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INTRODUCTION |
Apoptosis (or programmed cell death) is a fundamental and complex
biological process that enables an organism to eliminate unwanted or
defective cells through an orderly process of cellular disintegration
(1-3). Although apoptotic stimuli that elicit responses vary from cell
to cell, there seems to be a basic biochemical machinery underlying the
process of regulated programmed cell death. Growing evidence suggests
that apoptosis is mediated by the activation of a family of
cysteine proteases, known as caspases, which cleave target proteins
immediately after specific aspartic acid residues (4-6). To date, a
long list of caspase substrates has been identified (7-10). However,
the functional significance of the majority of these cleavage events
and their exact roles in the execution of apoptosis remain largely to
be unraveled.
Caspase-mediated cleavages can initiate their deadly assault on the
cell by inactivating proteins necessary for cell survival or structural
integrity. Meanwhile, caspase-dependent cell killing also
requires the activation of proapoptotic proteins. Protein kinases
emerge as the direct substrates and effectors of caspases (11, 12). For
example, two antiapoptotic protein kinases, Raf-1 and Akt, are
inactivated by caspase-mediated proteolytic degradation (13).
Proteolytic cleavage of ATM generates a kinase-inactive protein and
prevents DNA repair and DNA damage signaling (14). Cleavage of focal
adhesion kinase by caspases interrupts the assembly of the focal
adhesion complex, resulting in cell death (15, 16). By contrast, the
activities of several kinases are stimulated by caspase cleavage, such
as MEKK-1 (17), PAK2/human PAK65 (18, 19), MST1 (20), and protein
kinase C isoforms
(21, 22) and
(23). In each case, caspase
cleavage generates a constitutively active kinase by removing
inhibitory domains from the proteins. Importantly, the active fragments
then act as signals propagating the apoptosis processes.
Etk/Bmx (epithelial and endothelial tyrosine
kinase or bone marrow tyrosine
kinase gene in chromosome X) is a member of the Btk
(Bruton's tyrosine kinase)
tyrosine kinase family (24, 25). Etk and three other members of this
family, Btk (26, 27), Itk (28), and Tec (29), share a common domain
structure including a pleckstrin homology
(PH)1 domain, an Src homology
3 (SH3) domain, an SH2 domain, and a catalytic tyrosine kinase domain
(30, 31). Each kinase has a unique expression pattern, and Etk is
commonly expressed in epithelial cells, endothelial cells, and
monocytes/macrophages including prostate tissues (24, 25, 32-34).
Recent studies suggest that various signals transmitted by the Btk
family members play central but diverse modulatory roles in cell growth
and differentiation (35-38).
Several lines of evidence implicate Btk family members in the apoptosis
pathway. Interestingly, both Btk and Etk are reported to be able to
induce pro- as well as antiapoptotic signals. For instance, Btk and Itk
are known to play vital roles in B cell and T cell development by
protecting these cells from apoptosis (30, 39, 40). Btk is also shown
to be an inhibitor of Fas-mediated apoptotic signal in B cells by its
direct association with the death receptor Fas, blocking the Fas-FADD
interaction (41). Consistent with its antiapoptotic role is the finding
that Btk activates a survival signal pathway involving Akt and NF
B
(42-44). In contrast, Btk is also involved in transmitting apoptosis
signals, since an engineered variant of B cell line DT-40, which lacks both alleles of Btk, became resistant to UV-induced apoptosis (45). Etk
also seems to play dual roles in apoptosis. Overexpression of Etk
renders prostate cancer cell line LNCaP more resistant to photodynamic
therapy or thapsigargin-induced apoptosis (46), whereas it sensitizes
mast cell line 32D toward apoptosis upon treatment with G-CSF (47). How
the Btk family kinases assume such a paradoxical role in apoptosis is
unclear. One possibility we considered is that posttranslational
modification converts these kinases into functionally opposite isoforms.
In this report, we explore the possibility that Etk can be converted to
a proapoptotic form after caspase cleavage. We provide evidence that
Etk is an in vitro and in vivo substrate for
caspase 3 during apoptosis. Using point mutants, we mapped the cleavage site to be Asp242 in the SH3 domain. The cleavage separates
the N-terminal PH and SH3 domains from the C-terminal SH2 and catalytic
domains. The C-terminal fragment of Etk exhibits increased kinase
activity and sensitizes prostate cancer PC3 cells toward apoptosis in
response to apoptosis-inducing stimuli. To our knowledge, this is the
first case where Btk family member is shown to be a caspase target. Our
findings provide a mechanistic explanation as to how Etk can both be
anti- and proapoptotic.
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EXPERIMENTAL PROCEDURES |
DNA Constructs--
Plasmids encoding the full-length Etk
(Etk/wt, residues 1-674) (24), C terminus-truncated Etk (Etk/N-I
residues 1-242), and N terminus-truncated Etk (Etk/C, residues
243-675) were constructed in pcDNA3.1/Myc-His vector
(Invitrogen). The kinase-dead mutant, Etk/K445Q, and two mutants for
testing caspase cleavage, Etk/D242A and Etk/D295A, were generated by
site-directed mutagenesis (Stratagene) employing Etk/wt as the
template. All of the Etk constructs have the T7 epitope at N termini
and the Myc epitope at C termini except Etk/C, which only has the Myc
epitope at the C terminus.
Cell Culture and Transfections--
All cell lines were
purchased from the ATCC and maintained at 37 °C with 5%
CO2. Jurkat and A431 cells were grown in RPMI and DMEM
medium, respectively, supplemented with 10% heat-inactivated fetal
calf serum and 100 µg/ml penicillin/streptomycin. Human umbilical
vein endothelial cells (HUVEC) were grown in an endothelial cell
culture medium supplemented with epidermal growth factor and
endothelial cell growth supplement (E-STIM; Becton Dickinson). Human
prostate cancer PC3 cells were grown in RPMI medium supplemented with
10% fetal bovine serum and 100 µg/ml penicillin/streptomycin. Subconfluent PC3 cells were transfected with FuGENE 6 reagent according
to the manufacturer's instructions (Roche Molecular Biochemicals). PC3
cells stably expressing various Etk constructs were selected in
medium containing 400 µg/ml G418. Individual clones were
picked up and analyzed for Etk expression by Western blotting using
anti-c-Myc monoclonal antibody (9E10, Babco) as described below.
Preparation of Cell-free Apoptotic Extracts--
To
induce apoptosis, Jurkat cells were treated with 250 ng/ml
anti-Fas monoclonal antibody (clone CH11; Upstate Biotechnology, Inc.)
(48) or 1 µM staurosporine (Calbiochem) (49) for 5 h. To trigger UV-induced apoptosis in A431 cells, the culture medium was removed, and cells were then irradiated (180 J/m2) with
UV Stratalinker (Stratagene, model 2400) (50). After irradiation, the
culture medium was added back to the cells and incubated for another
24 h. To detect endogenous Etk degradation during apoptosis,
HUVEC were treated with 1 µM staurosporine or 100 ng/ml
actinomycin D (Calbiochem) for 24 h (51, 52). To prepare cell-free
extracts, cells were lysed in lysis buffer (20 mM PIPES, pH
7.2, 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, 10%
sucrose, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 mM Na3VO4, 1 mM NaF, and 10 µg/ml
each of leupeptin, aprotinin, chymostatin, and pepstatin). After
incubation at 4 °C for 30 min, cellular debris was removed by
centrifugation. Protein concentrations were determined by Bradford
assay, and equal amounts of total lysates were used for further analyses.
In Vitro Cleavage with Apoptotic Cell Extracts or Recombinant
Caspases--
The TNT Quick Coupled Transcription/Translation System
(Promega) was used to synthesize [35S]methionine-labeled
proteins. 35S-labeled Etk proteins were incubated with
various apoptotic extracts or recombinant caspases (Pharmingen) at
37 °C for 1 h. For caspase inhibitor treatment, apoptotic
extracts were preincubated with Ac-DEVD-CHO or Ac-YVAD-CHO (Calbiochem)
at 37 °C for 15 min before 35S-labeled Etk proteins were
added. To perform the protein dephosphorylation experiment,
35S-labeled Etk proteins were first incubated with caspase
3 at 37 °C for 30 min. After that, reaction mixtures were incubated with 400 units of
phosphatase (New England BioLabs) in
phosphatase reaction buffer (50 mM Tris, pH 7.8, 5 mM dithiothreitol, and 2 mM
MnCl2) at 30 °C for another 30 min.
Immunoprecipitation and Western Blot
Analysis--
Immunoprecipitations were performed with anti-T7 tag
monoclonal antibody (Novagen) or anti-Etk polyclonal antibody (against Etk N terminus) (24) at 4 °C for 2-4 h. Protein A-agarose beads (Upstate Biotechnology, Inc., Lake Placid, NY) were added and incubated
for another 2 h at 4 °C. Beads were washed three times with
buffer containing 10 mM HEPES, pH 7.0, 2 mM
MgCl2, 50 mM NaCl, 5 mM EGTA, 0.1%
Triton X-100, and 60 mM 2-glycerophosphate. Immune
complexes or total cell lysates were resuspended in SDS sample buffer
and resolved by SDS-PAGE. Proteins were transferred to polyvinylidene
difluoride membranes (Millipore Corp.) and detected with anti-T7
antibody, anti-Etk antibody, or anti-
-tubulin antibody (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA). The complexed IgGs were detected
by incubation with secondary antibodies conjugated to horseradish
peroxidase and developed using the ECL system (Amersham Pharmacia Biotech).
Proteolytic Cleavage of Etk in PC3 Cells--
PC3 cells were
transiently transfected with plasmids encoding Etk/wt or Etk/D242A
proteins. Twenty-four hours after transfection, PC3 cells were treated
with 1 µg/ml anti-Fas antibody for various time periods. Both
the adherent and detached cells were harvested, washed with
phosphate-buffered saline, and lysed in lysis buffer as described
before. Equal amounts of total lysates were immunoprecipitated with
anti-Etk antibody. The immune complexes were resolved by SDS-PAGE
followed by immunoblotting with anti-T7 antibody.
In Vitro Kinase Assay--
Etk/wt, Etk/K445Q, Etk/D242A, and
Etk/C were in vitro translated in the presence of
[35S]methionine. Etk/N-I was in vitro
translated in the absence of [35S]methionine. Various Etk
proteins were immunoprecipitated with anti-Myc antibody. These immune
complexes were washed three times with wash buffer as described above.
Etk kinase activity assays were carried out in kinase reaction buffer
containing 30 mM PIPES, pH 7.0, 10 mM
MnCl2, 30 µM ATP, 1 µCi of
[
-32P]ATP, 1 mM
Na3VO4, and immunoprecipitated Etk/N-I as the
substrate. Following incubation for 10 min at 30 °C, kinase
reactions were terminated by adding SDS sample buffer and analyzed by
SDS-PAGE and autoradiography.
Flow Cytometry--
Parental PC3 and various PC3 Etk stable
clones were either untreated or treated with 250 ng/ml anti-Fas
antibody, UV irradiation (180 J/m2), or 10 ng/ml TNF-
(Upstate Biotechnology) plus 10 µg/ml cycloheximide (Sigma) for
24 h. Both floating and adherent cells were collected and washed
once with phosphate-buffered saline. Apoptotic cells were quantified by
flow cytometry analysis (Becton Dickinson FACStar Plus flow cytometer)
after cells were stained with fluorescein isothiocyanate-conjugated
annexin V and counterstained with propidium iodide as described by the
manufacturer (ApoAlert Annexin V Apoptosis Kit,
CLONTECH).
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RESULTS |
Etk Is Proteolytically Cleaved in Apoptotic Extracts--
To
examine whether Etk might serve as a substrate for caspases during
apoptosis, we first prepared the cell-free apoptotic extracts from
either Jurkat T cells or A431 cells as the source of caspases.
Previously, Jurkat T cells were shown to be hypersensitive to a wide
variety of apoptotic inducers, such as anti-Fas antibody (48) and
staurosporine (49). Similarly, apoptosis and caspase activation were
observed when human epidermoid carcinoma A431 cells were UV-irradiated
(50). In our study, treatment of Jurkat T cells with either anti-Fas
antibody or staurosporine and A431 cells with UV irradiation resulted
in ~40-60% cell death, with concurrent elevation of the activities
(3-5-fold) of caspases 3, 6, 8, and 9 (data not shown). The in
vitro translated [35S]methionine-labeled Etk was
incubated, respectively, with these apoptotic extracts. As shown in
Fig. 1, Etk was proteolytically degraded
in all three apoptotic extracts but remained intact in untreated
extracts. Strikingly, the cleavage patterns of Etk were identical in
all three extracts, as exemplified by a doublet around 50 kDa and a
single fragment around 35 kDa. We presumed that the elevated caspase
activities in these apoptotic extracts were responsible for the
cleavage of Etk.

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Fig. 1.
Specific Etk degradation in apoptotic
extracts is dependent on caspase activity. Jurkat T cells were
either left untreated ( ), treated with 250 ng/ml anti-Fas antibody,
or treated with 1 µM staurosporine for 5 h,
respectively. A431 cells were either left untreated ( ) or
UV-irradiated with 180 J/m2, and cells were harvested
24 h later. Cytoplasmic extracts were incubated with in
vitro translated [35S]methionine-labeled Etk at
37 °C for 1 h. Cleavage reactions were analyzed by SDS-PAGE
followed by autoradiography. The arrows indicate full-length
Etk (85 kDa) and cleaved products (a doublet at 50 kDa and a 35-kDa
band). C, C terminus; N-I, N terminus.
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To test whether the cleavage of endogenous Etk also occurs in cells
undergoing apoptosis, the HUVEC were examined. The reason is that
although Etk is expressed in multiple tissues, its protein level is
generally low. Among all of the cell lines we surveyed, HUVEC contain
the highest level of endogenous Etk, which increased the sensitivity of
our assay. HUVEC were treated with either staurosporine or actinomycin
D to induce apoptosis (51, 52). Cell lysates were prepared and
immunoprecipitated with anti-Etk antibody raised against the PH domain
of Etk (24). Immunoblot analysis of immunoprecipitated complexes with
the same Etk antibody revealed a dramatic decrease of full-length Etk
in staurosporine and actinomycin D-treated cells (Fig.
2). However, we were unable to detect the
cleavage products, presumably due to the instability of the fragments
in this cell type under the experimental conditions.

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Fig. 2.
Endogenous Etk cleavage in staurosporine- or
actinomycin D-induced apoptotic HUVEC. HUVEC were treated with 1 µM staurosporine or 100 ng/ml actinomycin D for 24 h
to induce apoptosis. Cell lysates were immunoprecipitated by
polyclonal antibody against the PH domain of Etk. Immunocomplexes were
separated by SDS-PAGE followed by Western blot analysis with the same
Etk antibody. Equal amounts of proteins applied for immunoprecipitation
were confirmed by immunoblotting with anti- -tubulin antibody
(lower panel).
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Etk Is a Direct Substrate for Caspase 3--
Given the existence
of at least 14 caspases of diverse specificity (6), the specific
cleavage pattern of Etk (Fig. 1) points to the action of a selected set
of caspases. This prompted us to investigate which caspase(s) is
responsible for Etk degradation during apoptosis. Two approaches were
taken. First, caspase inhibitors at different concentrations were used
to discern the possible caspase(s) involved. As shown in Fig.
3A, cleavage of
[35S]methionine-labeled Etk in anti-Fas antibody or
staurosporine-treated apoptotic extracts (not shown) could be
totally blocked by Ac-DEVD-CHO (an inhibitor for caspase 3) at a
concentration as low as 0.1 µM, whereas it took a higher
concentration of caspase 1 inhibitor (Ac-YVAD-CHO) to achieve the same
goal. These data suggest that Etk cleavage is preferentially carried
out by DEVD-sensitive caspase(s) such as caspase 3 or caspase 3-like
proteases.

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Fig. 3.
Etk is sensitive to cleavage by caspase 3 in vitro. A, anti-Fas antibody-treated
Jurkat cell extracts were preincubated with the indicated
concentrations of Ac-DEVD-CHO or Ac-YVAD-CHO for 15 min at 37 °C.
Reaction mixtures were then incubated with
[35S]methionine-labeled Etk for 1 h at 37 °C.
Reaction products were analyzed by SDS-PAGE followed by
autoradiography. B, [35S]methionine-labeled
Etk was incubated with recombinant caspase 3, 6, 7, or 8 in the
indicated concentrations for 1 h at 37 °C, respectively.
Samples were analyzed by SDS-PAGE followed by autoradiography. When Etk
was incubated with higher concentrations of caspase 3 (20 and 100 ng),
the 50-kDa doublet (Etk/C) remained the same, but the 35-kDa (Etk/N-I)
cleavage product was further truncated to a 26-kDa band (Etk/N-II) as
indicated by the arrows.
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We also examined whether Etk could be cleaved by purified recombinant
caspases in vitro and, if so, whether the degradation patterns mirror that observed in apoptotic extracts. Fig. 3B
showed that [35S]methionine-labeled Etk could be cleaved
by caspase 3 at a concentration as low as 2 ng. By contrast, caspases 6 and 7 have no effect on Etk at the concentrations tested, and caspase 8 is able to cleave Etk only at a relatively high concentration. These
results strongly suggest that caspase 3 (or caspase 3-like proteases)
is more likely the enzyme that cleaves Etk and that the proteolytic
cleavage of Etk is a general feature in apoptotic cells.
In addition, it has been shown previously that phosphorylation of
presenilin-2 regulates its cleavage by caspases (53). To verify if the
autophosphorylation of Etk is required for caspase-mediated cleavage,
the invariant lysine in the ATP binding pocket of Etk was mutated to
glutamine. The resultant mutant, Etk/K445Q, has neither kinase activity
(see Fig. 8) nor autophosphorylation ability (data not shown) as
determined by an in vitro kinase activity assay. This mutant
was in vitro translated and treated with either apoptotic
extracts or recombinant caspases. The kinetics of Etk/K445Q degradation
was identical to that when Etk/wt was used as the target (data not
shown), indicating that neither the kinase activity nor
autophosphorylation of Etk was required for caspases cleavage.
To address the relative sensitivity of Etk cleavage by caspase 3, we
compared the cleavage kinetics with one of the well known caspase 3 targets, D4GDI (49). [35S]methionine-labeled Etk and
D4GDI (with similar molar ratio) were incubated respectively with
increasing amounts of recombinant caspase 3. The results showed that
both Etk and D4GDI were partially cleaved under low concentrations of
caspase 3. Etk was almost completely cleaved by 5 ng of caspase 3, whereas the cleavage of D4GDI was only about 30% under the same
conditions. Even with an excess amount of caspase 3 (10 ng), only about
50% of D4GDI was cleaved (Fig. 4). The
data indicate that Etk is a better substrate for caspase 3 when
compared with D4GDI.

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Fig. 4.
Relative caspase 3 cleavage kinetics of Etk
and D4GDI. [35S]Methionine-labeled Etk and D4GDI
were incubated alone or with increasing amounts (0.5-10 ng) of
recombinant caspase 3 for 1 h at 37 °C, respectively. Reaction
mixtures were separated by SDS-PAGE followed by autoradiography.
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Mapping the Caspase Cleavage Site in Etk--
When
[35S]methionine-labeled Etk was incubated with either
apoptotic extracts or recombinant caspase 3, a closely spaced 50-kDa doublet was generated (Figs. 1 and 3). The doublet may represent two
distinct cleavage products with similar sizes or the same product with
a different extent of posttranslational modification such as
phosphorylation. To distinguish these possibilities,
-phosphatase treatment was performed on either uncleaved or recombinant caspase 3-cleaved [35S]methionine-labeled Etk. Both the doublets
at 85 kDa (full-length Etk) and 50 kDa (cleavage products) became
single bands, indicating that the upper bands are the phosphorylated
forms of Etk (Fig. 5A). These
data suggest that the the 50-kDa doublet and the 35-kDa fragment were
derived from a single caspase cleavage event.

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Fig. 5.
Identification of the
caspase 3 cleavage site in Etk. A, to examine the
effect of phosphatase treatment on Etk electrophoretic mobility,
[35S]methionine-labeled Etk was first incubated with
recombinant caspase 3 for 30 min at 37 °C. Reaction mixtures were
then treated with or without phosphatase for 30 min at 30 °C.
The arrows indicate full-length Etk (a doublet at 85 kDa)
and cleaved products (a doublet at 50 kDa and a 35-kDa band).
Asterisks show the phosphorylation forms of Etk.
B, single mutations of Asp to Ala at amino acids 242 and 295 were introduced into Etk, respectively.
[35S]Methionine-labeled Etk/wt or mutants (D242A and
D295A) were incubated with 10 ng of recombinant caspase 3 for 1 h
at 37 °C. Both Etk/wt and Etk/D295A, but not Etk/D242A, had cleavage
products, Etk/C and Etk/N-I, as indicated by arrows.
C, [35S]methionine-labeled Etk/wt or Etk/D242A
was incubated with anti-Fas antibody-induced apoptotic Jurkat cell
extracts for 1 h at 37 °C. Etk/D242A showed no cleavage
products.
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We noticed that Etk contains three potential caspase 3 recognition
motifs, DXXD, which were found at the cleavage site
of many proteins during apoptosis (8). These sequences are
DFPD242
W, DDYD295
W, and
DLYD617
N (the arrows indicate the potential
cleavage sites in the amino acid sequence). A diagrammatic
representation of the protein domain structure and potential cleavage
sites of Etk is shown in Fig. 6A. Based on the size of
cleavage products, D242
W and D295
W are
likely candidate cleavage sites. These two sites were therefore individually mutated to alanines, and their susceptibility to cleavage
by caspases was tested. Etk/wt and these two mutants, Etk/D242A and
Etk/D295A, were in vitro translated and incubated with
recombinant caspases. Etk/wt and Etk/D295A, but not Etk/D242A, were
cleaved by either caspase 3 or 8 (Fig. 5B and data not
shown). Similar experiments were carried out using cell-free apoptotic extracts. Again, Etk/wt and Etk/D295A, but not Etk/D242A, were proteolyzed in these apoptotic extracts (Fig. 5C and data
not shown).

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Fig. 6.
Schematic diagram of putative caspase 3 cleavage sites in Etk and Btk. Several key domains including the
PH, SH3, SH2, and tyrosine kinase domains in Etk (A) and Btk
(B) are illustrated. Three putative caspase 3 cleavage sites
at DFPD242 W, DDYD295 W, and
DLYD617 N are present in Etk (A). There
are two putative caspase 3 cleavage sites, DPKD401 L and
DVMD656 E, in Btk (B).
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To further examine whether Asp242 is the bona
fide cleavage site in vivo during apoptosis, Etk/wt and
Etk/D242A, which were N-terminally tagged with T7 epitope, were
ectopically expressed in cultured cells. Due to the low transfection
efficiency of HUVEC, we used PC3, a prostate cancer cell line known to
express a low level of Etk, as the test system. Transfected PC3 cells
were treated with anti-Fas antibody for various time points to induce
apoptosis. Cell lysates prepared from different time points were
immunoprecipitated with anti-Etk antibody and immunoblotted with
anti-T7 antibody. The time course data revealed that Etk/wt but not
Etk/D242A was degraded within 2 h upon anti-Fas antibody treatment
as illustrated by the appearance of cleavage products, Etk/N-I and
Etk/N-II (Fig. 7). The sizes of Etk/N-I
and Etk/N-II were identical to those bands cleaved by a high
concentration of recombinant caspase 3 (Fig. 3B). Taken
together, the data confirm the identity of the cleavage site mapped
in vitro. The absence of Etk/N-II in D242A-expressing samples suggests that caspase cleavage of Etk after Asp242
is necessary for the exposure of the second caspase cleavage site. The
second cleavage site, however, remains unclear at this time.

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Fig. 7.
Etk/D242A mutant is resistant to degradation
in PC3 cells by anti-Fas antibody stimulation. PC3 cells were
transiently transfected with Etk/wt or caspase 3-resistant Etk/D242A
mutant. Both Etk proteins were T7 epitope-tagged at N termini.
Twenty-four hours after transfection, cells were left untreated or
treated with anti-Fas antibody (1 µg/ml) for different time intervals
(0-8 h). Cell lysates were prepared and immunoprecipitated with
anti-Etk antibody. Samples were separated by SDS-PAGE and analyzed by
Western blot with anti-T7 antibody.
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Activation of Etk after Caspase 3-mediated
Proteolysis--
Caspase 3-mediated cleavage of Etk generated two
fragments. The N-terminal fragment contains the complete PH and partial
SH3 domains (amino acids 1-242, referred to as Etk/N-I). The
C-terminal fragment contains partial SH3, complete SH2, and tyrosine
kinase domains (amino acids 243-675, termed Etk/C) (Fig.
6A). Based on the current model of regulation of Btk family
kinases (54) in the uninduced state, the C-terminal catalytic domain
interacts with the N-terminal PH domain, thus assuming a "closed"
form. The removal of the N-terminal 1-242 residues from the C-terminal catalytic domain by caspase cleavage might result in kinase activation of Etk. To test this possibility, in vitro kinase activity
assays were performed by using a peptide (Etk/N-I), which carries the autophosphorylation site of Etk, as a substrate. This experiment was
modeled after a similar one used successfully to measure the kinase
activity of Btk (55, 56). A series of C-terminally Myc-tagged Etk
constructs, including Etk/wt, Etk/K445Q, Etk/D242A, and Etk/C (Fig.
6A), were in vitro translated in the presence of
[35S]methionine. This strategy overcame the difficulty
encountered in the immunoprecipitation experiment (57) that was due to
comigration of IgG heavy chain with Etk/C in SDS-PAGE. This system
permits quantification of the amount of each sample as illustrated by Fig. 8. All of these samples were
immunoprecipitated with anti-Myc antibody, and the immunocomplexes were
subjected to kinase assay. The kinase activity of Etk/C was about
4-fold higher than that of Etk/wt or Etk/D242A (after calibration of
the methionine contents of Etk/wt and Etk/C) (Fig. 8). The most
plausible explanation of these results is that caspase cleavage removes
an inhibitory N-terminal regulatory domain (containing the PH domain)
from Etk, thereby generating an active kinase. This is consistent with
our earlier data showing that deletion of the PH domain leads to a constitutively active Etk (24, 58).

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Fig. 8.
Caspase 3-mediated proteolytic activation of
Etk. Myc epitope-tagged Etk proteins, including Etk/wt
(wt), catalytic inactive mutant Etk/K445Q (KQ),
Etk/C (amino acids 243-675) (C/WT), and Etk/D242A
(D242A) were in vitro translated in the presence
of [35S]methionine and immunoprecipitated with anti-Myc
antibody. Equal amounts of each immunoprecipitate were used for kinase
assay as judged by 35S intensity (upper panel).
A C-terminal truncated Etk, Etk/N-I (containing amino acids 1-242),
with T7 epitope tag was in vitro translated in the absence
of [35S]methionine and immunoprecipitated with anti-T7
antibody. These Etk/N-I immunoprecipitates were served as substrates
for various Etk proteins during kinase activity assays. Kinase activity
assays were proceeded in the presence of [ -32P]ATP for
10 min at 30 °C. Reactions were resolved by SDS-PAGE followed by
autoradiography.
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Proteolytic Activation of Etk Potentiates Apoptosis--
We next
investigated whether constitutively active Etk/C might contribute to
the demise of the cells. Initial experiments showed that ectopic
expression of Etk/C alone either in PC3, HeLa, or 293T cells did not
induce apoptosis, suggesting that the truncated Etk itself is not a
trigger for apoptosis (data not shown). As a result, we were able to
isolate stable clones of PC3 transfected with either Etk/wt or Etk/C.
Three clones with comparable protein expression levels (as judged by
Western blotting) were selected for each construct. PC3 is known to be
rather resistant to apoptosis (59). We therefore asked whether Etk/C
could sensitize PC3 cells toward apoptosis induced by Fas antibody, UV,
or TNF-
plus cycloheximide. Cells were either untreated or treated
with these agents, and the extent of apoptosis was determined by
annexin V and propidium iodide binding assay. Results showed that the
Etk/C-overexpressing clones are significantly more sensitive to
apoptotic stimuli than Etk/wt with a consistent 63-73% increase in
the number of dying cells (Fig. 9). The
fractional increase of apoptotic cells in this experiment is comparable
with those reported previously with other caspase-activated kinases
(19, 20). The finding suggests that the truncated Etk is proapoptotic,
at least under the conditions we analyzed.

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Fig. 9.
Proteolytic activation of Etk potentiates
apoptotic stimuli induced cell death in PC3 cells. Prostate cancer
cell line PC3-derived stable clones, Etk/wt and Etk/C, were either left
untreated or treated with anti-Fas antibody, TNF- plus cycloheximide
(CHX), or UV irradiation for 24 h. Both attached and
floating cells were collected and stained with fluorescein
isothiocyanate-conjugated annexin V and propidium iodide.
Light bars show the percentages of cells in the
early phase of apoptosis, which are annexin V-positive and propidium
iodide-negative. Dark bars show the percentages
of cells in the late phase of apoptosis, which are annexin V- and
propidium iodide-double positive. The data represent means of three
stable clones from each Etk construct and four separate culture
experiments.
|
|
 |
DISCUSSION |
There is very strong evidence that caspases participate in the
apoptotic process. The identification of caspase substrates provides a
means to understand the often complex and diverse apoptotic pathways.
In this study, we describe a tyrosine kinase Etk as a direct substrate
for caspases. Both recombinant caspases 3 and 8 cleave Etk, although
with different efficiency, and generate an identical cleavage pattern,
suggesting some degree of functional redundancy within the caspase
family (8). The data also raise the possibility that Etk can
alternatively be proteolyzed by caspase 8 in some caspase 3-deficient
cells. A single amino acid change from Asp242 to Ala
abolishes such a cleavage and unambiguously identifies the cleavage
site to be DFPD242
W. While this sequence does not match
any known caspase cleavage sequence, DXXD is a consensus
recognition motif of caspase (8, 60). It is interesting that among the
Btk family kinases, the DFPD
W sequence is uniquely present in Etk
despite the overall structural homology of Btk family kinases. There
are, however, two putative caspase 3 cleavage motifs in Btk. One of
them, DPKD401
L, is located in the proximity of the
kinase domain (Fig. 6B), the cleavage of which should in
theory also generate an active kinase without a PH domain. The kinase
activity of Etk/C is 4 times higher than that of the wild type. This
activation is similar in magnitude to that caused by either deletion of
the PH domain (24) or competitive binding of PTPD1 to the PH domain
(61). All of these data are consistent with the model that the PH
domain negatively regulates the kinase activity of Etk in a manner
similar to that proposed for Itk (54).
The proteolysis of ectopically expressed Etk/wt occurs within 2 h
in response to anti-Fas antibody treatment (Fig. 7). However, at this
time point, no biochemical or morphological manifestations of apoptotic
cell death could be observed, suggesting that Etk cleavage and its
activation may be involved in the apoptosis process. This is
reminiscent of several other protein kinases, such as MEKK-1 (17) and
PAK2/human PAK65 (18, 19), that are also activated by caspase-mediated
cleavage. A common feature of these kinases is that overexpression of
caspase-truncated kinase induces apoptosis of cells. In our case,
however, introduction of caspase-truncated Etk/C does not commit the
cell to apoptotic demise but enhances apoptosis induced by
extracellular stimuli (Fig. 9). These data suggest that Etk might not
play a role in the initiation of apoptosis but contributes to the
propagation or amplification of the apoptosis signal. How Etk does this
is presently unclear. It is conceivable that the cleavage and
activation of Etk might deregulate the activity of downstream targets.
This is supported by the findings that apoptosis induced by Fas
ligation (62, 63) and TNF (64, 65) results in rapid tyrosine kinase
activation and tyrosine phosphorylation of multiple cellular proteins
and that tyrosine kinase inhibitors can significantly reduce apoptosis
(62, 63).
Etk and Btk were shown to have both antiapoptotic (41-44,
46) and proapoptotic (45) potential (37). This suggests that Btk family
kinases may act as apoptotic switches and that their activities can
influence both pro- and antiapoptotic signal molecules. Depending on
the cell contexts and the forms of the kinases, as the present study
suggests, the balance could be tilted in either direction. In this
regard, it is interesting to note that Btk was shown to inhibit B cell
apoptosis induced by Fas ligation (41). This inhibition is due to a
direct interaction between Fas and Btk, via the PH domain, which
interferes with Fas-FADD association. The activated and associated Btk
kinase is apparently important in this inhibition process. If, however,
Btk is cleaved by a caspase, which separates the PH domain from the
kinase domain, the apoptosis process will be enhanced. This could be
one reason why, in Fas-mediated apoptosis, Btk family kinases could be
both anti- and proapoptotic.
In summary, we show in this report that a tyrosine kinase, Etk/Bmx, can
be a substrate of a caspase(s). The resulting truncated molecule
contains an intact SH2 domain and kinase domain. This molecule has an
enhanced kinase activity and, while not apoptotic on its own, possesses
an ability to enhance apoptosis induced by other agents. Our findings
offer an alternative mechanism to explain the dual roles of Etk (or Btk
family kinases in general) in apoptosis. The identification of new
substrate(s) and interacting protein(s) as the consequence of Etk
cleavage should provide insights into the apoptosis mediated by these
family kinases.