Involvement of Hematopoietic Progenitor Kinase 1 in T Cell
Receptor Signaling*
Pin
Ling
,
Christian F.
Meyer
,
Lisa P.
Redmond
§,
Jr-Wen
Shui
,
Beckley
Davis
,
Robert R.
Rich
,
Mickey C.-T.
Hu¶,
Ronald L.
Wange
, and
Tse-Hua
Tan
§**
From the
Department of Immunology and the
§ Interdepartmental Program in Cell and Molecular Biology,
Baylor College of Medicine, Houston, Texas 77030, ¶ Department of
Molecular and Cellular Oncology, The University of Texas M.D. Anderson
Cancer Center, Houston, Texas 77030, and the
Laboratory of
Biological Chemistry, NIA, National Institutes of Health,
Baltimore, Maryland 21224
Received for publication, February 16, 2001
 |
ABSTRACT |
Hematopoietic progenitor kinase 1 (HPK1), a
mammalian Ste20-related serine/threonine protein kinase, is a
hematopoietic-specific upstream activator of the c-Jun N-terminal
kinase. Here, we provide evidence to demonstrate the involvement of
HPK1 in T cell receptor (TCR) signaling. HPK1 was activated and
tyrosine-phosphorylated with similar kinetics following TCR/CD3 or
pervanadate stimulation. Co-expression of protein-tyrosine kinases, Lck
and Zap70, with HPK1 led to HPK1 activation and tyrosine
phosphorylation in transfected mammalian cells. Upon TCR/CD3
stimulation, HPK1 formed inducible complexes with the adapters Nck and
Crk with different kinetics, whereas it constitutively interacted with
the adapters Grb2 and CrkL in Jurkat T cells. Interestingly, HPK1 also
inducibly associated with linker for activation of T cells (LAT)
through its proline-rich motif and translocated into
glycolipid-enriched microdomains (also called lipid rafts) following
TCR/CD3 stimulation, suggesting a critical role for LAT in the
regulation of HPK1. Together, these results identify HPK1 as a new
component of TCR signaling. T cell-specific signaling molecules Lck,
Zap70, and LAT play roles in the regulation of HPK1 during TCR
signaling. Differential complex formation between HPK1 and adapters
highlights the possible involvement of HPK1 in multiple signaling
pathways in T cells.
 |
INTRODUCTION |
A key event in the regulation of immune responses is the
activation of T cells. Optimal T cell activation requires two signals. A primary signal is delivered by the engagement of the T cell antigen
receptor (TCR)1 with the
major histocompatibility complex-antigen complex on antigen-presenting
cells. Ligation of CD28 on T cells with the B7 proteins (B7-1 and
B7-2) on antigen-presenting cells provides a costimulatory signal.
Engagement of the TCR initiates tyrosine phosphorylation of immune
receptor tyrosine-based activation motifs within the TCR-associated CD3
subunits by the Src family protein-tyrosine kinases, Lck and Fyn (1,
2). This leads to the subsequent recruitment and activation of the
cytoplasmic tyrosine kinases, Zap70 and Syk (1, 2). These
protein-tyrosine kinases further activate several signaling molecules
to transmit the TCR-induced proximal signals to downstream effectors.
One of these critical signaling molecules is the linker for activation
of T cells (LAT) (3). LAT is a 36-38-kDa palmitoylated transmembrane
protein that localizes to specific plasma membrane compartments known as glycolipid-enriched microdomains (GEMs) or detergent-insoluble lipid
rafts, which are critical for T cell signaling (4, 5). Following TCR
stimulation, LAT becomes heavily tyrosine-phosphorylated and serves as
an anchor protein for association with a number of SH2
domain-containing signaling molecules, including Grb2, Gads,
phospholipase C-
1, and the p85 subunit of phosphatidylinositol 3-kinase (6, 7). Engagement of the CD28 receptor initiates multiple
signaling pathways through Itk tyrosine kinase, Grb2, and
phosphatidylinositol 3-kinase to facilitate TCR signaling (8, 9). Thus,
costimulation of TCR and CD28 triggers a series of biochemical events,
ultimately leading to the activation of downstream targets, including
NF-AT, NF-
B, and AP-1, which in turn mediate interleukin-2 production.
One important downstream pathway mediating T cell costimulatory events
is the c-Jun N-terminal kinase (JNK) cascade (10). The JNK family, a
group of serine/threonine protein kinases, belongs to the
mitogen-activated protein kinase (MAPK) superfamily, which consists of
two other groups: the extracellular signal-regulated kinase and p38
kinase families (11). Activation of these MAPKs is achieved through
evolutionarily conserved signaling cascades, which comprise MAPK
kinases (MAP2Ks), MAPK kinase kinases (MAP3Ks), and sometimes MAPK
kinase kinase kinases (MAP4Ks) (12). Down-regulation of JNK activity is
correlated to the onset of T cell anergy (13). Studies of
jnk1- and jnk2-null mice showed that both JNK1
and JNK2 are required for the differentiation of CD4+ T
cells into effector Th1 cells (14, 15). Although the importance of JNK
in T cells has been established, the links between receptor engagement
and JNK activation remain poorly defined. Recently, several mammalian
Ste20-related MAP4Ks have been identified as upstream activators of JNK
(12). They include germinal center kinase (GCK) (16), hematopoietic
progenitor kinase 1 (HPK1) (17, 18), HPK/GCK-like kinase (also referred
to as NIK) (19, 20), GCK-like kinase (21), and the kinase homologous to
SPS1/STE20 (KHS, also referred to as GCKR) (22, 23). It is likely that one or more of these MAP4Ks may mediate JNK activation during T cell costimulation.
HPK1 is a 97-kDa serine/threonine protein kinase expressed only in
hematopoietic cells and tissues (17, 18). HPK1 consists of an
N-terminal kinase domain followed by four proline-rich motifs, and a
citron homology domain at its distal C terminus (12). Previously, HPK1 was shown to interact with several SH2/SH3 adapters, including Crk, CrkL, Grb2, and Nck (24-26). These adapters play an
important role in the formation of signaling complexes following TCR/CD3 or CD28 stimulation (27). Because of HPK1's restricted tissue
expression, activation of the JNK pathway, and association with
important T cell signaling adapters, we explored the role of HPK1 in
TCR and CD28 signaling. We found the activation and tyrosine
phosphorylation of HPK1 upon TCR/CD3 stimulation in Jurkat T cells.
CD28 stimulation, however, did not induce HPK1 activation and tyrosine
phosphorylation. In addition, protein-tyrosine kinases Lck and Zap70
were involved in the regulation of HPK1. We found that HPK1
constitutively interacted with the adapters, Grb2 and CrkL, in Jurkat T
cells and formed inducible complexes with the adapters, Nck and Crk,
upon TCR/CD3 stimulation. More interestingly, HPK1 was inducibly
associated with LAT and was recruited into the GEMs following TCR/CD3
stimulation. This work demonstrates the involvement of HPK1 in TCR
signaling and provides possible links from the TCR to HPK1.
 |
EXPERIMENTAL PROCEDURES |
Antibodies--
The anti-CD3 monoclonal antibody (OKT3) was
purified from hybridoma cell supernatants on a protein G affinity
column using standard protocols. The anti-CD28 mAb (clone 9.3) was
kindly provided by Dr. C. June (University of Pennsylvania,
Philadelphia, PA). Anti-HPK1 (N-19) and anti-CrkL (C-20) antibodies
were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Another anti-HPK1 antibody (Ab484) was described previously (17). The anti-phosphotyrosine mAb (4G10), anti-Grb2, anti-LAT and anti-Nck antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-Crk and anti-Nck mAbs were purchased from Transduction Laboratories (Lexington, KY). The anti-LAT antibody was a
gift from Drs. W. Zhang and L. E. Samelson (National Institutes of
Health, Bethesda, MD). The anti-HA (12CA5) and anti-FLAG (M2) mAbs were
purchased from Roche Molecular Biochemicals and Sigma, respectively.
Cell Culture, Stimulation, and Lysate Preparation--
COS-1 and
human embryonic kidney 293T (HEK293T) cells were maintained in
Dulbecco's modified Eagle's medium with 10% fetal calf serum,
penicillin, and streptomycin. Jurkat T cells, Jurkat TAg cells
expressing SV40 large T antigen, P116 (Zap70-deficient Jurkat), and
JCaM1.6 (Lck-deficient Jurkat) cells were maintained in RPMI medium
supplemented with 10% fetal calf serum, penicillin, and streptomycin.
P116 and JCaM1.6 were generous gifts from Dr. R. T. Abraham (Duke
University, Durham, NC) and Dr. A. Weiss (University of California, San
Francisco, CA), respectively (28, 29). Human T cells were isolated by
negative selection from human peripheral blood mononuclear cell buffy
coats (Gulf Coast Regional Blood Center, Houston, TX) as described
previously (30). Peripheral blood T cells were cultured for 24 h
in RPMI supplemented with 10% fetal calf serum before stimulation.
Cells were washed by phosphate-buffered saline once, resuspended in 1 ml of RPMI medium without fetal calf serum, and placed on ice for 10 min. Then, cells were mixed with anti-CD3, anti-CD28, or both together
at 4 °C for 10 min. Rabbit anti-mouse antibodies (10 µg/ml) were added to cross-link the primary antibodies for another 10 min. Cells
were placed in a 37 °C water bath for stimulation at the times
indicated in figures. Cells were harvested at indicated time points and
lysed by different lysis buffers based on assays as indicated in figure
legends. The lysis buffers include 1% Triton X-100 lysis buffer (20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM
-glycerophosphate, 1% Triton X-100, 10% glycerol) with protease
and phosphatase inhibitors (0.5 mM phenylmethylsulfonyl
fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 50 mM
NaF, 1 mM Na3VO4),
radioimmunoprecipitation assay buffer (25 mM Tris (pH 7.5),
150 mM NaCl, 1 mM EDTA, 0.5% deoxycholate, 1%
Nonidet P-40) with protease and phosphatase inhibitors, and 1% Nonidet
P-40 lysis buffer (1% Nonidet P-40, 50 mM Tris (pH 8.0),
150 mM NaCl, 2 mM EDTA) with protease and
phosphatase inhibitors.
Plasmids and Transfections--
The pCI-Flag-tagged vectors of
wild-type HPK1 (Flag-HPK1) and its mutants, including kinase-dead
mutant HPK1-M46 (Flag-HPK1(M46)) and the kinase domain only
(Flag-HPK1-KD) were described previously (17). The pCR-HA-tagged HPK1
mutant containing the HPK1 proline-rich region (HA-HPK1-PR) was
described previously (24). The constructs of HA-JNK, MKK4-AL (also
referred to as SEK1-AL), and MKK7-K76E have been described previously
(24, 31). Myc-tagged Zap70 and Lck constructs were gifts from Dr.
L. E. Samelson (National Institutes of Health, Bethesda, MD) (32,
33). Jurkat TAg cells (2 × 107/0.4 ml) were used for
electroporation by a BTX Electro square porator. The calcium phosphate
precipitation method was utilized for transfection of COS-1 and HEK293T
cells as described previously (24).
Immunocomplex Kinase Assays, Immunoprecipitation, and
Immunoblotting--
For the HPK1 immunocomplex kinase assays, cell
lysates (100 µg) were immunoprecipitated with an anti-HPK1 antibody
(Ab484) or anti-Flag mAb (M2). Kinase assays were performed as
described previously (24). Myelin basic protein (MBP) and GST-Crk were used as substrates where indicated. For the JNK kinase assays, cell
lysates were immunoprecipitated with an anti-HA mAb (12CA5). Kinase
assays were performed as described previously (17). For immunoprecipitation and immunoblotting, cell lysates were
immunoprecipitated by antibodies as indicated in figure legends.
Immunoprecipitates were resolved by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride
membranes. The membranes were blocked with either 5% milk in
Tris-buffered saline/Tween buffer (20 mM Tris (pH 7.5), 135 mM NaCl, 0.1% Tween 20) or 2% bovine serum albumin buffer
(10 mM Tris-Cl, (pH 8.0), 150 mM NaCl, 2%
bovine serum albumin fraction V) for anti-phosphotyrosine
immunoblotting. The membranes were probed with antibodies as described
in the figure legends, and then incubated with horseradish
peroxidase-conjugated secondary antibody according to the primary
antibody used.
Preparation of GEM Fractions--
GEM fractions were prepared as
described previously (3). In brief, Jurkat T cells (5 × 107) were either unstimulated or stimulated with an
anti-CD3 mAb (OKT3, 10 µg/ml) for 2 min. Cells were harvested and
lysed in 1 ml of 1% Trition X-100 lysis buffer. Cell lysates were
mixed well with 1 ml of 80% sucrose and transferred to ultracentrifuge tubes. These mixtures were overlaid with 2 ml of 30% sucrose and 1 ml
of 5% sucrose sequentially, and then subjected to ultracentrifugation for 16-18 h at 4 °C. Twelve 0.4-ml fractions were collected from the top of the sucrose gradient. These fractions were subjected to immunoblotting.
 |
RESULTS |
TCR/CD3 Stimulation Activates HPK1 in Jurkat T Cells--
To
investigate whether HPK1 is regulated by TCR or CD28 signaling, we
first performed an immunocomplex kinase assay to examine HPK1 kinase
activity in Jurkat T cells upon TCR/CD3 stimulation, CD28 stimulation,
or both in combination. We found that TCR/CD3 stimulation alone was
able to induce HPK1 activation 2 min after stimulation, and that this
activation decreased after 10 min. A similar pattern of HPK1 activation
was also observed in Jurkat T cells with TCR/CD3 plus CD28
costimulation (Fig. 1A).
However, CD28 stimulation alone did not result in significant HPK1
activation (data not shown). We also observed TCR/CD3-induced HPK1
activation in purified human peripheral blood T cells (Fig.
1B). To confirm the specificity of HPK1 activation by
TCR/CD3 stimulation, we transiently expressed the Flag-tagged wild-type
HPK1 (Flag-HPK1) or its kinase-dead mutant (Flag-HPK1(M46)) into Jurkat
TAg cells to examine their kinase activity following TCR/CD3
stimulation. By an immunocomplex kinase assay using an anti-Flag
antibody, we found that only Flag-HPK1, but not Flag-HPK1(M46), was
activated upon TCR/CD3 stimulation (Fig. 1C). These results
demonstrate that the engagement of TCR/CD3 elicits a signal to activate
HPK1.

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Fig. 1.
Activation of HPK1 following TCR/CD3
stimulation in Jurkat T cells. A, Jurkat T cells were
either untreated or treated with an anti-CD3 mAb (OKT3, 10 µg/ml) or
in combination with an anti-CD28 mAb (9.3, 1 µg/ml). Cells were
collected and lysed in 1% Triton X-100 lysis buffer at the times
indicated. Cell lysates (100 µg/sample) were used for anti-HPK1
immunocomplex kinase assays using MBP as a substrate. Phosphorylation
of MBP was detected by autoradiograghy (top
panel). Comparable HPK1 protein levels in individual lanes
were confirmed by immunoblotting (bottom panel).
IP, immunoprecipitation; WB, Western blot.
B, human peripheral blood T cells were purified from healthy
donors, then treated and analyzed similarly as described in
panel A. Results were representative of five
independent donors. C, Jurkat TAg cells (2 × 107) were transfected with 10 µg of empty vector
(pCI-neo), Flag-tagged wild-type HPK1 (Flag-HPK1), or kinase-dead HPK1
mutant (Flag-HPK1-M46). 40 h after transfection, cells were left
untreated or treated with an anti-CD3 mAb (OKT3, 10 µg/ml) for 5 min,
and then lysed in 1% Triton X-100 lysis buffer. Cell lysates (100 µg) were subjected to immunocomplex kinase assays using an anti-Flag
mAb (M2) (top panel). Expression levels of
Flag-HPK1 and Flag-HPK1(M46) were examined by immunoblotting
with an anti-Flag mAb (M2) (bottom panel).
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Tyrosine Phosphorylation of HPK1 upon TCR/CD3
Stimulation--
Early events in TCR signaling include protein-protein
interactions and tyrosine phosphorylation of signaling molecules.
Previous studies showed that transfected HPK1 in COS-1 cells is
tyrosine-phosphorylated upon EGF stimulation and is also recruited to
the EGF receptor tail through the adapters Grb2 and Crk (24, 25).
Recently, adapters Crk and CrkL were also shown to form inducible
complexes with tyrosine kinases Zap70 and Fyn, respectively, upon
TCR/CD3 stimulation (34, 35). These observations provide a possible link between protein-tyrosine kinases and HPK1 during T cell
activation. We therefore examined whether TCR signaling could induce
tyrosine phosphorylation of HPK1. HPK1 was immunoprecipitated from the TCR/CD3-stimulated Jurkat cell lysates by an anti-HPK1 antibody. The
immunoprecipitates were then subjected to anti-phosphotyrosine immunoblot analyses. Our results revealed that HPK1 was
tyrosine-phosphorylated as early as 2 min after TCR/CD3 stimulation,
and that this signal sustained to 10 min (Fig.
2, top panel).
Subsequent stripping and reprobing of the blots with another anti-HPK1
antibody confirmed that the position of HPK1 corresponded exactly to
that of the phosphotyrosine band. A similar result was observed in
Jurkat T cells treated with TCR/CD3 plus CD28 costimulation but not
CD28 stimulation alone (Fig. 2, bottom and middle
panels). Interestingly, the kinetics of HPK1 tyrosine
phosphorylation was similar to that of its kinase induction, suggesting
a correlation between these two biochemical events.

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Fig. 2.
Tyrosine phosphorylation of HPK1 following
TCR/CD3 stimulation in Jurkat T cells. Jurkat T cells (1 × 108) were left untreated or treated with an anti-CD3 mAb
(OKT3, 10 µg/ml), an anti-CD28 mAb (9.3, 1 µg/ml), or in
combination. Untreated and treated cells were collected at the times
indicated, lysed in radioimmunoprecipitation assay buffer, and
subjected to immunoprecipitation (IP) with an anti-HPK1
antibody (Ab484). Immunoprecipitation samples were then analyzed by
immunoblotting with an anti-phosphotyrosine mAb (anti-pTyr;
4G10), and subsequently reprobed by an anti-HPK1 antibody (N-19). An
inducible phosphotyrosine band appeared at 97 kDa (top
panel), and immunoblotting with an anti-HPK1 antibody showed
the position of endogenous HPK1, which comigrated with the 97-kDa
tyrosine-phosphorylated protein (bottom panel).
WB, Western blot.
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Activation and Tyrosine Phosphorylation of HPK1 in
Pervanadate-treated Jurkat T Cells--
The similar kinetics between
HPK1 tyrosine phosphorylation and its kinase induction led us to
further explore the possible link between these two biochemical events.
To that purpose, we examined the HPK1 response in Jurkat T cells
stimulated by pervanadate, a potent tyrosine phosphatase inhibitor
capable of activating protein-tyrosine kinases and mimicking T cell
activation (36). Our results showed that pervanadate treatment induced
substantial and prolonged tyrosine phosphorylation of HPK1 in Jurkat T
cells (Fig. 3A). The same
pervanadate-treated Jurkat lysates were further analyzed by
immunocomplex kinase assays using anti-HPK1 antibodies. HPK1 kinase
activity was potently stimulated after pervanadate treatment (Fig.
3B). These results strongly suggest that tyrosine phosphorylation of HPK1 is important for regulating its kinase activity
during T cell activation.

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Fig. 3.
Pervanadate treatment induces tyrosine
phosphorylation and kinase induction of HPK1 in Jurkat T cells.
A, Jurkat T cells were treated with 100 µM
pervanadate for the indicated times. Cells were collected, lysed in 1%
Nonidet P-40 lysis buffer, and subjected to immunoprecipitation with an
anti-HPK1 antibody (Ab484). The immunoprecipitates were analyzed by
immunoblotting with an anti-phosphotyrosine mAb (4G10). The same
membrane was reprobed with an anti-HPK1 antibody (N-19). B,
Jurkat T cells were treated by pervanadate as described for
panel A. Cell lysates (100 µg/sample) were
subjected to anti-HPK1 immunocomplex kinase assay using GST-Crk as a
substrate. The equal amount of HPK1 for kinase assays was demonstrated
by immunoblotting with an anti-HPK1 antibody (N-19). IP,
immunoprecipitation; WB, Western blot.
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Tyrosine Kinases Lck and Zap70 Are Involved in Regulation of
HPK1--
We next attempted to determine which protein-tyrosine
kinase(s) could regulate HPK1 during TCR stimulation. We compared HPK1 tyrosine phosphorylation in JCaM 1.6 (Lck-deficient Jurkat), P116 (Zap70-deficient Jurkat) cells, and wild-type Jurkat T cells. HPK1
tyrosine phosphorylation induced by costimulation of TCR/CD3 and CD28
was significantly decreased in JCaM 1.6 and P116 cells, whereas
pervanadate-induced HPK1 tyrosine phosphorylation still remained at a
substantial level in these two mutant cell lines (Fig.
4A). This suggests that T
cell-specific tyrosine kinases Lck and Zap70 are critical for HPK1
tyrosine phosphorylation during T cell activation. However, pervanadate
treatment could still induce HPK1 tyrosine phosphorylation through
activating other tyrosine kinases in addition to Lck and Zap70. By
cotransfection studies, we observed that ectopic expression of Lck and
Zap70 with the kinase-dead mutant HPK1(M46) led to the HPK1 tyrosine phosphorylation in COS-1 cells (Fig. 4B). Moreover,
wild-type HPK1, but not the kinase-dead mutant HPK1(M46), was activated in the presence of Lck and Zap70 in transfected HEK293T cells (Fig.
4C). These results suggest that protein-tyrosine kinases Lck
and Zap70 mediate TCR-induced HPK1 activation.

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Fig. 4.
Tyrosine kinases Lck and Zap70 regulate HPK1
tyrosine phosphorylation and kinase induction. A,
Jurkat T cells, JCaM1.6 (Lck-deficient), and P116 (Zap70-deficient)
were untreated or treated with an anti-CD3 antibody or pervanadate
(PV) for 5 min. Cell lysates were subjected to
immunoprecipitation with an anti-HPK1 antibody (Ab484). The
immunoprecipitation samples were examined by anti-phosphotyrosine
immunoblotting (top panel). The membrane was
reprobed with an anti-HPK1 antibody (N-19) (bottom
panel). B, COS-1 cells were transfected with
Flag-HPK1(M46) (5 µg) alone or in combination with Myc-Zap70 (7 µg) plus Lck (7 µg). 40 h after tranfection, Flag-HPK1(M46)
was immunoprecipitated by an anti-Flag antibody and resolved by 10%
SDS-PAGE. Anti-phosphotyrosine (anti-pTyr) immunoblotting
demonstrated the tyrosine phosphorylation of Flag-HPK1(M46) in the
presence of Lck and Zap70. C, Flag-HPK1 (0.4 µg) or
Flag-HPK1(M46) (0.4 µg) was transfected into HEK293T cells alone or
with Lck (1.0 µg) plus Zap70 (1.0 µg). Transfected cells were
collected 40 h after transfection and lysed in 1% Triton X-100
lysis buffer. The cell lysates (50 µg/sample) were subjected to
immunocomplex kinase assays using an anti-HPK1 antibody (Ab484) and MBP
as a substrate. IP, immunoprecipitation; WB,
Western blot.
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Differential Inducible Association of HPK1 with Adapters Nck and
Crk upon TCR/CD3 Stimulation--
HPK1-interacting SH2/SH3 adapters
(e.g. Grb2, Nck, Crk, and CrkL) may function to couple HPK1
to protein-tyrosine kinases or tyrosine phosphorylated proteins.
However, whether HPK1 interacts with these adapters in T cells was
unclear. Therefore, we were interested in studying complex formation
between HPK1 and these adapters before and after TCR/CD3 stimulation.
By a series of co-immunoprecipitation analyses, we first observed that
HPK1 formed an inducible complex with Nck in Jurkat T cells after 1 min
of TCR/CD3 stimulation, and this complex sustained until 10 min of TCR/CD3 stimulation (Fig. 5A).
The kinetics of HPK1 interaction with Nck correlated with that of HPK1
activation, suggesting the possible involvement of Nck in the
regulation of HPK1 during TCR activation. In addition, we found that
the adapter Crk, unlike Nck, inducibly bound to HPK1 after 10 min of
TCR/CD3 stimulation (Fig. 5B).

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Fig. 5.
Differential inducible complex formation of
HPK1 with adapters Nck and Crk upon TCR/CD3 stimulation. Jurkat T
cells (2 × 107) were left untreated or treated with
an anti-CD3 mAb (OKT3, 10 µg/ml). Cells were collected at the times
indicated, lysed by 1% Nonidet P-40 lysis buffer, and subjected to
immunoprecipitation with an anti-Nck antibody (A) or an
anti-Crk antibody (B). Association of HPK1 was examined by
immunoblotting with an anti-HPK1 antibody (N-19). The same membranes
were reprobed by anti-Nck and anti-Crk mAbs, respectively (shown in the
bottom panels). IP,
immunoprecipitation; WB, Western blot.
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Constitutive Association of HPK1 with Adapters Grb2 and CrkL in
Jurkat T Cells--
By similar co-immunoprecipitation approaches, we
noticed that HPK1 formed a constitutive complex with Grb2 in Jurkat T
cells before and after TCR/CD3 stimulation (Fig.
6A). In addition, a constitutive HPK1-CrkL complex was also observed in Jurkat T cells (Fig. 6B). Our results, shown in Figs. 5 and 6, demonstrate
the differential complex formation between HPK1 and several SH2/SH3 adapters during TCR/CD3 stimulation, indicating an important role for
HPK1 in T cell signaling.

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Fig. 6.
Constitutive interaction of HPK1 with
adapters Grb2 and CrkL in Jurkat T cells. Jurkat T cells (2 × 107) were left untreated or treated with an anti-CD3 mAb
(OKT3, 10 µg/ml). Cells were collected at the times indicated and
lysed by 1% Nonidet P-40 lysis buffer. A, cell lysates were
immunoprecipitated with an anti-HPK1 antibody (Ab484). The
immunoprecipitates were resolved by 10% SDS-PAGE and then probed
successively with anti-Grb2 and anti-HPK1 antibodies. B,
cell lysates were first immunoprecipitated with an anti-CrkL antibody
and followed by immunoblotting successively with anti-HPK1 (N-19) and
anti-CrkL antibodies. IP, immunoprecipitation;
WB, Western blot.
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Inducible Association of HPK1 with LAT upon TCR/CD3
Stimulation--
It is known that the linker protein LAT plays a
critical role in TCR signaling through interaction of its
phosphotyrosine residues with the SH2 domains of several signaling
molecules, including Grb2, Gads, phospholipase C-
1, and the p85
subunit of phosphatidylinositol 3-kinase (6, 7). Because of the LAT-Grb2 interaction, we attempted to examine the potential association between LAT and HPK1 in T cells. We performed the
co-immunoprecipitation analyses and found that LAT formed an inducible
complex with HPK1 as early as 1 min after TCR/CD3 stimulation (Fig.
7A). To confirm this
interaction, we also utilized a cotransfection system to demonstrate
that overexpression of LAT and HPK1 led to the LAT-HPK1 complex
formation in transfected COS-1 cells (Fig. 7B). We further determined the region(s) of HPK1 for LAT association. Two HPK1 truncated mutants, the HPK1 kinase domain (Flag-tagged HPK1-KD) and
proline-rich region (HA-tagged HPK1-PR), were transfected into
Jurkat-Tag cells to test their ability to interact with LAT. Interestingly, our data showed that only HPK1-PR inducibly associated with LAT upon TCR/CD3 stimulation (Fig. 7C). HPK1-KD did not
associate with LAT (data not shown).

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Fig. 7.
HPK1 forms a complex with LAT through its
proline-rich region upon TCR/CD3 stimulation. Jurkat T cells
(2 × 107) were left untreated or treated with an
anti-CD3 mAb (OKT3, 10 µg/ml). Cells were collected at the times
indicated, lysed by 1% Nonidet P-40 lysis buffer. A, cell
lysates were subjected to immunoprecipitation with an anti-LAT antibody
and followed by immunoblotting sequentially with anti-HPK1 (N-19) and
then anti-LAT antibodies. B, Flag-HPK1 (10 µg) and LAT (5 µg) were transfected into COS-1 cells separately or in combination.
Cells were harvested and lysed 40 h after transfection. Lysates
were immunoprecipitated with an anti-Flag antibody and then subjected
to the immunoblot analyses using anti-LAT and anti-HPK1 (N-19)
antibodies sequentially. C, Jurkat TAg cells (2 × 107) were transfected with 5 µg of HA-HPK1-PR. 24 h
after transfection, the cells were left untreated or treated with an
anti-CD3 mAb for 1 min. Cells were harvested, lysed in 1% Nonidet P-40
lysis buffer, and subjected to immunoprecipitation using an anti-HA
mAb. The immunoprecipitates and direct Jurkat lysates were resolved by
10% SDS-PAGE and followed by immunoblotting using an anti-LAT
antibody. Expression of HA-HPK1-PR was examined by immunoblotting with
an anti-HA mAb. D, Jurkat TAg cells (2 × 107) were transfected with 10 µg of HA-JNK alone, or
together with 30 µg of HPK1-PR, MKK7-K76E, or MKK4-AL. 24 h
after transfection, cells were left untreated or treated with a
combination of anti-CD3 and anti-CD28 mAbs for 20 min. Cells were then
harvested, lysed in 1% Triton X-100 lysis buffer, and subjected to
immunocomplex kinase assays using an anti-HA mAb and GST-c-Jun as a
substrate. Phosphorylation of GST-c-Jun was detected by
autoradiography. The result is representative of three independent
experiments. IP, immunoprecipitation; WB, Western
blot.
|
|
After exploring the regulation of HPK1 by TCR signaling, we next
investigated whether HPK1 mediates TCR signaling for JNK activation
during T cell costimulation. HPK1-PR and two MAP2K mutants, MKK4-AL and
MKK7-K76E, were cotransfected with HA-JNK, respectively, into Jurkat
TAg cells to examine their effect on JNK activation by TCR/CD3 and CD28
costimulation. We found that HPK1-PR failed to block JNK activation,
whereas two MAP2K mutants, MKK4-AL and MKK7-K76E, blocked JNK
activation effectively (Fig. 7D). Although the JNK kinase
assay was very sensitive to HA-JNK activity, we were unable to detect
HA-JNK protein levels in transfected Jurkat T cells due to low
transfection efficiency (data not shown). We did detect HPK1-PR
expression as shown in Fig. 7C. Thus, failure to block JNK
activation by HPK1-PR was not due to the low level of HPK1-PR expression.
Recruitment of HPK1 into GEMs (or Lipid Rafts) upon TCR/CD3
Stimulation--
Emerging evidence has indicated that GEMs (or lipid
rafts) play an important role in T cell signaling (5). Disruption of these lipid microdomains results in the down-regulation of TCR signaling and consequently attenuated T cell activation. In T cells,
many critical signaling molecules are enriched in GEMs constitutively
or upon TCR/CD3 stimulation. They include tyrosine kinases
(e.g. Lck and Syk), adapters (e.g. LAT and Grb2),
and other signaling molecules (e.g. Ras and phospholipase
C-
1) (5, 37). Given the evidence that HPK1 inducibly interacted with LAT, we tested whether HPK1 could be recruited into GEMs or lipid rafts
upon TCR/CD3 stimulation. Our results showed that HPK1 rapidly translocated into GEMs upon TCR/CD3 stimulation (Fig.
8, middle panel).
However, HPK1 from unstimulated Jurkat T cells only remained in the
Triton X-100-soluble fractions (Fig. 8, top
panel). We also showed the presence of LAT in GEMs or lipid
rafts as a control for this analysis (Fig. 8, bottom
panel). This result suggests that the inducible LAT-HPK1
complex may recruit HPK1 into GEMs, leading to activation of HPK1 by
protein-tyrosine kinases.

View larger version (27K):
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|
Fig. 8.
Localization of HPK1 to GEMs upon TCR/CD3
stimulation. Jurkat T cells (5 × 107)
were either left unstimulated or stimulated with an anti-CD3 mAb (OKT3,
10 µg/ml) for 2 min. Cells were harvested, lysed in 1 ml of 1%
Triton X-100 lysis buffer, and mixed with 1 ml of 80% sucrose. These
lysates were overlaid with 2 ml of 30% sucrose and 1 ml of 5% sucrose
sequentially and then subjected to ultracentrifugation overnight
(16-20 h) at 4 °C. Gradient fractions were collected in 0.4-ml
aliquots from the top. These aliquots were subjected to immunoblotting
using anti-HPK1 (Ab484) and anti-LAT antibodies. WB, Western
blot.
|
|
 |
DISCUSSION |
Advances have been made in the characterization of signaling
molecules (e.g. Lck, Zap70, and LAT) proximal to the TCR and downstream effectors (e.g. AP-1, NF-AT, NF-
B, and JNK)
involved in interleukin-2 production. However, which and how the
intermediate modulators integrate both the TCR and CD28 signals to
downstream effectors for T cell activation remain unclear. Here we have
investigated the role of a hematopoietic-specific JNK activator, HPK1,
in T cell signaling. We first noticed the early kinase induction (1-2 min) of HPK1 following TCR/CD3 engagement in both Jurkat and human peripheral blood T cells, suggesting a close link between HPK1 and
TCR/CD3-proximal signaling events. This idea is further supported by
the observations that HPK1 was tyrosine-phosphorylated at early time
points following TCR/CD3 stimulation, and that HPK1 was potently tyrosine-phosphorylated and activated by pervanadate stimulation.
By cotransfection studies and the analysis of two Jurkat mutant cell
lines JCaM 1.6 (Lck-deficient) and P116 (Zap70-deficient), we observed
that Lck and Zap70 were essential for TCR-mediated HPK1 tyrosine
phosphorylation. Moreover, we provided evidence that ectopic expression
of Lck and Zap70 with HPK1 led to both HPK1 tyrosine phosphorylation
and kinase induction. Our results are supported by a recent paper (39)
indicating that TCR-induced HPK1 activation is abolished in both
Lck-deficient and Zap70-deficient Jurkat T cells. Future studies
mapping the tyrosine phosphorylation site(s) within HPK1 will provide
more insight into the regulation of HPK1 by Lck and Zap70.
Interestingly, our data showed that HPK1 displayed a more significant
level of tyrosine phosphorylation in pervanadate-treated JCaM 1.6 (Lck-deficient) cells than pervanadate-treated P116 (Zap70-deficient)
cells, implying that Zap70 plays a critical role for HPK1 tyrosine
phosphorylation in T cells. It is likely that in addition to Lck and
Zap70, there are other protein-tyrosine kinases capable of activating
HPK1. For instance, c-Abl tyrosine kinase inducibly binds to HPK1 in
Jurkat T cells in response to DNA-damaging agents, and this c-Abl-HPK1
interaction results in HPK1 phosphorylation and activation (38).
Our results showed that CD28 stimulation was unable to induce HPK1
kinase induction and tyrosine phosphorylation and also had no further
effect on TCR/CD3-induced HPK1 activation. This suggests that, unlike
JNK, HPK1 is mainly activated by TCR/CD3 stimulation. However, we do
not exclude the possibility that CD28 may play a role in the regulation
of HPK1 by other mechanisms, such as HPK1 localization and complex
formation. Although HPK1 is a JNK activator and is implicated in TCR
signaling, our data from transfection studies showed that the HPK1
mutant HPK1-PR, responsible for interaction with LAT and SH2/SH3
adapters, failed to block JNK activation by TCR plus CD28
costimulation. Recently, Liou et al. (39) also showed
similar results by using a kinase-inactive HPK1 mutant. These results
suggest that HPK1 is not indispensable for JNK activation during T cell
costimulation. One possibility is that other MAP4Ks may play a
redundant role in JNK activation in T cells. For example, other
HPK1-related kinases, such as GCK and GCK-like kinase , are also
expressed in T cells and contain proline-rich motifs for interaction
with SH2/SH3 adapters, suggesting their potential roles in TCR or CD28 signaling.
Our result showed that HPK1 was recruited into GEMs after 2 min of
TCR/CD3 stimulation. This finding further suggests that HPK1
participates in the early events of TCR signaling. In addition, the
recruitment of HPK1 into GEMs is possibly involved in HPK1 activation
and tyrosine phosphorylation. The underlying mechanism by which HPK1 is
recruited into GEMs is still unclear. One possibility is that upon
TCR/CD3 stimulation, SH2/SH3 adapters couple HPK1 to
tyrosine-phosphorylated LAT or other signaling molecules localized in GEMs where HPK1 is activated and tyrosine-phosphorylated by protein-tyrosine kinases. The finding of early inducible association between HPK1 and LAT upon TCR/CD3 stimulation supports this possibility.
We found that HPK1 differentially interacted with various adapters,
including LAT, Nck, Crk, CrkL, and Grb2, in Jurkat T cells. HPK1
interaction with the adapters Grb2 and CrkL was found to be
constitutive at all time points we tested, whereas HPK1 interaction with Nck and LAT was induced after 1 min of TCR/CD3 stimulation. The
interaction of HPK1 with Crk, however, was induced at a later time
point 10 min after TCR/CD3 stimulation. The early inducible association
of HPK1 with Nck and LAT following TCR/CD3 stimulation suggests the
potential roles for Nck and LAT in TCR-mediated HPK1 activation. Since
LAT does not contain an SH3 domain for its direct binding to the HPK1
proline-rich motifs, the inducible HPK1- LAT complex formation is
likely through SH2/SH3 adapters. One possibility is that the
constitutive HPK1-Grb2 complex may be recruited to tyrosine-phosphorylated LAT upon TCR/CD3 stimulation. Recently, others
have shown that HPK1 interacts with adapters Grap and Gads in T cells
(39, 40, 41). Because these two adapters are also shown to interact
with LAT (7), they are potential candidates to couple HPK1 to LAT. More
studies are needed to reveal the underlying mechanism of LAT-HPK1
complex formation in T cells. The constitutive HPK1-CrkL complex may
participate in the TCR-mediated signaling events through CrkL binding
to Fyn upon TCR/CD3 stimulation (35). Unlike the previous finding that
CrkL binds and activates HPK1 in an overexpression system (24), the
constitutive HPK1-CrkL interaction in Jurkat T cells did not lead to
HPK1 activation. The possible reason is that in transfected cells,
overexpressed CrkL and HPK1 may circumvent regulation by other
endogenous or tissue-specific factors to amplify the signaling
pathways. In contrast, these factors may tightly regulate endogenous
CrkL and HPK1 in Jurkat T cells. Thus, this could be one reason why the constitutive CrkL-HPK1 interaction did not lead to HPK1 activation in
Jurkat T cells. Finally, we have reported the late inducible association between HPK1 and Crk. The functional relevance of this late
interaction is unclear. One possibility is that Crk couples HPK1 to
other downstream signaling events in T cells. In addition to the
inducible Crk-HPK1 complex, we observed a low level of constitutive
Crk-HPK1 complex in Jurkat T cells (data not shown). Therefore, Crk may
play a role in coupling HPK1 to Zap70 upon TCR/CD3 stimulation. HPK1
association with multiple adapters in T cells implies an important role
for HPK1 in T cell signaling. Future studies will focus on dissecting
the multiple HPK1-mediated signaling pathways in T cells.
 |
ACKNOWLEDGEMENTS |
We thank members of the Tan laboratory for
their critical reviews of the manuscript, S. Lee and A. Ashtari for
technical assistance, and S. Robertson for secretarial assistance. We
also thank Drs. R. T. Abraham, C. June, L. Samelson, A. Weiss, Z. Yao, and W. Zhang for gifts of materials.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants RO1-AI38649 and RO1-AI42532 (to T.-H. T.).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.
**
Scholar of the Leukemia and Lymphoma Society. To whom
correspondence should be addressed. Tel.: 713-798-4665; Fax:
713-798-3033; E-mail: ttan@bcm.tmc.edu.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M101485200
 |
ABBREVIATIONS |
The abbreviations used are:
TCR, T cell
receptor;
JNK, c-Jun N-terminal kinase;
HPK1, hematopoietic progenitor
kinase 1;
GEM, glycolipid-enriched microdomain;
LAT, linker for
activation of T cells;
mAb, monoclonal antibody;
SH, Src homology;
MBP, myelin basic protein;
HA, hemagglutinin;
GST, glutathione
S-transferase;
MKK, MAPK kinase;
GCK, germinal center
kinase;
MAPK, mitogen-activated protein kinase;
MAP2K, mitogen-activated protein kinase kinase;
MAP4K, mitogen-activated
protein kinase kinase kinase kinase.
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