From the Department of Medical Microbiology and Immunology,
University of Alberta, Edmonton, Alberta T6G 2H7, Canada
Antibodies to either CD3 or CD45 have been shown
to induce dramatic changes in cell morphology, increased tyrosine
phosphorylation of cellular proteins, and the association of a subset
of these proteins with the tyrosine kinase Lck. The current study was
initiated to determine the identity of the tyrosine-phosphorylated
70-80 kDa protein that becomes Lck-associated after stimulation with anti-CD45 or anti-CD3. We demonstrate that the cytoskeletal protein paxillin becomes tyrosine-phosphorylated when cells are plated on
immobilized antibodies specific for CD45 or CD3. Only
tyrosine-phosphorylated paxillin is associated with Lck, suggesting
that the association is through the SH2 domain of Lck. Consistent with
this we demonstrate that the SH2 domain of Lck binds
tyrosine-phosphorylated paxillin. In contrast, the association of
paxillin with the FAK-related kinase Pyk2 was found to be constitutive
and not altered by the phosphorylation of either protein. Finally, we
establish that the phosphorylation of paxillin is dependent on the
expression of Lck. Taken together, these results demonstrate that
paxillin is physically associated with kinases from two different
families in T cells and suggest that paxillin may function as an
adaptor protein linking cellular signals with cytoskeletal changes
during T cell activation.
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INTRODUCTION |
T cells have been shown to undergo dramatic changes in cell
morphology, coincident with cytoskeletal rearrangements, upon activation (1, 2). A number of studies over the past few years have
indicated that protein tyrosine phosphorylation and the formation of
focal adhesions, the sites of contact between integrins on the cell
surface and extracellular matrix, are tightly linked (3, 4). It is
plausible that some of the phosphorylation events that are observed
upon T cell activation regulate cytoskeletal reorganization, leading to
the observed changes in cell morphology.
CD45 is a protein-tyrosine phosphatase expressed on all cells of
hematopoietic origin (5). We have previously demonstrated that
antibodies to CD45 induce rapid and dramatic changes in T cell
morphology (6). Coincident with these changes in morphology is an
increase in the tyrosine phosphorylation of proteins at approximately
70 kDa and 120 kDa and an association of these proteins with the
Src-related protein-tyrosine kinase Lck (6). Phosphorylation of these
proteins also occurs upon stimulation through the T cell receptor (6).
Furthermore, phosphorylation of these proteins is enhanced when both
anti-CD45 and anti-CD3 are coimmobilized, suggesting that engagement of
CD45 or CD3 stimulates the phosphorylation of an overlapping set of
proteins (6). The identification of these proteins might therefore
provide insight into the regulation of T cell activation.
Phosphorylation of a Src-related kinase at its negative regulatory site
results in the intramolecular interaction of the phosphorylated residue
with the SH2 domain of the same molecule, rendering it inactive (7-9).
An implication of this model is that dephosphorylation of Tyr-505 would
not only allow the kinase to become activated but would also allow for
the interaction of other phosphorylated proteins with the newly
available SH2 domain of Lck, which may have important biological
consequences. Consistent with this notion, recent studies have
suggested that the SH2 domain of Lck is required for T cell activation
(10, 11).
Paxillin is a cytoskeletal protein that localizes to sites of adhesion
to extracellular matrix (12). It becomes tyrosine-phosphorylated upon
integrin engagement and associates with focal adhesions (12), possibly
as a consequence of its direct association with focal adhesion kinase
(FAK)1 (13, 14). As a
cytoskeletal protein that targets to focal adhesions, paxillin is
thought to be intimately involved in cell spreading. Very little is
known about the role, if any, of paxillin in T cell activation. Because
T cells undergo dramatic changes in cellular morphology upon T cell
activation we sought to examine the possible link of Lck with the
cytoskeleton. In the current study we establish that paxillin becomes
phosphorylated upon T cell action and associates with Lck and may
therefore function as an adaptor protein linking tyrosine
phosphorylation events with the cytoskeleton by way of its association
with Lck.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
The murine H-2b-specific CTL clones
11 and AB.1 have been described previously (15). Cells were stimulated
weekly with irradiated C57BL/6J spleen cells in media supplemented with
interleukin-2, and experiments were performed 4-6 days after
stimulation. All of the experiments that are shown were done with AB.1
and confirmed with clone 11. Jurkat and J.CaM1.6 were obtained from the
ATCC.
Antibodies and Reagents--
The monoclonal antibodies 145-2C11
(2C11, anti-mouse CD3) and OKT3 (anti-human CD3) were obtained
through the ATCC. I3/2 (anti-CD45) was obtained from Dr. I. Trowbridge, and PY-72 (anti-phosphotyrosine) was obtained
from Dr. B. Sefton, both of whom are at The Salk Institute (La Jolla,
CA). Hybridomas were grown in Protein-Free Hybridoma Medium-II (Life
Technologies, Inc.). The monoclonal antibodies were purified by
ammonium sulfate precipitation and, if necessary, by protein A or
protein G chromatography. The monoclonal antibody specific for paxillin
was obtained from Transduction Laboratories (Lexington, KY). Antiserum
to the carboxyl terminus of Lck was generated in our laboratory using a
bovine serum albumin-coupled peptide based on amino acids 476-509 of
human Lck (6). Many of the experiments have also been done with
anti-Lck (carboxyl terminus) antiserum obtained from Upstate
Biotechnology Inc. (Lake Placid, NY). Anti-Pyk2 antiserum was generated
in our laboratory as described previously (16). Anti-GST was obtained
from Sigma. Horseradish peroxidase-coupled goat anti-mouse antibody and
goat anti-hamster IgG were purchased from Jackson Immunologicals
(Mississauga, ON) and protein A from Pierce. The GST fusion proteins
used in this study have also been described previously (17).
Protein Immobilization--
60 mm Petri dishes were incubated
with 5 ml of phosphate-buffered saline containing 15 µg/ml of the
indicated antibody (2C11 or I3/2) overnight at 4 °C. Plates were
then washed twice with phosphate-buffered saline, blocked with 2%
bovine serum albumin in phosphate-buffered saline at 37 °C for 30 min, washed twice with phosphate-buffered saline, then used immediately
for assay.
Immunoprecipitation, Polyacrylamide Gel Electrophoresis, and
Immunoblotting--
Cells (7.5 × 106 per plate) were
incubated for 20 min at 37 °C and were lysed directly on the plates
by adding 1.5× lysis buffer to give a final concentration of 1%
Nonidet P-40 in 20 mM Tris, pH 7.6, 150 mM
NaCl, 1 mM sodium vanadate. Postnuclear lysates were
incubated with the indicated antibody for 30 min on ice after which
protein A-Sepharose (Pharmacia, Ste-Anne-de-Bellevue, QC) was added
prior to a 90-min incubation with rotation. If a monoclonal antibody
was used, rabbit anti-mouse antiserum was added to facilitate immunoprecipitation. Immunoprecipitates were washed 4 times with RIPA
buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1%
Nonidet P-40, 1% deoxycholate, 0.1% SDS) before SDS-PAGE on 7.5%
gels. Immunoblotting was performed by either anti-phosphotyrosine or
anti-paxillin followed by rabbit anti-mouse coupled to horseradish
peroxidase. Blots were developed by chemiluminescence (NEN Life Science
Products) as described in the product bulletin.
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RESULTS |
Immobilized Antibodies to CD45 or CD3 Induce the Tyrosine
Phosphorylation of a 70-80-kDa Cellular Protein and Its Association
with Lck--
When T cell clones are plated on immobilized anti-CD45
or anti-CD3, but not on anti-Class I MHC, the cells undergo dramatic changes in cellular morphology typified by extensive spreading (6).
Coincident with cell spreading induced by these antibodies there is an
increase in the tyrosine phosphorylation of proteins in the ranges of
70 to 80 kDa and 115 to 120 kDa (Fig. 1).
The extent of phosphorylation induced with anti-CD3 is significantly greater than that seen with anti-CD45 (Fig. 1). Tyrosine-phosphorylated proteins are found in Lck immunoprecipitates following anti-CD3 stimulation (Fig. 1) as we have previously shown for the CD45-induced phosphoproteins (6). These proteins are not detected in control ZAP-70
immunoprecipitates (Fig. 1) or in preimmune serum (6, 17). We have
identified two of the approximately 120-kDa Lck-associated proteins as
the related tyrosine kinases FAK and Pyk2 (17). In the current study,
we turn our attention to the identification of the 70-80-kDa
tyrosine-phosphorylated proteins.

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Fig. 1.
Antibodies to CD45 or CD3 induce the tyrosine
phosphorylation and association of an 80-kDa protein with Lck.
Anti-phosphotyrosine blots of lysates or anti-Lck immunoprecipitates
from AB.1 CTL cells plated on BSA as a control or on immobilized
anti-CD45 (I3/2) or anti-CD3 (2C11). Antiserum to ZAP-70 was used as a
negative control for the associations. The closed arrowhead
indicates the 80-kDa protein that associates with Lck after either
anti-CD45 or anti-CD3 stimulation, and the open arrowhead
indicates the position of Lck.
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The Cytoskeletal Protein Paxillin Becomes Tyrosine-phosphorylated
and Associates with Lck after Stimulation with Anti-CD45 or
Anti-CD3--
Paxillin is an approximately 75-kDa cytoskeletal protein
that undergoes tyrosine phosphorylation during actin reorganization coincident with cell spreading (12). Because paxillin appears to fit
the characteristics of the Lck-associated protein detected in Fig. 1,
we determined whether the 70-80-kDa protein is indeed paxillin.
Paxillin immunoprecipitates from AB.1 cells that had been plated on
immobilized anti-CD3 or anti-CD45 or on BSA as a control were blotted
with anti-phosphotyrosine. Paxillin clearly undergoes a significant
increase in tyrosine phosphorylation after stimulation with either
antibody (Fig. 2A). Consistent
with the data presented in Fig. 1, the phosphorylation is much more
extensive in anti-CD3-stimulated cells. This phosphorylation is
coincident with a significant shift in the migration of paxillin as can
be seen in the anti-paxillin blot in Fig. 2B, which has been
confirmed with anti-phosphotyrosine immunoblotting (data not shown). A
large proportion of the paxillin, about 60-70% in most experiments, becomes phosphorylated upon anti-CD3 stimulation (Fig.
2B).

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Fig. 2.
Paxillin becomes tyrosine-phosphorylated in
response to immobilized anti-CD3 or anti-CD45. A,
anti-phosphotyrosine blot of paxillin immunoprecipitated from cells
plated on the indicated antibody or on BSA as a control. B,
anti-paxillin blot of postnuclear lysates from cells stimulated as in
A. The open arrow indicates the position of
non-tyrosine-phosphorylated paxillin and the closed arrow
the position of tyrosine-phosphorylated paxillin.
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To directly determine if paxillin is present in Lck immunoprecipitates
they were probed with anti-paxillin antibodies. Paxillin can be easily
detected in Lck immunoprecipitates from stimulated, but not
unstimulated, cells, and there is significantly more paxillin in the
Lck immunoprecipitates from the anti-CD3-stimulated than the
anti-CD45-stimulated cells (Fig. 3).
Interestingly, the associated paxillin is only of the more slowly
migrating species suggesting that the association with Lck requires
tyrosine phosphorylation of paxillin.

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Fig. 3.
Paxillin can be detected in Lck
immunoprecipitates from I3/2- or 2C11-stimulated cells. AB.1 T
cells were plated on BSA, I3/2, or 2C11 followed by Lck
immunoprecipitation and anti-paxillin immunoblotting. The open
arrow indicates the position where non-tyrosine-phosphorylated paxillin would run, and the closed arrow the position of
tyrosine-phosphorylated paxillin.
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Paxillin Associates with the SH2 Domain of Lck--
That the
association between Lck and paxillin requires the tyrosine
phosphorylation of paxillin implies that the interaction may be
mediated through the SH2 domain of Lck. We are able to compete for
binding of essentially all of the Lck-associated proteins with phenyl
phosphate (data not shown), which is consistent with what has been
shown previously with Lck immunoprecipitates prepared from activated T
cells (18). Anti-phosphotyrosine immunoblotting of paxillin
immunoprecipitates prepared from phenyl phosphate eluates of Lck
immunoprecipitates reveal that tyrosine-phosphorylated paxillin is
bound to Lck; however no tyrosine-phosphorylated paxillin is observed
in the control rabbit anti-mouse Ig immunoprecipitate from
2C11-stimulated cells, and very little is detected in paxillin immunoprecipitates from unstimulated cells (Fig.
4). These results further confirm that
paxillin associates with Lck in CTL clones and suggest that these
proteins associate in a phosphotyrosine-dependent manner
and possibly a SH2-dependent manner.

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Fig. 4.
Paxillin can be eluted by phenyl phosphate
from Lck immunoprecipitates. Cells were plated on BSA, I3/2, or
2C11, and Lck immunoprecipitates were prepared. The immunoprecipitates
were incubated with phenyl phosphate, and the eluted material was
reimmunoprecipitated with either paxillin or with rabbit anti-mouse Ig
as a control (C) and blotted with anti-phosphotyrosine. The
arrowhead indicates the position of tyrosine-phosphorylated
paxillin.
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To directly address the question of whether paxillin associates with
the SH2 domain of Lck, we generated GST fusion proteins containing
either the SH2 or the SH3 domain of Lck. The GST fusion proteins were
mixed with lysates from AB.1 stimulated with either anti-CD45 or
anti-CD3, and the complexes were captured with glutathione beads. An
80-kDa tyrosine-phosphorylated protein can associate with the Lck-SH2
domain GST fusion protein after stimulation of cells with 2C11 and to a
low level after plating of cells on immobilized anti-CD45 (Fig.
5). This phosphoprotein does not
associate with our SH3 domain preparations or with GST alone (Fig. 5).
When this same blot is probed with anti-paxillin (Fig. 5), it is clear
that paxillin specifically associates with the SH2 domain of Lck.
Although not apparent on this blot, only the more slowly migrating
phosphorylated form of paxillin is associating with the fusion protein
(data not shown).

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Fig. 5.
GST fusion proteins containing the SH2 domain
of Lck can associate with paxillin in cell lysates from anti-CD45- or
anti-CD3-induced cells. Lysates from control cells or anti-CD45-
or anti-CD3-stimulated cells were incubated with purified GST,
SH2-GST-Lck, or SH3-GST-Lck. The GST fusion proteins were incubated at
either 10 or 50 µg/ml. After incubation, complexes were recovered
with glutathione-coupled Sepharose beads, and the associated proteins
were detected by anti-phosphotyrosine immunoblotting. The blot was
stripped and reprobed with anti-paxillin.
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The argument could still be made that paxillin does not interact with
the SH2 domain of Lck directly but rather forms a complex with a
phosphoprotein that itself interacts with Lck. To assess whether
paxillin binds directly to the SH2 domain of Lck, a Far Western blot of
paxillin immunoprecipitates using GST-SH2-Lck as a probe was completed.
The data presented in Fig. 6 show that the SH2 domain of Lck binds to paxillin immunoprecipitated from 2C11-stimulated cells. Interestingly, the GST-SH2-Lck probe fails to
interact with immunoprecipitates from unstimulated cells, which contain
only nonphosphorylated paxillin (Fig. 6). Since anti-CD45 does not
induce extensive phosphorylation of paxillin, we did not include it in
these studies. These experiments confirm that there can be a direct
interaction between the SH2 domain of Lck and tyrosine-phosphorylated
paxillin.

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Fig. 6.
The SH2 domain of Lck binds to phosphorylated
paxillin. AB.1 were plated on BSA or 2C11, and paxillin was
immunoprecipitated. The blots were then probed with GST-SH2-Lck
followed by anti-GST antiserum and protein A coupled to horseradish
peroxidase (left). A parallel blot was performed with
anti-paxillin antibodies (right). The open arrow
indicates the position of non-tyrosine-phosphorylated paxillin and the
closed arrow the position of tyrosine-phosphorylated paxillin.
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Paxillin Is Constitutively Associated with Pyk2 in CTL--
It has
been demonstrated that paxillin can directly associate with FAK through
an interaction that is phosphotyrosine independent (13, 14). Recently
paxillin has also been shown to constitutively associate with the
FAK-related kinase Pyk2, also known as related adhesion focal tyrosine
kinase (RAFTK), in T cells with only a modest increase in the level of
association after T cell activation (19). Since both FAK and Pyk2 are
expressed in our CTL clones (16) we determined if either FAK or Pyk2
preferentially associates with paxillin in these clones. We prepared
immunoprecipitates of FAK, Pyk2, and paxillin and probed these with
anti-paxillin. Interestingly, we did not see a significant interaction
between paxillin and FAK; however we saw a strong association with Pyk2 (Fig. 7). Even when the blot was
considerably overexposed, paxillin could not be detected in the FAK
immunoprecipitates (data not shown). We know that the FAK antiserum is
functional as it immunoprecipitates tyrosine-phosphorylated FAK in
parallel experiments (data not shown and Ref. 17). The level of
association between Pyk2 and paxillin did not significantly change upon
T cell receptor complex stimulation; however the Pyk2-associated
paxillin did become phosphorylated after stimulation (Fig. 7). These
results indicate that paxillin is constitutively associated with Pyk2
but not with FAK in these T cell clones.

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Fig. 7.
Paxillin is associated with Pyk2. AB.1
were plated on either BSA or 2C11. After lysis, immunoprecipitates were
prepared with anti-FAK, anti-Pyk2, or anti-paxillin antibodies followed by anti-paxillin immunoblotting.
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Lck Is Required for the Tyrosine Phosphorylation of
Paxillin--
Since paxillin is physically associated with at least
two different tyrosine kinases in T cells, it is of interest to
determine if either of these kinases might be responsible for its
phosphorylation. To address this question we made use of the
Src-related tyrosine kinase inhibitor PP1 (20). When cells are treated
with this drug prior to anti-CD3 stimulation, as previously observed
(20), no increased tyrosine phosphorylation is detected (Fig.
8A). In this experiment cells
were stimulated with cross-linked anti-CD3 in solution since we have
observed that PP1 prevents cell spreading thereby limiting the ability
of cells to contact immobilized antibody (data not shown). When the
phosphorylation status of paxillin was examined, we found that PP1
completely inhibited its tyrosine phosphorylation (Fig. 8B),
suggesting that a Src-related kinase is required upstream of paxillin
phosphorylation. However, this inhibition could be either direct or
indirect because PP1 also inhibits the induction of Pyk2
phosphorylation, although PP1 does not directly inhibit Pyk2 kinase
activity as measured by autophosphorylation (data not shown).

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Fig. 8.
Paxillin phosphorylation is inhibited by the
Src-related kinase inhibitor PP1. AB.1 cells were stimulated for
the indicated times with cross-linked 2C11 in the presence or absence
of 10 µM PP1. A, total cell lysates were
blotted with anti-phosphotyrosine antibodies. B, paxillin
immunoprecipitates were prepared and blotted with anti-phosphotyrosine
antibodies.
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To more directly assess the role of Lck in the induction of tyrosine
phosphorylation of paxillin we examined paxillin phosphorylation in the
Lck-deficient variant of Jurkat called J.CaM1.6. We have previously
demonstrated that Lck is not required for the induction of FAK or Pyk2
phosphorylation (17). However, examination of paxillin phosphorylation
reveals that it does not become tyrosine-phosphorylated in the J.CaM1.6
cells (Fig. 9). Taken together these
results demonstrate that Lck is required, either directly or
indirectly, for paxillin phosphorylation.

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Fig. 9.
Paxillin does not become
tyrosine-phosphorylated in Lck-deficient Jurkat cells. Jurkat
cells or Lck-deficient J.CaM1.6 were stimulated for the indicated times
with 10 µg/ml OKT3 (anti-CD3) and then lysed. Paxillin
immunoprecipitates were prepared and subjected to PAGE, along with
aliquots of total cell lysates, followed by immunoblotting with
anti-phosphotyrosine antibodies. The same membrane was then stripped
and reprobed with anti-paxillin.
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DISCUSSION |
We have provided the first demonstration that antibodies to CD45
or CD3 stimulate the phosphorylation of paxillin leading to its
association with the SH2 domain of Lck. Paxillin contains a number of
potential tyrosine phosphorylation sites, five of which conform to
SH2-binding motifs (21), and it binds to the SH2 domain of Src upon
tyrosine phosphorylation (22). Since Lck is the predominant Src-related
kinase found in T cells it could be predicted that it might bind to
paxillin via its SH2 domain. Paxillin also contains a proline-rich
domain that may be important for linking it to other signaling
molecules or cytoskeletal components (21). Because paxillin is a major
component of the cytoskeleton and can bind to multiple signaling
molecules it may function as an adaptor molecule linking intracellular
signals with morphologic changes known to accompany T cell activation. The phosphorylation of paxillin may therefore be important for regulating its adaptor function during T cell activation.
In addition to the association with Lck, we have also found that
paxillin constitutively associates with Pyk2 in T cells, as previously
shown (19). We could detect little or no association between FAK and
paxillin. These results show that paxillin is able to bind to at least
two kinases from different tyrosine kinase families in T cells; however
it is not clear which kinase, if either, is directly responsible for
phosphorylating paxillin. Both FAK and Pyk2 have been shown to
phosphorylate paxillin in vitro (22, 23). It is therefore
possible that one or both of these kinases is responsible for paxillin
phosphorylation in CTL clones. It is also possible that Lck might
phosphorylate paxillin since paxillin has also been shown to be a
substrate for Src (22). Consistent with this we have demonstrated that
the Src kinase inhibitor PP1 is able to inhibit paxillin
phosphorylation. Although we cannot formally exclude a role for FAK or
Pyk2 in phosphorylating paxillin, we reason that paxillin is unlikely
to be a direct substrate of these kinases since both FAK and Pyk2
become phosphorylated (17) in the Lck-deficient cells while paxillin
does not become phosphorylated (Fig. 9). It is also conceivable that
both Pyk2 and Lck phosphorylate paxillin at different sites. Studies
are currently underway to distinguish these possibilities.
Adhesion through a number of different integrin receptors is known to
play an important role in T cell activation. The signals that are
required to induce the cytoskeletal changes required for increased
adhesion remain largely unknown. Our previous results show that cell
spreading, which is coincident with increased adhesion, correlates with
increased tyrosine phosphorylation (6). Which is cause and which is
effect remains to be determined. Interestingly, only those antibodies
or ligands that induce cell spreading induce tyrosine phosphorylation
of paxillin. So far this is limited, in our hands, to purified class I
MHC molecules (data not shown) and antibodies to the T cell receptor
complex or CD45. We have shown that antibodies to
3
integrins, which are expressed on these T cells, stimulate
phosphorylation of FAK and Pyk2 (16); however no significant cell
spreading or paxillin phosphorylation is stimulated by these
antibodies. Furthermore, antibodies to LFA-1 or class I MHC induce
limited phosphorylation of FAK and Pyk2; however little or no cell
spreading and paxillin phosphorylation is observed with these
antibodies (data not shown). Taken together these results suggest that
cell spreading correlates with paxillin phosphorylation. Paxillin
phosphorylation does not appear to require adhesion since
phosphorylation of paxillin occurs, albeit very transiently, upon
stimulation of CTL with soluble cross-linked antibody to CD3 (Fig. 8
and data not shown). We believe, therefore, that paxillin
phosphorylation leads to cell spreading and not vice versa.
That CD45 plays a role in regulating the phosphorylation of paxillin
suggests that it might be involved in the regulation of adhesion. It
has been clearly established that antibodies to CD45 can induce
homotypic adhesion in various cell types (24-26). We have observed
that CD45-deficient cell lines do not appear to be as adhesive as their
CD45-expressing
counterparts.2 Recently Roach
et al. (27) have shown that macrophages from CD45-deficient
mice are unable to maintain integrin-mediated adhesion. They further
demonstrated that this disregulation of adhesion correlates with the
hyperphosphorylation of Src-related kinases (27). It is possible that
CD45 might regulate adhesion in a global manner by dephosphorylating
Src-related kinases which in turn function to regulate the
phosphorylation of paxillin. Interestingly, a CD45-related
transmembrane tyrosine phosphatase, LAR, has been found to localize to
focal adhesions, where FAK and paxillin are located, and may induce
their disassembly (28). Taken together these studies support the
provocative idea that CD45 regulates adhesion by regulating FAK, Pyk2,
and paxillin phosphorylation, perhaps via Lck or other Src-related
kinases.
In summary, the phosphorylation of paxillin, and likely its function,
are regulated through the T cell receptor and can be modulated through
CD45 engagement. It appears that paxillin might act as a type of
adaptor protein that is able to bind to tyrosine kinases and
cytoskeletal proteins and may therefore be important for integrating
growth signals and cellular adhesion, both of which are essential for
mitogenic signals in T cells.
We thank Samuel Ho for generating the SH2-GST
fusion protein and Dr. Kevin Kane for critically reviewing the
manuscript.