From the Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, November 20, 2002, and in revised form, January 3, 2003
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ABSTRACT |
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The chemokine receptor CXCR4
and its cognate ligand, stromal cell-derived factor-1 The chemokine CXCL12 and its corresponding receptor CXCR4 play an
important role in immune and inflammatory responses, lymphopoiesis in
bone marrow, and in embryonic developmental processes (1-7). Targeted
disruption of either CXCR4 or CXCL12 protein leads to severe defects
that are embryologically lethal (3-6). CXCR4 has also been shown to
act as a coreceptor for the T-cell tropic human immunodeficiency virus
(HIV),1 type 1 strain and to
play a crucial role in HIV pathogenesis (8-10).
Although CXCL12 acts as a potent chemoattractant for various cell types
including T-cells and regulates the directional movement of these
cells, relatively little is known about the signaling pathways that may
mediate these effects (1, 11). We and others (12-16) have recently
deciphered the molecular mechanisms involved in regulating CXCR4 and
CCR5-mediated chemotaxis. We have demonstrated that CXCL12 binding to
CXCR4 stimulates multiple signaling pathways including activation of
focal adhesion components such as the related adhesion focal tyrosine
kinase (RAFTK, also known as Pyk2 or Cak- CD45 is expressed exclusively on cells of hematopoietic lineage
(18-20). It is a key regulator of antigen receptor signaling in T- and
B-cells, playing a pivotal role in the activation and development of
lymphocytes (18-23). Studies using CD45-deficient mice and cell lines
revealed that this phosphatase is very important for thymocyte
differentiation (24, 25). CD45 is shown to influence the early events
in T-cell activation by operating as a positive, as well as negative,
regulator of the Src family kinases, p56 Lck and p59 Fyn. Recent
studies have also identified CD45 as a negative regulator of
cytokine-mediated signaling by acting as a JAK tyrosine
phosphatase (26). Thus, CD45 plays a crucial role in cytokine
receptor-mediated differentiation, proliferation, and anti-viral
responses. In addition, CD45 is also required for some
integrin-mediated adhesion events (27, 28). It has also been associated
with Alzheimer's disease and multiple sclerosis in humans (29,
30).
Although our understanding of the molecular mechanisms of CD45 in
regulating TCR and cytokine receptor signaling have increased substantially, its role in chemokine-mediated biological functions has
not been explored. In the present study, we have investigated the role
of CD45 in regulating CXCL12-induced chemotaxis and MAP kinase
activation in T-cells. Our data indicate a prominent role for CD45 in
these processes and thus provide new information regarding CXCR4-mediated chemotactic signaling pathways.
Reagents and Materials--
Purified antibodies to
phosphospecific p44/42 MAP kinase were obtained from New England
Biolabs (Beverly, MA). Antibodies to p44/42 protein, phosphotyrosine
(pTyr99), and p56Lck were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Phosphospecific antibodies to
RAFTK (Pyk2, pTyr402, pTyr881) and FAK
(pTyr397) were obtained from BIOSOURCE
International (Camarillo, CA). Phosphotyrosine antibody (4G10), ZAP-70,
and SLP-76 were from Upstate Biotechnology (Lake Placid, NY). Paxillin
antibody was from Transduction Laboratory (San Diego, CA).
Electrophoresis reagents and nitrocellulose membrane were obtained from
Bio-Rad. The protease inhibitors leupeptin and antitrypsin, and
all other reagents, were obtained from Sigma.
Cells and Cell Culture--
The human T-cell line Jurkat, clone
JE6.1, and the CD45-deficient variant of this clone, J45.01, were
obtained from ATCC (Manassas, VA). J45/CH11 (expressing a chimeric
protein containing the extracellular and transmembrane domains of
HLA-A2 and the cytoplasmic domain of CD45), its control J45/A2
(expressing the extracellular and transmembrane domains of HLA-A2), and
J45/LB3 (J45.01 transfectants expressing normal human CD45) were kindly
provided by Dr. Gary A. Koretzky (University of Pennsylvania School of
Medicine) and Dr. Eric J. Brown (University of California, San
Francisco, CA) and have been described before (23). All the cell lines
were cultured at 37 °C in 5% CO2 in RPMI 1640 with 10%
fetal calf serum, 2 mM glutamine, 50 µg/ml penicillin,
and 50 µg/ml streptomycin.
Primary Lymphocyte Culture--
Primary lymphocytes were
isolated from heparinized venous blood as described before (31). Blood
collected from healthy donors was subjected to Ficoll-Hypaque density
gradient centrifugation at 3000 rpm for 25 min. The cells were
suspended in RPMI containing 15% fetal calf serum, 2 mM
glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin. Monocytes
were depleted by two rounds of adherence to plastic. Nonadherent cells
were stimulated with phytohemagglutinin (5 µg/ml) for 3 days. Cells
were then removed to a fresh medium supplemented with recombinant human
interleukin 2 (Advanced Biotechnologies, Columbia, MD). Two-week-old
cells were used for immunoprecipitation and Western blotting
experiments to study the association of CD45 with CXCR4, as described below.
Stimulation of Cells--
CD45-negative (J45.01), Jurkat
(JE6.1), J45/CH11, J45/A2, and J45/LB3 cells were washed twice with 1×
Hanks' salt solution (Mediatech Co.), suspended at 10 × 106 cells/ml in the same solution, and starved for 1 h
at 37 °C in 5% CO2. The cells were next stimulated with
100 ng/ml CXCL12. After stimulation, the cells were microfuged for
10 s and lysed with modified radioimmune precipitation assay
buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mM NaCl, 0.5% sodium deoxycholate, 200 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml each of
leupeptin and pepstatin, 2 mM each of sodium vanadate and
sodium fluoride, and 0.25 M sodium pyrophosphate). Total
cell lysates were clarified by centrifugation at 10,000 × g for 10 min. Protein concentrations were determined by a
Bio-Rad protein assay kit. The cell lysates were used for
immunoprecipitation, immunoblotting, and kinase assay as described below.
Immunoprecipitation--
Immunoprecipitation analysis was done
as described (12, 15). Briefly, equivalent amounts of protein from each
sample were precleared by incubation with protein-A-Sepharose
CL-4B/protein G-Sepharose (Amersham Biosciences) for 1 h at
4 °C. The supernatant from each sample was collected after brief
centrifugation. Different primary antibody was added for each
experiment, and samples were incubated at 4 °C for 4 h. The
immune complexes were precipitated with 50 µl of protein-A-Sepharose
CL-4B (50% suspension) or protein-G-Sepharose (10% suspension)
overnight at 4 °C or for 36 h for anti-CXCR4 immunoprecipitations. The nonspecific bound proteins were removed by
washing the Sepharose beads three times with modified radioimmune precipitation assay buffer and once with 1× phosphate-buffered saline
(PBS). The immune complexes bound to the beads were either subjected to
kinase assay or solubilized in 40 µl of 2× Laemmli buffer and
further analyzed by Western blotting, as described below.
Western Blotting--
Western blot analyses were done as
described previously. Briefly, equivalent amounts of protein from each
sample were run on 8% SDS-PAGE and transferred to nitrocellulose
membranes. The membranes were blocked with 5% non-fat dry milk and
incubated with primary antibody for 2 h at room temperature or
overnight at 4 °C. The blots were washed and incubated with
secondary antibody coupled to horseradish peroxidase for 2 h at
room temperature or overnight at 4 °C. The bands were visualized by
using the enhanced chemiluminescent system (Amersham Biosciences). The
data are representative of findings from three experiments. The
activity of the bands was quantified by densitometric analysis using a
Bio-Rad Imager. The mean densities of the bands are represented as the
optical density in units/mm2.
Chemotaxis Assays--
Assays were done as described previously
(15, 16). Briefly, J45.01, JE6.1, J45/CH11, J45/A2, or J45/LB3 cells
were washed twice, and 10 × 106 cells/ml were
suspended in medium containing RPMI 1640 with 2.5% fetal calf serum.
The chemotaxis assay was performed in 24-well plates containing 5-µm
porosity inserts (Co-Star Corporation, Kennebunk, ME). 100 µl (1 × 106 cells) from each cell line (J45.01, JE6.1, J45/CH11,
J45/A2, J45/LB3) was loaded onto the upper well. 0.6 ml of medium
containing CXCL12 (0, 10, 50, 100 ng/ml) was added to the lower
chamber. The plates were incubated for 3 h at 37 °C in 5%
CO2. After incubation, the inserts were removed carefully,
and the viable cells were counted using standard procedures. The
results are expressed as the number of cells migrating to the bottom
chamber. Each experiment was performed three or four times in triplicate.
Kinase Assay--
Kinase assay of the Src family member, Lck,
was done as described (32). Briefly, the immune complexes obtained by
immunoprecipitating the cell lysates with antibodies to Lck were washed
twice with radioimmune precipitation assay buffer and twice with kinase
buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 10 µM Na3VO4, 5 mM
MgCl2, 5 mM MnCl2). Finally, the
immune complexes were incubated in a total volume of 25 µl of kinase
buffer containing enolase as a substrate and 5 µCi of
[ Confocal Microscopy--
Confocal microscopy studies were done
as described earlier (33). Briefly, Jurkat (JE6.1 clones) cells were
washed twice with Hank's-buffered salt solution (Cellgro) and
resuspended in Hank's-buffered salt solution at a density of
107 cells/ml for 1 h at 37 °C. Serum-starved cells
were stimulated with 100 ng/ml CXCL12 at 37 °C for various time
periods. Following stimulation, the cells were washed with ice-cold
phosphate-buffered saline and fixed in 4% paraformaldehyde for 10 min
at room temperature. Next, the cells were permeabilized with 0.1%
Triton X-100 and 0.1% sodium citrate for 2 min on ice. The cells were
washed and blocked with 5% bovine serum albumin for 30 min at 4 °C.
CXCR4 or CD45 was stained with anti-CXCR4 or anti-CD45 antibodies
overnight at 4 °C, followed by staining with secondary antibody
coupled to Texas red (Vector Laboratories) or phosphatidylethanolamine (Amersham Biosciences). The cells were cytofuged on slides, and the
expression of these proteins was visualized using a Leica TCS
confocal microscope.
Flow Cytometry--
The CXCR4 or CD45 receptor on the JE6.1,
J45.01, J45/CH11, J45/A2, or J45/LB3 cells was stained with
phycoerythrin-coupled anti-CXCR4 or CD4 antibodies for 1 h at
4 °C. For CXCR4 down-modulation analysis, JE6.1 or J45.01 cells were
stimulated with CXCL12 (1 µg/ml) or HIV gp120 (1.2 µg/ml) for
various time periods (0, 2, 4, 6 h). Following stimulation, the
cells were washed with ice-cold PBS and fixed with 2% formaldehyde for
15 min at room temperature. The cells were stained with anti-CXCR4
antibody as discussed above and then washed with PBS, suspended in 1%
formaldehyde in PBS, and subjected to flow cytometric analysis.
Statistical Analysis--
The results are expressed as the
mean ± S.D. of data obtained from three or four experiments
performed in duplicate or triplicate. The statistical significance was
determined by the Student's t test.
CXCL12-induced and CXCR4-mediated Chemotaxis Is Reduced in
CD45-deficient Cells--
CXCL12-induced chemotaxis has been shown to
be regulated by tyrosine phosphatases SHP1 and SHP2 (15, 17). We have
further extended these studies to evaluate the role of CD45 in
CXCR4-mediated and CXCL12-induced chemotaxis. To examine the
involvement of CD45 in the regulation of CXCL12-induced chemotaxis, the
CD45-deficient Jurkat cell line J45.01 and CD45-expressing JE6.1 cells
were assessed for their ability to migrate in response to different
concentrations of CXCL12. The CD45-deficient J45.01 cells showed a
reduced response (>75%) toward CXCL12-induced (50 ng/ml) chemotaxis
in comparison to the control JE6.1 Jurkat cells (Fig.
1A).
The difference in chemotactic response toward CXCL12 was consistent
over a concentration range of 10-100 ng/ml, levels at which optimal
migration has been reported (16). However, migratory response was less
significant at higher concentrations of CXCL12 (500-1000 ng/ml) (data
not shown). Next, we identified the region of CD45 important for
CXCL12-induced chemotaxis by assaying the ability of transfectants
expressing hybrid CD45 cDNA to restore chemotaxis. As shown in Fig.
1, B and D, CD45-deficient Jurkat cells
expressing a chimeric protein consisting of the extracellular domain
and transmembrane domain of the class I major histocompatibility complex protein and the cytoplasmic domain of CD45 (J45/CH11) partially
restored the CXCL12-induced chemotaxis as compared with cells
expressing only the extracellular and transmembrane regions of the
class I major histocompatibility complex protein (J45/A2). Furthermore,
reconstitution of J45.01 cells with normal human CD45 (J45/LB3)
significantly restored the CXCL12-induced chemotaxis (Fig.
1C). These results show that expression of the full-length CD45 (J45/LB3) restored the CXCL12-induced chemotaxis to about 90%,
whereas cells expressing only the cytoplasmic domain of CD45 (J45/CH11)
restored the chemotaxis to about 65% as compared with the JE6.1
parental cell line (Fig. 1D). Similar levels of CXCR4 receptor were expressed by all cell lines (data not shown). Therefore, the differences in chemotaxis observed in the various Jurkat clones and
transfectants are not because of variation in CXCR4 levels.
CD45 Does Not Regulate CXCR4 Internalization--
CXCL12 and gp120
at higher concentrations have been shown to induce CXCR4 receptor
internalization (33). CXCR4 trafficking is important in HIV infection
and immune regulation. Recently, we have shown that the proteasome
pathway regulates CXCL12-induced down-modulation and chemotaxis (33).
In Fig. 1, we have shown that CD45 regulates CXCL12-induced chemotaxis.
Thus, we next explored the role of CD45 in the ligand-induced
down-modulation of the CXCR4 receptor. No significant difference in
CXCL12 or HIV gp120-induced down-modulation of CXCR4 was observed
between the CD45-positive JE6.1 (Fig.
2A) and CD45-negative J45.01
(Fig. 2B) cells. These results suggest that CD45 does not
regulate the CXCL12- or gp120-induced pathway leading to CXCR4
internalization.
CXCL12-induced Tyrosine Phosphorylation of CD45 and Its Association
with the CXCR4 Receptor--
To investigate further CD45-regulated
chemotactic signaling mechanisms, we first determined the tyrosine
phosphorylation status of CD45 upon CXCL12 treatment in the
CD45-positive JE6.1 cells. As shown in Fig.
3A, CXCL12 stimulation induced
the increased tyrosine phosphorylation of CD45. This phosphorylation
was rapid and reached a maximum level between 0.5 to 2.5 min. Equal
amounts of CD45 protein were present in each lane (Fig.
3A, bottom panel). We also investigated whether
the CXCR4 receptor associates with CD45 by coimmunoprecipitation and
immunoblotting studies. Cell lysates obtained from CD45-positive JE6.1
cells (Fig. 3B) or peripheral blood lymphocytes (Fig.
3C) stimulated with CXCL12 were immunoprecipitated with
anti-CXCR4 antibody and subjected to immunoblot analysis with anti-CD45
antibody. As shown in Fig. 3, B and C, CXCL12
stimulation induced the association of the CXCR4 receptor with CD45.
Equal amounts of protein were present in each lane as
detected by immunoblotting the lysates with anti-actin antibody (Fig.
3, B and C, bottom panels). The
association of CXCR4 with CD45 was further confirmed by confocal
microscopy. CD45-positive cells (JE6.1) were stimulated with CXCL12
(100 ng/ml) for different time periods. The cells were fixed and
labeled with fluorescein isothiocyanate-conjugated anti-CD45
(green) or phosphatidylethanolamine-conjugated anti-CXCR4 (red) antibody. As shown in Fig. 3D, a marked
colocalization (yellow) of the two proteins was observed
upon CXCL12 stimulation. CXCL12 treatment also induced the clustering
of CXCR4 and CD45 into pseudopodia-like structures (Fig.
3D).
Effect of Lipid Raft Inhibitor on the Association of CD45 with
CXCR4 and CXCL12-induced Chemotaxis--
Plasma membranes of many cell
types, including T-cells, contain microdomains referred to as lipid
rafts (34-36). These domains are rich in sphingolipids and
cholesterol, which form a lateral assembly in a saturated
glycerophospholipid environment. The domains are known to serve as
moving platforms on the cell surface and are more ordered and resistant
to detergents like Triton X-100. The domains also act as good sites for
cross-talk between various proteins. These include cytoskeletal
proteins, Src family kinases, protein kinase C, actin-binding proteins,
G proteins, and various molecules involved in TCR signaling (35, 36).
Lipid rafts have also been shown to be important for T-cell
polarization and chemotaxis (37, 38). To characterize the role of lipid
rafts in CXCR4-mediated signaling, we examined the effect of the lipid raft inhibitor, methyl- CD45 Regulates CXCR4-induced Src-related Kinases--
Src kinases
have been shown to play an important role in cell migration and
adhesion (39, 40). p56 Lck, a member of the Src family of
protein-tyrosine kinases, is a physiological substrate of CD45
(18-20). It has been shown that CD45-mediated dephosphorylation of
Tyr505 (Lck) activates this kinase. Therefore, we compared
the CXCL12-induced tyrosine phosphorylation and kinase activity of Lck
in CD45-positive and -negative cell lines. As shown, CXCL12 increased
the kinase activity of Lck (Fig. 5) as
compared with the untreated cells in the CD45-positive cell line.
However, no significant change in Lck kinase activity was observed in
the CXCL12-stimulated CD45-negative cells. We also observed that Lck
protein was hyperphosphorylated at tyrosine residues in the
CD45-negative cells as compared with the CD45-positive cells. Equal
amounts of Lck were present in the cell lysates.
CXCR4-mediated Tyrosine Phosphorylation of Focal Adhesion
Components Is Regulated by CD45--
Several components of focal
adhesion complexes are known to regulate chemokine-mediated chemotaxis
(41, 42). These include RAFTK/Pyk2, FAK, paxillin, and p130Cas.
These proteins have also been shown to be involved in
integrin-triggered cell adhesion and cell spreading. Therefore, we
examined the importance of CD45 in regulating the tyrosine
phosphorylation of these molecules upon stimulation with CXCL12 by
using phosphorylation site-specific antibodies. As shown in Fig.
6A, the CXCL12-induced
tyrosine phosphorylation of RAFTK at tyrosine residues 402 and 881 was
slightly reduced in the CD45-negative cells (J45.01) as compared with
the CD45-positive cells (JE6.1). Equal amounts of RAFTK were present in
each sample. Similarly, the tyrosine phosphorylation of FAK was
slightly reduced at tyrosine residue 397 in the CD45-negative cells as
compared with the CD45-positive cells (Fig. 6B).
Furthermore, we found that tyrosine phosphorylation of other focal
adhesion components, paxillin (Fig. 6C) and p130Cas (Fig.
6D), was also reduced in the CD45-negative cells as compared
with the CD45-positive cells.
CD45 Regulates CXCR4-mediated Activation of TCR Signaling Molecules
ZAP-70 and SLP-76--
ZAP-70 activation and SLP-76 tyrosine
phosphorylation are critical events in TCR signal transduction (43).
Recently, CXCL12-mediated T-cell chemotaxis and transendothelial
migration were shown to be regulated by ZAP-70 (44). Therefore, we
examined the CXCL12-induced tyrosine phosphorylation of ZAP-70 kinase
(Fig. 7A) and SLP-76 (Fig.
7B) in CD45-positive cells as compared with CD45-negative cells. As shown, the phosphorylations of ZAP-70 and SLP-76 were impaired in the CD45-negative cells in comparison to the positive variants. No change in ZAP-70 and SLP-76 protein levels was observed in
the CD45-positive and CD45-negative cells (Fig. 7, A and
B, bottom panels). These results are in
correlation with the observation that CD45 regulates ZAP-70 activity,
which in turn regulates SLP-76 phosphorylation.
The Role of CD45 in CXCL12-induced Mitogen-activated Protein Kinase
(MAPK) Activation--
CXCR4 has been shown to activate the MAPK
pathway (12). Thus, we examined the role of CD45 in CXCL12-induced
p44/42 MAP kinase activation. The activation of MAP kinase was
determined by examining the phosphorylation of p44/42 components using
phosphospecific (Tyr202 of p44 and Tyr204 of
p42) monoclonal antibodies. As shown in Fig.
8, A and B, absence of CD45 in the J45.01 and J45/A2 cells moderately increased the CXCL12-induced phosphorylation of p44/42 MAPK at early time periods. Equal amounts of p44/42 MAPK were present in each sample (Fig. 8,
A and B, lower panels).
This study indicates the central role of the membrane-bound
tyrosine phosphatase, CD45, in CXCL12-induced and CXCR4-mediated chemotactic signaling, which plays a critical role in the immune system
by regulating the trafficking and positioning of lymphocytes (11-17,
45). CXCR4 and its cognate ligand, CXCL12, have also been shown to play
an important role in HIV gene product nef-mediated chemotaxis
and breast cancer metastasis (46, 47). However, CXCR4-mediated
chemotactic mechanisms are complex and have not been completely
defined. We and others (12-17, 44, 45) have shown that CXCR4-mediated
chemotaxis involves activation of multiple signaling molecules
including tyrosine phosphatases SHP1 and SHP2, focal adhesion
components, Src-related kinases, and the T-cell activating molecule
ZAP-70. In the present study, we have shown that another important
component of the T-cell receptor signaling complex, the membrane-bound
tyrosine phosphatase, CD45, also regulates CXCL12-induced and
CXCR4-mediated chemotaxis. We observed reduced migration of
CD45-negative T-lymphocytes in response to optimal concentrations of
CXCL12 (10-100 ng/ml). However, migratory response was less
significant at higher concentrations of CXCL12 (500-1000 ng/ml) (data
not shown). The effects observed at higher CXCL12 concentrations were
similar to those observed by other investigators (48). Reconstitution
of full-length CD45 into J45.01 cells almost completely restored the
chemotactic response induced by CXCL12. Furthermore, transfection of
the cytoplasmic domain of CD45 (containing tyrosine phosphatase
activity) into CD45-negative cells was also able to moderately restore
the migratory response, suggesting that CD45 phosphatase activity is
important for mediating CXCL12-induced chemotactic signaling. The
cytoplasmic domain of CD45 has also been shown to be required for
TCR-mediated signaling events (22, 23).
The role of CD45 in cell spreading and chemotaxis is controversial. One
report (49) indicates that in T-cells, the presence of CD45 prevents
cell spreading in response to the binding of CD44, a cell adhesion
molecule. However, other studies (27, 50) indicate that CD45 positively
regulates integrin-mediated adhesion and spreading in macrophages and
the chemotaxis of neutrophils.
Our data show that CXCL12 treatment increases the tyrosine
phosphorylation of CD45. Similarly, T-cell activation has been shown to
result in the phosphorylation of CD45 on tyrosine and serine residues
located in its cytoplasmic domain (51, 52). Phosphorylation of CD45
might regulate its functions by altering its phosphatase activity or by
providing docking sites for its interaction with other proteins (52).
In the present studies, we observed by immunoprecipitation that CXCL12
treatment induced the association of CD45 with CXCR4. This result was
further confirmed by confocal microscopy, which showed that the CXCR4
receptor colocalized with CD45. CD45 has been shown to interact with
other cell surface molecules such as CD2, CD4, and TCR (53). We
obtained a somewhat diminished response to CXCL12 in cells transfected
with a chimeric molecule containing the cytoplasmic domain of CD45 and
the transmembrane and extracellular domains of the HLA-A2 molecule as
compared with cells transfected with full-length CD45. Thus, the
extracellular and transmembrane domains of CD45 may regulate the
magnitude of the CXCL12-induced chemotactic response through efficient
coupling of CXCR4 with its signaling complex via CD45.
We have also shown that CD45 interaction with CXCR4 can be inhibited by
pretreatment of cells with the lipid raft inhibitor, MBC. Furthermore,
MBC was also shown to abrogate CXCR4-mediated chemotaxis in medium
deprived of serum. Recently, MBC was shown to inhibit CXCL12-induced
chemotaxis and T-cell polarization in cholesterol-depleted cells (38).
These results suggest that lipid rafts play an important role in
CXCL12-induced chemotaxis. Colocalization of CD45 to lipid rafts in
T-cell receptor signaling is still not clear. It has been observed that
CD45 is excluded from the lipid raft domain upon TCR signaling (54).
However, cross-linking of the CD26 receptor induced an interaction
between CD26 and the cytoplasmic domain of CD45 that resulted in the
coaggregation of CD45 and CD26 in lipid rafts (55). The role of CD45 in
CXCR4-mediated lipid raft formation is a subject for further studies.
CD45 has been shown to regulate the activity of the Src-related kinase,
Lck (18-20). Moreover, Src kinases have been shown to be involved in
the CXCL12-induced signaling that regulates chemotaxis (15, 56).
Therefore, one possible explanation for the CXCL12-induced inhibition
of T-cell migration in CD45-negative cells is the effect of CD45 on
Lck. The Lck kinase was hyperphosphorylated at tyrosine residues and
possessed reduced kinase activity in the CD45-negative cells. The above
data suggest that CXCL12 may stimulate Lck kinase by activating CD45,
which, in turn, mediates the dephosphorylation of this kinase at its
negative regulatory carboxyl-terminal sites.
In addition to the Src-related kinases, CD45 was also shown to modulate
the phosphorylation of focal adhesion components such as RAFTK, FAK,
paxillin, and p130Cas. These proteins form signaling complexes that are
involved in chemotaxis (41, 42). Our data indicate that CD45 may
partially regulate the tyrosine phosphorylation of RAFTK at residues
402 and 881, whereas it regulates FAK phosphorylation at residue 397. These data indicate that CD45 differentially regulates the
phosphorylations of RAFTK and FAK at various tyrosine motifs. However,
tyrosine residues that bind to Src-related kinases located at positions
402 and 397 of RAFTK and FAK, respectively, were regulated by CD45. We
also observed reduced phosphorylation of the cytoskeletal proteins
paxillin and p130Cas in CD45-negative cells. Tyrosine phosphorylation
of these proteins regulates cytoskeletal reorganization, leading to the
morphological changes observed during chemotaxis (41, 42, 57).
Treatment of CD45-positive cells with anti-CD45 antibody has been shown
to induce the phosphorylation of paxillin and its association with Lck.
Furthermore, CD45 has been shown to colocalize to focal adhesion sites
(58).
Our results suggest that the tyrosine phosphorylation of T-cell
receptor signaling components ZAP-70 and SLP-76 are clearly impaired in
CD45-negative cells as compared with CD45-positive cells. Similarly,
the TCR-mediated activation of ZAP-70 and SLP-76 is also regulated by
CD45 (48). Moreover, it was shown that ZAP-70 regulates CXCR4-mediated
chemotaxis (44) and is involved in the HIV gene product nef-mediated
inhibition of CXCL12-induced chemotaxis (46).
CXCL12 is also known to stimulate the MAPK pathway (12). CXCL12 and HIV
gp120 at higher concentrations also induce internalization of CXCR4
receptors (33). Absence of CD45 resulted in a slight increase in
CXCL12-induced MAP kinase activity and had no effect on CXCR4
internalization. HIV gp120-induced CXCR4 internalization was also not
affected by the absence of CD45. These results suggest that CD45 is
linked more specifically and positively to the CXCL12-induced chemotactic signaling pathway, has a slight negative effect on the MAP
kinase pathway and no effect on CXCR4 internalization processes. In our
previous studies, we found that CXCL12-induced chemotaxis was not
related to MAPK activation (16). However, thrombin-induced MAP kinase
activation was shown to be negatively regulated by CD45 in T-cells
(59).
Taken together, our studies demonstrate a novel function of CD45 in
regulating chemokine-induced T-cell chemotaxis. These findings also
provide new information on the possible cross-talk between TCR and
CXCR4-mediated pathways. Specifically, these pathways modulate T-cell
chemotaxis through the regulation of various shared signaling
substrates such as CD45, Lck, RAFTK, and FAK. Thus, the molecular
mechanisms that regulate T-cell activation and migration may involve
common signaling molecules, and hence the coordinated integration of
both pathways is likely to play an important role in immune regulation
and inflammation.
(CXCL12),
regulate lymphocyte trafficking and play an important role in host
immune surveillance. However, the molecular mechanisms involved in
CXCL12-induced and CXCR4-mediated chemotaxis of T-lymphocytes are not
completely elucidated. In the present study, we examined the role of
the membrane tyrosine phosphatase CD45, which regulates antigen
receptor signaling in CXCR4-mediated chemotaxis and mitogen-activated
protein kinase (MAPK) activation in T-cells. We observed a
significant reduction in CXCL12-induced chemotaxis in the CD45-negative
Jurkat cell line (J45.01) as compared with the CD45-positive control
(JE6.1) cells. Expression of a chimeric protein containing the
intracellular phosphatase domain of CD45 was able to partially restore
CXCL12-induced chemotaxis in the J45.01 cells. However, reconstitution
of CD45 into the J45.01 cells restored the CXCL12-induced chemotaxis to about 90%. CD45 had no significant effect on CXCL12 or human
immunodeficiency virus gp120-induced internalization of the
CXCR4 receptor. Furthermore, J45.01 cells showed a slight enhancement
in CXCL12-induced MAP kinase activity as compared with the JE6.1 cells.
We also observed that CXCL12 treatment enhanced the tyrosine
phosphorylation of CD45 and induced its association with the CXCR4
receptor. Pretreatment of T-cells with the lipid raft inhibitor,
methyl-
-cyclodextrin, blocked the association between CXCR4 and CD45
and markedly abolished CXCL12-induced chemotaxis. Comparisons of
signaling pathways induced by CXCL12 in JE6.1 and J45.01 cells revealed
that CD45 might moderately regulate the tyrosine phosphorylation of the
focal adhesion components the related adhesion focal tyrosine
kinase/Pyk2, focal adhesion kinase, p130Cas, and paxillin. CD45 has
also been shown to regulate CXCR4-mediated activation and
phosphorylation of T-cell receptor downstream effectors Lck, ZAP-70,
and SLP-76. Our results show that CD45 differentially regulates
CXCR4-mediated chemotactic activity and MAPK activation by
modulating the activities of focal adhesion components and the
downstream effectors of the T-cell receptor.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), focal adhesion kinase
(FAK), paxillin, and p130Cas (12). Furthermore, protein-tyrosine
phosphatases SHP1 and SHP2 have also been shown to be involved in
CXCR4- or CCR5-mediated chemotaxis (13, 15, 17). Hematopoietic cells
derived from mice lacking SHP1 showed altered patterns of chemotactic
response to CXCL12 (17). SHP2 was shown to associate with CXCR4 and to regulate the CXCL12-induced migration of T- and pre-B-cells (15). In
the present investigation, we further delineated the role of tyrosine
phosphatases and showed that the membrane-bound tyrosine phosphatase
CD45 is a key regulator of CXCL12-induced and CXCR4-mediated chemotaxis.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-32P]ATP for 30 min at 30 °C. The proteins
were separated on 12% SDS-PAGE. Bands were detected by autoradiography.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (32K):
[in a new window]
Fig. 1.
CD45 regulates the chemotactic response
induced by CXCL12. A, cells lacking CD45 antigen
(J45.01) or the positive variant control (JE6.1)
were subjected to chemotaxis assay in the presence of CXCL12 (0-100
ng/ml) as described under "Experimental Procedures." B,
cells expressing the chimeric protein HLA-A2/cytoplasmic domain CD45
(J45/CH11) or control cells expressing HLA-A2
(J45/A2) were subjected to chemotaxis assay in
the presence of CXCL12 (0-100 ng/ml). C, J45.01 cells
reconstituted with CD45 (J45/LB3) were analyzed
for chemotactic response toward varying concentrations of CXCL12 as
indicated and compared with the response of the J45.01 cells.
D, shows the difference in cell migration in response to
CXCL12 between CD45-positive JE6.1 and CD45-negative
(J45.01) cells and between transfectants containing the
cytoplasmic domain of CD45 (J45/CH11),
transfectants lacking the cytoplasmic domain
(J45/A2), and the CD45 reconstituted J45.01 cells
(J45/LB3). Results presented in the
graph are representative of three experiments;
p < 0.005.
View larger version (16K):
[in a new window]
Fig. 2.
CD45 does not regulate CXCL12- or
gp120-induced internalization of the CXCR4 receptor. CD45-positive
variant cells (JE6.1) (A) or CD45-negative cells
(J45.01) (B) were unstimulated (- -) or
stimulated with CXCL12 (1 µg/ml) (-
-) or gp120 (1.2 µg/ml)
(-
-) for the indicated time periods. The cells were stained with
CXCR4-phosphatidylethanolamine antibody and subjected to flow
cytometric analysis as described under "Experimental Procedures."
Results are representative of three separate experiments.
View larger version (34K):
[in a new window]
Fig. 3.
Tyrosine phosphorylation of CD45 and its
association with CXCR4 upon stimulation with CXCL12.
CD45-positive variant (JE6.1) cells (A and
B) or peripheral blood lymphocytes (C) were
either unstimulated (0) or stimulated with CXCL12 (100 ng/ml) for the indicated time periods. Cells were lysed and
immunoprecipitated (IP) with CD45 (A) or CXCR4
(B and C) antibody. The immune complexes were
separated on 7% SDS-PAGE gel, transferred to nitrocellulose membrane,
and immunoblotted with anti-phosphotyrosine antibody (4G10,
pTyr99) (A, top panel) followed by
anti-CD45 antibody (A, bottom panel).
B and C, the blots were probed with anti-CD45
antibody. Protein loading was analyzed by running 50 µg of lysates on
SDS-PAGE and immunoblotting with anti-actin antibody (bottom
panels). D, the CD45-positive Jurkat cell clone (JE6.1)
was stimulated with CXCL12 (100 ng/ml) for the indicated time point.
The cells were fixed with paraformaldehyde and subjected to confocal
microscopic analysis using anti-CD45 (green) and anti-CXCR4
(red) antibodies, as described under "Experimental
Procedures." Yellow represents the colocalization of CD45
and CXCR4. P-Tyrosine, phosphotyrosine;
WB, Western blot; TCL, total cell lysates;
PBL, peripheral blood lymphocyte; UN,
unstimulated.
-cyclodextrin (MBC), on the association of
CXCR4 with CD45. As shown in Fig.
4A, pretreatment of JE6.1 cells with MBC (10 mM) inhibited the CD45 and CXCR4
association, as detected by immunoprecipitation followed by Western
blotting. An equal amount of protein was present in each
lane as detected by immunoblotting the lysates with
anti-actin antibody (Fig. 4A, bottom panel).
Furthermore, we observed that MBC treatment attenuated CXCL12-induced
chemotaxis in a dose-dependent manner in medium without
serum (Fig. 4B). The maximum inhibition observed was at a 10 mM concentration. However, no effect on chemotaxis at
various MBC concentrations was observed in medium containing 2.5%
serum (Fig. 4C). The lack of effect may be because of
cholesterol replenishment under this condition.
View larger version (20K):
[in a new window]
Fig. 4.
Lipid raft inhibitor disrupts the interaction
between CD45 and CXCR4 and blocks CXCL12-induced chemotaxis.
A, JE6.1 cells, untreated or pretreated with MBC (10 mM) for 1 h, were stimulated with CXCL12 (100 ng/ml)
for the indicated time points. The cells were lysed, and the lysates
were subjected to immunoprecipitation (IP) with anti-CXCR4
antibody. The immunoprecipitates were separated on 7% SDS-PAGE
followed by immunoblot analysis with anti-CD45 antibody. Protein
loading was analyzed by running 50 µg of lysates on SDS-PAGE and
immunoblotting with anti-actin antibody (bottom panel).
B and C, JE6.1 cells were preincubated with MBC
at different concentrations (mM) in the absence
(B) or presence (C) of 2.5% serum. The
chemotactic activity of the pretreated cells toward CXCL12 (100 ng/ml)
was monitored as described under "Experimental Procedures";
p < 0.005. TCL, total cell lysates;
WB, Western blot.
View larger version (40K):
[in a new window]
Fig. 5.
CD45 modulates CXCL12-induced Lck
kinase activity. Lysates obtained from unstimulated (0)
or CXCL12-stimulated (100 ng/ml) CD45-negative and -positive cells were
immunoprecipitated (IP) with anti-Lck antibodies. The immune
complex was subjected to an in vitro kinase assay, as
described under "Experimental Procedures" (upper panel)
by using enolase (acid-denatured) as a substrate. The immune complex
was also separated on SDS-PAGE and immunoblotted with
anti-phosphotyrosine antibody (4G10) (middle panel). The
same blot was stripped and reprobed with anti-Lck antibody
(bottom panel). C, antibody control;
P-Tyrosine, phosphotyrosine.
View larger version (38K):
[in a new window]
Fig. 6.
CD45 regulates the tyrosine phosphorylation
of focal adhesion components. Lysates, obtained from CD45-negative
(J45.01) or -positive (JE6.1) cells unstimulated
(0) or stimulated with CXCL12 (100 ng/ml) for the indicated
time periods, were run on SDS-PAGE and subjected to serial
immunoblotting with phosphospecific antibodies of RAFTK
pTyr402, pTyr881, or anti-RAFTK antibody
(A) or phosphospecific FAK pTyr397 or anti-FAK
antibody (B). The immunoblots were stripped and
reblotted with RAFTK or FAK, respectively. Cell lysates prepared from
unstimulated (0) or CXCL12-stimulated (100 ng/ml) cells for
the indicated time periods were immunoprecipitated (IP) with
anti-paxillin (C) or anti-p130Cas (D) antibodies.
The immune complexes were separated on SDS-PAGE and immunoblotted with
anti-phosphotyrosine antibody (4G10) (C and D,
upper panels). The same blots were stripped and reprobed
with anti-paxillin (C, bottom panel) or
anti-p130Cas (D, bottom panel) antibody.
P-RAFTK or P-FAK, tyrosine phosphorylation at the
indicated site; C, antibody control; TCL, total
cell lysates; P-Tyrosine, phosphotyrosine;
WB, Western blot; MW, molecular
weight.
View larger version (50K):
[in a new window]
Fig. 7.
CD45 modulates ZAP-70 and SLP-76
phosphorylation. Lysates, prepared from unstimulated
(0) or CXCL12-stimulated (100 ng/ml) CD45-negative or
-positive variant cells for the indicated time periods, were
immunoprecipitated (IP) with anti-ZAP-70 (A) or
anti-SLP-76 (B) antibody. The immune complexes were
separated on SDS-PAGE and immunoblotted with anti-phosphotyrosine
antibody (4G10) (upper panels). The same blot was stripped
and reprobed with anti-ZAP-70 antibody (A) or anti-SLP-76
antibody (B) (bottom panels). C,
antibody control; TCL, total cell lysates;
P-Tyrosine, phosphotyrosine; WB, Western
blot; MW, molecular weight.
View larger version (30K):
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Fig. 8.
CD45 negatively modulates CXCL12-induced MAP
kinase activity. Lysates were prepared from unstimulated
(0) or CXCL12-stimulated (100 ng/ml) cells for the indicated
time periods. Proteins were separated on SDS-PAGE and immunoblotted
with phosphospecific antibody for p44/42 MAPK (upper left
panels). The optical density (O.D.) values obtained
after densitometric scanning of the p44/42 MAPK bands are presented as
bar graphs (right panels). The same blots were
stripped and reprobed with p44/42 MAPK antibody (bottom left
panels). P-p44/42 MAPK, phospho-p44/42 MAPK;
WB, Western blot; MW, molecular
weight.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Gary A. Koretzky (University of Pennsylvania School of Medicine) and Dr. Eric J. Brown (University of California, San Francisco, CA) for the generous gift of CD45 transfectants. We also thank Janet Delahanty for editing the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants AI49140 and CA76950 and by a grant from the American Foundation for AIDS Research (to R. K. G).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Contributed equally to this work.
§ To whom correspondence should be addressed: Harvard Inst. of Medicine-BIDMC, 4 Blackfan Circle, Rm. 343, Boston, MA 02115. Tel.: 617-667-0060; Fax: 617-975-5243; E-mail: rganju@caregroup.harvard.edu.
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M211803200
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ABBREVIATIONS |
---|
The abbreviations used are:
HIV, human
immunodeficiency virus;
FAK, focal adhesion kinase;
MAP, mitogen-activated protein;
MAPK, MAP kinase;
MBC, methyl--cyclodextrin;
RAFTK, related adhesion focal tyrosine kinase;
TCR, T-cell receptor;
pTyr, phosphotyrosine;
PBS, phosphate-buffered
saline.
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