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INTRODUCTION |
Constant turnover and the capacity to adapt efficiently to a
changing environment are hallmarks of the hematopoietic system. Inflammatory cytokines like tumor necrosis factor
(TNF
)1 and interleukin 1 trigger intracellular pathways resulting in the activation of the
stress-activated protein kinases SAPKs/JNKs and p38s as well as of
NF
B family transcription factors (reviewed in Refs. 1 and 2). SAPKs
are executing enzymes acting at the basal level of a hierarchical
three-tiered kinase cascade (3), which upon activation enter the
nucleus and phosphorylate nuclear transcription factors (4, 5).
In mammals, two families of serine/threonine kinases have been
identified that contain a catalytic domain with extensive homology to
Sterile 20 (Ste20) kinase of the yeast Saccharomyces
cerevisiae. Kinases prototypically represented by p21-activated
kinase (PAK) are characterized by a C-terminal kinase domain and an
N-terminal p21-binding domain, flanked by proline-rich sequences that
serve as SH3 domain-binding sites (6). PAKs are activated by the GTP-bound forms of the small GTPases Rac/Cdc42 and have been implicated in regulation of cytoskeletal dynamics, cell cycle, and oxidant generation in neutrophils (7).
The second family comprises kinases related to germinal center kinase
(GCK), which are defined by a N-terminally located kinase domain. Based
on homologies within their C-terminal domains GCKs can be grouped into
six subfamilies. Four kinases, GCK, GCKR/KHS, GLK, and HPK1, which will
be referred to as subfamily I, share a C-terminally located regulatory
domain, called citron homology domain (CNH) (8, 9). Of this group
GCKR/KHS (10, 11) and GLK (12) are ubiquitously expressed, whereas GCK
(13) and HPK1 (14, 15) display tissue specificity, with HPK1 being exclusively expressed in the hematopoietic cells of the adult. Upon
overexpression subfamily I kinases are rendered active and activate
potently and selectively the SAPK/JNK pathway via MAP3Ks. Kinase
activity of endogenous GCK, GCKR/KHS, and GLK is stimulated in response
to TNF
(11, 12, 16). In addition GCK and GCKR/KHS have been shown to
bind to the TNF receptor-associated factor 2 (TRAF2) (17, 18). In
contrast to GCK, GCKR/KHS, and GLK are responsive to UV light. HPK1,
which already displays significant kinase activity when
immunoprecipitated from nonstimulated tissues or cell lines, has been
reported to be activated in response to erythropoietin receptor
engagement (19) as well as T and B cell immunoreceptor cross-linking
(20, 21).
Four proline-rich stretches located between the kinase domain and CNH
domain of HPK1 contain a PXXP motif, the minimal sequence requirement for SH3 domain ligands. Three of these have been shown to
interact with small adaptor proteins including Grb2 (22), Nck (22), HS1
(19), and Crk (23, 24), providing a possible link to activated
transmembrane receptors. SH3 domain-mediated coupling to possible
downstream MAP3Ks like mixed lineage kinase 3 (MLK3) has also been
described (14). Despite their structural similarity, the SH3 domain
ligand motifs are poorly conserved between subfamily I kinases.
Therefore, subfamily I kinases appear to be subject to different
regulatory mechanisms and most likely serve distinct physiological functions.
NF
B/Rel proteins are dimeric, sequence-specific transcription
factors that control many important biological processes, including development, immune responses, cell growth, and apoptosis. NF
B family transcription factors are rendered inactive within the cytoplasm
by interaction with I
B inhibitory proteins. In response to
extracellular signals, a high molecular weight I
B kinase (IKK) complex is activated resulting in I
B phosphorylation followed by
ubiquitinylation and degradation. De-repressed NF
B proteins translocate to the nucleus, where they bind and transactivate
B
sites within the promoter region of NF
B-regulated genes (25).
Tissue-specific signaling molecules such as HPK1 are likely to provide
specific inputs in ubiquitous transduction pathways and may function as
signaling integrators or branch points. The increasing number of
kinases that activate both SAPKs and NF
B family transcription
factors prompted us to investigate a possible function of HPK1 in
NF
B signaling. Here we report a robust stimulation of NF
B
activity by HPK1 in hematopoietic cells. In apoptotic cells HPK1 was
cleaved by a caspase 3-like activity, resulting in the generation of a
dominant-negative C-terminal fragment that inhibited NF
B stimulation.
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EXPERIMENTAL PROCEDURES |
Generation of NF
B Reporter and Expression Plasmids--
The
pGL8xNF
B-fos reporter plasmid contains 8 repeats of the mouse
major histocompatibility complex class I h2dk gene
B site fused to the mouse c-fos minimal promoter driving
a luciferase reporter gene. For normalization we generated pfos-LacZ
that contains the identical mouse c-fos minimal promoter
lacking the
B-binding sites. pSP64T-HPK1(D383E):HA and
pSP64T-HPK1(D383N):HA were generated by site-directed mutagenesis using
the U.S.E. Mutagenesis Kit (Amersham Pharmacia Biotech) according to
the manufacturer's protocol. pMT2-based HPK1 expression plasmids were
described previously (14). For detection by immunoblotting T7-tagged
versions of the GC family kinases GCK, GCKR, and GLK were generated in
pCAT7. For pCAT7:FL:GLK an NcoI/Bsp120I fragment
from pCR3.1-FL:GLK was inserted into the SmaI site of
pCAT7-neo. For pCAT7:GCKR an EcoRI/SmaI fragment
from pFLAG-GCKR was inserted into pCAT7-neo, and pCAT7:GCK was created
by blunt insertion of an EcoRV/XbaI fragment from pRC/CMV-GCK into the EcoRI site of pCAT7-neo.
Generation of Deletion Mutants in the Proline-rich Motifs
P1, P2, and P4 of mHPK1--
A KpnI fragment comprising
1233 base pairs of mHPK1 cDNA containing the proline-rich motifs P1
to P4 was subcloned into pBluescript giving rise to the plasmid
pB-mHPK1:KpnI. The following primer combinations were used
to introduce deletions into the proline-rich motifs P1, P2, and P4 by
polymerase chain reaction technology. For motif P1, deletion of the
amino acids PPP: 5'-CTAGAACTAGTGGATCCCCCGGGCTGCAGG-3' and
5'-GGTAGATCTGATCCGCCGAGGGATGGCCTCAGGCTCCTCATC-3'; for motif P2,
deletion of the amino acids PPPLPPKPKF:
5'-CTAGAACTAGTGGATCCCCCGGGCTGCAGG-3' and
5'-GCTCAATTGCCCATCGTCCCTAATTCCTCCAGAACCATCATCTGATGGAGACCGAGGTATGTTCTCTGAAGGGGC-3'; and for motif P4, deletion of the amino acids PPLVP,
5'-CTAGAACTAGTGGATCCCCCGGGCTGCAGG-3' and
5'-CTCCATCTTTCCCCTCATCTTTTCCTTCCTTGGCTGGCCTGGCTCCGGAGCAGC-3'.
Polymerase chain reaction products containing the desired mutations
were cut with XhoI/BglII (
P1),
XhoI/MfeI (
P2), and
XhoI/StyI (
P4) and reinserted into
pB-mHPK1:KpnI. Insertion of a
P1 containing XhoI/BglII fragment into pB-mHPK1:KpnI
P2 and pB-mHPK1:KpnI
P4 gave rise to the double
mutants pB-mHPK1:KpnI
P1/P2 and pB-mHPK1:KpnI
P1/P4. Insertion of a
P4 containing
MfeI/StyI fragment into pB-mHPK1:KpnI
P1/P2 resulted in pB-mHPK1:KpnI
P1/P2/P4. The respective mutations were then reintroduced into pcDNA3-mHPK1 by
swapping of the corresponding KpnI fragments.
Tissue Culture and Cell Lines--
COS1 cells were cultured in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 5% fetal bovine serum (Sigma), 100 µg/ml
penicillin (Life Technologies, Inc.), 100 µg/ml streptomycin (Life
Technologies, Inc.), and 20 mM L-glutamine (Life Technologies, Inc.).
The IL-3-dependent hematopoietic progenitor lines FDC-P1
(26) and 32D-Cl3 (27) were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 5% fetal bovine serum (Sigma), 100 µg/ml penicillin (Life Technologies, Inc.), 100 µg/ml streptomycin (Life Technologies, Inc.), 20 mM
L-glutamine (Life Technologies, Inc.), 0.001%
monothioglycerol (Sigma), and 1.5% conditioned medium of the myeloma
cell line X63 Ag8-653 IL-3 as a source of IL-3 (28).
Transient Transfections, NF
B Reporter Assays, and Retroviral
Infection of FDC-P1 Cells--
For reporter assays 2 × 105 COS1 cells were transfected by the
Ca2+-phosphate coprecipitation method with 2 µg of
expression plasmid, 1 µg of pGL8x NF
B-fos, and 0.2 µg of
pfos-LacZ. After 48 h NF
B activity was determined using the
Tropix, Inc. Dual-LightTM Chemiluminescent Reporter Assay
System following the manufacturer's protocol. All experiments were at
least repeated three times. Bars depict the average of duplicate
transfections. Values presented within a diagram are derived from a
single transfection experiment.
Jurkat cells were transiently transfected by electroporation of 20 µg
of NF
B luciferase reporter plasmid, 2 µg of
-galactosidase reporter construct, and 0-10 µg of wild-type or kinase-inactive HPK1
as indicated in 400 µl of serum-free RPMI 1640 medium.
FDC-P1 cells were infected using transient supernatants of the
ecotropic packaging cell line GP+E 86 transfected with 10 µg of
retroviral vector. After 48 h successfully infected cells were selected by a 12-day selection period at 1 mg/ml G418 (Life
Technologies, Inc.).
In Vitro Kinase Assays, SAPK Activation, and
Immunoblotting--
HPK1 and p54-SAPK
kinase assays were performed
as described previously (14). For Western blotting the polyclonal
rabbit anti-HPK1 sera 3, 5, 6, and 7 were used. Sera 5 and 6 have been described previously (14), and serum 3 was raised against the peptide KSGYQPPRLKEKSRWSSSC located in sub-domain X of the HPK1 kinase
domain and serum 7 against the C-terminal peptide TRPTDDPTAPSNLYIQE. The HA tag was detected using the mouse monoclonal antibody 12CA5. A
phosphospecific antibody against serine 32 of I
B
was obtained from Cell Signaling Technology, the I
B
(antibody 2/PC142)
antibody from Oncogene Research Products. The T7 tag was detected by
the monoclonal antibody T7·Tag (Novagen).
Generation of Apoptotic HL60 Cell Extracts and in Vitro Cleavage
of HPK1--
Cytoplasmic extracts from apoptotic HL60 cells were
prepared as described (29, 30). Lysates were centrifuged for 50 min at
100,000 × g, and the resulting clear supernatant was
stored in aliquots at
80 °C until further usage. The presence of a
caspase 3-like activity in the cytoplasmic extract was verified by
cleavage of Ac-DEVD-7-amino-4-methylcoumarin substrate peptide.
In vitro translated HPK1:HA, HPK1(D383E):HA, and
HPK1(D383N):HA were generated as described (14). 2 µl of
[35S]methionine-labeled reaction product were mixed with
5 µl of apoptotic HL60 cell extract and incubated at 37 °C for the
indicated times. Caspase inhibitors (Peptide Institute, Osaka, Japan)
were added in Me2SO at a final concentration of 0.1 µM (Ac-DEVD-CHO) and 1.0 µM
(Ac-YVAD-CHO).
Electrophoretic Mobility Shift Assays--
Soluble nuclear
proteins were prepared and used for electrophoretic mobility shift
assays as described (31, 32). For each shift reaction 10-20 fmol of
32P-labeled
B-binding oligonucleotide
(5'-TCGAGATGGGGAATCCCCAGCCTCGA-3') were employed.
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RESULTS |
HPK1 Activates the NF
B Pathway in Hematopoietic Cell
Lines--
We tested the capacity of HPK1 to activate NF
B in Jurkat
T cells, which endogenously express HPK1. Cotransfection of increasing amounts of HPK1 expression plasmid and a NF
B-dependent
reporter resulted in HPK1-dependent NF
B activation (Fig.
1A), whereas expression of the
kinase-deficient mutant HPK1(K46E) failed to activate NF
B. These
results suggest that HPK1 is an activator of NF
B in Jurkat T
cells.

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Fig. 1.
Activation of NF B
transcription factors by HPK1 and the related kinases GLK, GCKR/KHS,
and GCK. A, upper panel, Jurkat T cells were
transiently cotransfected with expression plasmids encoding full-length
HPK1, the kinase-deficient mutant HPK1(K46E), or empty vector DNA
(control) and an NF B-dependent reporter system. Values
are averages of triplicates ± S.D. Lower panel,
expression of HPK1 was assessed by anti-HA Western blotting. B,
upper panel, NF B mobility shift activity was determined in
nuclear extracts of wild-type (WT) FDC-P1 myeloid progenitor
cells (FDC-P1/WT) or retrovirally transduced FDC-P1 clones
stably overexpressing HA-tagged HPK1 (FDC-P1/C9 and
FDC-P1/D4). Equal amounts of nuclear extracts prepared from
4 × 106 cells were incubated with a
32P-labeled DNA fragment comprising an NF B-binding site.
Nucleoprotein complexes were separated by native PAGE, visualized by
autoradiography, and quantified using image analysis software.
Middle panel, expression of endogenous and HA-tagged HPK1 in
FDC-P1/WT, FDC-P1/C9, and FDC-P1/D4 cells was assessed by anti-HPK1
Western blotting using rabbit serum 7. Lower panel,
endogenous and HA-tagged HPK1 was immunopurified from FDC-P1/WT,
FDC-P1/C9, and FDC-P1/D4 cells using anti-HPK1 antisera 5/6 and tested
for the ability to autophosphorylate in vitro.
Electrophoretic mobilities of endogenous and HA-tagged HPK1 are
indicated by arrows. C, dual reporter plasmid system used to
assess activation of NF B in transiently transfected COS1 cells.
Transcription of a luciferase reporter gene driven by 8 NF B-binding
sites, fused to a c-fos minimal promoter, was normalized on
the basis of -galactosidase expression from an identical
c-fos promoter lacking NF B-binding sites. D, upper
panel, COS1 cells were transiently cotransfected with plasmids
encoding HPK1, the kinase-deficient version HPK1(K46E), or the
C-terminal deletion mutant HPK1-Ko in the presence of the double
reporter system depicted in C. After 36 h control cells
were stimulated with 2 nM recombinant hTNF and incubated
for additional 12 h followed by cell lysis. After 48 h
NF B-driven luciferase activity was determined and normalized against
-galactosidase activity using a chemiluminescence assay system.
Relative activation of NF B-driven luciferase activity normalized for
transfection efficacy is shown. Depicted are averages of a
representative experiment, in which all transfections were performed in
duplicate. Middle panel, in parallel identical amounts of
the same plasmids were assayed for p54-SAPK activation. After
48 h cells were lysed, and p54-SAPK was immunopurified.
SAPK/JNK activation was determined by in vitro
phosphorylation of a bacterially expressed c-Jun N-terminal fragment
fused to GST. Phosphoproteins were separated on SDS-PAGE and visualized
by autoradiography. Lower two panels, expression of the
different proteins was visualized by Western blotting using the
polyclonal anti-HPK1 rabbit serum 3 (directed against kinase domain
subdomain XI) or 7 (directed against the HPK1 C terminus). E,
upper panel, increasing amounts of expression plasmids for the
indicated GCK-related kinases or empty vector DNA were cotransfected
into COS1 cells. The total amount of transfected DNA was kept constant.
Lower panel, expression of the different proteins was
visualized by Western blotting using the polyclonal anti-HPK1 rabbit
serum 7 or anti-T7 tag antibody. F, identical amounts of
expression plasmids for the GCK-related kinases were cotransfected in
COS1 cells and assayed for their ability to undergo autophosphorylation
(upper panel) or activate an HA-tagged p54-SAPK
(middle panel). p54-SAPK expression levels were
visualized by anti-HA Western blotting (lower panel).
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To investigate HPK1-dependent NF
B activation in a
myeloid progenitor cell line, FDC-P1 cells (26) were used, which also endogenously express HPK1. We demonstrated HPK1-dependent
NF
B activation taking advantage of two retrovirally transduced
FDC-P1 clones FDC-P1/C9 and FDC-P1/D4 that stably express an exogenous, HA-tagged variant of HPK1. FDC-P1/D4 cells harbor approximately double
the amount of HPK1 kinase activity present in FDC-P1/C9 cells (Fig.
1B). FDC-P1/C9 cells contain about double the amount of HPK1
kinase activity of FDC-P1 wild-type cells. Comparing nuclear extracts
derived from FDC-P1 wild-type cells and the cell clones C9 and D4, we
observed an increase in NF
B bandshift activity that paralleled HPK1
kinase expression levels. These results provide an independent
demonstration of HPK1-mediated NF
B transcription factor activation.
Activation of NF
B Depends on Full-length Kinase-active
HPK1--
To analyze the HPK1-driven NF
B activation in more detail
and to circumvent the presence of endogenous HPK1, we used COS1 cells
for further analysis. For subsequent transfection experiments we
generated a double reporter gene system, in which
NF
B-dependent transcription of a luciferase reporter
gene was normalized against basal transcription of a galactosidase
reporter gene (Fig. 1C). To delineate the requirements for
NF
B activation, we transiently expressed wild-type HPK1, the
kinase-deficient variant HPK1(K46E), or the isolated kinase domain
HPK1-Ko in COS1 cells. NF
B activity levels were compared with the
activity level observed after treatment with the inflammatory cytokine
TNF
, a well established inducer of NF
B (Fig. 1D). HPK1
caused a robust activation of NF
B, comparable to TNF
stimulation,
whereas HPK1(K46E) was not able to activate NF
B. Interestingly,
HPK1-Ko failed to stimulate NF
B, although it still activated the
SAPK/JNK pathway.
These results confirm our observation in Jurkat T cells demonstrating
again that HPK1 kinase activity is essential for NF
B activation.
Furthermore, NF
B activation required the presence of full-length
HPK1, whereas the C-terminal regions of HPK1 are dispensable for
SAPK/JNK activation.
The Ste20-related Kinases of the GC Family Show Variable Capacities
to Activate NF
B--
We next addressed the question whether the
potential to activate NF
B is shared by other members of subfamily I
GCK-related kinases, besides HPK1. After transient expression of
increasing amounts of HPK1, GLK, GCKR/KHS, or GCK in COS1 cells, we
found a dose-dependent and profound activation of NF
B by
HPK1 closely followed by GCKR/KHS (Fig. 1E). Moderate NF
B
activation by GCK was only seen at the highest expression level,
whereas NF
B activation by GLK was largely blunted.
At expression levels that resulted in comparable levels of
autophosphorylation activity (Fig. 1F, upper panel), the
capacity of the GCK-related kinases to activate NF
B correlated with
their ability to activate the SAPK/JNK p54
(Fig. 1F, middle
panel).
These experiments clearly demonstrate that within the GCK subfamily I
of Ste20 kinases HPK1 and the most closely related GCKR/KHS are potent
activators of NF
B.
HPK1-mediated NF
B Activation Is Independent of HPK1-mediated
SAPK Activation--
We wondered whether NF
B activation by HPK1 was
secondary to HPK1-mediated SAPK/JNK activation or caused by a component
of the SAPK/JNK pathway. Three MAP3Ks, MEKK1 (15), MLK3 (14), and TAK1
(33), have been implicated as potential downstream elements of HPK1 in
SAPK/JNK activation. In our assay system HPK1 and the established
NF
B activator MEKK1 (34, 35) displayed comparable potency in
activating NF
B, whereas MLK3 failed to activate NF
B (Fig.
2A, upper panel).
By using identical conditions HPK1, MEKK1, and MLK3 all activate the
SAPK/JNK p54
to a comparable extent (Fig. 2A, lower
panel). Furthermore, we detected no synergism between HPK1 and
MEKK1 in NF
B nor in SAPK/JNK p54
activation. Surprisingly, we
found that coexpression of MLK3 potently inhibits HPK1-mediated NF
B
activation, whereas it had no influence on SAPK/JNK p54
activation,
suggesting that NF
B activation by HPK1 does not involve MLK3.

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Fig. 2.
HPK1-mediated NF B
activation is independent of its SAPK/JNK activation and involves the
IKK complex. A, COS1 cells were transiently
cotransfected with expression plasmids for HPK1, MEKK1, and MLK3,
either alone or in combination. 48 h after transfection NF B
activation was determined. Lower panel, in parallel,
identical amounts of the indicated plasmids were cotransfected with a
p54-SAPK expression plasmid, and p54-SAPK kinase activity was
assayed. B, expression plasmids for MEKK1, HPK1, and
SEK1(AL) were transfected into COS1 cells alone or in combination, and
their capacity to activate NF B was determined. Lower
panel, the same plasmids were tested for their capacity to
activate p54-SAPK . Relative increase of p54-SAPK activity
(numerical values) was determined using a phosphorimaging system.
C, the indicated expression plasmids were transiently
transfected into COS1 cells, and after 48 h NF B activation was
determined. Inset, expression of HPK1 was visualized by
Western blotting using the polyclonal anti-HPK1 rabbit serum 7.
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To demonstrate formally that HPK1-mediated NF
B and SAPK/JNK
activation utilize distinct effector pathways, we took advantage of a
mutant form of SEK1/MKK4, SEK1(S220A,T224L), which we will refer to as
SEK1(AL). Mutations of the critical activation loop residues Ser-220
and Thr-224 render SEK1 refractory to upstream activating kinases and
turn it into a potent dominant-negative inhibitor of SAPK/JNK
activation at the MAP2K level. SEK1(AL) potently inhibits SAPK/JNK
activation in a dominant-negative fashion at the MAP2K level (36).
Whereas SEK1(AL) caused no change in HPK1 or MEKK1-induced NF
B
activation (Fig. 2B, upper panel), SAPK/JNK activation was
profoundly inhibited (Fig. 2B, lower panel), demonstrating
that HPK1-mediated NF
B activation is independent of HPK1-mediated
SAPK/JNK activation.
Kinase-deficient I
B Kinase
(IKK
) Abrogates HPK1-mediated
Activation of NF
B--
To define further the level of HPK1 action
and to test whether IKK functions downstream of HPK1, we coexpressed
HPK1 and dominant-negative forms of NIK and IKK
, NIK(KK429,430AA),
and IKK
(K44A). The dominant-negative variants of NIK and IKK
both
inhibited TNF
-stimulated as well as HPK1-mediated NF
B activation
(Fig. 2C). Kinase-active NIK displayed an NF
B activation
potential comparable to that of HPK1, whereas no synergy between both
kinases was detectable. Blockage of HPK1 signaling to NF
B by
overexpression of dominant-negative NIK or IKK
protein suggested
that HPK1 might act upstream of the IKK complex.
HPK1 kinase activity and phosphorylation status were not responsive to
TNF
, neither did overexpression of HPK1 augment TNF
-mediated NF
B activation (not shown). Taken together these findings argue against a direct involvement of HPK1 in TNF
signaling.
HPK1-mediated NF
B Activation Is Blocked by Overexpression of SH3
Domain-containing Molecules--
The surprising finding that MLK3
potently inhibited HPK1-mediated NF
B activation (Fig. 2A)
led us to speculate that the SH3 domain-driven interaction between MLK3
and HPK1 (14) could result in sequestration of HPK1 from an
NF
B-activating complex. To test this hypothesis we coexpressed
MLK3
, a truncation mutant of MLK3 consisting of the N terminus,
which includes the SH3 domain and 21 amino acids of the adjacent kinase
domain (14) with HPK1 (Fig.
3A). According to our
hypothesis we detected a potent suppression of HPK1-driven NF
B
activation. Therefore we reasoned that other HPK1-binding SH3
domain-bearing molecules should also interfere with NF
B activation.
The small adaptor Grb2, which consists of a central SH2 domain flanked
by SH3 domains, has been shown to associate with HPK1 (22).
Coexpression of HPK1 and Grb2 resulted in a strong inhibition of
HPK1-mediated NF
B activation, whereas MEKK1-mediated NF
B
activation was not affected (Fig. 3B). These results lend
further support to our notion that SH3 domain interactions are likely
critically involved in HPK1-mediated NF
B activation.

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Fig. 3.
NF B activation is
dependent on the proline-rich SH3-binding sites in HPK1. A,
upper panel, COS1 cells were transiently cotransfected with
expression plasmids for HPK1 or HA-tagged MLK3 alone or in
combinations. 48 h after transfection NF B activation was
determined. Lower two panels, expression of the proteins was
visualized by Western blotting using the polyclonal anti-HPK1 rabbit
serum 7 or anti-HA antibody. B, the indicated expression
plasmids were transiently transfected into COS1 cells, and after
48 h NF B activation was determined. Lower panel,
expression of the HA-tagged forms of HPK1 and Grb2 was visualized by
anti-HA Western blotting. C, HPK1 or HA-tagged deletion
mutants lacking the indicated SH3 domain-binding motifs and the NF B
reporter system were cotransfected into COS1 cells. After 48 h
NF B activation was determined. Expression of HPK1 proteins was
demonstrated by anti-HPK1 Western blotting using rabbit serum 7. Localization of the proline-rich sites (P1, P2, and P4) and the amino
acid sequence DDVD on the HPK1 protein is depicted (inset).
D, HPK1 polyproline stretch deletion mutants are not
impaired in their ability to activate p54-SAPK . Top
panel, the HPK1 mutants assayed in C were transiently
coexpressed in COS1 cells with p54-SAPK , immunopurified, and tested
for their ability to autophosphorylate in vitro. Middle
panel, equal expression was demonstrated by anti-HA Western
blotting. Bottom panel, p54-SAPK activation was
determined as described in Fig. 1D.
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NF
B Activation Is Dependent on the Proline-rich SH3-binding
Sites in HPK1--
Nonspecific inhibition of HPK1-mediated NF
B
activation could be excluded by cotransfection of an SH3
domain-containing molecule that does not bind to HPK1. Because of the
possibility of residual interactions between overexpressed SH3 domains
and HPK1, we decided to generate HPK1 mutants, in which the three
proline-rich SH3 domain-binding sites (P1, P2, and P4) were deleted
either singularly or in combination. When we tested the HPK1 proline
deletions for their ability to activate NF
B, none of them displayed
an activity comparable to wild-type HPK1 (Fig. 3C). The
mutations did not impair protein stability, as they did not decrease
HPK1-associated kinase activity or the capacity of HPK1 to activate
SAPK/JNK (Fig. 3D). These results demonstrate a strong
dependence of NF
B activation on the proline-rich SH3-binding sites
in HPK1, whereas those sites were dispensable for SAPK/JNK activation.
Furthermore, we found a C-terminally HA-tagged version of HPK1 to be
less efficient in activating NF
B as compared with the native protein
(Fig. 3C), indicating a critical role of the HPK1 C terminus
in NF
B activation.
Taken together our findings demonstrate that SH3 domain-mediated
interactions are a prerequisite for NF
B activation and that HPK1-mediated SAPK/JNK and NF
B activation differ significantly in
their molecular requirements.
HPK1 Is Proteolytically Degraded in FDC-P1 Cells Rendered Apoptotic
by IL-3 Withdrawal--
NF
B target genes have been implicated in a
plethora of pro- and anti-apoptotic processes, and a number of NF
B
regulators have been shown to be subject to caspase cleavage in
apoptotic cells. Growth and survival of the hematopoietic progenitor
cell line FDC-P1 is strictly IL-3-dependent (37). We tested
HPK1 stability in apoptotic FDC-P1/D4 hematopoietic progenitor cells after induction of apoptosis by IL-3 withdrawal for 18 h. At this time point DNA fragmentation, a hallmark of the apoptotic cell death
program, was maximal (not shown). By using Western blot analysis, we
detected reduced HPK1 levels after IL-3 deprivation and observed the
appearance of HPK1 cleavage products (Fig.
4A). These results were
reproduced under identical conditions using the
IL-3-dependent myeloid progenitor cell line 32D-Cl3
indicating that HPK1 is proteolytically degraded in hematopoietic cells
rendered apoptotic after growth factor withdrawal.

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Fig. 4.
HPK1 is cleaved in vitro and
in apoptotic cells by a caspase 3-like activity at a DDVD motif
immediately preceding the P2 region. A, FDC-P1/D4
(left panel) or 32D-Cl3 (right panel) cells were
rendered apoptotic by IL-3 withdrawal and lysed, and proteins were
separated by SDS-PAGE. HPK1 was visualized by Western blotting with the
polyclonal anti-HPK1 rabbit serum 7 directed against the C terminus of
HPK1. Electrophoretic mobilities of full-length HPK1 and its cleaved
forms are indicated. B, sequence comparison of the hinge
region between the N-terminal kinase domain and the C-terminal Citron
homology (CNH) domain of subfamily I GCK-related kinases. Kinase and
CNH domains are indicated by gray boxes. The proline-rich
motifs P1, P2, and P4 as well as the DDVD cleavage site are indicated
by bars. Black boxed amino acids are conserved in
all four kinases, and gray boxed amino acids identify
conservative exchanges. C, top panel and
middle panel, [35S]methionine-labeled HPK1 was
generated by in vitro translation using cell-free
reticulocyte lysates. Aliquots of the in vitro translation
reaction were incubated at 37 °C for the indicated intervals with
apoptotic cell extracts derived from HL60 cells. Where indicated
apoptotic extract plus Me2SO or heat-pretreated apoptotic
extract was added. Addition of an Ac-YVAD-CHO caspase inhibitor had no
influence on the cleavage of in vitro translated
[35S]methionine-labeled HPK1. In contrast, an Ac-DEVD-CHO
caspase inhibitor blocked HPK1 cleavage completely. Bottom
panel, the [35S]methionine-labeled HPK1 mutants
HPK1(D383E) and HPK1(D383N) were incubated at 37 °C for 60 min in
the absence ( ) or presence (+) of apoptotic extracts. Reaction
products were separated by SDS-PAGE and visualized by autoradiography.
Electrophoretic mobilities of HPK1 and two predominant cleavage
products are indicated by asterisks.
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|
The Hinge Region between the HPK1 Kinase and the Citron Homology
Domain Contains a Caspase Recognition Motif--
Caspases, the
effector proteases during apoptosis (38), display overlapping substrate
specificity, with the four N-terminal amino acids preceding their
cleavage site being most important for substrate recognition and
turnover. An Asp residue at the position N-terminally flanking the
cleavage site is indispensable for all caspases. Caspase 3/7 cleavage
sites are defined by a DX
D motif, where X
denotes a wide variety of amino acids and
a hydrophobic amino acid
(39, 40). Inspection of the primary sequence of the HPK1 hinge region
between kinase and CNH domain, which also contains the proline-rich
motifs, revealed several potential caspase recognition sites with a
DDVD motif immediately preceding the P2 proline-rich stretch being most
prominent (Fig. 4B).
HPK1 Is Efficiently Cleaved by Apoptotic Cell Extracts in
Vitro--
To address the question, if the HPK1 DDVD motif is
recognized by caspases, we first employed an in vitro test
system, in which [35S]methionine-labeled HPK1 generated
by in vitro translation was exposed to cytoplasmic extracts
of apoptotic HL60 cells. Such extracts contain abundant activated
caspases. During a 60-min incubation HPK1 was efficiently cleaved into
two fragments, the size of which correlated well with the calculated
fragment sizes of 43 and 48 kDa. Heat pretreatment of the apoptotic
extracts completely abolished proteolytic degradation (Fig. 4C,
top panel).
HPK1 Cleavage Is Blocked by the Caspase 3/7 Inhibitor Ac-DEVD-CHO
and Depends on a DDVD Motif--
Caspases 3 and 7 can be blocked by
incubation with the inhibitory peptide Ac-DEVD-CHO, whereas the
Ac-YVAD-CHO inhibitor blocks caspases 1 and 4 preferentially. When HPK1
was incubated with apoptotic HL60 extracts in the presence of
Ac-DEVD-CHO, proteolytic cleavage was efficiently blocked, whereas the
Ac-YVAD-CHO inhibitor showed no effect (Fig. 4C, middle
panel). Addition of the vehicle Me2SO alone had no
inhibiting effect on HPK1 cleavage (Fig. 4C, top panel).
Two point mutants HPK1(D383N) and HPK1(D383E), in which the DDVD motif
was either changed to DDVN or DDVE, were found to be completely
cleavage-resistant (Fig. 4C, bottom panel). These data identify the DDVD motif in the hinge region as a relevant target site
for HPK1 cleavage by apoptotic extracts in vitro.
Our data indicated that in apoptotic myeloid progenitor cells
proteolytic cleavage of HPK1 occurs after growth factor withdrawal.
The Isolated HPK1 C Terminus Suppresses NF
B Activation by
HPK1--
To address possible implications of HPK1 proteolytic
cleavage during apoptosis, we tested the signaling capacity of the
isolated HPK1 kinase domain (HPK1-Ko) or the HPK1 C-terminal fragment
(HPK1-
N). HPK1-
N comprises the proline-rich motifs P2 and P4 as
well as the CNH domain. Whereas expression of full-length HPK1 caused robust stimulation of NF
B-mediated transcription, none of the two
fragments caused a detectable activity (Fig.
5A, upper panel). In agreement
with our previous results the intact kinase domain HPK1-Ko was
necessary and sufficient for SAPK/JNK activation (14), whereas
HPK1-
N failed to activate SAPK/JNK (Fig. 5A, 2nd panel from top). We did not detect an augmented kinase activity of
HPK1-Ko as a result of the removal of the C-terminal part (not
shown).

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Fig. 5.
The isolated C-terminal portion of HPK1
inhibits NF B activation. A, top
panel, HPK1, the isolated kinase domain (HPK1-Ko) or
the C-terminal portion (HPK- N) were
transiently expressed alone or in combination and tested for their
ability to activate NF B in COS1 cells. 2nd panel from
top, the HPK1 proteins assayed in A were
transiently coexpressed in COS1 cells with p54-SAPK , immunopurified,
and tested for their ability to phosphorylate a c-Jun N-terminal
fragment fused to GST in vitro. Lower two panels, expression
of the different proteins was visualized by anti-HPK1 Western blotting
using rabbit serum 3 or 7. B, increasing amounts of
HPK1- N cDNA were expressed either alone or in combination with
NIK and the NF B reporter system in COS1 cells. Luciferase activity
was measured as described above. Inset, expression of
HPK- N was visualized by anti-HPK1 Western blotting using rabbit
serum 7. C, FDC-P1 myeloid progenitor cell pools infected
with either the empty retroviral vector MSCV (FDC-P1/vector only) or a
virus transducing HPK1- N (FDC-P1/HPK1- N) were stimulated with 2 nM recombinant hTNF and incubated for 2, 5, or 10 min
followed by cell lysis. Endogenous phospho-I B was visualized by
Western blotting using a phospho-I B (Ser-32)-specific antibody
(upper panel). Total levels of I B and HPK1
proteins were visualized by anti-I B and by anti-HPK1 (rabbit
serum 7) Western blotting.
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When we coexpressed HPK1 fragments in combination with full-length
HPK1, HPK1-
N inhibited HPK1-induced NF
B stimulation, whereas
HPK1-Ko failed to exert an effect. Under identical conditions activation of SAPK/JNK by full-length HPK1 was not altered.
These data indicate that the C-terminal part of HPK1 was capable of
inhibiting HPK1-driven NF
B activation, although it did not alter
HPK1-mediated SAPK/JNK activation.
HPK1-
N Inhibits NF
B Activation by NIK--
To investigate
whether the C-terminal part of HPK1 was able to inhibit NF
B
activation by stimuli other than HPK1 itself, we tested a potential
effect on NIK that displayed an NF
B activation capacity comparable
to HPK1 (Fig. 2C). Coexpression of increasing amounts of NIK
and HPK1-
N resulted in a dramatic dose-dependent decrease in NF
B activation (Fig. 5B). This observation
clearly showed that a C-terminal fragment of HPK1, like HPK1-
N, is a potent inhibitor of NF
B activation.
HPK1-
N Reduces Phosphorylation of I
B
in FDC-P1
Cells--
To assess the capacity of HPK1 cleavage products to act as
inhibitors of NF
B activation at physiological levels, we derived retrovirally infected FDC-P1 cells that stably expressed the C-terminal fragment HPK1-
N. Selected clones were pooled to avoid effects due to
clonal variation. These clones expressed endogenous full-length HPK1
and HPK1-
N at a similar ratio to that observed in FDC-P1/D4 cells
rendered apoptotic by growth factor withdrawal (see Fig. 4A
and Fig. 5C, bottom panel). To test for a possible impact of HPK1-
N on NF
B activation, we compared the appearance of I
B
phosphorylated on serine 32 in FDC-P1/HPK1
N cells and FDC-P1 cells
infected with the parental retroviral vector pMSCV (FDC-P1/vector only)
after application of TNF
.
In FDC-P1/vector only cells I
B
phosphorylation sharply peaked at
5 min after stimulation and was barely detectable after 10 min (Fig.
5C, top panel), at the same time a decrease in total I
B
levels became apparent (Fig. 5C, middle panel). In
FDC-P1/HPK1-
N cells the accumulation of phospho-I
B
was found
to be significantly reduced indicating a suppression of NF
B
activation (Fig. 5C, top panel). Therefore, it appears that
cleavage of HPK1 in apoptotic hematopoietic cells may be utilized
as an effective tool to block NF
B activation.
 |
DISCUSSION |
Hematopoietic progenitor kinase (HPK1), a GC kinase-related
mammalian Ste20 homologue, has been implicated as an upstream regulator
of SAPK activity. We show here that HPK1 also potently activates NF
B
transcription factors in hematopoietic and COS1 cells. These findings
corroborate a previous report showing stimulation of
IKK
/
-mediated I
B
phosphorylation after forced expression of
HPK1 (41). Among the GCK subfamily I kinases, we also observed NF
B
activation by the closest HPK1 homologue GCKR/KHS, whereas the more
distantly related GCK caused only moderate NF
B activation, and GLK
failed to elicit any activity in COS1 cells. NF
B activation by
GCK-related kinases may be highly cell type-dependent as
GCK and GCKR/KHS failed to affect NF
B in HEK293 cells (11), whereas in melanoma cells GCK activated NF
B moderately (42).
The molecular requirements for NF
B activation differed substantially
from those for activation of SAPK, which appear to necessitate the HPK1
kinase domain mainly (14). Our data suggest the existence of distinct
HPK1-containing complexes responsible for SAPK/JNK and NF
B
activation. The scaffolding protein JIP1 has been described to
coordinate a SAPK/JNK-activating complex that contains SAPK/JNK, MKK7/SEK2, MLK3, and HPK1 and likely facilitates the interaction of the
pathway compounds (43). NF
B activation has been shown to be
dependent on the activity of a high molecular mass complex of
700-900 kDa containing the I
B-kinases (IKKs)
and
(44). Whether HPK1 is physically associated with this complex remains to be
established. Kinase-deficient mutants of NF
B-inducing kinase (NIK)
and IKK
efficiently inhibited HPK1-mediated NF
B activation, suggesting that HPK1 acts upstream of the IKK complex. Suppression of
NIK-induced NF
B activation by an N-terminal HPK1 deletion mutant
implicated HPK1 downstream of NIK. The apparent paradox of HPK1 being
able to act upstream and downstream of NIK might result from the action
of overexpressed mutant forms of both proteins on the same
NF
B-inducing complex. Furthermore, SH3 domain interactions are
likely to contribute crucially to the formation of the HPK1-containing NF
B-activating complex, as coexpression of SH3 domain-containing molecules strongly interfered with this process, and NF
B activation was dependent on the presence of intact polyproline sites in HPK1.
MLK3, which has been shown to interact with HPK1 via its SH3 domain
(14), was recently reported to phosphorylate directly IKK
and IKK
(45) raising the interesting possibility that MLK3 might mediate NF
B
stimulation by HPK1. Although we and others (46) failed to detect
NF
B activation by MLK3, this could be a consequence of the different
cell types used in the assay systems (HeLa versus COS1) or
different amounts of DNA transfected (45).
In the adult, HPK1, which is exclusively hematopoietic, displays the
most restricted expression pattern of the GCK subfamily I kinases. In
contrast to GCK, GCKR/KHS and GLK, which appear to be elements of TNF
signaling pathways, HPK1 is not activated by TNF
in vivo.
GCK and GCKR/KHS were reported to bind TRAF2 (11, 18) and therefore are
likely to act at a receptor proximal position in TNF signaling.
However, GCK binding is dispensable for TRAF2-mediated p38, SAPK/JNK,
and NF
B activation (47). The TRAF2-GCK complex was recently
described to protect melanoma cells against UV-induced apoptosis (42).
Increasing expression of TRAF2 and GCK during melanoma progression was
positively linked to JNK and NF
B activity.
In T cells HPK1 has been shown to be constitutively associated with the
adaptor protein Grb2 and its homologue Grap (20), whereas association
with the Grb2 family member Gads was only observed after T cell
receptor ligation (21). Both B and T cell receptor engagement cause
stimulation of HPK1 kinase which is dependent on Src and Syk/ZAP-70
tyrosine kinases and the adaptor proteins LAT, SLP76/BLNK (20). Taken
together these studies suggest that HPK1, which is dependent on
inducible tyrosine phosphorylation of immunospecific adaptors like LAT
and SLP76/BLNK, mediates immunoreceptor signals in a receptor proximal
position resulting in the stimulation of SAPK/JNK and NF
B effector
pathways. The subfamily I GC kinase HPK1 therefore appears to have
adopted a specific function in hematopoietic cells coupling cell
type-specific receptor systems to ubiquitous effector pathways.
The precise role of HPK1 during apoptosis of murine hematopoietic cells
is not well understood. We found HPK1 to be cleaved by a caspase 3-like
activity in vitro, and we detected corresponding proteolytic
products in apoptotic cells in vivo. The resulting C-terminal fragment inhibited NF
B activation by HPK1 and NIK in a
dominant-negative manner and strongly reduced phosphorylation of
I
B
in FDC-P1 cells. Although HPK1 is not likely to be involved in
the induction of apoptotic processes in hematopoietic cells, its
conversion from an activator of NF
B to an inhibitor of NF
B may
contribute significantly to the efficient execution of the apoptotic
program. While our manuscript was in preparation, caspase-mediated cleavage of human HPK1 was demonstrated in Jurkat T cells following Fas
ligation (48).
Caspase cleavage has been reported for several mammalian Ste20 kinases.
Proteolysis of SPAK/PASK has been implicated in the regulation of
subcellular localization (49), whereas caspase 3 cleavage of MST/Krs
(50, 51), SLK (52) and PAK2 (53, 54) results in the generation of an
activated apoptosis-inducing kinase domain fragment. Interestingly, we
did not detect an enhanced kinase activity of the isolated HPK1 kinase
domain, suggesting that the HPK1 regulatory C terminus fulfills no
autorepressive function as has been described for PAK2.
Mammalian GCK-related kinases are a rapidly growing family of
cytoplasmic serine/threonine kinases. Here we have presented data that
show activation of NF
B by HPK1 and proteolytic cleavage of HPK1
during apoptosis in growth factor-deprived myeloid progenitor cells. Proteolytic cleavage converts HPK1 into an inhibitor of NF
B.
These findings significantly enhance our knowledge on HPK1 activity and
open novel avenues to test the biological roles of HPK1 which is likely
to fulfill yet undefined functions that rely on NF
B activation.