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Prepublished online as a Blood First Edition Paper on April 30, 2002; DOI 10.1182/blood-2002-01-0089.
IMMUNOBIOLOGY
From the Department of Molecular Pathology, Institute
of Pathology, Institute of Virology and Immunobiology, and Molecular
Epigenetics Group, Institute of Medical Radiation and Cell Research
(MSZ), D-97080 Wuerzburg, Germany.
Hematopoietic progenitor kinase 1 (HPK1) is a member of germinal
center kinases that is predominantly expressed in hematopoietic cells
and transiently activated by T-cell receptor (TCR) triggering. We show
here that HPK1 supports apoptosis of T cells. When HPK1 was
overexpressed in murine CD4+ T cells, a substantial
increase was observed in spontaneous and TCR/CD3-mediated apoptosis as
well as in Fas ligand (FasL) expression. In
H2O2-treated EL-4 thymoma cells, which
show an increase in reactive oxygen species (ROS) and apoptosis,
overexpression of HPK1 enhanced ROS-mediated apoptosis, whereas
expression of HPK1 antisense (AS) RNA impaired apoptosis. HPK1
expression also led to a sustained increase in c-Jun N-terminal kinase
(JNK) activity, suggesting that JNK activation contributes to the
HPK1-mediated apoptosis in H2O2-treated EL-4
cells. Under the same conditions, a rapid cleavage of HPK1 was
observed, and overexpression of N- and C-terminal cleavage products in
CD4+ T cells resulted in, similar to full-length HPK1, an
increase in apoptosis. In agreement with published data, we show that
the C-terminal portion of HPK1 suppresses I Activation of peripheral T-effector cells by
presented antigens can result in diverging effects for the affected
cell. Depending on the "strength" of interaction between the
antigen/major histocompatibility complex (MHC) and the T-cell
receptor (TCR) complex and the involvement of coreceptors, a resting T
cell can be stimulated to produce lymphokines and to proliferate, to
become anergic, or to die by apoptosis. Activation Induced Cell Death
(AICD) is a particular form of apoptosis that is of pivotal importance
for the termination of the immune response and, therefore,
homeostasis of the immune system. One important pathway of AICD
in lymphocytes is the activation of caspase cascade through
so-called death receptors, that is, a subgroup of the tumor
necrosis factor (TNF)/nerve growth factor (NGF)-receptor
family that comprise Fas, TNF-receptor I, TNF-related apoptosis-inducing ligand (TRAIL), and further
receptors.1 Their stimulation leads to the activation of
caspase cascade and, in turn, to cleavage of death substrates and,
finally, to apoptosis of T cells.
However, the activation of caspase cascade through death receptors is
not the only mechanism by which AICD of lymphocytes is executed. Thus,
superantigen (SAg) injection into triple-mutant mice deficient in Fas
and TNF-receptor I + II expression led to rapid AICD of
V In spite of numerous studies on apoptosis of lymphocytes, relatively
little is known about lymphoid-specific signaling molecules and
pathways involved in AICD induced by TCR triggering or other mechanisms. The observation that TCR/CD3 stimulation of Jurkat T cells,
which induces AICD, also induces activity of HPK1, whereas CD28
stimulation, which provides a survival signal for T cells, did not show
any effect on HPK1 activity6,7 prompted us to investigate
whether HPK1 regulates apoptosis of T cells. We show here that
overexpression of HPK1 accelerates both spontaneous and Cells, DNA transfections, reporter gene assays, and retroviral
infections
EL-4 cells stably transduced with recombinant retroviruses were
selected with zeocin (250 nM) for 7 days. HPK1 expression was tested in
Western blots, and enhanced green fluorescent protein (EGFP)
fluorescence was checked by a fluorescence-activated
cell-sorter scanner (FACS). DO11.10 TCR transgenic (tg) BALB/c
mice11 were killed between 6 and 8 weeks
of age. CD4+ T cells from lymph nodes (LNs) were isolated
by passing them through CD4 T-cell recovery columns (Cedarlane, Hornby,
Canada) according to the manufacturer's protocol. The cells were
cultured at 5 × 105/mL in X-VIVO15 (Bio
Whittaker) supplemented with 5% FCS, Gln, nonessential amino acids,
pyruvate (all 2 mM), antibiotics (penicillin, streptomycin), and
5 × 10-5 M 2-mercaptoethanol. Dendritic cells were
prepared from irradiated spleen and used as antigen-presenting cells
(APCs). CD4+ T cells were stimulated by the cognate
ovalbumin peptide (OVA)-peptide (323-339) presented on APCs
and cultured in the presence of interleukin (IL)-2 as
described.12 After 24 hours, they were infected with retroviruses as described previously.13
Induction and determination of apoptosis
Retroviral constructs The bicistronic retroviral vector pEGZ-HA is based on the vector pczCFG2 hCD8 EYZ in which the cytomegalovirus (CMV) enhancer replaces the U3 region of the 5' long terminal repeat (LTR) of murine leukemia virus (for further details, see Berberich-Siebelt et al13). An oligonucleotide was cloned into its EcoRI site, creating a start codon and HA-Flag epitope followed by ClaI, AscI, BsiWI, RsrlI, HpaI, and SacII sites, which facilitate "in frame" cloning of cDNAs. The retroviral vector expressing wild-type (wt) HPK1, pHA-HPK1 wt contains the cDNA (amino acids 2-833) of human HPK1,15 which was amplified by polymerase chain reaction (PCR) and cloned between the ClaI/HpaI sites of pEGZ-HA. A mutated version of HPK1, pHA-HPK1 M(46) containing a K46 to M46 substitution15 was used to test a kinase-inactive version of HPK1. The pHA-HPK1 wt N-terminus (encoding amino acid [aa] 2-385) and C-terminus constructs (aa 386-833) were created by cloning PCR products into pEGZ-HA. In addition, a kinase-inactive (K46 to M46) version of pHA-HPK1 wt N-terminus was also constructed. The retroviral AS vector, pHA-HPK1 AS, bears the sequences 386-2 between its ClaI/HpaI sites. All DNA modifying enzymes were purchased from MBI Fermentas (St Leon-Rot, Germany).Antibodies and Western blot assays Antibodies (Abs) raised against HPK1 (N-19; sc-6231), phospho-Tyr (PY99; sc-7020), I B (C-21; sc-371), Erk1 (K23;
sc-94), JNK1 (C-17; sc-474), p-JNK (G-7; sc-6254), and p38 (C-20;
sc-535) were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Abs detecting phospho-Erk1/2 (Thr202/Tyr204) or
phospho-p38 (Thr180/Tyr182) were purchased from Cell Signaling
Technology (Beverly, MA), a mAb against  -actin (AC-15) was
obtained from Sigma-Aldrich (Taufkirchen, Germany). A
biotin-conjugated mAb against murine FasL was obtained from Alexis
Corporation (Grünberg, Germany), and PE-conjugated streptavidin from PharMingen (Heidelberg, Germany). The mAb
HA.11 raised against HA tag was purchased from BAbCO (Richmond, VA).
In Western blots, an indicated amount of whole cellular protein lysate was fractionated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membrane. For detection of proteins, appropriate peroxidase-coupled secondary Abs were used with a standard enhanced chemiluminescence system (Amersham, Freiburg, Germany). Protein kinase assays For in vitro phosphorylation with immunoprecipitated protein kinases, 500 µg of whole cell protein extract from 293 HEK cells transfected with retroviral vectors was incubated with the HA.11 mAb. Immune complexes were collected onto protein G-sepharose beads and, after washing with immunoprecipitation (IP) buffer, used to phosphorylate 10 µg GST/ c-Jun5-89 in 30 µL kinase buffer containing 20 µM adenosine 5-triphosphate (ATP) and 5 µCi (1.8 × 105 Bq) of 32P- -ATP.16 After 20 minutes at 37°C,
proteins were fractionated by 12.5% SDS-PAGE followed by autoradiography.
HPK1 enhances CD3Abs), a proapoptotic stimulus, for 3 hours
leads to a more than 3-fold increase in HPK1 protein levels (Figure
1A). This finding and the observation
that CD3+CD28Abs but not CD28Ab stimulation alone enhanced HPK1
activity7 prompted us to investigate whether HPK1 plays a
physiological role in T-cell apoptosis. To this end, we transduced
primary murine CD4+ T cells with retroviruses expressing wt
HPK1 (pHA-HPK1 wt) or a kinase-inactive version of HPK1 bearing a K46
to M46 substitution (pHA-HPK1 M[46]).15 As
shown in Figure 1B, the viral vectors encoding HPK1 protein bear a
fusion gene consisting of an N-terminally tagged HA epitope and HPK1
cDNA, which is linked via an internal ribosomal entry site (IRES) to
EGFP and zeocin gene (Zeo). Therefore, HPK1, the EGFP
fluorescence marker and zeocin resistance genes are coordinately
expressed. In control experiments, transfection of 293T cells with
constructs encoding the wt but not the kinase-inactive HPK1 mutant led
in in vitro kinase assays to the autophosphorylation of HPK1 and
phosphorylation of GST-cJun5-89, which was
added as an exogenous substrate (Figure 1C).
CD4+ T cells from DO 11.10 TCR tg mice were infected with
HPK1-expressing retroviruses 1 day after primary stimulation, and 4 days later spontaneous and HPK1 enhances apoptosis induction of EL-4 thymoma cells by ROS In order to elucidate molecular mechanisms of HPK1-mediated apoptosis, we established lines of retrovirally infected EL-4 thymoma cells overexpressing different versions of HPK1 protein. These were treated with H2O2, which enhances the levels of free ROS that are important mediators of apoptosis in T cells.2 Treatment of EL-4 cells with 1 mM H2O2 for 5 to 120 minutes increased the level of endogenous HPK1 (Figure 2A), whereas those of other protein kinases, such as of Erk, JNK and p38 mitogen-activated protein (MAP) kinases, remained constant (Figure 3). In addition, H2O2 treatment led to an increase in level of N-terminal HPK1 peptide containing the HPK1 kinase domain (Figure 1B). This released N-terminal peptide was shown to exhibit an enhanced kinase activity compared to full-size HPK1.17
When EL-4 cells overexpressing HPK1 were treated with 1 mM H2O2, a 40% increase in apoptosis was detected after 8 hours compared to cells infected with the pEGZ-HA control vector (Figure 2B). While cells overexpressing the kinase-inactive HPK1 M(46) showed a moderate increase in apoptosis (of approximately 15%, data not shown), cells expressing HPK1 AS-RNA were markedly more resistant against H2O2-mediated apoptosis than control cells (Figure 2B). Since expression of HPK1 AS-RNA suppressed HPK1 levels (Figure 2C) one may conclude that a lower HPK1 level results in a decrease in H2O2-mediated apoptosis. Taken together, these results demonstrate that HPK1 expression and activity significantly influence ROS-mediated apoptosis of EL-4 cells, which represents an important pathway of AICD in T cells.2 HPK1 increases JNK kinase activation in
H2O2-treated EL-4 cells and B
activity in various cell types.8,15,18,19 To elucidate
which signaling pathways might be affected by HPK1 in EL-4 cells and primary CD4+ T cells, we studied first Tyr protein
phosphorylation and found that expression of wt HPK1 strongly enhanced
Tyr phosphorylation of numerous proteins in EL-4 cells treated with
H2O2 (results not shown). In addition,
H2O2 exerted a strong stimulatory effect on
JNK activation.
H2O2 treatment leads to a weak but measurable activation of JNK1, p38, and Erk1/Erk2 MAP kinases in EL-4 cells transduced with the control pEGZ-HA virus (Figure 3, panel pEGZ-HA). As expected, HPK1 expression exerted a strong and persistent enhancement of JNK1 activity (Figure 3, panel pHA-HPK1 wt). Albeit expression of mutant HPK1 M(46) kinase also enhanced JNK1 activity, that of wt HPK1 resulted in a pronounced longer-lasting JNK1 activation. This indicates that at least a part of JNK activation was due to HPK1 kinase activity and not to its adaptor function. Compared to JNK1 activation, a more moderate increase in p38 and Erk activation was observed in cells expressing HPK1. However, almost the same inducible effect was detected in cells expressing HPK1 M(46), suggesting that HPK1 activity plays a minor role for p38 and Erk induction. The increased activity of all 3 MAP kinases but not their expression could be suppressed by HPK1 AS-RNA (Figure 3, panel pHA-HPK1 AS), indicating that overexpression of HPK1 stimulates all 3 MAP kinase signaling cascades, although to a very different extent. A marked increase in JNK activation by HPK1 was also observed in
primary CD4+ T cells infected with a retrovirus expressing
HPK1. This is shown in Figure 4, where a
distinct, stronger JNK1 phosphorylation can be seen in untreated cells
and cells stimulated with
Both N- and C-terminal HPK1 peptides promote apoptosis of CD4+ T cells The rapid cleavage of HPK1 after H2O2 treatment in EL-4 cells (Figure 2A, middle panel) prompted us to investigate whether the cleavage products of HPK1 (presumably generated by caspase 38,17) support apoptosis of primary T cells. To this end, we constructed viruses expressing either the N-terminal cleavage product comprising aa 2-385 or the C-terminal product comprising aa 386-833 (Figure 5B) and infected CD4+ T cells after primary stimulation. As summarized in Figure 5A, similar to cells infected with HA-HPK1 wt virus, infection with viruses expressing one of the two cleavage products resulted in an increase of spontaneous apoptosis from approximately 20% to 30-33% and ofCD3-mediated apoptosis from
50.5% to 70-75% 3 days after primary stimulation. Interestingly,
expressing a kinase-inactive version of N-terminal cleavage product
(pH-HPK1 M(46) N-terminus) was without effect on apoptosis. In
addition, infection with virus-expressing HPK1 AS-RNA impaired slightly
spontaneous and CD3-mediated apoptosis (Figure 5A). These data
demonstrate that in HPK1-promoted apoptosis, both the N- and C-terminal
portions of HPK1 are involved.
HPK1 modulates NF- B activation under
the conditions of apoptosis induction in EL-4 and primary T cells, we
cotransfected first EL-4 cells with a luciferase reporter gene driven
by multiples of NF-B binding sites from the c-myb promoter, with
vectors expressing HPK1 wild-type or HPK1 C-terminal peptide. Treatment
of HPK1 wt-transfected cells with 500 µM
H2O2 led to a 3- to 4-fold increase in
NF-B-driven luciferase activity compared to cells transfected with
a control "empty" vector. In contrast, a 2- to 3-fold decrease in
NF-B activity was observed for cells transfected with a HPK1
C-terminus-expressing construct (Figure
6A). This shows that HPK1 wt protein
exerts a stimulatory and HPK1 C-terminus peptide a suppressive effect
on NF-B activation.
A suppressive effect of HPK1 C-terminus peptide on NF- HPK1 enhances FasL expression of primary T lymphocytes FasL is a key molecule in AICD of peripheral T cells. Therefore, we also investigated if HPK1 can modulate the expression level of FasL. After 3 days of infection with pEGZ-HA control virus, approximately 20% of primary T cells expressed FasL, whereas approximately 40% of cells infected with HPK1 virus were found to express FasL (Figure 7). Induction of both cell types through CD3 led to an increase in number of FasL-expressing cells to approximately 40% and 60%, respectively. HPK1 AS-RNA, on the other hand, only slightly affected FasL expression. These findings show that HPK1 can enhance FasL expression, but mechanisms in addition to the FasL/Fas signaling cascade are involved in HPK1-mediated apoptosis of primary T cells.
HPK1 is a member of the family of germinal center kinases that is predominantly expressed in adult hematopoietic tissues. It has originally been described as an upstream Ser/Thr protein kinase of the JNK signaling cascade.15,18 Recently, it was shown that TCR stimulation leads to a rapid but transient activation of HPK1 and its binding to the linker of activated T cells (LAT) and the adaptor proteins Nck, Crk, and Gads. HPK1, which also binds to the signaling molecules SLP-76, Grb2, Grap, and CrkL is localized in lipid rafts and needs lck and ZAP70 for its activation.6,7,20 These data suggested an involvement of HPK1 in the signaling transfer from the TCR to downstream signaling cascades. However, the role for HPK1 in T-cell physiology remained to be shown. We show here that one of the functions of HPK1 activation upon T-cell
stimulation is to control the termination of immune response by
supporting apoptosis of T cells. This is demonstrated by overexpressing
wt HPK1, which led to a substantial increase in spontaneous and
Two prominent downstream signaling cascades, the JNK and NF- H2O2 treatment exerted a strong and, in particular, sustained JNK1 activation in EL-4 cells infected with HPK1-expressing virus (Figure 3). In primary murine embryo fibroblasts deficient for JNK1 and 2, the defect in inducible apoptosis was localized to the mitochondria.26 Therefore, one may assume that effector molecules involved in mitochondrial apoptosis resistance, such as Bcl-2, are the targets of enhanced JNK activity in EL-4 cells. Both Bcl-2 and Bcl-XL, a further member of antiapoptotic group of Bcl-2-like proteins, were described to be phosphorylated and inactivated by JNKs in vivo.27,28 In spite of disagreement with other studies, the Bcl-2-like proteins, including the BH3-only protein Bim and its relatives, appear to be potential targets of JNK in regulating mitochondrial apoptosis induction (see Davis29 for a discussion). Independent of JNK activation, HPK1 is able to induce NF- Taken together, these results lead to a model in which, during the
initial phase of T-cell activation, the rapid and transient HPK1
induction provides both proapoptotic and antiapoptotic signals by
activating JNK and NF-
We are indebted to Christa Kraus, Doris Michel, Ilona Pietrowski, and Olga Reimer for excellent technical assistance. We thank Drs Ingolf Berberich, Stefan M. Feller, and Friedemann Kiefer for gifts of materials and support; and Rajesh Kumar Singh and Stefan Klein-Hessling for critically reading the manuscript.
Submitted November 28, 2001; accepted April 2, 2002.
Prepublished online as Blood First Edition Paper, April 30, 2002; DOI 10.1182/blood-2002-01-0089.
Supported by the W.-Sander-Stiftung (E.S.), the Österreichische Akademie der Wissenschaften (providing a PhD fellowship to B.S.-N.), and the Deutsche Forschungsgemeinschaft through SFB 465 (Würzburg), project B5; SFB 466 (Erlangen), project B3; the FOR 303, projects A2 (E.S.) and C1 (A.A.); and the graduate college (GK) "Cell growth" (A.S. and E.S.).
A.A. and E.S. share senior authorship of this manuscript.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Edgar Serfling, Dept of Molecular Pathology, Institute of Pathology, Josef-Schneider-St 2, D-97080 Würzburg, Germany; e-mail: path015{at}mail.uni-wuerzburg.de.
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Top
Abstract
Introduction
. After 8 hours, cells were
stained with Annexin V-PE (PharMingen) according to the
manufacturer's protocol. The effect of HPK1 on apoptosis was
determined by FACS dividing the Annexin V-PE positive and infected
cells by all infected cells (× 100). Apoptosis of EL-4 cells was
induced, after careful washing, by incubation of 5 × 105
cells per milliliter fresh medium containing 1 mM
H2O2. Apoptosis was determined by FACS either
using Annexin V-PE staining or a slightly modified SubG1
technique.
essential initiators of apoptotic cell death.
Cell.
2000;103:839-842