4G10
anti-phosphotyrosine antibody (Fig. 1
D, compare Lanes
2 with 3 where a "kinase dead" (KD) Src, lacking
the conserved lysine important for ATP binding, was coexpressed). Equal
loading of the lysates was shown by immunoblotting with the FAK C-20
antibody (Fig. 1G)
, which recognizes endogenous Cos-7 FAK
but does not recognize the FAKHA variants because of disruption of the
C-20 epitope by the HA tag. The near-equal expression of the FAKHA
variants was shown by immunoblotting with the 12CA5 antibody against
the HA epitope (Fig. 1F)
.
|
,
compare Lanes 2 and 3), indicating that the
antibodies recognize FAK tyrosine sites phosphorylated by Src. Although
total cell lysates were used in this analysis, at the exposures shown
in Fig. 1
the immunoreactivity of each of the three pFAK antibodies was
limited to the region of the blots corresponding to the position of
FAKHA, and no signal was detected in the mock-transfected sample
(Lane 1), although the endogenous Cos-7 FAK could be weakly
detected upon prolonged exposures. The specificities of the pFAK
antibodies for the individual phosphotyrosine sites were demonstrated
by lack of recognition of the relevant FAKHA mutants. Thus, the pFAK397
antibody failed to detect F397-FAKHA, although coexpression of KA Src
resulted in some tyrosine phosphorylation of this variant, as detected
by 4G10 (Fig. 1, C
and D, Lane 5). Similarly, the
pFAK576 antibody failed to recognize either F576-FAKHA or
F576/F577-FAKHA but still recognized F577-FAKHA (Fig. 1
B, Lanes
4, 6, and 7). The pFAK576/577 antibody provided no
detectable signal when either WT-FAKHA was coexpressed with KD Src or
when any of the phosphorylation site mutants was expressed with KA Src
(Fig. 1A)
. The signal generated by the pFAK576/577 antibody
was weak, relative to that of pFAK576. This may be attributable in part
to the lower stoichiometry of dually phosphorylated Tyr-576 and Tyr-577
residues compared with singly phosphorylated Tyr-576 (10)
.
The Cos-7 immunoblot analyses also provided evidence for FAK activation
loop and autophosphorylation sites being regulated in a manner
partially dependent upon one another. Thus, pFAK576 and pFAK576/577
antibody recognition of the F397-FAKHA variant was much reduced,
relative to WT-FAKHA (Fig. 1, A
and B, compare
Lanes 2 and 5). Similarly, pFAK397 antibody
recognition of FAKHA was reduced by the F576 and/or F577 mutations
(Fig. 1
C, compare Lane 2 with Lanes 4,
6, and 7).
FAK Activation Loop Phosphorylation Is Elevated in Src-transformed
Cells.
Our pFAK576 and pFAK576/577 antibodies were next used to determine
whether the FAK activation loop sites were phosphorylated to higher
levels in Src-transformed cells. Immunoprecipitates were prepared from
lysates of normal NIH 3T3 mouse fibroblasts versus their
counterparts transformed by F527-Src using antibody C-20 against the
FAK COOH terminus, and immunoblot analyses were performed using either
of the phosphospecific antibodies. Control immunoblots revealed the
equal recovery of FAK from the lysates and the expected elevation of
total FAK phosphotyrosine and retarded electrophoretic mobility in the
transformed cells (Fig. 2, C and D)
. Both the pFAK576 and the pFAK576/577
antibodies gave significantly stronger signals in the Src-transformed
cells relative to nontransformed cells (Fig. 2, A and B)
, indicating that FAK activation loop phosphorylation is
elevated by expression of the constitutively active Src mutant. The
pFAK576/577 antibody was unable to detect FAK from the nontransformed
cells (Fig. 2A)
, suggesting that very little dual
phosphorylation of the activation loop occurs in these cells under
normal growth conditions.
|
. Near-equal FAK recovery from the samples was shown by immunoblot
analysis of the immunoprecipitates using the C-20 antibody (Fig. 3D)
. Immunoblot detection with the pFAK576 antibody
indicated that FAK Tyr-576 is phosphorylated under attached conditions,
becomes dephosphorylated upon cell suspension, and then becomes rapidly
rephosphorylated, recovering to the levels of the attached growing
cells within 30 min of fibronectin replating (Fig. 3B)
. The
dynamics of Tyr-576 phosphorylation during this experiment was similar
to that of total FAK tyrosine phosphorylation, as assessed by analysis
of the same samples with 4G10 antibody (Fig. 3C)
. The
pFAK576/577 antibody detected FAK poorly, but weak reactivity was
observed in the adherent samples (Fig. 3A)
. These results
indicate that FAK activation loop phosphorylation, primarily of the
Tyr-576 site, is part of the initial rapid FAK tyrosine phosphorylation
in response to integrin-mediated cell adhesion.
|
These cells allow a direct test of the hypothesis that FAK Tyr-397
phosphorylation and subsequent Src recruitment to this site are
required to achieve phosphorylation of the FAK activation loop during
an integrin-stimulated signaling response. The Tet-FAK cells were
induced to express WT-FAK or F397-FAK to near-equal levels; then the
FAK variants were immunoprecipitated from lysates of attached,
suspended, and fibronectin-replated cells, and immunoblot analyses were
performed (Fig. 4)
. Similar to results obtained from NIH 3T3 fibroblasts, WT-FAK
expressed in the Tet-FAK cells exhibited adhesion-dependent elevation
of phosphotyrosine content including the Tyr-576 site in the activation
loop (Fig. 4
, right panel). In striking contrast, F397-FAK
accumulated little or no phosphotyrosine, including at the activation
loop site, under adherent conditions (Fig. 4
, left panel).
These results provide convincing evidence that FAK Tyr-397
phosphorylation and Src-family kinase recruitment to this site are
necessary steps leading to phosphorylation of other FAK tyrosine sites,
including those in the activation loop.
|
. Consistent with our previous report (12)
, F576/F577-FAK
shows reduced phosphotyrosine content in adherent cells, relative to
WT-FAK (Fig. 5B)
. This is most evident for the early time
points after fibronectin replating. Immunoblot analysis of the
immunoprecipitates with the pFAK397 antibody indicated that Tyr-397
phosphorylation is also reduced in the F576/F577-FAK mutant, relative
to WT-FAK. These results further support the hypothesis that FAK
activation loop phosphorylation elevates autophosphorylation activity
as part of an integrin signaling response.
|
or pFAK397 antibody (not shown) readily detected the
nascent punctate adhesive contacts enriched at the cell periphery. In
overnight cultures, fully spread cells commonly exhibited the
characteristic polarized migrating morphology, and both the pFAK576
(Fig. 6C)
and pFAK397 (Fig. 6E)
antibodies
stained the prominent peripheral focal adhesions of these cells.
Notably, the pFAK antibodies did not distinguish between focal
adhesions in the broad leading edge lamellipodia and those in the
narrower trailing tails. In all cells examined, tyrosine-phosphorylated
FAK recognized by the pFAK antibodies had a cellular localization
closely resembling that of total FAK detected by the FAK-TL antibody
(Fig. 6
, compare panels A, C, and E with B,
D, and F). Similar data were obtained when the pFAK
antibodies were used to stain NIH-3T3 cells (data not shown). In
uninduced Tet-FAK(WT) cells not expressing FAK, neither the pFAK576 nor
pFAK397 antibodies detected nascent or mature focal adhesions, although
these structures were readily recognized by an antibody against
vinculin (data not shown). Taken together, these results provide
further evidence for the specificity of the pFAK antibodies and
indicate that tyrosine-phosphorylated forms of FAK (those likely to be
actively engaged in signaling functions) are present in both newly
forming and mature focal adhesions.
|
In this report, we describe phosphospecific polyclonal antibodies generated against the FAK kinase domain activation loop and their use in experimental procedures involving immunoblot analysis and cell immunostaining. The antibodies, designated pFAK576 and pFAK576/577, were generated by immunizing rabbits with conjugated peptides corresponding to mouse FAK residues 571–582 (MEDSTYYKASKG), in which phosphotyrosines were incorporated in place of either Tyr-576 or both Tyr-576 and Tyr-577 residues. This sequence is absolutely conserved in all known FAK orthologues described in other vertebrate species including Xenopus, chicken, rat, and human. Thus, antibodies recognizing this region should detect phosphorylated FAK from a variety of vertebrate species. Moreover, the antibodies may not cross-react with the FAK-related kinase Pyk2/Cakß/RAFTK/CADTK, which is significantly divergent in this region, with the corresponding sequence being IEDEDYYKASVT. Because FAK Tyr-576 and Tyr-577 have been recognized as sites of phosphorylation by Src (10) , rather than autophosphorylation sites, phosphospecific antibodies targeting these residues will detect FAK molecules that have been activated in their signaling capacity through their association with and phosphorylation by Src-family kinases.
The specificity of the pFAK576 and pFAK576/577 polyclonal antibodies was demonstrated in Cos-7 cell expression experiments, where enhanced immunoblot recognition of HA-tagged FAK was observed upon coexpression of a catalytically active Src kinase and by the complete loss of this immunoreactivity when the relevant FAK activation loop tyrosines were mutated to phenylalanine. Data from Src-transformed NIH 3T3 cells further demonstrated a relationship between Src activity and pFAK576 and pFAK576/577 antibody recognition. In Cos-7 cells, the pFAK576 and pFAK576/577 antibodies failed to recognize the FAK activation loop mutants, although these mutants were significantly phosphorylated on other tyrosine sites, as shown by their efficient detection with the 4G10 anti-phosphotyrosine antibody. Thus, the FAK phosphospecific antibodies do not recognize phosphotyrosine-containing motifs in a nonspecific manner. Further indicating specificity of the pFAK576 antibody was the observation that no significant immunoreactivity was observed when the antibody was used in immunostaining experiments of uninduced Tet-FAK cells in which FAK was not expressed. This is also evidence for a lack of cross-reactivity with Pyk2/Cakß/RAFTK/CADTK, which is highly expressed and tyrosine phosphorylated in these cells (12) . In immunoblot detection experiments, the pFAK576 antibody consistently demonstrated a much greater recognition of FAK as compared with the pFAK576/577 antibody. This is likely a reflection of higher in vivo stoichiometric levels of singly phosphorylated Tyr-576 relative to doubly phosphorylated Tyr-576/Tyr-577. This conclusion is consistent with FAK phosphopeptide mapping studies, which indicated that Tyr-576 is a major site of FAK phosphorylation in vivo and a preferred in vitro Src site, relative to Tyr-577 (10) . The pFAK576/577 antibody may also have a relatively low affinity for the doubly phosphorylated activation loop site, which could contribute to the weak signal observed.
Our data showing a striking reduction of pFAK576 and pFAK576/577 antibody immunoreactivity with FAK from both Cos-7 and Tet-FAK cells expressing the F397-FAK mutant indicate that FAK activation loop phosphorylation is largely dependent on the autophosphorylation site. This observation, considered together with our other findings that immunoblot detection with the pFAK576 and pFAK576/577 antibodies is enhanced under conditions of high Src kinase activity, strongly indicate that in vivo FAK activation loop phosphorylation is primarily achieved through the direct SH2-mediated association of Src-family kinases with the Tyr-397 site, as speculated previously (10) . Our data from Cos-7 and Tet-FAK cells expressing the F576/F577-FAK mutant further indicate that the FAK activation loop tyrosines are required to achieve maximal Tyr-397 phosphorylation. The dependency of Tyr-397 phosphorylation upon the activation loop tyrosines was not as dramatic as that of activation loop phosphorylation dependency on Tyr-397. This is consistent with past observations that FAK autophosphorylation activity is only partially lost in F576/F577-FAK mutants (12) . FAK detection with the pFAK397 antibody could also reflect the phosphorylation of this site by Src-family kinases bound elsewhere within the complex.
In fibronectin-replating experiments, both pFAK576 and pFAK397 antibodies detected FAK from spreading cells with kinetics indistinguishable from total FAK tyrosine phosphorylation, as detected by the 4G10 antibody. Moreover, both pFAK576 and pFAK397 antibodies recognized the early focal contacts apparent during initial cell spreading on fibronectin. Thus, phosphorylation of both the FAK activation loop and autophosphorylation sites appear to be part of the initial integrin-stimulated FAK signaling response that occurs at new sites of cellular contact with the ECM. Our results also indicate that FAK phosphorylated at both the activation loop and autophosphorylation sites is present in mature focal adhesions, as evidenced by immunostaining analyses of growing polarized cells, where both pFAK576 and pFAK397 antibodies detected focal adhesions at both the leading and trailing edges of apparently motile cells. These findings indicate that FAK signaling complexes are not uniquely distributed among the contacts of polarized migrating fibroblasts. Rather, they raise the possibility that FAK signaling occurs at many areas of cell-ECM contact, including new contacts made as lamellipodia extend from the leading edge and at fully developed contacts at both the leading edges and trailing tails. This is consistent with previous studies suggesting a role for FAK signaling in regulating both focal adhesion formation associated with cell spreading and focal adhesion turnover associated with cell motility (12 , 24 , 25) . Further studies will be needed to determine whether distinct FAK signaling functions occur during focal adhesion assembly and maturation as a result of differences in stoichiometry of phosphorylation at the activation loop, autophosphorylation, and other FAK tyrosine phosphoacceptor sites and their association with downstream effector proteins.
In summary, our findings provide new experimental support for a model of reciprocal catalytic activation of FAK and Src-family kinases (12) . According to this model, association with the FAK autophosphorylation site promotes catalytic activation of Src-family kinases, leading to phosphorylation of the FAK activation loop. This in turn enhances FAK kinase activity and intermolecular autophosphorylation, resulting in signal amplification at sites of integrin-mediated cell adhesion. FAK activation loop phosphospecific antibodies and antibodies directed against other sites of FAK phosphorylation represent a new tool for study of FAK signaling. The antibodies should be particularly useful in revealing areas of active FAK signaling in intact tissues including developing embryos, tumors, and other normal or diseased tissues undergoing dynamic changes resulting from cell adhesion and other extracellular stimuli.
Materials and Methods
Antibodies.
Anti-FAK rabbit polyclonal antibody C-20 was obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-FAK monoclonal antibody,
designated FAK-TL, was obtained from Transduction Laboratories
(Lexington, KY). Anti-Src monoclonal antibody 327 was kindly provided
by A. Reynolds, Vanderbilt University. Anti-phosphotyrosine monoclonal
antibody 4G10 was obtained from Upstate Biotechnology (Lake Placid,
NY). Anti-FAK pTyr-397 (pFAK397) was from Biosource
International, Inc. (Hopkinton, MA). Polyclonal antibodies, recognizing
phosphorylated FAK activation loop tyrosines, were raised in rabbits
immunized with peptides conjugated to diphtheria toxoid. The peptide
immunogens were either MEDSTpYYKASKGC or MEDSTpYpYKASKGC (single letter
code, pY standing for phosphoTyr) for production of pFAK576 or
pFAK576/577 antisera, respectively. Rabbits were initially injected
s.c. with 100 µg of peptide conjugate in RIBI (ImmunoChem Research,
Inc., Hamilton, MT) adjuvant and subsequently boosted (biweekly
or monthly) with 100 µg of peptide conjugate in PBS.
Cells and Cell Culture.
Cos-7 cells were obtained from Steve Hann, Vanderbilt University. NIH
3T3 and F527 Src-transformed NIH 3T3 fibroblasts were obtained from A.
Reynolds, Vanderbilt University. These cell lines were cultured in DMEM
supplemented with 10% calf serum (Hyclone, Logan, UT), 100 units/ml
penicillin, and 100 µg/ml streptomycin and were maintained at 37°C
in a humidified 5% CO2 incubator.
Tet-FAK(WT), Tet-FAK(F397), and Tet-FAK(F576/577) are mouse embryo
fibroblasts derived from FAK-null embryos and engineered to inducibly
express either WT-FAK, F397-FAK, or F576/577-FAK, respectively, under
the control of the tetracycline repression system (12)
.
All Tet-FAK cells were maintained at 37°C in a humidified 5%
CO2 incubator in DMEM containing 4500 mg/l
D-glucose and 584 mg/l L-glutamine and
was further supplemented with 1 mM sodium pyruvate, 100
units/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml
Amphotericin B, 1 mM nonessential amino acids (all from
Life Technologies, Inc., Grand Island, NY), and 10% fetal bovine serum
(Atlanta Biologicals, Norcross, GA). Tetracycline (Calbiochem, La
Jolla, CA) was included in the culture media to maintain Tet-FAK cells
in the "uninduced" state. FAK expression was induced by removal of
tetracycline from the culture medium. FAK expression levels were
controlled by varying the duration cells were cultured in the absence
of tetracycline. Tet-FAK(WT) and Tet-FAK(F576/577) cells were induced
for 
2 days to achieve maximal expression levels of the FAK variants,
which are 2–3-fold higher than FAK levels observed in normal mouse
fibroblasts (12)
. Tet-FAK(F397) cells were induced for
only 12–14 h to achieve F397-FAK expression levels comparable with
WT-FAK.
Cos-7 Cell Expression and Immunoblot Analysis of HA-tagged FAK.
Plasmids for expression of mouse FAK variants tagged at their COOH
termini with the HA epitope and mouse c-Src variants have been
described previously (12
, 26)
. Plasmid DNA (2 µg total)
was transfected with Lipofectamine (Life Technologies) into
subconfluent Cos-7 cells, and cells were maintained in culture 2 days
prior to lysis in 2x SDS-PAGE sample buffer. Proteins in the
whole-cell lysates were resolved on 7% polyacrylamide SDS-PAGE,
transferred to Immobilon-P (Millipore Corp., Bedford, MA), and
subjected to immunoblot analysis with C-20, 4G10, 12CA5, Src-327,
pFAK397, pFAK576, or pFAK576/577 antibodies. Blots were blocked 2 h in Tris-buffered saline containing 0.2% Tween 20 (TBST) and either
3% BSA/1% ovalbumin (for 4G10 antibody) or 5% nonfat dry milk (for
all others). After blocking, the blots were incubated 2 h in
primary antibody solutions prepared in blocking buffer and then washed
extensively in TBST prior to detection by enhanced chemiluminescence
using horseradish peroxidase-conjugated secondary antibodies and
detection reagents from Amersham Pharmacia Biotech (Piscataway, NJ).
Concentrations of primary antibodies were as follows: C-20, 0.4
µg/ml; 4G10, 1.0 µg/ml; 12CA5, 1.0 µg/ml; Src-327, 1.0 µg/ml;
pFAK397, 0.5 µg/ml; pFAK576 and pFAK576/577, 1:200 dilution of whole
antiserum.
Analysis of FAK Phosphorylation in Src-transformed Cells and in
Cells Plated onto Fibronectin.
Subconfluent cultures of NIH 3T3 and F527 Src-transformed NIH 3T3
fibroblasts were maintained in serum-containing medium prior to lysis.
Fibronectin-replating experiments were performed essentially as
described (12)
, except that cells (NIH 3T3 or Tet-FAK)
were maintained in the absence of serum for 2 h prior to
suspension and were replated onto fibronectin for times ranging from
0.5 to 4 h. Cells were lysed in RIPA buffer [50 mM
Tris-Cl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1%
NP40, 1% sodium deoxycholate, 0.1% SDS, 50 mM NaF, 1%
aprotinin, and 0.1 mM
Na3VO4], and protein
concentrations were determined using the BCA assay (Pierce, Rockford,
IL) and adjusted to equal amounts of total protein for all samples
within a given experiment in 1 ml of final volume. FAK was
immunoprecipitated from the lysates using 1 µg of C-20 antibody,
resolved by 7% polyacrylamide SDS/PAGE, transferred to Immobilon-P
transfer membrane (Millipore Corp., Bedford, MA), and subjected to
immunoblot analysis with C-20, 4G10, pFAK397, pFAK576, or pFAK576/577
antibodies, as indicated above.
Immunofluorescence Microscopy.
Tet-FAK(WT) fibroblasts were induced for 2 days and then either plated
onto glass coverslips and allowed to adhere and grow overnight in
serum-containing medium or plated onto coverslips coated with 5 µg/ml
fibronectin and allowed to adhere and spread for 30 min. The cells were
fixed 10 min in 3.7% formaldehyde in PBS, permeabilized 10 min in
0.5% Triton X-100 in PBS, and then blocked 1–2 h in 5% milk in PBS.
After blocking, the coverslips were incubated 1 h at 37°C in
primary antibody mixtures prepared in 5% milk/PBS. The primary
antibody mixtures consisted of FAK-TL (25 µg/ml) and either pFAK576
(1:20 dilution of whole antiserum) or pFAK397 (5 µg/ml). After
repeated washes with PBS, secondary antibodies of rhodamine-conjugated
goat antimouse IgG and FITC-conjugated goat antirabbit IgG (Molecular
Probes, Eugene, OR) were applied at 7–10 µg/ml each in 5% milk/PBS
and incubated for 1 h at 37°C. After repeated washes with PBS,
coverslips were mounted onto glass microscope slides and viewed with a
Zeiss Axioplan photomicroscope.
Acknowledgments
We thank Eric Schaeffer for providing the pFAK397 antibody, Tara Gower and Barney Graham for assistance with immunofluorescence microscopy, and Samyuka Reddy for general technical assistance.
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.
1 Supported by NIH Grants GM49882 (to S. K. H.)
and P03-CA68485 (funding the Molecular Recognition Shared Resource of
the Vanderbilt-Ingram Cancer Center). 
2 To whom requests for reprints should be
addressed, at Department of Cell Biology, Vanderbilt University School
of Medicine, Medical Center North, 1161 21st Avenue South, Nashville,
TN 37232. Phone: (615) 343-8502; Fax: (615) 343-4539; E-mail;
hankss@ctrvax.vanderbilt.edu.
3 The abbreviations used are: ECM, extracellular
matrix; FAK, focal adhesion kinase; SH2, Src homology 2; WT, wild
type.
Received for publication 9/13/99. Revision received 12/ 1/99. Accepted for publication 12/ 2/99.
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 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]
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 N. P.Y. Lee, D. D. Mruk, C.-h. Wong, and C. Y. Cheng Regulation of Sertoli-Germ Cell Adherens Junction Dynamics in the Testis Via the Nitric Oxide Synthase (NOS)/cGMP/Protein Kinase G (PRKG)/{beta}-Catenin (CATNB) Signaling Pathway: An In Vitro and In Vivo Study Biol Reprod, September 1, 2005; 73(3): 458 - 471. [Abstract] [Full Text] [PDF]
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A. Polykratis, P. Katsoris, J. Courty, and E. Papadimitriou Characterization of Heparin Affin Regulatory Peptide Signaling in Human Endothelial Cells J. Biol. Chem., June 10, 2005; 280(23): 22454 - 22461. [Abstract] [Full Text] [PDF]
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 C.-H. Wong, W. Xia, N. P. Y. Lee, D. D. Mruk, W. M. Lee, and C. Y. Cheng Regulation of Ectoplasmic Specialization Dynamics in the Seminiferous Epithelium by Focal Adhesion-Associated Proteins in Testosterone-Suppressed Rat Testes Endocrinology, March 1, 2005; 146(3): 1192 - 1204. [Abstract] [Full Text] [PDF]
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G. P. van Nieuw Amerongen, K. Natarajan, G. Yin, R. J. Hoefen, M. Osawa, J. Haendeler, A.J. Ridley, K. Fujiwara, V. W.M. van Hinsbergh, and B. C. Berk GIT1 Mediates Thrombin Signaling in Endothelial Cells: Role in Turnover of RhoA-Type Focal Adhesions Circ. Res., April 30, 2004; 94(8): 1041 - 1049. [Abstract] [Full Text] [PDF]
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I. Hunger-Glaser, E. P. Salazar, J. Sinnett-Smith, and E. Rozengurt Bombesin, Lysophosphatidic Acid, and Epidermal Growth Factor Rapidly Stimulate Focal Adhesion Kinase Phosphorylation at Ser-910: REQUIREMENT FOR ERK ACTIVATION J. Biol. Chem., June 13, 2003; 278(25): 22631 - 22643. [Abstract] [Full Text] [PDF]
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A. Pace, L. J. Garcia-Marin, J. A. Tapia, M. J. Bragado, and R. T. Jensen Phosphospecific Site Tyrosine Phosphorylation of p125FAK and Proline-rich Kinase 2 Is Differentially Regulated by Cholecystokinin Receptor Type A Activation in Pancreatic Acini J. Biol. Chem., May 23, 2003; 278(21): 19008 - 19016. [Abstract] [Full Text] [PDF]
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K. A. Hotchkiss, A. W. Ashton, and E. L. Schwartz Thymidine Phosphorylase and 2-Deoxyribose Stimulate Human Endothelial Cell Migration by Specific Activation of the Integrins {alpha}5{beta}1 and {alpha}V{beta}3 J. Biol. Chem., May 23, 2003; 278(21): 19272 - 19279. [Abstract] [Full Text] [PDF]
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H. Mao, F. Li, K. Ruchalski, D. D. Mosser, J. H. Schwartz, Y. Wang, and S. C. Borkan hsp72 Inhibits Focal Adhesion Kinase Degradation in ATP-depleted Renal Epithelial Cells J. Biol. Chem., May 9, 2003; 278(20): 18214 - 18220. [Abstract] [Full Text] [PDF]
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M. K. Y. Siu, D. D. Mruk, W. M. Lee, and C. Y. Cheng Adhering Junction Dynamics in the Testis Are Regulated by an Interplay of {beta}1-Integrin and Focal Adhesion Complex-Associated Proteins Endocrinology, May 1, 2003; 144(5): 2141 - 2163. [Abstract] [Full Text] [PDF]
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L. Kwong, M. A. Wozniak, A. S. Collins, S. D. Wilson, and P. J. Keely R-Ras Promotes Focal Adhesion Formation through Focal Adhesion Kinase and p130Cas by a Novel Mechanism That Differs from Integrins Mol. Cell. Biol., February 1, 2003; 23(3): 933 - 949. [Abstract] [Full Text]
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 L. Zeng, X. Si, W.-P. Yu, H. T. Le, K. P. Ng, R. M.H. Teng, K. Ryan, D. Z.-M. Wang, S. Ponniah, and C. J. Pallen PTP{alpha} regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration J. Cell Biol., January 2, 2003; 160(1): 137 - 146. [Abstract] [Full Text] [PDF]
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M. Toutant, A. Costa, J.-M. Studler, G. Kadare, M. Carnaud, and J.-A. Girault Alternative Splicing Controls the Mechanisms of FAK Autophosphorylation Mol. Cell. Biol., November 15, 2002; 22(22): 7731 - 7743. [Abstract] [Full Text] [PDF]
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T. Akagi, K. Murata, T. Shishido, and H. Hanafusa v-Crk Activates the Phosphoinositide 3-Kinase/AKT Pathway by Utilizing Focal Adhesion Kinase and H-Ras Mol. Cell. Biol., October 15, 2002; 22(20): 7015 - 7023. [Abstract] [Full Text] [PDF]
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L. A. Cary, R. A. Klinghoffer, C. Sachsenmaier, and J. A. Cooper Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading Mol. Cell. Biol., April 15, 2002; 22(8): 2427 - 2440. [Abstract] [Full Text] [PDF]
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P. J. Ruest, N.-Y. Shin, T. R. Polte, X. Zhang, and S. K. Hanks Mechanisms of CAS Substrate Domain Tyrosine Phosphorylation by FAK and Src Mol. Cell. Biol., November 15, 2001; 21(22): 7641 - 7652. [Abstract] [Full Text] [PDF]
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K. A. West, H. Zhang, M. C. Brown, S. N. Nikolopoulos, M.C. Riedy, A. F. Horwitz, and C. E. Turner The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL) J. Cell Biol., July 9, 2001; 154(1): 161 - 176. [Abstract] [Full Text] [PDF]
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D. Vial, H. Okazaki, and R. P. Siraganian The NH2-terminal Region of Focal Adhesion Kinase Reconstitutes High Affinity IgE Receptor-induced Secretion in Mast Cells J. Biol. Chem., September 1, 2000; 275(36): 28269 - 28275. [Abstract] [Full Text] [PDF]
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M. Achison, C. M. Elton, P. G. Hargreaves, C. G. Knight, M. J. Barnes, and R. W. Farndale Integrin-independent Tyrosine Phosphorylation of p125fak in Human Platelets Stimulated by Collagen J. Biol. Chem., January 26, 2001; 276(5): 3167 - 3174. [Abstract] [Full Text] [PDF]
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E. P. Salazar and E. Rozengurt Src Family Kinases Are Required for Integrin-mediated but Not for G Protein-coupled Receptor Stimulation of Focal Adhesion Kinase Autophosphorylation at Tyr-397 J. Biol. Chem., May 18, 2001; 276(21): 17788 - 17795. [Abstract] [Full Text] [PDF]
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