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*
Department of Medical Biophysics and The Arthur and Sonia Labatt Brain Tumor Research Center, Hospital for Sick Children Research Institute, University of Toronto, Toronto, Ontario, Canada; and
Max Planck Institute for Physiological and Clinical Research W. G. Kerckhoff Institute, Bad Nauheim, Germany
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 (PLC-1) (10),
c-Cbl (11), and the linker for activation of T cells (LAT)
(12, 13, 14, 15). The three members of the Grb2 family of adaptor proteins, Grb2, Grap, and Gads, have also been implicated in the formation of protein complexes at the TCR and are important for linking specific pathways, such as Ras to TCR activation. The Grb2 family of adaptors possess amino- and carboxyl-terminal SH3 domains that flank a central SH2 domain (7, 16, 17, 18, 19, 20, 21). Gads also has a 120-aa unique region between the SH2 domain and carboxyl-terminal SH3 domain (20). This family of proteins couples tyrosine-phosphorylated membrane-associated proteins bound by the SH2 domain with cytoplasmic proteins associated through the SH3 domains (7, 16, 17, 18, 19, 20, 21, 22). Grb2 is ubiquitously expressed, while expression of both Grap and Gads is restricted to hemopoietic cells (18, 19, 20, 23, 24, 25). It has been proposed that Grb2 and Grap may serve overlapping functions, since their SH2 and SH3 domains appear to bind to similar proteins in T cells and since Grap null mice display no apparent phenotype (26). In contrast, Gads is likely to have a unique function because it binds to distinct SH3 domain targets (20, 22).
The function of the Grb2 adaptor is best defined in the context of growth factor receptor signaling, where it couples activated receptors with downstream effector pathways, such as the evolutionarily conserved pathway involved in Ras activation (16, 26, 27, 28, 29, 30, 31, 32, 33, 34). The SH2 domain of Grb2 binds to activated growth factor receptors either directly or indirectly via the docking protein Shc, localizing Grb2 and the stably associated Ras guanine nucleotide exchange factor, Sos, to the membrane, resulting in the activation of Ras (28, 29, 30, 31, 32, 34, 35). Within T cells, Grb2 is proposed to couple TCR engagement to Ras activation through the recruitment of Grb2-Sos complexes to tyrosine-phosphorylated LAT, a membrane-bound linker protein (14, 15, 36). By analogy, Grap may function to amplify TCR activation of Ras through recruitment of additional pools of Sos to membrane-associated LAT.
Gads also inducibly associates with LAT in T cells, but unlike Grb2 and Grap, it does not form complexes with Sos (20, 22). Instead, Gads is bound to the SLP-76 adaptor protein, which plays a critical role in signaling from both pre-TCR and mature TCR (8, 37, 38, 39). Gads synergizes with SLP-76 to augment both NF-AT and IL-2 activation, and this augmentation is dependent on a functional Gads SH2 domain (22). Hence, through its association with SLP-76 and LAT, Gads appears to play a central role in signaling from the activated TCR (22, 24, 25).
To date, SLP-76 is the only Gads SH3 domain-binding protein that has been identified. In an effort to identify additional signaling effectors downstream of Gads, we used an expression screening strategy and identified hemopoietic progenitor kinase-1 (HPK1) as a Gads-binding protein. HPK1 belongs to a family of Ste20 homologous serine/threonine kinases that includes germinal center kinase (GCK), GCKR/KHS (GCK-related kinase/kinase homologous to SPS1/Ste20), and GLK (GCK-like kinase) (40). The family is defined by their N-terminally located kinase domain and the regulatory C-terminal domain, and all have been implicated as upstream effectors in activation of the stress-activated protein kinase (SAPK/c-Jun NH2-terminal kinase) pathway (40, 41, 42, 43, 44). Here we show that HPK1 is activated by the TCR, that Gads and HPK1 interact in vivo, and that Gads can function to link HPK1 to signaling from the activated TCR complex.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 16-day mouse embryo expression library (Novagen, Madison, WI)
was plated and protein expression induced according to the
manufacturer’s directions. The nitrocellulose filters were washed in
TBST (20 mM Tris-base (pH 7.5), 150 mM NaCl, and 0.05% (v/v) Tween 20)
and blocked at 4°C in blocking buffer (1% nonfat milk powder (w/v)
and 1 mM DTT in TBST) for 2 h. Radiolabeled GST-Gads fusion
protein was prepared by incubating 15 µg of GST-Gads fusion protein
bound to glutathione-Sepharose beads with heart muscle kinase (0.9
U/µl; Sigma, St. Louis, MO) and [-32P]ATP
(0.7 µCi/µl) in 170 µl of kinase buffer (20 mM Tris-HCl (pH 7.5),
100 mM NaCl, 12 mM MgCl2, and 1 mM DTT) at 4°C
for 40 min. The beads were washed five times with PBS (137 mM NaCl, 2.7
mM KCl, 4.3 mM Na2HPO4, and
1.4 mM KH2PO4, pH 7.4), and
the radiolabeled GST-Gads fusion protein was eluted with 300 µl of
elution buffer (100 mM Tris-HCl (pH 8.0), 120 mM NaCl, and 20 mM
reduced glutathione (Sigma)), followed by an additional elution with
400 µl of elution buffer. The filters were incubated overnight with
radiolabeled GST-Gads fusion protein in blocking buffer at 4°C and
washed four times for 10 min each time in TBST, and the filters were
exposed to film.
Plasmids and Abs
The pEF-FLAG wild-type Gads and mutant Gads (N*/C, N/C*, N*/C*,
and SH2*) constructs were previously described (22). The
GST-Gads fusion constructs and polyclonal anti-Gads Ab (H1-A) were
used as previously described (20). The pcDNA3-HPK1:HA and
pcDNA3-KD HPK1:HA contain the full-length murine wild-type and
kinase-dead HPK1 cDNA, respectively, with an in-frame fusion of a
triple HA epitope tag. Deletion mutants lacking the proline-rich
regions (P1, P2, and P4), individually or in combination, were
generated as follows. A KpnI fragment comprising 1233 bp of
murine HPK1 (mHPK1) cDNA containing the proline-rich motifs P1 to P4
was subcloned into pBluescript, giving rise to the plasmid
pB-mHPK1:KpnI. Deletion mutants of aa 309–311 (
P1), aa
392–401 (P2), and aa 467–471 (P4) were generated by PCR. PCR
products containing the desired mutations were reinserted into
pB-mHPK1:KpnI, and then reintroduced into pcDNA3-HPK1:HA by
swapping the corresponding KpnI fragments.
Synthetic peptides representing the N-terminus (MALVDPDIFNKDPREHYD) and C-terminus (TRPTDDPTAPSNLYIQE) of mHPK1 were coupled to keyhole limpet hemocyanin via a carboxyl-terminal cysteine residue and used to immunize rabbits and generate antisera 2 (N-terminus) and 7 (C terminus). The anti-HPK1 antiserum 5 has been previously described (41). For anti-HPK1 immunoprecipitations, a mixture of 2.5 µl of HPK1 2 antisera and 2.5 µl of HPK1 5 antisera was used. A 1/500 dilution of HPK1 7 antisera was used for immunoblotting. Monoclonal anti-phosphotyrosine Ab (4G10; Upstate Biotechnology, Lake Placid, NY) was used at a dilution of 1/1000 for immunoblotting. Four micrograms of anti-HA mAb (12CA5; Roche, Indianapolis, IN) was used for immunoprecipitations. Ten micrograms of soluble monoclonal anti-mouse CD3 (145–2C11; PharMingen, San Diego, CA) was used for stimulations.
Cell culture and transient transfections
COS1 cells were maintained in DMEM supplemented with 10% (v/v) FBS, 200 mM L-glutamine, 5 U/ml penicillin C, and 5 mg/ml streptomycin sulfate. Ten micrograms of wild-type or proline-deficient HPK1 plasmids were transfected into a 100-mm dish of 50% confluent COS1 cells with lipofectin reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions, and cell lysates were prepared for in vitro binding experiments as detailed below. The murine T cell hybridoma, DO11.10, was maintained in RPMI 1640 supplemented with 10% FBS (v/v), 200 mM L-glutamine, 5 U/ml penicillin C, 5 mg/ml streptomycin sulfate, and 55 µM 2-ME. For electroporations, 20 million cells were resuspended in 400 µl of RPMI 1640 and electroporated with a Gene Pulser (Bio-Rad, Hercules, CA) set at 250 V and 960 µF. Optimal expression of transfected constructs was obtained with 15 µg of pcDNA3-HPK1:HA and 45 µg of pEF-FLAG-Gads constructs. Primary murine T cells were isolated from dissected lymph nodes and maintained in RPMI for 4 h before stimulation with anti-CD3.
In vitro binding experiments
GST-Gads wild-type and mutant fusion proteins were prepared as previously described (20), and purified proteins were quantified by Coomassie staining. Wild-type or proline mutant pcDNA3-HPK1:HA-transfected COS1 cells were lysed in 1 ml of PLC lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM NaPPi, 100 mM NaF, 1 mM Na3VO4, and Complete protease inhibitors (Roche)), and the cellular lysates were clarified by centrifugation at 14,000 rpm for 10 min at 4°C. Five hundred microliters of lysate was mixed with 2 µg of GST and either wild-type GST-Gads or GST-Gads mutants fusion protein conjugated to glutathione-Sepharose beads for 1 h at 4°C. The beads were washed five times with Nonidet P-40 lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM EDTA, 100 mM ZnCl2, 1% (v/v) Nonidet-P40, 10% (v/v) glycerol, and Complete protease inhibitors (Roche)) and 1 mM DTT, resuspended in SDS-Laemmli sample buffer, resolved on a 10% SDS-PAGE gel (NOVEX, San Diego, CA), and electrophoretically transferred to an Immobilon-P membrane (Millipore, Bedford, MA). The membranes were then immunoblotted with anti-HPK1 7 antiserum.
Immunoprecipitations and Western blotting analysis
DO11.10 or lymph node-derived T cells were prewarmed at 37°C for 10 min, left unstimulated, or stimulated with soluble anti-CD3 Ab for 2 min at 37°C, followed by the addition of 700 µl of an ice-cold phosphatase inhibitor mix (100 mM NaF, 10 mM NaPPi, and 1 mM Na3VO4 in PBS, pH 7.4). Cells were collected by centrifugation and lysed in 1 ml of PLC lysis buffer. The clarified lysates were incubated with the indicated Ab and 50 µl of a 20% (v/v) protein A-Sepharose bead slurry (Sigma) for 90 min at 4°C. The immune complexes were washed five times with Nonidet P-40 lysis buffer, eluted in SDS-Laemmli sample buffer, and separated on a 10% SDS-PAGE gel. Proteins were electrophoretically transferred to an Immobilon-P membrane and incubated in BLOTTO (5% nonfat milk powder (w/v) in TBST) or 1% (w/v) BSA in TBST (for anti-phosphotyrosine blotting), for 30 min before the addition of primary Ab for 1 h at room temperature. The membranes were washed three times for 10 min each time in TBST and were incubated at room temperature for 1 h with the appropriate secondary Ab conjugated to HRP. Membranes were washed three times in TBST and developed using enhanced chemiluminescence (Amersham-Pharmacia Biotech, Piscataway, NJ). Where necessary, the membranes were stripped (according to the manufacturer’s instructions) and reblotted.
In vitro kinase assays
DO11.10 cells or lymph node-derived T cells (5 x
106 cells/sample) were left unstimulated or were
stimulated with soluble anti-CD3 Ab for 5 min at 37°C and lysed,
and anti-HPK1 immunoprecipitates were performed as outlined above.
The immunoprecipitates were washed three times with 140 mM NaCl, 50 mM
Tris-HCl (pH 8.0), 5 mM EDTA, and 1% Nonidet P-40 (v/v), twice with
140 mM NaCl, 50 mM Tris-HCl (pH 8.0), and 5 mM EDTA, and once
with 50 mM Tris-HCl (pH 7.5), 8 mM MgCl2, 2 mM
MnCl2, and 1 mM DTT, and kinase reactions were
performed in 20 µl of 50 mM Tris-HCl (pH 7.5), 10 mM
MgCl2, and 1 mM EDTA at 30°C for 15 min in the
presence of 10 µCi of [-32P]ATP and 20
µg of myelin basic protein (MBP) as outlined previously
(41). The kinase reactions were stopped by adding 2x
SDS-Laemmli sample buffer and boiled for 5 min, and proteins were
resolved by SDS-PAGE. The proteins were electrophoretically transferred
to Immobilon-P membrane, and the incorporated radiolabeled phosphate
was assessed by PhosphorImager analysis using the ImageQuant program
(Molecular Dynamics, Sunnyvale, CA). To confirm that equivalent levels
of HPK1 were present in each kinase assay, the membrane was
subsequently immunoblotted with anti-HPK1 antisera as described
above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify Gads-binding proteins, radiolabeled GST-Gads fusion protein was used to screen an embryonic day 16 mouse expression library. A 2.6-kb cDNA clone was isolated from this screen that encoded the full-length murine hemopoietic progenitor kinase-1 (HPK1, GenBank accession no. Y09010).
Since both Gads and HPK1 proteins are expressed in T cells, (22, 41, 45), we examined whether Gads binds to HPK1 in the DO11.10
murine T cell hybridoma. DO11.10 cells were left unstimulated or were
stimulated with anti-CD3 Ab for 2 min, lysates were prepared, and
immunoprecipitations were performed with R
M IgG, anti-Gads, and
anti-HPK1 Abs. Anti-phosphotyrosine immunoblotting revealed the
presence of a tyrosine-phosphorylated protein of 
100 kDa in the
anti-HPK1 immunoprecipitate following TCR activation (Fig. 1
). Tyrosine-phosphorylated SLP-76 (76
kDa) and LAT (36 kDa), and weaker tyrosine-phosphorylated proteins of
100, 130, and 180 kDa were detected in anti-Gads
immunoprecipitates from anti-CD3-stimulated DO11.10 lysates. The
membranes were stripped and reprobed with anti-HPK1 Ab, identifying
the 100-kDa phosphoprotein in both anti-HPK1 and anti-Gads
immunoprecipitates as HPK1 and demonstrating that HPK1 is inducibly
tyrosine-phosphorylated and associated with Gads in vivo following TCR
ligation (Fig. 1). Following prolonged exposure of the blot to film,
only a very small amount of HPK1 was detected in Gads
immunoprecipitates before stimulation (data not shown). The identities
of the 130- and 180-kDa phosphoproteins present in the anti-Gads
immunoprecipitate from anti-CD3-stimulated lysate are unknown at
this time. Gads was not detected in anti-HPK1 immunoprecipitates,
suggesting that the anti-HPK1 Abs do not recognize HPK1 when
complexed with Gads. HPK1 was also detected in Gads immunoprecipitates
from primary lymph node-derived murine T cells following anti-CD3
stimulation (data not shown).
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It has been previously reported that activation of the
TGF-ß and erythropoietin receptors leads to an increase in HPK1
kinase activity (46, 47). In addition, Ling et al. have
previously provided evidence that HPK1 may be involved in regulating
IL-2 activation in Jurkat T cells (48). To determine
whether HPK1 kinase activity is regulated by TCR engagement, in vitro
kinase assays were performed on anti-HPK1 immunoprecipitates from
unstimulated and anti-CD3-stimulated DO11.10 hybridoma and primary
lymph node-derived T cells, using MBP as a substrate (Fig. 2A). The level of
phosphorylated MBP increased by 2-fold, on the average, following TCR
activation in both DO11.10 and lymph node-derived T cells, indicating
that HPK1 kinase activity is up-regulated by TCR activation.
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P1/2/4; Fig. 4A). The third (P3; aa
430–441) motif was not included in the deletion because of its poor
fit to the SH3 domain-binding consensus motif, and since Anafi et al.
(49) previously demonstrated that a P3 peptide was unable
to compete recombinant SH3 domain binding to HPK1. HA-epitope
tagged wild-type, kinase-dead, and P1/2/4 HPK1 constructs were
transiently expressed in DO11.10 cells, the cells were left
unstimulated or were stimulated with anti-CD3 and lysed, and
anti-HA immunoprecipitation was performed, followed by kinase
assays using MBP as substrate. Following TCR activation, an 2-fold
increase in HPK1 kinase activity was observed with the wild-type HPK1,
while a marginal increase was observed for the kinase-dead HPK1 (Fig. 2
B). The induced kinase activity of the P1/2/4 HPK1
mutant was markedly reduced following TCR ligation compared with that
of wild-type HPK1. The defect in activation of P1/2/4 HPK1 in vivo
is probably due to its inability to couple to the activated TCR rather
than to a structural defect that affects the kinase domain, since the
basal in vitro kinase activity of this mutant is equivalent to that of
wild-type HPK1.5
Therefore, the proline-rich regions of HPK1 and probably SH3
domain-containing adaptors are important in coupling regulation of HPK1
activity to TCR activation.
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To map the domain of Gads responsible for interacting with HPK1,
recombinant full-length GST-Gads fusion proteins with inactivated SH3
or SH2 domains were assayed for their ability to precipitate HPK1 from
transfected COS1 cells. Inactivation of both SH3 domains (N*/C*) or
inactivation of the carboxyl-terminal SH3 domain (N/C*) eliminated the
interaction between Gads and HPK1, whereas mutation of the SH2 (SH2*)
domain had no effect on the ability of Gads to precipitate HPK1 in
vitro (Fig. 3). Similar experiments
performed in DO11.10 cells also show that the carboxyl-terminal SH3
domain is required for binding to endogenous HPK1 following activation
of the TCR (data not shown).
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A). The binding of GST-Gads
fusion protein to HPK1 mutants that possessed individual or combined
deletions of the first, second, and fourth proline-rich regions was
assayed; the third proline-rich region was not removed due to reasons
described above (Fig. 4B). Deletion of the first or second
proline-rich region of HPK1, either individually or together, did not
eliminate the Gads-HPK1 interaction. However, all HPK1 mutants that
lacked the fourth proline-rich region of HPK1 were no longer
precipitated by GST-Gads, suggesting that the major Gads binding site
on HPK1 is within the fourth proline-rich region (Fig. 4B).
Deletion of the second proline-rich region reduced binding to HPK1,
whereas loss of the fourth proline-rich region abolished the
interaction. Together, these results suggest that the while the fourth
proline-rich region is critical for association between Gads and HPK1,
the second proline-rich region may partially contribute to the
interaction. To confirm the role of the Gads SH3 domains and the HPK1
proline-rich regions in the Gads-HPK1 interaction in vivo, full-length
epitope-tagged Gads and HPK1 cDNAs with mutations in the domains
important for the interaction were cotransfected into DO11.10 cells.
Wild-type HA-HPK1 was efficiently coimmunoprecipitated with FLAG-Gads;
however, deletion of all three proline-rich regions (P1/P2/P4) or the
P4 region alone resulted in loss of HPK1-Gads association (Fig. 5A, left panel). An
inactivating mutation in the Gads SH2 domain (Gads-SH2*) had no effect
on the ability of FLAG-Gads to coprecipitate HA-HPK1, while mutation of
the SH3 domains of Gads (Gads-N*/C*) abolished the interaction (Fig. 5A, right panel). Equivalent expression of the
HA-HPK1 and FLAG-Gads proteins was confirmed by Western blots of whole
cell lysates with anti-HA and anti-FLAG Abs (Fig. 5B).
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Recruitment of HPK1 to the proximity of activated receptors by
SH2/SH3 domain-containing adaptor proteins has been shown to facilitate
the subsequent tyrosine phosphorylation of HPK1 in response to
activation of the epidermal growth factor and erythropoietin receptors
(47, 49). To test whether the Gads-binding region, P4, is
required for HPK1 tyrosine phosphorylation following TCR activation, we
transiently expressed HPK1 mutants lacking the various
proline-rich regions in DO11.10 cells and examined the tyrosine
phosphorylation status of these HPK1 mutants following anti-CD3
stimulation (Fig. 6). Loss of the second
or fourth proline-rich region in HPK1 led to a substantial decrease in
the tyrosine-phosphorylation status of HPK1 following TCR ligation,
while loss of the first proline-rich region had no effect. Together,
these data suggest that the second and fourth proline-rich regions are
important for the efficient tyrosine phosphorylation of HPK1.
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Since a Gads-HPK1 complex was detected in anti-CD3-stimulated
DO11.10 cells, we examined the effect of overexpression of Gads mutant
proteins on the tyrosine phosphorylation of HPK1 following TCR
activation. Gads mutant proteins that possessed inactivated SH3 or SH2
domains were transiently coexpressed with HA-tagged HPK1 in DO11.10
cells, and the relative tyrosine phosphorylation level of HPK1
following anti-CD3 stimulation was assessed (Fig. 7). Expression of wild-type Gads or
mutant Gads with inactivated SH3 domains (N*/C*) did not alter the
tyrosine phosphorylation level of HPK1 relative to the control level.
However, the expression of mutant Gads with an inactivated SH2 domain
(SH2*) appreciably reduced tyrosine phosphorylation of HPK1, suggesting
that Gads may be directly involved in coupling HPK1 to tyrosine kinases
that are activated by the TCR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Substantial research focused on elucidating the pathways downstream of HPK1 has led to a common model that HPK1 initiates a kinase cascade involving several intermediates including mixed lineage kinase 3 (41), MEKK1 (42), and TGF-ß-activated kinase 1 (50), which can all phosphorylate and activate SAPK/ERK kinase 1 leading to the activation of SAPK. In contrast, less is known about signaling events upstream of HPK1 activation. Several groups have described the association of SH2/SH3 domain-containing adaptor proteins, including Crk, CrkL, and Grb2 with HPK1, and have implicated these adaptors in connecting HPK1 to activated receptors (45, 48, 49). Additionally, it has been previously demonstrated that expression of the HPK1 proline-rich regions in Jurkat T cells prevents association with Crk and Grb2 adaptor proteins and disrupts IL-2 activation (48). We have found that the Gads adaptor protein inducibly associates with HPK1 in T cells, and that HPK1 kinase activity is regulated by TCR activation, which is dependent on the presence of its proline-rich regions for efficient activation. Taken together, this supports a model for HPK1 kinase regulation in T cells, where SH2/SH3 domain-containing adaptor proteins such as Grb2, Crk, CrkL, and Gads are able to recruit HPK1 to the proximity of the TCR, thus facilitating subsequent activation of the HPK1 kinase. These adaptor proteins probably provide a means of coupling the activated TCR complex to kinase cascades downstream of HPK1, which may ultimately regulate the induction of a downstream effector response.
HPK1 has been previously identified as a substrate for tyrosine kinases. In experiments using ectopically expressed HPK1 in COS1 cells, the activated epidermal growth factor receptor, platelet-derived growth factor receptor, and constitutively activated cytoplasmic tyrosine kinases v-Src and v-Fps induce the tyrosine phosphorylation of HPK1 (49). Our studies provide the first report that endogenous HPK1 is tyrosine phosphorylated following TCR activation. It is not known whether tyrosine phosphorylation of HPK1 directly regulates its kinase activity, although based upon the small proportion of tyrosine-phosphorylated HPK1 observed in anti-HPK1 immunoprecipitates from TCR-activated cells, this would appear unlikely. At this time, the functional significance of HPK1 tyrosine phosphorylation, as observed in response to a wide variety of activated receptors and cytoplasmic tyrosine kinases, remains elusive.
Within the proline-rich region, murine HPK1 harbors three consensus SH3 domain binding sites, and human HPK1 potentially has four. One of the motifs in mHPK1 (P2) and two in human HPK1 conform with the binding consensus for Crk proteins, PxLPxK (where x is any amino acid) (41, 45, 49). The remaining two motifs in mHPK1 (P1, P4) have arginine at the P-3 specificity position, which is usually preferred by Grb2 SH3 domains (41, 49). We have demonstrated that the Gads carboxyl-terminal SH3 domain preferentially interacts with the P4 motif. Furthermore, our results indicate that P2 and P4 are required for the efficient tyrosine phosphorylation of HPK1, supporting a model where tyrosine phosphorylation of HPK1 is dependent on the recruitment of HPK1 to the proximity of activated tyrosine kinases via SH2/SH3 domain-containing adaptor proteins. This model is strengthened by our observations that coexpression of a Gads SH2 mutant also attenuated the tyrosine phosphorylation of HPK1. This effect might result from the ability of this mutant to effectively bind HPK1 through an intact SH3 domain, while sequestering it from the proximity of the activated TCR by preventing association with proteins such as LAT. In contrast, the Gads SH3 mutant was found to have only a marginal effect on HPK1 tyrosine phosphorylation. This is not entirely unexpected, since the Gads SH3 mutant can no longer interact with HPK1. Tyrosine phosphorylation of HPK1 is not completely abolished in the presence of the Gads SH2 mutant, suggesting that other SH2/SH3 domain-containing adaptor proteins such as Crk, CrkL, and Grb2, probably serve redundant roles in recruiting of HPK1 to the proximity of the activated TCR complex. This redundancy might also be perceived as a mechanism for amplifying the level of HPK1 kinase activation.
Although the interaction of Gads and HPK1 is SH3 domain mediated, we have found that in vivo the association in DO11.1 is not constitutive, but is induced following TCR activation. The inducible nature of the Gads-HPK1 interaction is analogous to the interactions reported for the Grb2 and Grap adaptor proteins with Sos and for Grap with dynamin in activated T cells (13, 19, 51). In keeping with those reports, our results suggest that certain events must be initiated by the TCR for the Gads-HPK1 interaction to occur. For example, phosphorylation of HPK1 or engagement of the Gads SH2 domain could induce a conformational change resulting in an increased accessibility of the Gads SH3 domain to the fourth proline-rich region of HPK1. In support of the latter, Ravichandran et al. have shown that binding of the Grb2 SH2 domain to a Shc-derived phosphopeptide significantly enhances the Grb2-Sos interaction (51). Alternatively, TCR activation may result in the relocalization of proteins that facilitates their interaction. The inducible association of Grb2-family members with Sos, dynamin, and HPK1 suggests that the formation of these signaling complexes is highly regulated within T cells.
We have previously described a potential role for Gads in coupling TCR activation to the distal activation of NF-AT and IL-2 through its interactions with the SLP-76 and LAT adaptor proteins (22). We now propose that Gads also functions in an additional pathway downstream of the activated TCR that regulates the kinase activity and tyrosine phosphorylation of HPK1.
Note:
While this manuscript was in revision, Liou et al. (54) published similar results demonstrating that HPK1 is activated by the TCR.
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Acknowledgments |
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Footnotes |
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2 S.K.L. and C.A.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. C. Jane McGlade, The Arthur and Sonia Labatt Brain tumor Research Center, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8.
4 Abbreviations used in this paper: SH, Src homology; PLC, phospholipase C; Gads, Grb2-related adaptor downstream of Shc; Grap, Grb2-related adaptor protein; SAPK, stress-activated protein kinase; GCK, germinal center kinase; GLK, GCK-like kinase; GCKR/KHS, GCK-related kinase/kinase homologous to SPS1/Ste20; SLP-76, SH2 domain-containing leukocyte protein of 76 kDa; LAT, linker of activated T cells; HPK1, hemopoietic progenitor kinase 1; mHPK1, murine HPK1; pY, phosphotyrosine; HA, hemagglutinin; PVDF, polyvinylidene difluoride; MBP, myelin basic protein.
5 R. Arnold and F. Kiefer. Hematopoietic progenitor kinase 1 (HPK1) activates NF-
B and promotes apoptosis after growth factor withdrawal. Submitted for publication.
Received for publication January 28, 2000. Accepted for publication May 19, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1. Proc. Natl. Acad. Sci. USA 88:5484.1, and other phosphoproteins with Grb2 and the 
-chain of the TCR. J. Biol. Chem. 270:18428.B, by the tumor necrosis factor (TNF) receptor-associated factor 2- and germinal center kinase related-dependent pathway. J. Biol. Chem. 272:32102.This article has been cited by other articles:
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  D. Brenner, M. Brechmann, S. Rohling, M. Tapernoux, T. Mock, D. Winter, W. D. Lehmann, F. Kiefer, M. Thome, P. H. Krammer, et al. Phosphorylation of CARMA1 by HPK1 is critical for NF-{kappa}B activation in T cells PNAS, August 25, 2009; 106(34): 14508 - 14513. [Abstract] [Full Text] [PDF]
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 H. Wang, X. Song, C. Logsdon, G. Zhou, D. B. Evans, J. L. Abbruzzese, S. R. Hamilton, T.-H. Tan, and H. Wang Proteasome-Mediated Degradation and Functions of Hematopoietic Progenitor Kinase 1 in Pancreatic Cancer Cancer Res., February 1, 2009; 69(3): 1063 - 1070. [Abstract] [Full Text] [PDF]
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 D. Brenner, A. Golks, M. Becker, W. Muller, C. R. Frey, R. Novak, D. Melamed, F. Kiefer, P. H. Krammer, and R. Arnold Caspase-cleaved HPK1 induces CD95L-independent activation-induced cell death in T and B lymphocytes Blood, December 1, 2007; 110(12): 3968 - 3977. [Abstract] [Full Text] [PDF]
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 S. Sawasdikosol, S. Pyarajan, S. Alzabin, G. Matejovic, and S. J. Burakoff Prostaglandin E2 Activates HPK1 Kinase Activity via a PKA-dependent Pathway J. Biol. Chem., November 30, 2007; 282(48): 34693 - 34699. [Abstract] [Full Text] [PDF]
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 S. C. Bunnell, A. L. Singer, D. I. Hong, B. H. Jacque, M. S. Jordan, M.-C. Seminario, V. A. Barr, G. A. Koretzky, and L. E. Samelson Persistence of cooperatively stabilized signaling clusters drives T-cell activation. Mol. Cell. Biol., October 1, 2006; 26(19): 7155 - 7166. [Abstract] [Full Text] [PDF]
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R. Chen, S. Latour, X. Shi, and A. Veillette Association between SAP and FynT: Inducible SH3 Domain-Mediated Interaction Controlled by Engagement of the SLAM Receptor Mol. Cell. Biol., August 1, 2006; 26(15): 5559 - 5568. [Abstract] [Full Text] [PDF]
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R. Arnold, I. M. Patzak, B. Neuhaus, S. Vancauwenbergh, A. Veillette, J. Van Lint, and F. Kiefer Activation of Hematopoietic Progenitor Kinase 1 Involves Relocation, Autophosphorylation, and Transphosphorylation by Protein Kinase D1 Mol. Cell. Biol., March 15, 2005; 25(6): 2364 - 2383. [Abstract] [Full Text] [PDF]
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G. Zhou, J. S. Boomer, and T.-H. Tan Protein Phosphatase 4 Is a Positive Regulator of Hematopoietic Progenitor Kinase 1 J. Biol. Chem., November 19, 2004; 279(47): 49551 - 49561. [Abstract] [Full Text] [PDF]
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M. Lewitzky, M. Harkiolaki, M.-C. Domart, E. Y. Jones, and S. M. Feller Mona/Gads SH3C Binding to Hematopoietic Progenitor Kinase 1 (HPK1) Combines an Atypical SH3 Binding Motif, R/KXXK, with a Classical PXXP Motif Embedded in a Polyproline Type II (PPII) Helix J. Biol. Chem., July 2, 2004; 279(27): 28724 - 28732. [Abstract] [Full Text] [PDF]
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S. Yamasaki, K. Nishida, M. Sakuma, D. Berry, C. J. McGlade, T. Hirano, and T. Saito Gads/Grb2-Mediated Association with LAT Is Critical for the Inhibitory Function of Gab2 in T Cells Mol. Cell. Biol., April 1, 2003; 23(7): 2515 - 2529. [Abstract] [Full Text] [PDF]
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J. Schulze-Luehrmann, B. Santner-Nanan, M. K. Jha, A. Schimpl, A. Avots, and E. Serfling Hematopoietic progenitor kinase 1 supports apoptosis of T lymphocytes Blood, July 18, 2002; 100(3): 954 - 960. [Abstract] [Full Text] [PDF]
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C. Bourgin, R. P. Bourette, S. Arnaud, Y. Liu, L. R. Rohrschneider, and G. Mouchiroud Induced Expression and Association of the Mona/Gads Adapter and Gab3 Scaffolding Protein during Monocyte/Macrophage Differentiation Mol. Cell. Biol., June 1, 2002; 22(11): 3744 - 3756. [Abstract] [Full Text] [PDF]
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 S. F. Hussain, C. F. Anderson, and D. L. Farber Differential SLP-76 Expression and TCR-Mediated Signaling in Effector and Memory CD4 T Cells J. Immunol., February 15, 2002; 168(4): 1557 - 1565. [Abstract] [Full Text] [PDF]
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J. Yu, C. Riou, D. Davidson, R. Minhas, J. D. Robson, M. Julius, R. Arnold, F. Kiefer, and A. Veillette Synergistic Regulation of Immunoreceptor Signaling by SLP-76-Related Adaptor Clnk and Serine/Threonine Protein Kinase HPK-1 Mol. Cell. Biol., September 15, 2001; 21(18): 6102 - 6112. [Abstract] [Full Text] [PDF]
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 S. Tsuji, M. Okamoto, K. Yamada, N. Okamoto, R. Goitsuka, R. Arnold, F. Kiefer, and D. Kitamura B Cell Adaptor Containing Src Homology 2 Domain (Bash) Links B Cell Receptor Signaling to the Activation of Hematopoietic Progenitor Kinase 1 J. Exp. Med., August 20, 2001; 194(4): 529 - 540. [Abstract] [Full Text] [PDF]
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R. Arnold, J. Liou, H. C. A. Drexler, A. Weiss, and F. Kiefer Caspase-mediated Cleavage of Hematopoietic Progenitor Kinase 1 (HPK1) Converts an Activator of NFkappa B into an Inhibitor of NFkappa B J. Biol. Chem., April 27, 2001; 276(18): 14675 - 14684. [Abstract] [Full Text] [PDF]
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P. Ling, C. F. Meyer, L. P. Redmond, J.-W. Shui, B. Davis, R. R. Rich, M. C.-T. Hu, R. L. Wange, and T.-H. Tan Involvement of Hematopoietic Progenitor Kinase 1 in T Cell Receptor Signaling J. Biol. Chem., May 25, 2001; 276(22): 18908 - 18914. [Abstract] [Full Text] [PDF]
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K. Sauer, J. Liou, S. B. Singh, D. Yablonski, A. Weiss, and R. M. Perlmutter Hematopoietic Progenitor Kinase 1 Associates Physically and Functionally with the Adaptor Proteins B Cell Linker Protein and SLP-76 in Lymphocytes J. Biol. Chem., November 21, 2001; 276(48): 45207 - 45216. [Abstract] [Full Text] [PDF]
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