(Received for publication, February 28, 1995; and in revised form, May 3, 1995)
From the
The concomitant tyrosine phosphorylation of the focal adhesion
protein, paxillin, and the tyrosine kinase, focal adhesion kinase
(FAK), in response to multiple stimuli including integrin-mediated cell
adhesion suggests that paxillin phosphorylation is closely coupled to
FAK activity. In the present study, we have identified a specific
tyrosine residue within paxillin, tyrosine 118 (Tyr-118), that
represents the principle site of phosphorylation by FAK in
vitro. The identification of this site as a target for FAK
phosphorylation was accomplished by immunoprecipitating FAK and
performing in vitro kinase assays, using as substrate either
glutathione S-transferase (GST)-paxillin fusion proteins
containing truncations in paxillin sequence or fusion proteins with
phenylalanine substitutions for tyrosine residues. GST-paxillin
containing a phenylalanine substitution at Tyr-118 (Y118F) was not
phosphorylated by FAK immunoprecipitates; however, this mutant was
shown to bind FAK equally as well as the wild type fusion protein. As a
first step toward assessing the function of paxillin phosphorylation on
Tyr-118, a Y118F paxillin cDNA construct was transiently transfected
into NIH 3T3 cells. Similar to wild type paxillin, mutated paxillin
localized to focal adhesions, indicating that the phosphorylation of
paxillin on Tyr-118 is not essential for the recruitment of paxillin to
sites of cell adhesion.
Cell adhesion to an extracellular matrix plays a fundamental
role in regulating cellular behaviors such as migration, proliferation,
and differentiation(1, 2) . The organization of cell
adhesion sites is directed by a family of transmembrane receptors known
as integrins(3) . The binding of integrins to extracellular
matrix ligands catalyzes the recruitment of multiple cytoskeletal
associated proteins to the cytoplasmic face of cell attachment sites
(focal adhesions) and thereby organizes the actin
cytoskeleton(4, 5) . The molecular mechanisms by which
cell adhesion triggers various cellular events have not yet been
defined. However, evidence is emerging that implicates the tyrosine
phosphorylation of certain focal adhesion proteins in at least one
aspect of integrin-meditated signaling, that of cytoskeleton
reorganization. In particular, the binding of integrins to
extracellular matrix ligands results in an increase in
tyrosine-phosphorylated forms of several focal adhesion
proteins(6, 7) . Two focal adhesion proteins that
demonstrate a high stoichiometry of tyrosine phosphorylation upon
integrin activation are the nonreceptor tyrosine kinase, focal adhesion
kinase
(FAK)
The coordinate phosphorylation of
paxillin and FAK on tyrosine residues has also been observed in other
systems. Cells transformed by Rous sarcoma virus (14) and
tissues undergoing embryogenesis (15, 16) have been
shown to contain enriched populations of tyrosine-phosphorylated forms
of paxillin and FAK. Additionally, tyrosine-phosphorylated paxillin and
FAK have been detected in cultured cells that have been treated with a
variety of stimuli, including lysophosphatidic acid (17) ,
angiotensin II(18) , sphingosine(19) , low doses of
platelet-derived growth factor(20) , and the neuropeptides
bombesin, endothelin, and vasopressin(21, 22) . The
multiple observations of concomitant phosphorylation of FAK and
paxillin suggest that tyrosine phosphorylation of paxillin may be a
direct consequence of FAK activity. Additional support for this
hypothesis is provided by the finding that purified paxillin can be
phosphorylated in vitro by a FAK
immunoprecipitate(16) .
The paxillin cDNA has been recently
cloned(23, 24) , and a region of the paxillin molecule
has been identified that supports the binding of both vinculin and
FAK(24) . An analysis of the paxillin amino acid sequence
reveals the presence of additional domains, which are thought to
function in protein-protein interactions. Among these domains are a
proline-rich, putative SH3 binding
motif(25, 26, 27) , four LIM domains (28) , and multiple tyrosine residues, which are likely to
represent sites of phosphorylation, given that these residues lie
within good consensus sequences for binding to proteins that contain
SH2 domains (29) . In the present study, we have determined
that one of the tyrosine residues that potentially comprises part of an
SH2 binding domain for the adapter protein, Crk, is a primary site of
paxillin phosphorylation by FAK in vitro.
To perform kinase assays with
native paxillin, anti-paxillin monoclonal antibody 165 (32) and
anti-FAK monoclonal antibody 2A7 were added simultaneously to gizzard
lysates. Following a 90-min incubation at 4 °C, paxillin and FAK
were coprecipitated by the addition of rabbit anti-mouse saturated
protein A-Sepharose. The precipitates were washed and then resuspended
in kinase buffer containing 10 µCi of
[
Transient transfections of NIH 3T3 cells, plated on coverslips, were
performed using the Ca
Previously, a GST-paxillin fusion protein containing paxillin
sequence from amino acids 54 through 313 was shown to support the
binding of FAK (24) (see ``Materials and Methods''
regarding a change in nomenclature). This same fusion protein,
GST-paxillin 54-313 (Fig. 1), was used in in vitro kinase assays with FAK immunoprecipitates. Phosphorylation of the
GST-paxillin fusion protein (Fig. 2A, lane3), but not GST alone (Fig. 2A, lane2), was observed, confirming that the phosphorylation of
the fusion protein was specific to the paxillin domain. Phosphorylation
of immunoprecipitated FAK (i.e. no fusion protein added) was
also observed (Fig. 2A, lanes4 and 5). The phosphorylation of FAK most likely represents an
autophosphorylation event(37) . Excision of phosphorylated
GST-paxillin 54-313 from SDS-PAGE gels and subsequent
phosphoamino acid analyses revealed that the phosphorylation of the
fusion protein was restricted to tyrosine residues (data not shown).
Additionally, the phosphorylation of both paxillin and FAK was
eliminated by the inclusion of a tyrosine kinase inhibitor,
tyrphostin(38) , in the assay buffer (data not shown).
Tyrphostin has been previously shown to inhibit FAK activity in
vitro and in vivo(39) .
Figure 1:
Synthesis of GST-paxillin 54-313.
Schematic representation of the paxillin molecule and the region of the
protein incorporated into a GST-paxillin fusion protein(24) . Numbers correspond to amino acid position. All of the tyrosine
residues included within this fusion protein are
indicated.
Figure 2:
A, phosphorylation of GST-paxillin
54-313 and truncated variants of GST-paxillin 54-313. FAK
was immunoprecipitated from embryonic chicken gizzard lysates with
monoclonal antibody 2A7. After washing the immunoprecipitates, fusion
proteins (previously dialyzed into kinase buffer) were added along with
10 µCi of [
To confirm that tyrosine 118 was the primary
site of phosphorylation by the FAK immunoprecipitate, site-directed
mutagenesis was used to alter individual tyrosine residues within the
fusion protein to phenylalanine. Mutated fusion proteins were then used
in in vitro kinase assays with one additional modification.
All of the mutated fusion proteins contained paxillin sequence from
amino acids 54-191 instead of the 54-313 fusion protein
used in Fig. 1. The truncation of paxillin sequence from amino
acid 313 to 191 was instituted because fusion protein 54-313
migrated to the same position as IgG heavy chain on SDS-PAGE gels. This
factor made it difficult to quantify the fusion protein by Coomassie
staining and thereby ensure that equivalent amounts of mutated fusion
proteins were added to each kinase assay. GST-paxillin 54-191
contained all of the tyrosine residues that were present in
54-313 and, furthermore, no diminution of phosphorylation of
54-191 was observed with respect to 54-313 (Fig. 2A, lanes6 and 3,
respectively).
In vitro kinase assays performed with
mutated fusion proteins revealed that the phosphorylation of paxillin
by the FAK immunoprecipitate was eliminated by introducing a
phenylalanine substitution at tyrosine 118 (Fig. 3). In
contrast, no diminution in the level of phosphorylation was observed in
any of the other mutated proteins, indicating that the phosphorylation
of GST-paxillin by FAK occurred on a single tyrosine residue, Tyr-118.
It is unlikely that the inhibition in phosphorylation of the Y118F
mutant was due to a mutation-induced perturbation in the folding of the
paxillin molecule because the Y118F mutant was shown by Western blot
analysis to bind both FAK and FRNK, a truncated variant of
FAK(6) , equally as well as the wild type fusion protein (Fig. 4).
Figure 3:
In vitro kinase assays with
mutated GST-paxillin. Site-directed mutagenesis was used to alter
individual tyrosine residues within GST-paxillin 54-191 to
phenylalanine. Four mutated fusion proteins were synthesized,
corresponding to the four tyrosine residues within GST-paxillin
54-191, Y76F, Y88F, Y118F, and Y182F. Mutated fusion proteins
were used in in vitro kinase assays with FAK
immunoprecipitates as described under ``Materials and
Methods.''
Figure 4:
Western blot of FAK precipitated by
GST-paxillin fusion proteins. GST (lane2),
GST-paxillin 54-313 (lane3), and GST-paxillin
54-313 containing a phenylalanine substitution at tyrosine 118 (lane4) were incubated with embryonic chicken
gizzard lysates for 90 min at 4 °C. GST and GST-fusion proteins
were precipitated with GSH-Sepharose, washed, and resolved by SDS-PAGE.
A sample of the embryonic chicken gizzard lysate was also
electrophoresed (lane1). Following electrophoresis,
proteins were transferred to nitrocellulose, and both FAK and FRNK, a
truncated variant of FAK(6) , were detected by Western blotting
using anti-FAK polyclonal antisera (BC3). The position of molecular
weight standards are indicated on the left.
Figure 5:
Phosphopeptide analysis of native paxillin
and GST-paxillin fusion protein. Both native paxillin and GST-paxillin
54-313 were phosphorylated in vitro by FAK
immunoprecipitates. The phosphorylated proteins were excised from
SDS-PAGE gels and subjected to thin layer electrophoresis (E)
in pH 1.9 buffer(35) , followed by thin layer chromatography (C) in isobutyric acid
buffer(35) .
In view of the previous observation that focal adhesion
formation is correlated with the tyrosine phosphorylation of paxillin
and FAK(8) , we were interested in determining if the
phosphorylation of paxillin on tyrosine 118 was necessary for the
localization of paxillin to focal adhesions. Consequently, paxillin
cDNA and paxillin cDNA containing a phenylalanine substitution at
tyrosine 118 were transfected into NIH 3T3 cells (Fig. 6). These
cDNA constructs contained chicken paxillin sequence coding for amino
acids 54-559. Similar to nonmutated paxillin (panelA), the Y118F mutant paxillin was detected in focal
adhesions by the chicken-specific antibody (panelC),
indicating that phosphorylation at tyrosine 118 was not required for
the recruitment of paxillin to adhesion sites. It is also noteworthy
that the proline-rich, putative SH3 binding domain of paxillin (amino
acids 46-55) does not appear to be necessary for localization,
since the amino acids coding for this domain were not contained within
the transfected cDNA constructs. This is of interest because previous
studies have shown an interaction between paxillin and the SH3 domain
of Src(41) , a tyrosine kinase that has been identified in
focal adhesions(42) . Paxillin is a multi-domain protein
containing binding sites for several focal adhesion proteins. It is
therefore likely that the transfected paxillin constructs are recruited
to focal adhesions through associations with vinculin and/or FAK or via
interactions involving the paxillin LIM domains. Clearly, the
phosphorylation of paxillin on Tyr-118 is not essential for the
localization of paxillin to pre-existing focal adhesion sites; however,
the possible requirement for phosphorylation of Tyr-118 in the initial
assembly of focal adhesions remains to be determined, given that
endogenous paxillin is present in NIH 3T3 cells.
Figure 6:
Transfection of paxillin containing a
phenylalanine substitution at Tyr-118. NIH 3T3 cells were transiently
transfected with either chicken paxillin cDNA encoding amino acids
54-559 (panelsA and B) or with the
same construct containing a phenylalanine substitution at tyrosine 118
(Y118F) (panelsC and D). PanelsE and F represent nontransfected NIH 3T3 cells.
Cells in panelsA, C, and E were
labeled with chicken-specific anti-paxillin antisera (see
``Materials and Methods''), followed by incubation with a
fluorescein-conjugated secondary antibody. Cells in panelsB, D, and F were double labeled with
rhodamine-conjugated phalloidin to visualize actin-containing stress
fibers. Bar, 5 µm.
In summary, we have identified a specific tyrosine
residue within paxillin that is a primary target for phosphorylation by
FAK and have further demonstrated that the phosphorylation of this
residue is not essential for the localization of paxillin to focal
adhesion sites. Additional experiments will be required to elucidate
signaling events that may lie downstream of paxillin phosphorylation by
FAK.
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank®/EMBL Data Bank with accession
number(s) L30099[GenBank® Link].
We are indebted to Dr. J. Tom Parsons (University of
Virginia) for the generous gift of antibodies against FAK. We also
thank Dr. Michael Brown for critical reading of this manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)(8, 9, 10, 11, 12) ,
and the 68-kDa protein, paxillin(8, 13) . The tyrosine
phosphorylation of these two proteins has been suggested to be involved
in both the formation of focal adhesions and the assembly of actin
stress fibers(8) .
Synthesis of GST-Paxillin Fusion
Proteins
Selected regions of paxillin cDNA were amplified
from paxillin clone 10 (24) by polymerase chain reaction (PCR)
using oligonucleotides containing 5`-BamHI or
3`-EcoRI restriction sites. The PCR products were subcloned
into BamHI/EcoRI-digested pGEX-2T (Pharmacia Biotech
Inc.). Verification that paxillin was subcloned into pGEX-2T in the
correct reading frame was provided by dideoxy chain termination
sequencing (Sequenase 2, U. S. Biochemical Corp.). The GST-paxillin
fusion proteins were expressed and purified using glutathione
(GSH)-Sepharose as described elsewhere(30) . For use in kinase
assays, fusion proteins were eluted from GSH-Sepharose beads with 20
mM GSH in 50 mM Tris-HCl buffer (pH 8.0), then
dialyzed overnight into kinase buffer (10 mM HEPES, 3
mM MnCl, pH 7.3).
In Vitro Kinase
Assays
14-16-day-old embryonic chicken gizzards (a
source of smooth muscle focal adhesion proteins) were homogenized in 10
volumes of buffer containing 50 mM Tris-HCl (pH 7.6), 150
mM NaCl, 0.1% Triton X-100, 0.1% deoxycholate, 10 µg/ml
leupeptin, and 50 µM sodium orthovanadate (lysis buffer).
Lysates were centrifuged at 100,000 g to remove
insoluble material. FAK was immunoprecipitated by incubating the
lysates for 90 min at 4 °C with anti-FAK monoclonal antibody 2A7
(gift from Dr. J. T. Parsons, University of Virginia), followed by the
addition of rabbit, anti-mouse IgG complexed to protein A-Sepharose.
The immune complexes were pelleted, washed extensively with lysis
buffer, washed once with 10 mM HEPES, 3 mM
MnCl
, pH 7.3 (kinase buffer), then resuspended into 20
µl of kinase buffer. Approximately 5-20 µg of
GST-paxillin fusion protein (previously dialyzed into kinase buffer)
were added to the FAK immunoprecipitate with 10 µCi of
[
-
P]ATP. In some kinase assays, 100
µM tyrphostin (Life Technologies, Inc.) was also added.
Following a 20-min incubation at room temperature, kinase reactions
were terminated by boiling the samples in SDS-PAGE sample buffer.
Samples were resolved on 10% acrylamide gels by SDS-PAGE (31) and then visualized by autoradiography. Protein amounts
were determined by using a Bio-Rad protein assay kit, as well as by
Coomassie staining of SDS-PAGE gels.
-
P]ATP. As described above, kinase
reactions proceeded for 20 min at room temperature, and the
phosphorylated samples were resolved by electrophoresis.
Site-directed Mutagenesis
Phenylalanine
substitutions for tyrosine residues were introduced into the paxillin
sequence using a PCR method described by Landt et
al.(33) . Mutated paxillin cDNA was subcloned into pGEX-2T
as described above. The correct sequence of the mutated GST-paxillin
fusion protein was verified by sequencing.
Western Blotting
GST, GST-paxillin
54-313, and GST-paxillin 54-313/Y118F, each coupled to
GSH-Sepharose beads(30) , were incubated with embryonic chicken
gizzard lysates for 90 min at 4 °C. Samples were pelleted and
washed extensively with lysis buffer. Samples were resolved by
electrophoresis on 10% acrylamide gels and transferred to
nitrocellulose using standard procedures(34) . FAK and FRNK, an
alternately spliced FAK isoform(6) , were detected by
incubating nitrocellulose blots with anti-FAK polyclonal antibody, BC3
(gift from Dr. Tom Parsons, University of Virginia), followed by
incubation with I-labeled goat anti-rabbit IgG.
Phosphopeptide
Mapping
P-Labeled fusion proteins were eluted
from dried SDS-PAGE gels, trypsinized, and subjected to two-dimensional
phosphopeptide mapping according to standard methods(35) .
Phosphopeptides were separated in the first dimension by thin layer
electrophoresis in pH 1.9 buffer (35) and then resolved by thin
layer chromatography in isobutyric acid buffer(35) . Performic
acid oxidation of phosphopeptides was not performed prior to the
two-dimensional analysis.
Transfection and
Immunofluorescence
Chicken paxillin cDNA encoding amino
acid residues 54-559 was amplified by PCR using paxillin clone 10 (24) as a template. PCR primers contained 5`-BamHI and
3`-EcoRI restriction sites to permit force cloning into the BamHI/EcoRI restriction sites of the eukaryotic
expression vector, pcDNA3 (Invitrogen), a plasmid which contains a
cytomegalovirus promoter. In addition, residue 58 of the paxillin
sequence was mutated to methionine since transcription from the pcDNA3
vector is initiated at the first methionine residue within the insert.
Mutation of tyrosine 118 to phenylalanine was as described above.
PO
precipitation method
of Solowska et al.(36) . 24 h post-transfection, the
cells were fixed and processed for immunofluorescence microscopy as
previously described(32) . Expression and localization of
transfected paxillin were detected using a chicken-specific polyclonal
antisera, Pax 2(24) . Cells were double-labeled with
rhodamine-phalloidin to visualize actin-containing stress fibers.
Nomenclature
Following our initial report
of the cloning of paxillin and the identification of FAK binding to a
partial paxillin GST-fusion protein(24) , we have identified,
using 5`-rapid amplification of cDNA ends, an additional chicken cDNA
sequence encoding 5 amino acids (MDDLD) at the amino terminus. Thus,
all nomenclature used in this report is based on a full-length protein
of 559 amino acids. The additional cDNA sequence (including
5`-untranslated sequence) has been deposited in GenBank. An identical
5-amino acid sequence was reported for human paxillin (23) following submission of this manuscript.
-
P]ATP. Kinase reactions
proceeded for 20 min at room temperature and were terminated by boiling
in SDS-PAGE sample buffer. Proteins were resolved by electrophoresis on
10% acrylamide gels. The following protein samples were added to the
FAK immunoprecipitates: GST (lane2), GST-paxillin
54-313 (lane3), no fusion protein added (lanes4 and 5), and truncated variants of
GST-paxillin 54-313 (lane1 and lanes6-12). B, summary of results from kinase
assays with truncated GST-paxillin. Kinase assays performed with
truncated variants of GST-paxillin 54-313 indicated that the
primary site of phosphorylation lies within amino acids
114-133.
To map the site(s) of
paxillin phosphorylation by FAK, a series of truncated GST-paxillin
fusion proteins was synthesized and used in in vitro kinase
assays with FAK immunoprecipitates (Fig. 2, A and B). The original fusion protein, GST-paxillin 54-313,
contained four tyrosine residues, Tyr-76, Tyr-88, Tyr-118, and Tyr-182. In vitro kinase assays using truncated variants of this
original fusion protein indicated that phosphorylation of the fusion
protein by the FAK immunoprecipitate occurred within a region of the
paxillin molecule spanning amino acids 114-133. This finding
implicated Tyr-118 as the most likely site of phosphorylation by the
FAK immunoprecipitate.
The segment of paxillin used in the previous
experiments represents approximately 50% of the paxillin sequence (the
expression of full-length paxillin as a GST-fusion protein resulted in
an insoluble product). Because a significant portion of the paxillin
sequence was excluded from the fusion proteins used in our assays, it
was important to determine if the phosphorylation of the fusion protein
by the FAK immunoprecipitate reflected the in vitro phosphorylation of native paxillin by FAK. To address this
question, both GST-paxillin 54-313 and native paxillin were
phosphorylated in vitro by the FAK immunoprecipitate. The
phosphorylated proteins were excised from SDS-PAGE gels, trypsinized,
and subjected to two-dimensional phosphopeptide analysis. As shown in Fig. 5, multiple radiolabeled spots were observed in the maps of
both native paxillin and GST-paxillin fusion protein. Given that
previous data ( Fig. 2and 3) suggested that phosphorylation
occurred at a single site (Tyr-118), it is likely that multiple spots,
at least in the case of the fusion protein, resulted from incomplete
tryptic digests and/or differences in the oxidation state of a single
phosphopeptide. A comparison of the two phosphopeptide maps revealed
that the patterns of spots were superimposable, confirming that the
phosphorylation of native paxillin occurred at essentially the same
site(s) as the fusion protein. Variation in the relative intensities of
individual phosphopeptides are likely due to the differential
accessibility of trypsin sites in the native paxillin versus the GST-paxillin fusion protein. Alternatively, these differences
may reflect the phosphorylation of paxillin at a second site, which was
labeled more efficiently in the native molecule.
The phosphopeptide
analysis yielded two important findings. First, phosphopeptide 2
represented one of the most intensely labeled phosphopeptides in both
maps, implicating the same tyrosine residue, Tyr-118, as a principle
site of phosphorylation in both native paxillin and the GST-paxillin
fusion protein. Second, no novel phosphopeptides were noted in the map
of native paxillin as compared with the fusion protein, suggesting that
all of the primary sites of paxillin phosphorylation by the FAK
immunoprecipitate were present within fusion protein 54-313. To
further consider the possibility that tyrosine residues not included in
GST-paxillin 54-313 might be substrates for phosphorylation by
FAK, we performed in vitro kinase assays with several
additional fusion proteins: GST-paxillin 11-79 (proline-rich
region), GST-paxillin 323-383 (LIM 1), GST-paxillin 382-445
(LIM 2), GST-paxillin 439-496 (LIM 3), and GST-paxillin
501-559 (LIM 4). No significant phosphorylation of any of these
fusion proteins by FAK immunoprecipitates was observed (data not
shown), supporting our hypothesis that Tyr-118 is the primary site of
phosphorylation by FAK invitro. While this
manuscript was under review, the identification of Tyr-118 as a
substrate for phosphorylation by FAK was corroborated by a report
indicating that Tyr-118 of paxillin is phosphorylated in cultured cells
that overexpress FAK(40) . In this same report, however, Tyr-31
was also implicated as a potential phosphorylation site. In contrast
with this finding, we have not detected any phosphorylation of paxillin
on Tyr-31 under the conditions used in our in vitro kinase
assays.
Another potential
function of paxillin phosphorylation on Tyr-118 may involve the
recruitment of other signaling molecules to focal adhesion sites.
Phosphotyrosine residues typically serve to provide docking sites for
proteins containing SH2 domains and thereby facilitate the formation of
signaling complexes. It has been established that the three amino acids
immediately following a phosphotyrosine residue confer a specificity
for binding to particular SH2 domains(29) . An examination of
the amino acid sequence following tyrosine 118 of paxillin (Y118SFP)
suggests that phosphorylation at this site is likely to be responsible
for mediating the known association between paxillin and the SH2 domain
of the oncogenic protein, v-Crk(43) . This hypothesis is
supported by a recent report demonstrating an interaction between
paxillin phosphorylated on Tyr-118 and the SH2 domain of Crk (40) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.