(Received for publication, December 13, 1995)
From the
The macrophage colony-stimulating factor (M-CSF) is able to
induce the expression of the integrin receptor on the surface of cultured human macrophages
(De Nichilo, M. O., and Burns, G. F.(1993) Proc. Natl. Acad. Sci.
U. S. A. 90, 2517-2521). In the present study, we establish
that the adhesion of M-CSF-treated macrophages to vitronectin is
mediated by the integrin
, and show
by indirect immunofluorescence analysis that
and the cytoskeletal protein
paxillin localize to focal contacts upon adhesion to vitronectin.
Immunoprecipitation and Western blot analysis revealed that
M-CSF-treated macrophages do not express focal adhesion kinase (FAK),
thereby providing direct evidence for integrin-dependent localization
of paxillin to focal contacts in the absence of FAK expression.
Investigation of paxillin phosphorylation by two-dimensional
phosphoamino acid analysis indicates that paxillin is 99%
phosphorylated on serine residue(s) in response to vitronectin
adhesion, and only 1% phosphorylated on tyrosine. Stimulation of
protein kinase C (PKC) activity with the phorbol ester phorbol
12-myristate 13-acetate enhances paxillin phosphorylation, while two
selective inhibitors of PKC, GF109203X and chelerythrine chloride,
effectively block the phosphorylation of paxillin induced in response
to vitronectin adhesion. Taken together, these data demonstrate that in
M-CSF-treated macrophages adherent to vitronectin, paxillin localizes
to focal contacts in the absence of FAK expression and is predominantly
phosphorylated on serine residue(s) in a PKC-dependent manner.
Interactions with the extracellular matrix (ECM) ()are known to have profound influence on cell growth and
differentiation, as well as migration(1) . The receptors that
have received most study in this regard belong to a large family of
heterodimers termed integrins(2) . Integrins
function as cell surface receptors for a variety of ECM proteins
(including vitronectin, fibronectin, laminin, and the collagens) and
are thought to transmit signals to the cell upon ligand occupancy, and
thereby regulate cell behavior(3, 4, 5) . The
integrin family is now known to consist of over 20 distinct receptors,
with the combination of a particular
and
subunit
determining ligand specificity(2) . In general, upon binding
their specific ligands, the integrins associate with the actin
cytoskeleton and organize into structures known as focal contacts
(focal adhesions or adhesion plaques)(6) . Focal contacts are
thought to act not only as structural links between the ECM and the
cytoskeleton, but also as sites of signal transduction from the ECM (6) . Despite their importance, the underlying molecular
mechanisms orchestrating the formation of focal contacts upon integrin
ligation remain poorly defined.
FAK (focal adhesion kinase) is a 125-kDa cytoplasmic protein tyrosine kinase that was named for its ability to localize to focal contacts(7, 8) . Published data obtained primarily using cultured fibroblasts have shown that engagement of integrins with ECM ligands or cross-linking of cell surface integrins with antibodies leads to a pronounced increase in the tyrosine phosphorylation of FAK (9, 10, 11, 12) and a concomitant increase in its intrinsic tyrosine kinase activity in vitro(13, 14) . The presence of tyrosine-phosphorylated proteins in focal contacts and the regulation of their tyrosine phosphorylation in response to integrin-mediated cell adhesion has led to the suggestion that FAK plays a central role in regulating focal contact assembly (10) .
Paxillin is a 68-kDa vinculin-binding protein that also colocalizes with FAK and integrins to focal contacts (15, 16) and is phosphorylated on tyrosine residues during integrin-mediated adhesion of fibroblasts to ECM substrates(10) . Paxillin has been identified as a target substrate for FAK phosphorylation in vitro(17, 18, 19) . On the basis of their colocalization to focal contacts and coordinate phosphorylation, it has been hypothesized that paxillin phosphorylation is closely coupled to FAK activation(10, 16, 19) . Recent evidence, however, suggests that the tyrosine phosphorylation of paxillin mediated by FAK may not be a critical determinant in paxillin localization to focal contacts(20) , suggesting that FAK phosphorylation of paxillin may serve an additional function possibly related to cell signaling(19, 20) .
The
serine/threonine kinase, protein kinase C (PKC) is another regulatory
enzyme that has been localized to focal
contacts(21, 22) . Woods and Couchman (23) previously provided evidence in support of PKC involvement
in the regulation of focal contact formation in fibroblasts. While it
is well established that compounds that activate PKC such as phorbol
12-myristate 13-acetate (PMA) enhance cell adhesion and spreading on
ECM substrates(24, 25, 26) , the identity of
cytoskeletal proteins within focal contacts that serve as targets for
PKC-mediated phosphorylation in response to integrin-dependent cell
adhesion remain poorly defined. In the present study, we show that in
M-CSF-treated macrophages, paxillin localizes to focal contacts in the
absence of FAK expression and is predominantly serine-phosphorylated in
response to integrin -mediated
adhesion to vitronectin. PMA stimulation of PKC activity was found to
enhance paxillin phosphorylation, whereas selective inhibitors of PKC
activity effectively block the phosphorylation of paxillin induced in
response to vitronectin adhesion. These latter results indicate that
serine phosphorylation of paxillin observed in response to vitronectin
adhesion is thus PKC-dependent.
Figure 1:
Flow cytometric
analysis of surface integrin expression.
M-CSF-treated macrophages were stained with mAb LM142 to the
subunit (
), mAb SZ-21 to the
subunit (
), mAb B5-IVF2 to the
subunit (
), or mAb P1F6 to the
complex
(
) (solid peaks). Cells were
then treated with fluorescein-conjugated goat F(ab`)
anti-mouse IgG, washed, and analyzed by flow cytometry. Control
cells (open peaks) were stained with the secondary antibody
alone. Results are depicted as histograms with fluorescence intensity
on the abscissa and cell number on the ordinate.
Figure 2:
Inhibition of macrophage adhesion to
vitronectin. A, M-CSF-treated macrophages were allowed to
attach and spread on wells coated with vitronectin (0-100
µg/ml) for 30 min at 37 °C. Nonadherent cells were removed by
gentle washing, and cell adhesion was quantitated as described under
``Experimental Procedures.'' The data represent the mean
± S.E. of triplicate determinations. B, M-CSF-treated
macrophages were pretreated with various dilutions of either mAb P1F6 (anti-) or mAb
LM142 (control) for 30 min on ice, then added to wells coated
with 1 µg/ml vitronectin and allowed to attach and spread for 30
min at 37 °C. Cell adhesion was quantitated as described under
``Experimental Procedures.'' Results are shown as percent of
maximum adhesion, as defined by cell binding to wells in the absence of
antibodies. Data are expressed as mean ± S.E. of triplicate
determinations.
Figure 3:
Localization of integrin
, paxillin, and vinculin distribution by indirect
immunofluorescence. M-CSF-treated macrophages were allowed to attach
and spread on vitronectin-coated coverslips under serum-free conditions
for 60 min. Cells were fixed, permeabilized, and stained with: A, secondary fluorescein-conjugated antibody alone; B, mAb B5-IVF2 to the
subunit; C,
mAb VII-F9B11 to vinculin; D, mAb to paxillin. Focal contacts
are highlighted by arrowheads. Bar = 5
µm.
Figure 4: Analysis of FAK protein expression. Left, total cell lysates (1 mg) from human foreskin fibroblasts (lanes 1 and 2) or M-CSF-treated macrophages (lanes 3 and 4) were immunoprecipitated with either an isotype control mAb (lanes 1 and 3) or an anti-FAK mAb (lanes 2 and 4). The precipitated immunocomplexes were analyzed for FAK expression by Western immunoblotting. Right, as a control, total cell lysates (1 mg) from M-CSF-treated macrophages were immunoprecipitated with either an isotype control mAb (lane 5) or an anti-paxillin mAb (lane 6). The precipitated immunocomplexes were analyzed for paxillin expression by Western immunoblotting.
Figure 5: Analysis of paxillin tyrosine phosphorylation. M-CSF-treated macrophages were allowed to attach and spread on uncoated (A and C) or vitronectin-coated (B) coverslips in serum-free conditions for 60 min. Cells were fixed, permeabilized and stained with: A and B, mAb to paxillin; or C, secondary fluorescein-conjugated antibody alone. Bar = 10 µm. D, M-CSF-treated macrophages were harvested and maintained in a nonadherent state for 60 min (lane 1), or allowed to attach and spread on either uncoated (lane 2) or vitronectin-coated (lane 3) Petri dishes for 60 min. Cells were solubilized in RIPA lysis buffer, and paxillin was immunoprecipitated from equal concentrations and volumes of cell lysate. The precipitated immunocomplexes were separated by SDS-PAGE and probed by Western immunoblotting with the anti-phosphotyrosine mAb PY-20 (anti-PY). The filter was then stripped and reprobed with the anti-paxillin mAb (anti-paxillin). Note that in lane 3, staining for paxillin reveals substantial broadening of the band by more slowly migrating material. It should also be noted that the exposure time of the anti-PY blot to autoradiographic film was 10 min, whereas the anti-paxillin blot was exposed for 10 s.
Zachary et al. (34) reported a similar mobility
shift of paxillin in Swiss 3T3 fibroblasts stimulated with bombesin.
These authors noted that even though paxillin was strongly
tyrosine-phosphorylated in response to bombesin stimulation, inhibition
of the PKC pathway either by down-regulation of PKC or treatment with
the selective PKC inhibitor GF109203X blocked this characteristic
mobility shift of paxillin without influencing the levels of tyrosine
phosphorylation, thus suggesting the potential involvement of
serine/threonine phosphorylation. Based on these observations, we
examined directly whether paxillin could be phosphorylated on
serine/threonine residues in response to
-mediated adhesion to vitronectin. Fig. 6A shows that in M-CSF-treated macrophages labeled
with [
P]orthophosphate, paxillin was heavily
phosphorylated in response to vitronectin adhesion. This
phosphorylation appeared to be a specific integrin-mediated response,
because very little [
P]orthophosphate was
incorporated into paxillin that was immunoprecipitated from cells
either maintained in a nonadherent state or allowed to attach and
spread on plastic over the same period of time (Fig. 6A). Two-dimensional phosphoamino acid analysis
revealed that 99% of the [
P]orthophosphate
incorporated into paxillin upon adhesion to vitronectin was on serine
residue(s) (Fig. 6B). In contrast, phosphorylation on
tyrosine residue(s) accounted for only 1% of the total paxillin
phosphorylation, a figure that is consistent with the low levels of
paxillin tyrosine phosphorylation observed by Western immunoblotting (Fig. 5D). Phosphorylation on threonine residue(s) was
not detected by phosphoamino acid analysis under the conditions
described (Fig. 6B). These data provide the first
evidence for serine phosphorylation of paxillin in response to
integrin-mediated adhesion to vitronectin.
Figure 6:
Analysis of total paxillin
phosphorylation. A, M-CSF-treated macrophages were labeled
with [P]orthophosphate as described under
``Experimental Procedures,'' then maintained either in a
nonadherent state for 60 min (lane 1), or allowed to attach
and spread on uncoated (lane 2) or vitronectin-coated (lane 3) Petri dishes for 60 min. Cells were solubilized in
RIPA lysis buffer, and equal concentrations and volumes of cell lysates
were immunoprecipitated using an anti-paxillin mAb. The precipitated
immunocomplexes were visualized following SDS-PAGE and autoradiography. B, the phosphorylated band corresponding to paxillin was
excised and subjected to two-dimensional phosphoamino acid analysis as
described under ``Experimental Procedures.'' PT (phosphothreonine), PY (phosphotyrosine), and PS (phosphoserine) indicate the relative migration of the
phosphoamino acid standards. The smear running below and to the left of the PY area represents phosphopeptides generated by
incomplete acid hydrolysis.
Figure 7:
Effect of PMA on paxillin phosphorylation. A, M-CSF-treated macrophages were labeled with
[P]orthophosphate as described under
``Experimental Procedures.'' Cells were subsequently
maintained either in a nonadherent state for 60 min (lane 1),
or allowed to attach and spread on vitronectin-coated dishes for 60 min
in the absence (lane 2) or presence of 5 ng/ml PMA (lane
3). To assess paxillin phosphorylation, cells were solubilized in
RIPA lysis buffer and equal concentrations and volumes of cell lysates
were immunoprecipitated using an anti-paxillin mAb. The precipitated
immunocomplexes were visualized following SDS-PAGE and autoradiography. B, M-CSF-treated macrophages were labeled with
[
P]orthophosphate as described under
``Experimental Procedures.'' Cells were then allowed to
attach and spread on vitronectin-coated dishes for time points up to
and including 60 min in the presence or absence of 5 ng/ml PMA.
Paxillin phosphorylation was assessed as described above in A.
Autoradiographs were scanned by laser densitometry and results
expressed as arbitrary units normalized against
background.
Figure 8: Effects of PMA and chelerythrine chloride on paxillin localization to focal contacts. M-CSF-treated macrophages were allowed to attach and spread on vitronectin-coated coverslips under serum-free conditions for 60 min in the absence (A) or presence (B) of 5 ng/ml PMA. Cells were fixed, permeabilized, and stained with mAb to paxillin. Focal contacts are highlighted by arrowheads. In panel C, cells were pretreated with 1 µM chelerythrine chloride for 60 min prior to attachment and spreading on vitronectin for an additional 60 min in the absence of PMA. Cells were then fixed, permeabilized, and stained with mAb to paxillin. Cells pretreated with GF109203X revealed a staining pattern that was virtually identical to that seen in panel C for chelerythrine chloride. Bar = 10 µm.
Figure 9:
Effect of PKC inhibitors GF109203X and
chelerythrine chloride on paxillin phosphorylation. M-CSF-treated
macrophages were labeled with [P]orthophosphate
and pretreated with either dimethyl sulfoxide (vehicle control) (lanes 1, 2, and 4), 10 µM GF109203X (lane 3), or 1 µM chelerythrine chloride (lane 5) for 60 min as described under ``Experimental
Procedures.'' Cells were then maintained either in a nonadherent
state for 60 min (lane 1), or allowed to attach and spread on
vitronectin-coated Petri dishes for 60 min (lanes 2-5).
To assess the phosphorylation status of both paxillin and Src, cells
were solubilized in RIPA lysis buffer and equal concentrations and
volumes of cell lysates immunoprecipitated using either an
anti-paxillin mAb (paxillin) or a polyclonal antisera raised
against c-src (src). The precipitated immunocomplexes
were visualized by autoradiography after
SDS-PAGE.
We previously reported that the colony-stimulating factor
M-CSF is able to induce expression of the integrin receptor on the surface of cultured human
macrophages(28) . Moreover, we found that in M-CSF-treated
macrophages adherent to vitronectin,
staining was
localized to focal contacts. However, the identity of the
subunit
associating with
in focal contacts could not be
demonstrated definitively(28) . Here, we confirm by using mAbs
to the
subunit and
complex that M-CSF-treated macrophages express
only in association with
, and extend these
observations to demonstrate that these cells adhere to vitronectin in
an
-dependent manner. Indirect
immunofluorescence analysis establishes definitively the localization
of
to focal contacts, suggesting the
surface expression of
on cultured
human macrophages is a determining factor in their morphology on
vitronectin.
We find that like the integrin
, paxillin, a vinculin-binding
protein (15, 16) also localizes to focal contacts in
M-CSF-treated macrophages adherent to vitronectin. FAK is thought to
play a central role in the organization of the cytoskeleton as cells
adhere to the ECM. In particular, it has been suggested that the
localization of paxillin to focal contacts and its phosphorylation on
tyrosine are tightly coupled to FAK activation(10) . We
demonstrate here by immunoprecipitation and Western blot analysis,
together with indirect immunofluorescence studies, that M-CSF-treated
macrophages do not express FAK. These observations are consistent with
previous reports in mouse bone marrow-derived macrophages(39) ,
and in human peripheral blood monocytes (33) that demonstrate
the absence of FAK expression at both the RNA and protein levels,
respectively. Our findings and particularly those described by
Juliano's group (33) are somewhat at variance with the
observations recently reported by Kharbanda et
al.(40) . These authors reported the presence of FAK
protein in human monocytes freshly isolated from peripheral blood. The
reason for this discrepancy is not known; one distinct possibility,
however, is that FAK observed in these cells is not of monocyte origin,
but is rather derived from contaminating platelet microparticles that
are rich in FAK and are known to bind to the surface of activated
monocytes as a direct result of monocyte purification by adhesion to
plastic(41) , the same method used by Kharbanda et
al.(40) . It is well established that monocyte
purification by counterflow centrifugal elutriation does not result in
the activation of these cells as assessed by the absence of
interleukin-2 receptor expression, a sensitive marker of monocyte
activation(42) . The monocytes prepared in this laboratory and
that of Juliano's (33) were isolated by counterflow
centrifugal elutriation, so it is therefore not surprising that both
studies reach the same conclusion that monocytes/macrophages do not
express FAK.
This study provides direct evidence for
integrin-dependent localization of paxillin to focal contacts in the
absence of FAK, and is consistent with a recent report demonstrating
focal contact formation in cells derived from FAK-deficient
mice(43) . Our observations expand the implications of recent
findings that the process of tyrosine phosphorylation of paxillin by
FAK is not a critical determinant in the localization of paxillin to
focal contacts (20) , and that paxillin can undergo tyrosine
phosphorylation in the absence of FAK(43) . Although the
underlying molecular mechanism responsible for mediating paxillin
localization to focal contacts is currently open to speculation,
Schaller et al.(44) recently established that
paxillin is able to bind specifically to synthetic peptides that mimic
integrin subunit cytoplasmic domains. Whether paxillin binding is
a direct or indirect interaction is presently unresolved, although it
is apparent from these studies that the interaction occurs independent
of FAK(44) . Our own observations, coupled with those of other
laboratories raise the distinct possibility that upon adhesion of
M-CSF-treated macrophages to vitronectin, the localization of paxillin
to focal contacts may occur via interaction with the integrin
subunit cytoplasmic domain.
One of the major
observations communicated in this report is the demonstration by
phosphoamino acid analysis that paxillin is 99% phosphorylated on
serine residue(s) in response to
-mediated adhesion to vitronectin.
While much attention in the literature has focused on the
phosphorylation of paxillin on tyrosine residues, serine
phosphorylation of paxillin in response to integrin-mediated cell
adhesion to the ECM was previously overlooked, despite the knowledge
that paxillin contains multiple consensus target sites for a number of
serine/threonine kinases, including PKC(18) , cAMP-dependent
protein kinase, casein kinase II, p34
, and S6 kinase.
Our observations indicate that in M-CSF-treated macrophages adherent to
vitronectin, the serine phosphorylation of paxillin is mediated via a
PKC-dependent mechanism. We provide evidence to show that direct
stimulation of PKC activity with the phorbol ester PMA enhances
paxillin phosphorylation, whereas two selective inhibitors of PKC,
GF109203X and chelerythrine chloride, specifically and effectively
block the phosphorylation of paxillin induced in response to
vitronectin adhesion. Whether the phosphorylation of paxillin on serine
is directly mediated by PKC, or alternatively, whether PKC acts
indirectly to modulate the activity of other serine/threonine kinase(s)
that phosphorylate paxillin is yet to be determined. Inhibition of
paxillin serine phosphorylation was found to correlate with inhibition
of focal contact formation in M-CSF-treated macrophages adherent to
vitronectin. PKC-mediated phosphorylation changes may be an important
mechanism in the assembly of focal contacts(23) . The
localization of PKC to focal contacts (21, 22) supports this potential role of PKC and
therefore serine/threonine phosphorylation in focal contact assembly
and cell spreading.
In addition to serine phosphorylation, we also
demonstrate the phosphorylation of paxillin on tyrosine residue(s),
albeit at very low levels, in response to
-mediated adhesion of macrophages to
vitronectin. In the absence of FAK expression, these data suggest that
paxillin serves as a substrate for a protein-tyrosine kinase other than
FAK. One obvious candidate to fulfill this role is the cytoplasmic
protein-tyrosine kinase pp60
, which was recently shown to
phosphorylate paxillin on tyrosine in vitro at sites that
become phosphorylated in vivo(19) . Our own
observations demonstrate concomitant phosphorylation of both Src and
paxillin in response to integrin-mediated adhesion of M-CSF-treated
macrophages to vitronectin, suggesting that paxillin tyrosine
phosphorylation is coupled to Src kinase activity. The precise role of
integrin-mediated tyrosine phosphorylation of paxillin remains unknown.
Recent evidence indicates that tyrosine phosphorylation of paxillin is
not essential for the localization of paxillin to focal
contacts(20) . Phosphorylation of paxillin on tyrosine
residues, however, has been reported to create binding sites for the
SH2 domains of the protein-tyrosine kinases Csk (45) and
Src(46) , as well as the oncoprotein
Crk(19, 47) , suggesting paxillin may serve a role in
signal transduction as an adaptor protein to facilitate the recruitment
of signaling molecules to focal contact sites(8) .
Our findings that paxillin localizes to focal contacts in the absence of FAK expression and is predominantly phosphorylated on serine residue(s) in response to vitronectin adhesion are intriguing, particularly in light of the recent report that tyrosine phosphorylation of paxillin is not essential for its recruitment to focal contacts(20) . Whether serine phosphorylation of paxillin alone is the critical determinant in its localization to focal contacts remains to be determined, but the 99:1 predominance of serine to tyrosine phosphorylation suggests a significant functional role for this major extracellular substrate-dependent modification.