The Journal of Immunology
Distinct Role of ZAP-70 and Src Homology 2
Domain-Containing Leukocyte Protein of 76 kDa in the
Prolonged Activation of Extracellular Signal-Regulated Protein
Kinase by the Stromal Cell-Derived Factor-1␣/CXCL12
Chemokine1
Kimberly N. Kremer, Troy D. Humphreys, Ashok Kumar, Nan-Xin Qian, and Karen E. Hedin2
Stimulation of T lymphocytes with the ligand for the CXCR4 chemokine receptor stromal cell-derived factor-1␣ (SDF-1␣/
CXCL12), results in prolonged activation of the extracellular signal-regulated kinases (ERK) ERK1 and ERK2. Because SDF-1␣
is unique among several chemokines in its ability to stimulate prolonged ERK activation, this pathway is thought to mediate special
functions of SDF-1␣ that are not shared with other chemokines. However, the molecular mechanisms of this response are poorly
understood. In this study we show that SDF-1␣ stimulation of prolonged ERK activation in Jurkat T cells requires both the
ZAP-70 tyrosine kinase and the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) scaffold protein. This
pathway involves ZAP-70-dependent tyrosine phosphorylation of SLP-76 at one or more of its tyrosines, 113, 128, and 145. Because
TCR activates ERK via SLP-76-mediated activation of the linker of activated T cells (LAT) scaffold protein, we examined the role
of LAT in SDF-1␣-mediated ERK activation. However, neither the SLP-76 proline-rich domain that links to GADS and LAT, nor
LAT, itself are required for SDF-1␣ to stimulate SLP-76 tyrosine phosphorylation or to activate ERK. Together, our results
describe the distinct mechanism by which SDF-1␣ stimulates prolonged ERK activation in T cells and indicate that this pathway
is specific for cells expressing both ZAP-70 and SLP-76. The Journal of Immunology, 2003, 171: 360 –367.
he chemokine, stromal cell-derived factor-1␣ (SDF-1␣/
CXCL12)3 and its receptor, CXCR4, are critical for the
regulation of T lymphocyte functions under normal conditions and disease (1). SDF-1␣ is a member of a superfamily of
⬃50 chemokines that are classified as C, CC, CXC, or CX3C
depending on the spacing and number of their conserved cysteine
residues (2). Interestingly, CXCR4, the sole known receptor for
SDF-1␣, is not unique to T lymphocytes, but rather is ubiquitously
expressed in mammalian cells. In the immune system, SDF-1␣
modulates T cell development in the thymus (3, 4), T cell migration, and the expression of genes controlling T cell signaling, migration, and survival (5). In addition, SDF-1␣ costimulates the
immune activation of T lymphocytes stimulated by the TCR (6).
CXCR4 has also elicited significant attention because it is exploited for HIV-1 infection, a function that is antagonized by
SDF-1␣ (1). Thus, whereas little is known regarding the function
T
Departments of Surgery and Immunology, Mayo Graduate and Medical Schools,
Mayo Clinic, Rochester, MN 55905
Received for publication June 24, 2002. Accepted for publication April 21, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by the philanthropy of Barbara Lipps to the Mayo
Clinic, and by National Institutes of Health Training Grant AI074525-06 (to T.D.H.)
and RO1 Grant GM59763 (to K.E.H).
2
Address correspondence and reprint requests to Dr. Karen E. Hedin, Department of
Immunology, Mayo Clinic, Medical Sciences Building 2nd Floor, 200 First Street
Southwest, Rochester, MN 55905. E-mail address: [email protected]
3
Abbreviations used in this paper: SDF-1␣, stromal cell-derived factor-1␣; [Ca2⫹]i,
intracellular calcium ion concentration; ERK, extracellular signal-regulated kinase;
LAT, linker of activated T cells; SH2, Src homology 2; MAP, mitogen-activated
protein; SLP-76, SH2 domain-containing leukocyte protein of 76 kDa; MEK, MAP/
ERK kinase.
Copyright © 2003 by The American Association of Immunologists, Inc.
of SDF-1␣ in most mammalian cells, the role of this chemokine
and its receptor in the regulation of T cell homeostasis and viral
infection is well established.
Unfortunately, the molecular mechanisms that mediate the effects of SDF-1␣ on T cells are not well understood. In particular,
the signaling pathways that permit SDF-1␣ to regulate T lymphocyte gene expression are incompletely characterized. However,
emerging data indicate that chemokine receptors use extracellular
signal-regulated kinase (ERK) mitogen-activated protein (MAP)
kinase for gene expression. SDF-1␣-mediated activation of the
ERK MAP kinase pathway is required for protection against serum-deprived apoptosis and for the expression of many genes, including those that inhibit apoptosis (5). In addition, because the
ERK pathway also contributes to T lymphocyte immune activation
(7) and critically regulates thymocyte development (8), ERK activation is likely involved in the regulation of these processes by
SDF-1␣. In contrast to most chemokines, SDF-1␣ stimulates ERK
activation in T cells for a prolonged period of time (9). Because
prolonged rather than transient ERK activation provokes distinct
cellular responses downstream of other cell-surface receptors (8,
10 –12), it is thought that prolonged SDF-1␣-dependent ERK activation may subserve a special biological role. Therefore, in this
study we have focused on delineating the molecular mechanisms
of SDF-1␣-mediated ERK activation in T cells.
CXCR4 and other chemokine receptors are members of the G
protein-coupled receptor superfamily. Although CXCR4-mediated
ERK activation has been extensively studied (9, 13–16), the molecular machinery that links CXCR4 signaling to ERK activation
in T cells has not been fully characterized. SDF-1␣ signaling via
CXCR4 uses pertussis toxin-sensitive Gi-type G proteins for both
migration and ERK activation. SDF-1␣-mediated ERK activation
0022-1767/03/$02.00
The Journal of Immunology
has also been shown to require phosphatidylinositol 3-kinase activity (15), Ras (16), and the ERK kinase, MAP/ERK kinase
(MEK)-1. In other cell types, tyrosine kinases can participate in Gi
protein-mediated activation of phosphatidylinositol 3-kinase and
the Ras-Raf-MEK-ERK cascade (17). Moreover, both tyrosine kinase- and Gi protein-mediated signal transduction often result in
increased intracellular calcium ion concentrations ([Ca2⫹]i), and
[Ca2⫹]i is elevated in T cells in response to SDF-1␣ (2, 9, 18).
Although SDF-1␣ has been shown to use the lymphocyte-specific
tyrosine kinase, ZAP-70, for adhesion and migration (19, 20), the
role of ZAP-70 in SDF-1␣-mediated ERK activation and [Ca2⫹]i
signaling has not been addressed. Similarly, beside MEK-1 and
ERK themselves, key tyrosine kinase substrates that link CXCR4
signaling with ERK activation remain to be identified.
In this study we show that ZAP-70 as well as the tyrosine kinase
substrate and scaffold protein, Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76), are required for the
distinctive prolonged ERK activation that occurs in response to
treatment of Jurkat T cells with SDF-1␣, but not other chemokines.
We further demonstrate that this pathway requires ZAP-70-dependent tyrosine phosphorylation of SLP-76, thereby implicating SH2
domain-containing signaling proteins that bind to this region of
SLP-76 in prolonged ERK signaling. However, SDF-1␣-dependent ERK activation does not require either the SLP-76 prolinerich region or linker of activated T cells (LAT), domains that were
previously shown to mediate ERK activation by SLP-76 downstream of the TCR. In addition to showing the unique roles that
ZAP-70 and SLP-76 play in ERK activation, we also found that
these two molecules are important for SDF-1␣-induced signaling
that results in increased [Ca2⫹]i. These findings significantly advance our understanding of the molecular mechanisms underlying
SDF-1␣ effects on T cells and begin to address the mechanisms
that distinguish SDF-1␣ signaling from that of other cell-surface
receptors that also activate ERK.
Materials and Methods
Cells
Jurkat T cells and mutant sublines were maintained in medium A (RPMI
1640 supplemented with 5% FCS, 5% calf serum, 10 mM HEPES (pH 7.4),
2 mM L-glutamine, and 2 ␮M 2-ME) at ⬍106 cells/ml. Robert T. Abraham
(Duke University, Durham, NC) generously provided Jurkat sublines that
are LAT-deficient (called ANJ-3 (21)), ZAP-70-deficient (called P116
(18)), or stably reconstituted with ZAP-70 (called P116-C40 (18)). SLP76-deficient Jurkat T cells (J14) were a generous gift from A. Weiss (University of California, San Francisco, CA) (22).
Assay for active phosphorylated ERK1 and ERK2
Cells were stimulated for the indicated times at 37°C, pelleted by rapid
centrifugation, lysed in SDS-PAGE sample buffer, fractionated on SDSPAGE, and transferred to Immobilon-P membrane (Millipore, Bedford,
MA). Unless otherwise indicated, cells were stimulated with 5 ⫻ 10⫺8 M
SDF-1␣ (R&D Systems, Minneapolis, MN) or 2 ⫻ 10⫺7 M CCL19, doses
determined to give maximum ERK responses. Both SDF-1␣ and CCL19
(macrophage inflammatory protein-3␤) were obtained from R&D Systems.
The presence of active ERK1 and ERK2 phosphorylated on threonine 202
and tyrosine 204 was detected by immunoblotting with phospho-specific
p44/p42 ERK1 and ERK2 (Thr202/Tyr204) antiserum, whereas total ERK2
protein was detected using p44 ERK kinase antiserum (both antisera from
New England Biolabs, Beverly, MA).
ERK MAP kinase assay
Jurkat cells (2– 4 ⫻ 106/point) were stimulated as above, then lysed and
analyzed for ERK1 plus ERK2 MAP kinase activity toward the epidermal
growth factor receptor 662– 681 peptide, as described (23, 24). ERK activity measured by this assay is completely inhibited by pretreatment with
the MEK-1 inhibitor, PD098059 (data not shown).
361
Transient transfections
Cells (107) were mixed with 3–20 ␮g of the indicated expression plasmid
plus irrelevant plasmid DNA (pcDNA3.1; Invitrogen, San Diego, CA) to
make a total of 30 ␮g, electroporated using a BTX electro-square-porator
model T820 (BTX, San Diego, CA), cultured in medium A for 18 –24 h,
then harvested and assayed as indicated. Under these conditions transient
transfection with a green fluorescent protein expression plasmid routinely
yielded high expression by 60 – 80% of Jurkat T cells. Expression plasmids
encoding FLAG-tagged wild-type or mutant SLP-76 proteins were generous gifts from G. A. Koretzky (University of Pennsylvania, Philadelphia,
PA (25)). An expression plasmid encoding myc-tagged ZAP-70 was a gift
from R. T. Abraham (Duke University, Durham, NC (18)). Expression of
transfected proteins was detected by immunoblotting with SLP-76 or
ZAP-70 antisera (gifts from P. J. Leibson, Mayo Clinic, Rochester, MN) or
anti-myc mAb 9E10 (Berkeley Antibody, Richmond, CA).
Detection of tyrosine phosphorylated SLP-76
Cells were stimulated with SDF-1␣ as indicated and lysed (26). Then,
either endogenous SLP-76 was immunoprecipitated with SLP-76 antiserum and protein A-Sepharose (Sigma-Aldrich, St. Louis, MO), or FLAGtagged transiently transfected SLP-76 was immunoprecipitated with antiFLAG agarose (Sigma-Aldrich). Phosphotyrosine incorporation into
SLP-76 was detected by immunoblotting with anti-phosphotyrosine mAb
4G10 (Upstate Biotechnology, Lake Placid, NY).
[Ca2⫹]i analysis
Cells were loaded with indo-1 (Molecular Probes, Eugene, OR), then
[Ca2⫹]i was assayed by using a FACStarPlus flow cytometer (BD Biosciences, San Jose, CA) to measure the ratio of 405:495 nm fluorescence,
as described (27). Stimuli were either 5 ⫻ 10⫺8 M SDF-1␣ or 500 ng/ml
anti-CD3 mAb (OKT3) cross-linked with 0.1 mg/ml goat-anti-mouse IgG
(ICN Pharmaceuticals, Costa Mesa, CA). Data were analyzed and plotted
by using FlowJo software (Tree Star, Palo Alto, CA).
Results
SDF-1␣ stimulates prolonged ERK activation in Jurkat T
lymphocytes via a mechanism requiring SLP-76
Treatment of Jurkat T cells with SDF-1␣ results in the accumulation of active phosphorylated ERK1 and ERK2 with high levels of
ERK activation extending to at least 10 min (Fig. 1A). To explore
the roles of different signaling proteins in SDF-1␣-mediated ERK
activation in T cells, we used well-characterized somatic mutants
of the Jurkat T cell line that are deficient in distinct signaling
proteins. Interestingly, SDF-1␣ evokes only transient ERK activation in J14 cells, which are deficient in the SLP-76 scaffold protein
(22) (Fig. 1, A and B). ERK activation at 8 or 10 min was significantly lower in J14 cells compared with the parental Jurkat T cell
line (Fig. 1B). In contrast, SDF-1␣-dependent ERK activation at 2
min did not significantly differ between J14 and wild-type Jurkat T
cells (n ⫽ 4, p ⫽ 0.6). Thus, only the later, more prolonged phase
of SDF-1␣-mediated ERK response is defective in J14 cells. The
control experiments demonstrate that the signaling defect in J14
cells results from a deficiency in SLP-76 and not some other mutation, because transient expression of SLP-76, but not vector
(pcDNA3.1) alone, significantly increased SDF-1␣-dependent
ERK activation at both 8 and 10 min (Fig. 1, C and D). The deficiency in SDF-1␣-dependent ERK activation is not caused by
decreased levels of CXCR4 on the surface of J14 cells, because
flow cytometric analyses detected no deficiencies in CXCR4 cellsurface expression by J14 cells compared with the parental Jurkat
T cell line (data not shown). Therefore, these results demonstrate
that SDF-1␣ stimulates prolonged ERK activation in Jurkat T cells
via a mechanism requiring SLP-76.
Phosphorylation of SLP-76 is required for prolonged ERK
activation in response to SDF-1␣
We next analyzed the molecular mechanisms that permit SLP-76
to mediate prolonged ERK activation by SDF-1␣. SLP-76 is a
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SDF-1␣ USES ZAP-70 AND SLP-76 TO STIMULATE PROLONGED ERK
FIGURE 1. SDF-1␣ stimulates prolonged ERK activation in Jurkat T cells via a mechanism requiring SLP-76. A, Cells were stimulated for the indicated
times with 5 ⫻ 10⫺8 M SDF-1␣ and assayed for active, phosphorylated ERK1 and ERK2 (P-ERK1 and P-ERK2) by immunoblotting (upper gel). The same
blot was stripped and immunoblotted with antisera to total ERK2 as a control (lower gel). B, Summary of three independent experiments as in A, where
the responses of SLP-76-deficient cells are represented as a percentage of the responses of the parental Jurkat cell line in the same experiment ⫾ SEM.
ⴱ, Significantly different (p ⬍ 0.05) from Jurkat control (100%). C, SLP-76-deficient J14 cells were transiently transfected with either vector alone
(pcDNA3.1) or a plasmid encoding wild-type SLP-76 (SLP-76-WT), then assayed for SDF-1␣-dependent ERK activation as in A. D, Summary of 12–15
experiments as in C. The responses of SLP-76-transfected cells are represented as a percentage of the responses of cells transfected with pcDNA3 in the
same experiment ⫾ SEM. ⴱ, Significantly different (p ⬍ 0.05) from pcDNA3.1 alone (100%).
scaffold protein composed of several domains. TCR signal transduction results in SLP-76 tyrosine phosphorylation at the aminoterminal tyrosines 113, 128, and 145 via a mechanism requiring
the ZAP-70 tyrosine kinase. Phosphorylated SLP-76 subsequently
binds SH2 domain-containing signaling proteins that contribute to
TCR signal transduction (7, 25, 28). Therefore, we asked if
SDF-1␣ signals to ERK via a mechanism involving SLP-76 tyrosine phosphorylation. SDF-1␣ treatment induced the tyrosine
phosphorylation of endogenous SLP-76 in Jurkat T cells (Fig. 2A)
as well as SLP-76 introduced into J14 Jurkat T cells by transient
transfection (Fig. 2B). Site-directed disruption of the previously
described (25, 29) tyrosine phosphorylation sites 113, 128, and 145
of SLP-76 abolished the tyrosine phosphorylation of SLP-76 in
response to SDF-1␣ (SLP-76-3YF; Fig. 2B). These results suggest
a role for SLP-76 tyrosine phosphorylation in mediating the ERK
response to SDF-1␣.
To test this hypothesis, we asked if SLP-76-3YF could rescue
ERK activation in response to SDF-1␣ treatment of J14 cells.
However, SLP-76-3YF failed to rescue ERK activation, despite
being expressed at equivalent or higher levels than wild-type
SLP-76 that did rescue prolonged ERK activation in the J14 cells
(Fig. 3A). In multiple experiments, the SDF-1␣-dependent prolonged ERK activation of J14 cells transfected with SLP-76-3YF
was significantly different from that of J14 cells transfected with
SLP-76-WT (n ⫽ 4, p ⬍ 0.05) (Fig. 3B) and was not significantly
different from J14 cells transfected with pcDNA3 alone (n ⫽ 4,
p ⫽ 0.4). Together, the results in this section indicate that one or
more of the previously described amino-terminal tyrosine phosphorylation sites of SLP-76 are required for prolonged ERK activation in response to SDF-1␣.
SDF-1␣ stimulates tyrosine phosphorylation of SLP-76 via a
mechanism requiring ZAP-70
Tyrosines 113, 128, and 145 of SLP-76 are targets of the ZAP-70
tyrosine kinase in the TCR signaling pathway (25, 29). We therefore used a ZAP-70-deficient somatic mutant of the Jurkat T cell
line (P116 (18)) to determine whether ZAP-70 is required for
SLP-76 tyrosine phosphorylation in response to SDF-1␣. SDF-1␣
treatment of P116 cells did not result in SLP-76 tyrosine phosphorylation (Fig. 4A). SDF-1␣-dependent tyrosine phosphorylation of SLP-76 was rescued by transient expression of ZAP-70
(Fig. 4A). Furthermore, stable expression of ZAP-70 (creating the
P116-C40 cell line (18)) also rescued SDF-1␣-dependent tyrosine
phosphorylation of SLP-76 (Fig. 4B). The deficiency in SDF-1␣dependent SLP-76 tyrosine phosphorylation is not caused by decreased levels of CXCR4 on the surface of P116 cells, because
P116 cells express normal cell-surface levels of CXCR4 (data not
The Journal of Immunology
FIGURE 2. One or more amino-terminal tyrosines of SLP-76 are required for SDF-1␣ to stimulate SLP-76 tyrosine phosphorylation. A, Jurkat
T cells were stimulated with SDF-1␣ for 10 min, and endogenous SLP-76
was immunoprecipitated and immunoblotted with anti-phosphotyrosine
mAb or SLP-76 antiserum. B, SLP-76-deficient (J14) cells were transiently
transfected with plasmids encoding either wild-type (SLP-76-WT) or mutant (SLP-76-3YF) SLP-76 and stimulated with SDF-1␣ for 8 min. SLP-76
proteins were immunoprecipitated and analyzed by immunoblotting with
anti-phosphotyrosine mAb or SLP-76 antiserum. Results in A and B are
each representative of three separate experiments.
shown). These results strongly support a role for ZAP-70 in the
SDF-1␣-dependent tyrosine phosphorylation of SLP-76.
Similar to SLP-76, ZAP-70 is required for SDF-1␣ to stimulate
prolonged ERK activation
Because phosphorylation of SLP-76 is required for prolonged
ERK activation in response to SDF-1␣, and because SLP-76 tyrosine phosphorylation correlates with prolonged ERK activation
in response to this chemokine, we examined the role of ZAP-70 in
SDF-1␣-dependent ERK regulation. Interestingly, the ZAP-70-deficient P116 cell line exhibits a defect in SDF-1␣-stimulated ERK
activation that closely resembles the ERK activation defect of
SLP-76-deficient J14 cells (Fig. 5, A and B, compared with Fig. 1,
A and B). ERK activation at 8 or 10 min was significantly lower in
P116 cells compared with the parental Jurkat T cell line. Only the
later phase of ERK activation requires ZAP-70, as ERK activation
at 2 min did not significantly differ between P116 and Jurkat cells
(n ⫽ 6, p ⫽ 0.1)(Fig. 5B). Thus, as for the SLP-76-deficient J14
cells, only the prolonged phase of SDF-1␣-dependent ERK activation is deficient in P116 cells. Genetic reconstitution experiments show that the signaling defect in P116 cells results from a
deficiency in ZAP-70, as stable re-expression of ZAP-70 in P116
cells (P116-C40 cells) restored prolonged ERK activation in response to SDF-1␣ (Fig. 5, A and B). Stable ZAP-70 expression in
P116 cells restored SDF-1␣-dependent ERK activation at 8 min to
levels indistinguishable from those of the parental Jurkat T cell
line (n ⫽ 3, p ⫽ 0.8), a significant increase from ERK activation
at 8 min of the ZAP-70-deficient P116 cells (n ⫽ 3, p ⬍ 0.05).
Together, the results in Figs. 4 and 5 indicate that ZAP-70 is required for SDF-1␣ to stimulate both SLP-76 tyrosine phosphorylation and prolonged ERK activation.
363
FIGURE 3. One or more amino-terminal tyrosines of SLP-76 are
required for prolonged ERK activation in response to SDF-1␣. A, SLP76-deficient J14 cells were transiently transfected with either wild-type
(SLP-76-WT) or mutant (SLP-76-3YF) SLP-76, then assayed for SDF-1␣dependent ERK activation as in Fig. 1A. B, A summary of four independent
experiments as in A, where the responses of SLP-76-3YF-transfected cells
are represented as a percentage of the responses of cells transfected with
SLP-76-WT in the same experiment ⫾ SEM. ⴱ, Significantly different from
100% (p ⬍ 0.05).
SLP-76 and ZAP-70 are required for [Ca2⫹]i mobilization in
response to SDF-1␣
In addition to ERK, both SLP-76 and ZAP-70 have been shown to
participate in signaling that results in increased [Ca2⫹]i (22, 21).
Therefore, we asked if these proteins participate in [Ca2⫹]i mobilization in response to SDF-1␣. Fig. 6 shows the results of stimulating cells and assessing [Ca2⫹]i responses by using a flow cytometer to measure changes in intracellular indo-1 fluorescence.
SDF-1␣ treatment induced a [Ca2⫹]i response in wild-type Jurkat
T cells. In contrast, SDF-1␣-dependent [Ca2⫹]i responses were
defective in both the SLP-76-deficient J14 and the ZAP-70-deficient P116 cell lines. As previously described, TCR/CD3-induced
[Ca2⫹]i responses were also deficient in J14 and P116 cells (18,
22). Thus, in addition to being important for SDF-1␣-induced
ERK activation, both SLP-76 and ZAP-70 are also required for
SDF-1␣-induced mobilization of [Ca2⫹]i.
Prolonged ERK activation in response to SDF-1␣ does not
require the SLP-76 GADS-LAT binding domain or LAT
In the TCR signaling pathway, SLP-76 binds to GADS-LAT via its
proline-rich domain (30). Because LAT could provide a link to Ras
and ERK activation (21, 31), we asked if SLP-76 stimulates prolonged ERK activation via LAT. Therefore, we used a SLP-76
deletion mutant lacking the proline-rich domain (SLP-76-⌬224244) that was previously shown to be unable to bind GADS-LAT
(30). Transient expression of SLP-76-⌬224-244 in J14 cells rescued SDF-1␣-mediated ERK activation in a manner similar to
364
SDF-1␣ USES ZAP-70 AND SLP-76 TO STIMULATE PROLONGED ERK
FIGURE 4. SDF-1␣ stimulates tyrosine phosphorylation of SLP-76 via
a mechanism requiring ZAP-70. A, ZAP-70-deficient (P116) cells were
transiently transfected with plasmids encoding FLAG-SLP-76 ⫾ mycZAP-70, then stimulated with SDF-1␣ for 8 min. FLAG-SLP-76 was immunoprecipitated and immunoblotted with anti-phosphotyrosine mAb,
SLP-76 antiserum, or anti-myc mAb. B, Tyrosine phosphorylation of endogenous SLP-76 in Jurkat, ZAP-70-deficient cells (P116), and P116 cells
stably reconstituted with ZAP-70 (P116-C40). Results are representative of
three independent experiments.
wild-type SLP-76 (Fig. 7, A and B)(p ⫽ 0.6). Consistent with this
result, SLP-76-⌬224-244 was also inducibly phosphorylated in response to SDF-1␣ (Fig. 7C, upper gel); moreover, phosphorylation
was approximately proportionate to protein expression levels (Fig.
7C, lower gel). Thus, the domain of SLP-76 that binds to GADSLAT is not required for SDF-1␣ to inducibly phosphorylate
SLP-76 and stimulate prolonged ERK activation.
To confirm that SDF-1␣ stimulates prolonged ERK activation
via a mechanism independent of SLP-76 binding to GADS-LAT,
we assayed ERK activation in a LAT-deficient Jurkat T cell line
(ANJ-3). The ANJ-3 LAT-deficient Jurkat T cell line exhibited no
defect in SDF-1␣ stimulation of prolonged ERK activation (Fig.
7D). ERK activation in ANJ-3 was not significantly different from
ERK activation in the parental Jurkat cell line at 2, 8, or 10 min
(n ⫽ 3; p ⫽ 0.4, 0.3, and 0.4 at 2, 8, and 10 min, respectively).
Thus, LAT is not required for SDF-1␣ to stimulate prolonged ERK
activation. Therefore, the pathway by which SLP-76 mediates prolonged ERK activation in response to SDF-1␣ differs from previously described SLP-76 signaling downstream of the TCR, which
involves both the SLP-76 GADS-LAT binding domain and LAT.
FIGURE 5. ZAP-70 is required for SDF-1␣ to stimulate prolonged
ERK activation. A, Jurkat, ZAP-70-deficient (P116) cells, or P116 cells
stably reconstituted with ZAP-70 (P116-C40) were stimulated for the indicated times with SDF-1␣, lysed in SDS sample buffer, and assayed for
active, phosphorylated ERK1 and ERK2 as in Fig. 1A. B, A summary of
three independent experiments as in A, comparing the SDF-1␣-dependent
ERK activation of the indicated cell lines. The levels of ZAP-70 expressed
by P116-C40 cells are approximately equivalent to those expressed by the
parental Jurkat cell line (18). The responses of ZAP-70-deficient P116 cells
and ZAP-70-reconstituted P116-C40 cells are represented as a percentage
of the responses of the parental Jurkat cell line in the same experiment ⫾ SEM. ⴱ, Significantly different from 100% (p ⬍ 0.05).
growth factor receptors, activation of the ERK MAP kinase pathway is important for gene regulation by CXCR4 (5). Other receptors use a large number of distinctive molecular mechanisms for
Discussion
Chemokines are emerging as pleiotropic regulators of immune cell
functions, acting as critical regulators of migration and gene expression important for the control of cell division, death, and differentiation, and for the synthesis and secretion of cytokines. For
example, in addition to stimulating chemotaxis, CXCR4 signaling
in T cells induces the expression of multiple genes, including those
that inhibit apoptosis (5), and contributes to T cell proliferation and
cytokine gene expression following TCR stimulation (6). As with
FIGURE 6. SLP-76 and ZAP-70 are required for [Ca2⫹]i mobilization
in response to SDF-1␣. The indicated cell lines were loaded with indo-1,
then [Ca2⫹]i was assayed by using a flow cytometer to measure the ratio of
405:495 nm fluorescence (which correlates with [Ca2⫹]i). Cells were stimulated at the times indicated by the arrows with either 5 ⫻ 10⫺8 M SDF-1␣
(top) or 0.5 ␮g/ml anti-CD3 mAb cross-linked with goat-anti-mouse IgG
(bottom).
The Journal of Immunology
365
FIGURE 7. SDF-1␣ signaling via SLP-76 does not require the SLP-76 region that links to GADS-LAT. A, SLP-76-deficient J14 cells were transiently
transfected with wild-type (SLP-76-WT) or mutant (SLP-76-⌬224-244) SLP-76 and then stimulated with SDF-1␣. Active ERK, total ERK2, and SLP-76
were assayed by immunoblotting as in Fig. 1A. B, A summary of 4 – 8 independent experiments as in A, where the responses of SLP-76- or SLP-76⌬224-244-transfected cells are represented as a percentage of the responses of cells transfected with pcDNA3 alone in the same experiment ⫾ SEM.
ⴱ, Significantly different from 100% (p ⬍ 0.05). C, SLP-76-deficient J14 cells were transiently transfected with plasmids encoding either SLP-76-WT or
SLP-76-⌬224-244. Following SDF-1␣ stimulation, SLP-76 proteins were immunoprecipitated and analyzed by immunoblotting with anti-phosphotyrosine
mAb or SLP-76 antiserum. Upper gel, anti-phosphotyrosine immunoblot showing inducible tyrosine phosphorylation of SLP-76-⌬224-244 in response to
SDF-1␣. Lower gel, SLP-76 immunoblot showing protein expression levels. The asterisk indicates a cross-reacting band that is apparent just below SLP-76
when this particular batch of SLP-76 antiserum is used. This band is also seen in J14 cells that are either untransfected or transfected with a control plasmid,
and is therefore not SLP-76. D, Cells were stimulated for the indicated times with 5 ⫻ 10⫺8 M SDF-1␣ and assayed for active, phosphorylated ERK1 and
ERK2 (P-ERK1 and P-ERK2) as in Fig. 1A.
ERK activation, a property that permits selective regulation and
cross-regulation of this important gene-regulatory pathway (17).
However, the pathways leading to ERK activation downstream of
chemokine receptors are incompletely characterized. In this study
we analyze the molecular mechanisms of CXCR4-mediated ERK
activation in T cells, focusing particularly on the mechanism by
which the CXCR4 ligand, SDF-1␣, but not other chemokines (9),
stimulates prolonged ERK activation in these cells.
Using Jurkat T cell lines deficient in different signaling proteins,
we show that normal prolonged ERK activation by the SDF-1␣
chemokine requires both the tyrosine kinase, ZAP-70, and the tyrosine kinase substrate and scaffold protein, SLP-76. We further
show that these molecules function in a signaling pathway wherein
SDF-1␣ treatment stimulates ZAP-70-dependent tyrosine phosphorylation of SLP-76, and subsequently, tyrosine phosphorylated-SLP-76 mediates prolonged ERK activation in response to
SDF-1␣. These results strongly suggest that SLP-76 mediates
SDF-1␣ signaling by binding to SH2 domain-containing signaling
molecules. When phosphorylated, SLP-76 tyrosines 113, 128,
and/or 145 recruit multiple signaling proteins, including the Rac/
CDC42 guanine-nucleotide exchange factor, Vav-1 (25, 28), the
adaptor, Nck (32), and the kinase, Itk (33, 34). Therefore, Vav-1,
Nck, Itk, and/or other proteins binding to this region of SLP-76
may mediate ERK activation in response to CXCR4 signaling.
Because we also found that both ZAP-70 and SLP-76 are required
for normal [Ca2⫹]i mobilization following treatment with SDF-1␣,
it is likely that SLP-76 binding proteins also function in other
pathways stimulated by SDF-1␣.
We found that both ZAP-70 and SLP-76 were required for
CXCR4-mediated ERK activation. Therefore, we initially hypothesized that CXCR4 activates ERK in a manner similar to the TCR.
The GADS-LAT binding region of SLP-76 plays a role in TCRmediated ERK activation (35), and TCR-mediated ERK activation
requires LAT (21, 31). However, in contrast to TCR signaling, we
found that neither the SLP-76 GADS-LAT binding region nor
LAT itself are necessary for SDF-1␣ to stimulate SLP-76 tyrosine
phosphorylation and to activate ERK. Therefore, our results indicate the existence of a new mechanism by which ZAP-70 and
SLP-76 collaborate in mediating ERK activation downstream of a
cell-surface receptor, in addition to characterizing the previously
unknown roles of ZAP-70 and SLP-76 in CXCR4-mediated ERK
activation.
Our results provide a molecular explanation for the distinctive
prolonged ERK activation that results from treating T cells with
SDF-1␣ (9). SDF-1␣ stimulates only transient ERK activation in
Jurkat T cells in the absence of either ZAP-70 or SLP-76, a result
366
SDF-1␣ USES ZAP-70 AND SLP-76 TO STIMULATE PROLONGED ERK
indicating that both ZAP-70 and SLP-76 are required for prolonged ERK activation in response to SDF-1␣. Therefore, we
asked if SLP-76 is also required for transient ERK activation by
other chemokines. However, CCL19, which induces only transient
ERK activation in Jurkat T cells (9), signals normally to ERK in
both SLP-76-deficient and ZAP-70-deficient Jurkat T cells (data
not shown). Thus, use of ZAP-70 and SLP-76 for prolonged ERK
activation is a characteristic of SDF-1␣ but not CCL19. Moreover,
our results indicate that prolonged ERK activation by SDF-1␣ results from CXCR4 using the ZAP-70 and SLP-76 signaling pathway described above, in contrast to CCL19, which does not use
this pathway. In addition to CCL19, CCL2, CCL4, CCL20, and
CXCL10 stimulate only transient ERK activation in T cells (9).
Therefore, prolonged ERK activation in response to SDF-1␣
seems likely to distinguish effects of SDF-1␣ from those of other
chemokines. In PC12 cells, nerve growth factor induces prolonged
ERK activation, which in turn leads to the expression of specific
genes that provoke neuronal differentiation (10 –12). Moreover, a
recent report suggests that positive selection during T cell development in the thymus requires prolonged rather than transient
ERK activation (8). Therefore, SDF-1␣ may use prolonged ERK
activation via ZAP-70 and SLP-76 as a way to modulate T cell
development in the thymus (3, 4).
CXCR4 is expressed by many different cell types in addition to
T cells, and the developmental defects of the vasculature, heart,
brain, and hematopoietic system in mice deficient in either SDF-1␣
or CXCR4 indicate a critical requirement for SDF-1␣/CXCR4 signaling in multiple cell types (1). Our results identify one mechanism that is apparently used by SDF-1␣ to coordinate the actions
of many different CXCR4-expressing cell types: the use of a tissue-specific signaling pathway. SLP-76 is selectively expressed in
platelets (36) and T-lymphoid, monocyte, and granulocyte lineages
(37, 38), whereas ZAP-70 expression is restricted to T cells and
NK cells (39), and the related Syk kinase is restricted to hematopoietic cell types (40). Therefore, CXCR4 regulation of ERK and
[Ca2⫹]i signaling via SLP-76 may underlie the distinctive effects
of SDF-1␣ on certain hematopoietic cell types compared with
other cell types that express CXCR4. Similarly, impaired CXCR4
signaling via SLP-76 may contribute to defects in developing T
cells, mast cells, and platelets that are seen in SLP-76-deficient
mice (14, 36, 41, 42), because all three cell types express CXCR4
and ZAP-70 and/or the related Syk kinase (3, 43, 44).
In summary, this study demonstrates that stimulation of prolonged, as opposed to transient, ERK activation by SDF-1␣/
CXCR4 signaling requires phosphorylation of the ZAP-70 phosphorylation sites at the amino-terminus of SLP-76. Other
previously described protein-binding regions of SLP-76, including
the SLP-76 157–223 domain that binds phospholipase C-␥1 (35)
and the SH2 domain that binds the SLAP-130/Fyb/ADAP scaffold
protein (45, 46), may also contribute to SDF-1␣-dependent ERK
activation in T cells. However, in contrast to the TCR, which also
uses SLP-76 for signaling, SDF-1␣-mediated ERK activation is
independent of the scaffold function of SLP-76 as it specifically
relates to the GADS-LAT binding domain. Together, these results
provide a molecular explanation for the prolonged ERK activation
that is characteristic of SDF-1␣/CXCR4 signaling in T cells and
indicate that this pathway is restricted to certain cell types.
Acknowledgments
We are grateful to Drs. G. A. Koretzky, P. J. Leibson, and R. T. Abraham
for providing critical reagents and cell lines.
References
1. Murdoch, C. 2000. CXCR4: chemokine receptor extraordinaire. Immunol. Rev.
177:175.
2. Thelen, M. 2001. Dancing to the tune of chemokines. Nat. Immunol. 2:129.
3. Suzuki, G., Y. Nakata, Y. Dan, A. Uzawa, K. Nakagawa, T. Saito, K. Mita, and
T. Shirasawa. 1998. Loss of SDF-1 receptor expression during positive selection
in the thymus. Int. Immunol. 10:1049.
4. Onai, N., Y. Zhange, H. Yoneyama, T. Kitamura, S. Ishikawa, and K. Matsushima.
2000. Impairment of lymphopoiesis and myelopoiesis in mice reconstituted with
bone marrow-hematopoietic progenitor cells expressing SDF-1-intrakine. Blood 96:
2074.
5. Suzuki, Y., M. Rahman, and H. Mitsuya. 2001. Diverse transcriptional response
of CD4⫹ T cells to stromal cell-derived factor (SDF)-1: cell survival promotion
and priming effects of SDF-1 on CD4⫹ T cells. J. Immunol. 167:3064.
6. Nanki, T., and P. E. Lipsky. 2000. Cutting edge: stromal cell-derived factor-1 is
a costimulator for CD4⫹ T cell activation. J. Immunol. 164:5010.
7. Koretzky, G. A., and P. S. Myung. 2001. Positive and negative regulation of
T-cell activation by adaptor proteins. Nat. Rev. Immunol. 1:95.
8. Welen, G., B. Hausmann, and E. Palmer. 2000. A motif in the ␣␤ T-cell receptor
controls positive selection by modulating ERK activity. Nature 406:422.
9. Tilton, B., L. Ho, E. Oberlin, P. Loetscher, F. Baleux, I. Clark-Lewis, and
M. Thelen. 2000. Signal transduction by CXC chemokine receptor 4: stromal
cell-derived factor 1 stimulates prolonged protein kinase B and extracellular signal-regulated kinase 2 activation in T lymphocytes. J. Exp. Med. 192:313.
10. Harada, T., T. Morooka, S. Ogawa, and E. Nishida. 2001. ERK induces p35, a
neuron-specific activator of CDK5, through induction of Egr1. Nat. Cell Biol.
3:453.
11. Woods, D., H. Cherwinski, E. Venetsanakos, A. Bhat, S. Gysin, M. Humbert,
P. F. Bray, V. L. Saylor, and M. McMahon. 2001. Induction of ␤3-integrin gene
expression by sustained activation of the Ras-regulated Raf-MEK-extracellular
signal regulated kinase signaling pathway. Mol. Cell. Biol. 21:3192.
12. Muller, J., A. M. Cacace, W. E. Lyons, C. B. McGill, and D. K. Morrison. 2000.
Identification of B-KSR1, a novel brain-specific isoform of KSR1 that functions
in neuronal signaling. Mol. Cell. Biol. 20:5529.
13. Ganju, R. K., S. A. Brubaker, J. Meyer, P. Dutt, Y. Yang, S. Zin, W. Newman,
and J. E. Groopman. 1998. The ␣-chemokine, stromal cell-derived factor-1␣,
binds to the transmembrane G protein coupled CXCR4 receptor and activates
multiple signal transduction pathways. J. Biol. Chem. 273:23169.
14. Vicente-Manzanares, M., M. Rey, D. R. Jones, D. Sancho, M. Mellado,
J. M. Rodriguez-Frade, M. A. del Pozo, M. Yanez-Mo, A. M. de Ana,
C. Martinez-A. et al. 1999. Involvement of phosphatidylinositol 3-kinase in stromal cell-cerived factor-1␣-induced lymphocyte polarization and chemotaxis.
J. Immunol. 163:4001.
15. Sotsios, Y., G. C. Whittaker, J. Westwick, and S. G. Ward. 1999. The CXC
chemokine stromal cell-derived factor activates a Gi-coupled phosphoinositide
3-kinase in T lymphocytes. J. Immunol. 163:5954.
16. Weber, K. S. C., G. Ostermann, A. Zernecke, A. Schroder, L. B. Klickstein, and
C. Weber. 2001. Dual role of H-Ras in regulation of lymphocyte function antigen-1 activity by stromal cell-derived factor-1␣: implications for leukocyte transmigration. Mol. Biol. Cell 12:3074.
17. Marinissen, M. J., and J. S. Gutkind. 2001. G protein-coupled receptors and
signaling networks: emerging paradigms. Trends Pharmacol. Sci. 22:368.
18. Williams, B. L., B. J. Irvin, S. L. Sutor, C. C. S. Chini, E. Yacyshyn,
J. B. Wardenburg, M. Dalton, A. C. Chan, and R. T. Abraham. 1999. Phosphorylation of Tyr319 in ZAP-70 is required for T-cell antigen receptor-dependent
phospholipase C-␥1 and Ras activation. EMBO J. 18:1832.
19. Soede, R. D. M., Y. M. Wijnands, I. V. Kouteren-Cobzaru, and E. Roos. 1998.
ZAP-70 tyrosine kinase is required for LFA-1-dependent T cell migration. J. Cell
Biol. 142:1371.
20. Ottoson, N. C., J. T. Pribila, A. S. H. Chan, and Y. Shimizu. 2001. Cutting edge:
T cell migration regulated by CXCR4 chemokine receptor signaling to ZAP-70
tyrosine kinase. J. Immunol. 167:1857.
21. Zhang, W., B. J. Irvin, R. P. Trible, R. T. Abraham, and L. E. Samelson. 1999.
Functional analysis of LAT in TCR-mediated signaling pathways using a LATdeficient Jurkat cell line. Int. Immunol. 11:943.
22. Yablonski, D., M. R. Kuhne, T. Kadlecek, and A. Weiss. 1998. Uncoupling of
nonreceptor tyrosine kinases from PLC-␥1 in an SLP-76-deficient T cell. Science
281:413.
23. Mitchell, F. M., L. E. Heasley, N. X. Qian, J. Zamarripa, and G. L. Johnson.
1995. Differential modulation of bombesin-stimulated phospholipase C␤ and mitogen-activated protein kinase activity by [D-Arg1,D-Phe5,D-Trp7,9,Leu11] substance P. J. Biol. Chem. 270:8623.
24. Gardner, A. M., C. A. Lange-Carter, R. R. Vaillancourt, and G. L. Johnson. 1994.
Measuring activation of kinases in mitogen-activated protein kinase regulatory
network. Methods Enzymol. 238:258.
25. Fang, N., D. G. Motto, S. E. Ross, and G. A. Koretzky. 1996. Tyrosines 113, 128,
and 145 of SLP-76 are required for optimal augmentation of NFAT promoter
activity. J. Immunol. 157:3769.
26. Kohn, A. D., K. S. Kovacina, and R. A. Roth. 1995. Insulin stimulates the kinase
activity of Rac-PK, a pleckstrin homology domain containing Ser/Thr kinase.
EMBO J. 14:4288.
27. Leibson, P. J., D. E. Midthun, K. P. Windebank, and R. T. Abraham. 1990.
Transmembrane signaling during natural killer cell-mediated cytotoxicity: regulation by protein kinase C activation. J. Immunol. 145:1498.
28. Fang, N., and G. A. Koretzky. 1999. SLP-76 and Vav function in separate, but
overlapping pathways to augment interleukin-2 promoter activity. J. Biol. Chem.
274:16206.
The Journal of Immunology
29. Wardenburg, J. B., C. Fu, J. K. Jackman, H. Flotow, S. E. Wilkinson,
D. H. Williams, R. Johnson, G. Kong, A. C. Chan, and P. R. Findell. 1996.
Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for
T cell receptor function. J. Biol. Chem. 271:19641.
30. Liu, S. K., N. Fang, G. A. Koretzky, and C. J. McGlade. 1999. The hematopoietic-specific adaptor protein Gads functions in T cell signaling via interactions
with the SLP-76 and LAT adaptors. Curr. Biol. 9:67.
31. Finco, T. S., T. Kadlecek, W. Zhang, L. E. Samelson, and A. Weiss. 1998. LAT
is required for TCR-mediated activation of PLC␥1 and the Ras pathway. Immunity 9:617.
32. Bubeck Wardenburg, J., R. Pappu, J. Y. Bu, B. Mayer, J. Chernoff, D. Straus, and
A. C. Chan. 1998. Regulation of PAK activation and the T cell cytoskeleton by
the linker protein, SLP-76. Immunity 9:607.
33. Bunnell, S. C., M. Diehn, M. B. Yaffe, P. R. Findell, L. C. Cantley, and L. J. Berg.
2000. Biochemical interactions integrating Itk with the T cell receptor-initiated
signaling cascade. J. Biol. Chem. 175:2219.
34. Su, Y. W., Y. Zhang, J. Schweikert, G. A. Koretzky, M. Reth, and J. Wienands.
1999. Interaction of SLP adaptors with the SH2 domain of tec family kinases.
Eur. J. Immunol. 29:3702.
35. Yablonski, D., T. Kadlecek, and A. Weiss. 2001. Identification of a phospholipase C-␥1 (PLC-␥1) SH3 domain-binding site in SLP-76 required for T cell
receptor-mediated activation of PLC-␥1 and NFAT. Mol. Cell. Biol. 21:4208.
36. Judd, B. A., P. S. Myung, L. Leng, A. Obergfell, W. S. Pear, S. J. Shattil, and
G. A. Koretzky. 2000. Hematopoietic reconstitution of SLP-76 corrects hemostasis and platelet signaling through ␣IIb␤3 and collagen receptors. Proc. Natl.
Acad. Sci. USA 97:12056.
37. Clements, J., S. E. Ross-Barta, L. T. Tygrett, T. J. Waldschmidt, and
G. A. Koretzky. 1998. SLP-76 expression is restricted to hemopoietic cells of
monocyte, granulocyte, and T lymphocyte lineage and is regulated during T cell
maturation and activation. J. Immunol. 161:3880.
367
38. Jackman, J. K., D. G. Motto, Q. Sun, M. Tanemoto, C. W. Turck, G. A. Peltz,
G. A. Koretzky, and P. R. Findell. 1995. Molecular cloning of SLP-76, a 76-kD
tyrosine phosphoprotein associated with Grb2 in T cells. J. Biol. Chem. 270:
7029.
39. Wange, R. L., A. N. T. Kong, and L. E. Samelson. 1992. A tyrosine-phosphorylated 70-kDa protein binds a photoaffinity analogue of ATP and associates with
both the ␨-chain and CD3 components of the activated T cell antigen receptor.
J. Biol. Chem. 267:11685.
40. Turner, M., E. Schweighoffer, F. Colucci, J. P. Di Santo, and V. L. Tybulewicz.
2000. Tyrosine kinase Syk: essential functions for immunoreceptor signalling.
Immunol. Today 21:148.
41. Clements, J. L., B. Yang, S. E. Ross-Barta, S. L. Eliason, R. F. Hrstka,
R. A. Williamson, and G. A. Koretzky. 1998. Requirement for the leukocytespecific adapter protein SLP-76 for normal T cell development. Science 281:416.
42. Pivniouk, V., E. Tsitsikov, P. Swinton, G. Rathbun, F. W. Alt, and R. S. Geha.
1998. Impaired viability and profound block in thymocyte development in mice
lacking the adaptor protein SLP-76. Cell 94:229.
43. Lin, T. J., T. B. Issekutz, and J. S. Marshall. 2000. Human mast cells transmigrate
through human umbilical vein endothelial monolayers and selectively produce
IL-8 in response to stromal cell-derived factor-1␣. J. Immunol. 165:211.
44. Abi-Younes, S., A. Sauty, F. Mach, G. K. Sukhova, P. Libby, and A. D. Luster.
2001. The stromal cell-derived factor-1 chemokine is a potent platelet agonist
highly expressed in atherosclerotic plaques. Circ. Res. 86:131.
45. Peterson, E. J., M. L. Woods, S. A. Dmowski, G. Derimanov, M. S. Jordan,
J. N. Wu, P. S. Myung, Q. H. Liu, J. T. Pribila, B. D. Freedman, et al. 2001.
Coupling of the TCR to integrin activation by SLAP-13-/Fyb. Science 293:2263.
46. Griffiths, E. K., C. Krawczyk, Y. Y. Kong, M. Raab, S. J. Hyduk, D. Bouchard,
V. S. Chan, I. Kozieradzki, A. J. Oliveira-dos-Santos, A. Wakeham, et al. 2001.
Positive regulation of T cell activation and integrin adhesion by the adapter Fyb/
SLAP. Science 293:2260.