From the Parker Hughes Cancer Center, Molecular Signal Transduction Laboratory, Departments of Immunology, Molecular Biology, and Biochemistry, Hughes Institute, St. Paul, Minnesota 55113
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ABSTRACT |
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Bruton's tyrosine kinase (BTK) is a
member of the Src-related Tec family of protein tyrosine kinases.
Mutations in the btk gene have been linked to severe
developmental blocks in human B-cell ontogeny leading to X-linked
agammaglobulinemia. Here, we provide unique biochemical and genetic
evidence that BTK is an inhibitor of the Fas/APO-1 death-inducing
signaling complex in B-lineage lymphoid cells. The Src homology 2, pleckstrin homology (PH), and kinase domains of BTK are all
individually important and apparently indispensable, but not
sufficient, for its function as a negative regulator of Fas-mediated
apoptosis. BTK associates with Fas via its kinase and PH domains and
prevents the FAS-FADD interaction, which is essential for the
recruitment and activation of FLICE by Fas during the apoptotic signal.
Fas-resistant DT-40 lymphoma B-cells rendered BTK-deficient through
targeted disruption of the btk gene by homologous
recombination knockout underwent apoptosis after Fas ligation, but
wild-type DT-40 cells or BTK-deficient DT-40 cells reconstituted with
wild-type human btk gene did not. Introduction of an Src
homology 2 domain, a PH domain, or a kinase domain mutant human
btk gene into BTK-deficient cells did not restore the
resistance to Fas-mediated apoptosis. Introduction of wild-type BTK
protein by electroporation rendered BTK-deficient DT-40 cells resistant
to the apoptotic effects of Fas ligation. BTK-deficient RAMOS-1 human
Burkitt's leukemia cells underwent apoptosis after Fas ligation,
whereas BTK-positive NALM-6-UM1 human B-cell precursor leukemia cells
expressing similar levels of Fas did not. Treatment of the
anti-Fas-resistant NALM-6-UM1 cells with the leflunomide metabolite
analog Apoptosis is a common mode of eukaryotic cell death which is
triggered by an inducible cascade of biochemical events leading to
activation of endonucleases that cleave the nuclear DNA into oligonucleosome-length fragments (1-3). Several of the biochemical events that contribute to apoptotic cell death as well as both positive
and negative regulators of apoptosis have recently been identified
(1-4). Apoptosis plays a pivotal role in the development and
maintenance of a functional immune system by ensuring the timely
self-destruction of autoreactive immature and mature lymphocytes as
well as any emerging target neoplastic cells by cytotoxic T-cells (1-7). Inappropriate apoptosis may contribute to the development as
well as chemotherapy resistance of human leukemias and lymphomas (5-7). Therefore, an improved understanding of the molecular basis of
apoptosis and the pro-apoptotic versus anti-apoptotic regulatory signals may provide further insights into the pathogenesis of human lymphoid malignancies and have important implications for
treatment of leukemias and lymphomas.
The Fas/APO-1 (CD95) cell surface receptor, a member of the tumor
necrosis factor (TNF)1
receptor family, is one of the major regulators of apoptosis in a
variety of cell types (8-11). Functional abnormalities of Fas have
been associated with pathologic conditions of the immune system
homeostasis, including lymphoproliferative disorders,
immunodeficiencies, and autoimmunity (10, 11). Identifying the
molecules that participate in the apoptotic death signal pathways
linked to the Fas receptor and finding ways to modulate the activity of
such molecules could provide the basis for innovative treatment
programs. Ligation of the cell surface Fas molecule rapidly and
dramatically induces apoptosis in many but not all Fas-positive cell
types (8). DT-40 is a chicken lymphoma B-cell line that we have used previously to elucidate the molecular mechanism of radiation-induced apoptosis (12). Despite their abundant surface expression of Fas, DT-40
cells, similar to human B-cell precursor leukemia cells, are very
resistant to the cytotoxic effects of Fas ligation, indicating the
existence of potent negative regulators of Fas-mediated apoptosis.
Bruton's tyrosine kinase (BTK) is a member of the Src-related Tec
family of protein tyrosine kinases (PTK) (13, 14). Mutations in the
btk gene have been linked to severe developmental blocks in
human B-cell ontogeny leading to human X-linked agammaglobulinemia (15,
16) and less severe deficiencies in murine B-cells leading to murine
X-linked immune deficiency (17). Recent studies implicated BTK as a
pro-apoptotic enzyme in B-lineage lymphoid cells exposed to
ionizing radiation (12) as well as mast cells deprived of growth
factors (18). In murine B-cells, BTK has also been shown to act as an
anti-apoptotic protein upstream of bcl-xL in the B-cell antigen
receptor (but not the CD40 receptor) activation pathway (19). Because
of the recently discovered but not well understood ability of BTK to
act both as a positive and negative regulator of apoptosis after
ionizing radiation, growth factor deprivation, or B-cell antigen
receptor signaling and its abundant expression in DT-40 cells (12), we
investigated whether BTK plays a role in the pronounced resistance of
DT-40 cells as well as human leukemic B-cell precursors against
Fas-mediated apoptosis. Our study provides biochemical and genetic
evidence that BTK is an inhibitor the Fas/APO-1 death-inducing
signaling complex (DISC) in B-lineage lymphoid cells. BTK associates
with Fas via its kinase and pleckstrin homology (PH) domains and
prevents the FAS-FADD interaction, which is essential for the
recruitment and activation of FLICE by Fas during the apoptotic signal.
Notably, treatment of human leukemic B-cell precursors with a potent
inhibitor of BTK abrogated the BTK-Fas association and sensitized the
cells to Fas-mediated apoptosis.
Cell Lines, Reagents, and Biochemical Assays--
The
establishment of BTK-deficient DT-40 lymphoma B-cell clones has been
described previously (12). To disrupt the btk gene, targeting constructs containing the neomycin resistance gene cassette (i.e. pcBTK-neo) or histidinol resistance gene cassette
(i.e. pcBTK-hisD) were sequentially transfected into DT-40
cells. The targeting vectors, pcBTK-neo and pcBTK-hisD, were
constructed by replacing the 0.7-kilobase
BglII-BamHI genomic fragment containing exons
that correspond to human BTK amino acid residues 91-124 with the
neo or hisD cassette. pcBTK-neo was linearized
and introduced into wild-type DT-40 cells by electroporation. Screening
was done by Southern blot analysis using a 3'-flanking probe
(0.5-kilobase BglII-Bgl-II fragment). The neo-targeted clone
was again transfected with pcBTK-hisD and selected with both G418 (2 mg/ml) and histidinol (1 mg/ml). Southern blot analysis of a
BTK-deficient DT-40 clone confirmed the homologous recombination at
both btk loci, and hybridization with a neo and hisD probe
indicated that the targeted clone had incorporated a single copy of
each construct. Lack of BTK expression in BTK-deficient DT-40 cells was
confirmed by both immune complex kinase assays and Western blot
analysis (12). Mutations in the human btk cDNA were
introduced by polymerase chain reaction using Pfu polymerase
(Strategene) and confirmed by sequencing. Wild-type and mutant
btk cDNAs were subcloned into pApuro expression vector and electroporated into BTK-deficient cells. The PTK activity of BTK
immune complexes, as measured by in vitro
autophosphorylation, was abrogated by the catalytic domain mutation,
reduced by the PH domain mutation, but not affected by the mutation in
the Src homology 2 (SH2) domain. Equal amounts of BTK protein were
detected by Western blot analysis in all of the BTK-deficient DT-40
clones transfected with wild-type or mutated human btk
genes, but no BTK protein was detectable in the untransfected
BTK-deficient DT-40 cells (12). The establishment and characterization
of LYN-deficient DT-40 clones were reported previously (12). In addition to these chicken lymphoma B-cells, we also used the following human B-lineage lymphoid cell lines: NALM-6-UM1, a BTK-positive human
B-cell precursor (pre-B acute lymphoblastic leukemia) cell line;
RAMOS-1, a BTK-deficient human Burkitt's/B-cell leukemia line; and
KL2, a BTK-positive human Epstein-Barr virus-transformed normal
B-lymphoblastoid cell line.
Antibodies directed against BTK, SYK, and LYN have been described
previously (12, 20, 21). Polyclonal antibodies to BTK were
generated by immunization of rabbits with glutathione S-transferase (GST) fusion proteins (Amersham Pharmacia
Biotech) containing the first 150 amino acids of BTK. In addition, we
used the following anti-BTK antibodies in Western blots of purified fusion proteins: polyclonal goat anti-BTK carboxyl terminus (Santa Cruz
Biotechnology), polyclonal goat anti-BTK amino terminus (Santa Cruz
Biotechnology), and polyclonal rabbit serum raised against the BTK
SH2-SH3 domains (amino acids 219-377). Polyclonal anti-MBP (maltose-binding protein) antibodies were generated by immunizing rabbits. The rabbit polyclonal anti-Fas (sc-715 mixed 1:1 with sc-714),
which cross-reacts with both human and chicken Fas proteins, goat
polyclonal anti-FADD (sc-1171), goat polyclonal anti-TRADD (sc-1163),
and goat polyclonal anti-FLICE (sc-6135) were purchased from Santa Cruz
Biotechnology and used according to the manufacturer's recommendations. The monoclonal anti-Fas antibody (F22120) was obtained
from the Transduction Laboratories, Inc. (Lexington, KY).
Immunoprecipitations, immune complex protein kinase assays, and
immunoblotting using the enhanced chemiluminescence (ECL) detection
system (Amersham Pharmacia Biotech) were conducted as described
previously (12, 20-25). The BTK inhibitor
Expression and Purification of MBP-BTK and GST-BTK Fusion
Proteins--
cDNAs encoding full-length BTK and its kinase or PH
domains with polymerase chain reaction-generated 5'- and
3'-BamHI sites were cloned into the Escherichia
coli expression vector pMAL-C2 with the isopropyl
1-thio- Confocal Laser Scanning Microscopy--
Wild-type and
BTK-deficient DT-40 cells treated with anti-Fas antibody (1 µg/ml,
24 h at 37 °C) were attached to
poly-L-lysine-coated coverslips and fixed in ice-cold
( Apoptosis Assays--
To induce apoptosis, cells were treated
with an agonistic anti-Fas/APO-1 antibody (Bender MedSystems) at a 0.1, 0.5, or 1.0 µg/ml final concentration. MC540 binding (as an early
marker of apoptosis) and propidium iodide (PI) permeability (as a
marker of advanced stage apoptosis) were measured simultaneously in
DT-40 cells 24 h after exposure to anti-Fas antibody, as described
previously (12). Whole cells were analyzed with a FACStar Plus flow
cytometer (Becton Dickinson, San Jose, CA). All analyses were done
using 488 nm excitation from an argon laser. MC540 and PI emissions were split with a 600 nm short pass dichroic mirror. A 575 nm band pass
filter was placed in front of one photomultiplier tube to measure MC540
emission, and a 635 nm band pass filter was used for PI emission.
To detect apoptotic fragmentation of DNA, DT-40, NALM-6-UM1, and
RAMOS-1 cells were harvested 24 h after exposure to anti-Fas. DNA
was prepared from Triton X-100 lysates for analysis of fragmentation (12, 24, 27). In brief, cells were lysed in hypotonic 10 mmol/liter
Tris-HCl, pH 7.4, 1 mmol/liter EDTA, 0.2% Triton X-100 detergent and
subsequently centrifuged at 11,000 × g. To detect apoptosis-associated DNA fragmentation, supernatants were
electrophoresed on a 1.2% agarose gel, and the DNA fragments were
visualized by ultraviolet light after staining with ethidium bromide.
In some experiments, MBP-BTK fusion proteins (100 µg/2.5 × 108 cells) were electroporated (420-V electrical field, 125 microfarads) into BTK-deficient DT-40 cells using a Bio-Rad gene pulser
and the procedures of Bergland and Starkey (28) with slight
modifications 4 h before Fas ligation and apoptosis assays.
Pull-down Assays with MBP-BTK and GST-BTK Fusion
Proteins--
GST-BTK fusion proteins were noncovalently bound to
glutathione-agarose beads (Sigma), and MBP-BTK fusion proteins were
noncovalently bound to amylose beads under conditions of saturating
protein, as described previously (21, 26). In brief, 50 µg of each protein was incubated with 50 µl of the beads for 2 h at
4 °C. The beads were washed three times with 1% Nonidet P-40
buffer. Nonidet P-40 lysates of BTK-deficient DT-40 cells, NALM-6-UM1 human leukemic B-cell precursors, and KL2 human Epstein-Barr
virus-transformed lymphoblastoid cells were prepared as described (12,
21), and 500 µg of the lysate was incubated with 50 µl of fusion
protein-coupled beads for 2 h on ice. The fusion protein
adsorbates were washed with ice-cold 1% Nonidet P-40 buffer and
resuspended in reducing SDS sample buffer. Samples were boiled for 5 min and then fractionated on SDS-polyacrylamide gel electrophoresis.
Proteins were transferred to Immobilon-P (Millipore) membranes, and the
membranes were immunoblotted with anti-Fas (F22120, Transduction
Laboratories), according to procedures described previously (12, 20,
21, 25).
In a series of experiments designed to examine the potential
negative regulatory role of BTK in Fas-mediated apoptosis, we first
compared the effects of Fas ligation on wild-type DT-40 cells with the
effects of Fas ligation on a BTK-deficient subclone of DT-40 cells
which was established by homologous recombination knockout (12). To
this end, we first used a quantitative flow cytometric apoptosis
detection assay (12). MC540 binding and PI permeability were measured
simultaneously before and after treatment with the agonistic anti-Fas
antibody (1 µg/ml × 24 h). Only 5.0% of wild-type DT-40
cells treated with the anti-Fas antibody showed apoptotic changes,
whereas 96.3% of BTK-deficient DT-40 cells underwent apoptosis, as
determined by MC540 single fluorescence (early apoptosis) or MC540/PI
double fluorescence (advanced apoptosis) at 24 h (Fig.
1). Notably, BTK-deficient DT-40 cells
reconstituted with a wild-type human btk gene displayed very
little flow cytometric evidence of apoptosis, which provided formal
evidence that BTK plays a pivotal role in preventing the apoptotic
death signal triggered by Fas ligation. In accordance with previously
published information regarding the pro-apoptotic function of Src
family PTK (29-31) and the reported impairment of Fas-mediated
apoptosis in B-cells from LYN-deficient mice (32), very little
apoptosis was found in an anti-Fas-treated LYN-deficient subclone of
DT-40 cells which was included as a control in these experiments (Fig. 1). As shown in Fig. 2A, no
BTK protein was detectable by Western blot analysis in the whole cell
lysates of BTK-deficient DT-40 cells, whereas BTK-deficient DT-40 cells
reconstituted with a wild-type human btk gene expressed
higher levels of BTK than the wild-type DT-40 cells. However, the Fas
protein expression levels in these three B-cell clones were virtually
identical (Fig. 2B). These findings were confirmed further
by confocal microscopy. As shown in panels C1-C3, all three
cell lines exhibited similar levels of punctate Fas staining.
Three-dimensional reconstructions of serial optical sections confirmed
the expression of Fas both in the cytoplasm and on the surface membrane
of all three cell lines without any detectable difference relative to
expression levels or pattern. Thus, the resistance of wild-type DT-40
cells or BTK-deficient DT-40 cells reconstituted with wild-type BTK against Fas-mediated apoptosis was not caused by lower expression levels of Fas protein, and the susceptibility of BTK-deficient DT-40
cells to Fas-mediated apoptosis was not caused by augmented Fas protein
expression.
-cyano-
-methyl-
-hydroxy-N-(2, 5-dibromophenyl)propenamide, a potent inhibitor of BTK, abrogated the
BTK-Fas association without affecting the expression levels of BTK or
Fas and rendered them sensitive to Fas-mediated apoptosis. The ability
of BTK to inhibit the pro-apoptotic effects of Fas ligation prompts the
hypothesis that apoptosis of developing B-cell precursors during normal
B-cell ontogeny may be reciprocally regulated by Fas and BTK.
INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References
-cyano-
-methyl-
-hydroxy-N-(2, 5-dibromophenyl)propenamide) (LFM-A13; IC50 for BTK,
2.5 µM; IC50 values for epidermal growth
factor receptor, insulin receptor, JAK-1, JAK-2, JAK-3, SYK, HCK, all
>300 µM) was a kind gift from Dr. Yaguo Zheng from the
Department of Chemistry at the Hughes Institute. The analytical
physicochemical data for LMA-13 were: yield: 78% from tetrahydrofuran;
mp: 148-150 °C; IR (KBr): 3,353, 2,211, 1,648, and 1,590 cm
1; UV-visible: 220, 245, and 296 nm; 1H NMR
(dimethyl sulfoxide-d6):
11.41 (s, 1H, NH),
8.57 (m, 1H, ArH), 7.55 (d, J = 8.7 Hz, 1H, ArH), 7.14 (q, J1 = 6.0 Hz, J2 = 2.4 Hz, 1H, ArH), 7.10 (s br, 1H, OH), 2.17 (s, 3H, CH3);
electron ionization mass spectrometry m/z
[M]+: 358.
-D-galactopyranoside-inducible Ptac promoter to
create an in-frame fusion between these coding sequences and the 3'-end
of the E. coli malE gene, which codes for MBP. cDNAs encoding the SH2, SH3, or SH2+SH3 domains with polymerase chain reaction-generated 5'- and 3'-BamHI sites were cloned
into the E. coli expression vector pGEX-2t with the
isopropyl 1-thio-
-D-galactopyranoside-inducible Ptac
promoter to create an in-frame fusion between these coding sequences
and the 3'-end of the E. coli GST gene. The generated recombinant plasmids were transformed into the E. coli
strain DH5
. Single transformants were expanded in 5 ml of
Luria-Burtain (LB) medium (1% tryptone, 1% NaCl, 0.5% yeast extract)
containing ampicillin (100 µg/ml) by overnight culture at 37 °C.
Expression of the fusion proteins was induced with 10 mM
isopropyl 1-thio-
-D-galactopyranoside. The cells were
harvested by centrifugation at 4,500 × g in a Sorvall RC5B centrifuge for 10 min at 4 °C, lysed in sucrose-lysozyme buffer
(20 mM Tris, pH 8.0, 150 mM NaCl, 10% sucrose,
1 mM EDTA, 20 mM lysozyme), and disrupted
further by sonication. After removal of the cell pellets by
centrifugation at 35,000 × g for 1 h at 4 °C,
GST-BTK fusion proteins were purified by gluthathione-Sepharose chromatography (21), whereas MBP-BTK fusion proteins were purified from
the culture supernatants by amylose affinity chromatography (26).
20 °C) methanol for 15 min. After fixation, the coverslips were
washed for 15 min in phosphate-buffered saline (PBS) + 0.1% Triton
X-100. Cells were stained with a rabbit polyclonal anti-tubulin
antibody according to the manufacturer's recommendations (Sigma) to
visualize their cytoplasms. DNA was labeled for 10 min with Toto-3, a
DNA specific dye (Molecular Probes, Eugene OR) to visualize the
apoptotic changes in the nuclei. MBP-BTK-electroporated BTK-deficient
DT-40 cells and nonelectroporated BTK-deficient DT-40 cells were
labeled with an antibody raised against MBP. The secondary antibody was
a goat anti-rabbit fluorescein-conjugated antibody. In some
experiments, cells were examined for Fas expression by confocal
microscopy. In brief, cells were attached to
poly-L-lysine-coated coverslips and fixed for 40 min in 2%
paraformaldehyde in PBS. Cells were rinsed in PBS + 115 mM
glycine to quench the formaldehyde and then blocked in PBS containing
2% bovine serum albumin (PBS+BSA). A monoclonal antibody raised
against the extracellular domain of Fas (Transduction Labs, Lexington,
KY) was added in PBS+BSA, and the coverslips were incubated for 40 min
at 37 °C before rinsing again in PBS. A fluorescein-labeled
secondary antibody (Zymed Laboratories Inc., San
Francisco) diluted in PBS+BSA was then added to the coverslips, and
they were again incubated for 40 min at 37 °C. After another wash,
cellular DNA was labeled by incubation in 1 µM Toto-3 for
20 min at room temperature. Coverslips were inverted and mounted onto
slides in Vectashield (Vector Labs, Burlingame, CA) to prevent
photobleaching and were sealed with nail varnish. Slides were examined
using a Bio-Rad MRC-1024 laser scanning confocal microscope mounted on
an Nikon Eclipse E-800 upright microscope equipped for epifluorescence
with high numerical aperture objectives (27). Optical sections were
obtained and turned into stereomicrographs using Lasersharp software
(Bio-Rad). Representative digital images were saved to Jaz disk and
processed using Adobe Photoshop software (Adobe Systems, Mountain View
CA). Images were printed with a Fuji Pictography thermal transfer
printer (Fuji Photo, Elmsford, NY). Digital data were archived and
stored on CD-ROM.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
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Fig. 1.
BTK is an inhibitor of Fas-mediated apoptosis
in DT-40 lymphoma B-cells. FACS correlated two-parameter displays
of wild-type (WT), BTK-deficient
(BTK ), LYN-deficient (LYN
) DT-40
cells as well as BTK-deficient DT-40 cells reconstituted with wild-type
human btk gene (BTK
, rBTK[WT]) stained with
MC540 and PI 24 h after treatment with the control mouse IgG MsIgG
(1 µg/ml) or anti-Fas (1 µg/ml). The percentages indicate the
fraction of cells at an early stage of apoptosis, as measured by single
MC540 fluorescence, and the fraction of cells at an advanced stage of
apoptosis, as measured by dual MC540/PI fluorescence (12).
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Fig. 2.
Fas protein expression levels in wild-type
and BTK-deficient DT-40 cells. Panel A, the expression
levels of BTK and actin in wild-type, BTK-deficient, and human
btk gene-reconstituted BTK-deficient DT-40 cells were
measured by Western blot analysis using appropriate monoclonal
antibodies and the ECL detection system (20, 25) according to the
manufacturer's recommendations. Panel B, the membranes
immunoblotted with anti-BTK, and anti-actin antibodies were stripped
and reblotted with the monoclonal anti-Fas antibody to compare the Fas
protein expression levels in the individual clones. Panel C,
1, 2, and 3, Fas expression levels of
WT and BTK-deficient DT-40 cells were examined by confocal microscopy
as detailed under "Experimental Procedures." Green,
anti-Fas labeling; blue, Toto-3 stained DNA in nucleus;
scale bar, 10 µm.
The comparative examination of the morphologic features of wild-type versus BTK-deficient DT-40 cells by laser scanning confocal microscopy showed no evidence of apoptosis for wild-type cells after treatment with the agonistic anti-Fas antibody, whereas BTK-deficient cells showed shrinkage and nuclear fragmentation consistent with apoptosis (Fig. 3A). On agarose gels, DNA from Triton X-100 lysates of anti-Fas-treated BTK-deficient DT-40 cells showed a ladder-like fragmentation pattern consistent with apoptosis, whereas no DNA fragmentation was observed in wild-type DT-40 cells (Fig. 3B). These results were reproduced in four independent experiments and provided direct evidence that BTK can inhibit Fas/APO-1-mediated apoptosis.
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BTK has a unique amino-terminal region that contains a PH and a Tec
homology domain, a single SH3 domain that contains the autophosphorylation site at tyrosine 223, a single SH2 domain, and a
catalytic kinase domain that contains the transphosphorylation site at
tyrosine 551 (13, 14). The PH domain of BTK interacts with various
isoforms of protein kinase C subunits of heterotrimeric G
proteins (13, 14, 33) as well as the BAP-135 protein (34). SH3 domains
have been shown to interact with proline-rich sequences of other
proteins, whereas SH2 domains interact with tyrosine-phosphorylated proteins (34). However, specific proteins interacting with the BTK SH2
or SH3 domains in B-lineage lymphoid cells have not been reported.
Mutations in the catalytic domain, SH2 domain, as well as PH domain of
the BTK have been found to lead to maturational blocks at early stages
of B-cell ontogeny in human X-linked agammaglobulinemia (35, 36).
BTK-deficient mice generated by introducing PH domain or catalytic
domain mutations in embryonic stem cells showed defective B-cell
development and function (37). Thus, different regions of BTK are
important for its physiologic functions. To examine the participation
of the various domains of BTK in negative regulation of Fas-mediated
apoptosis, we introduced wild-type human btk gene as well as
human btk genes harboring mutations either in the catalytic domain (Arg525
Gln), SH2 domain (Arg307
Ala), or PH domain (Arg28
Cys) into the BTK-deficient
DT-40 cells (12). As evidenced in Fig. 3, C and
D, BTK-deficient DT-40 cells reconstituted with wild-type
human btk gene (rWT) did not undergo apoptosis after treatment with the agonistic anti-Fas antibody, whereas Fas activation of reconstituted BTK-deficient DT-40 cells expressing human BTK with
mutations in the kinase (rK
), SH2 (rmSH2), or PH (rmPH)
domains induced apoptosis as it did in nonreconstituted BTK-deficient
DT-40 cells shown in Fig. 3B. Thus, the kinase, SH2, and PH
domains of BTK are all important and apparently indispensable for its
function as a negative regulator of Fas-mediated apoptosis.
To characterize further the anti-apoptotic function of BTK, we introduced by electroporation an MBP fusion protein containing full-length wild-type BTK into BTK-deficient cells 4 h before treatment with the anti-Fas antibody. Examination of these cells by confocal laser scanning microscopy (Fig. 4A) as well as Western blot analysis using anti-BTK and anti-MBP antibodies (Fig. 4B) confirmed the presence of the electroporated MBP-BTK protein. As shown in Fig. 4C, introduction of wild-type BTK protein by electroporation rendered the BTK-deficient DT-40 cells resistant to the apoptotic effects of Fas ligation, suggesting direct protein-protein interactions between BTK and members of the Fas signal transduction pathway as a possible mechanism for the anti-apoptotic function of BTK.
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The downstream pro-apoptotic events initiated by the ligation of Fas or TNF receptor-1 are beginning to be illuminated (3, 38-45). Both Fas and TNF receptor-1 contain a homologous intracellular "death domain," which plays a pivotal role in ligand-dependent assembly of a pro-apoptotic DISC (38). The death domains of p55 TNF receptor-1 and Fas/CD95 serve as docking sites that mediate ligand-dependent recruitment of and heteroassociation with other death domain-containing multivalent adaptor proteins: Fas-associated protein with death domain (FADD) and receptor-interacting protein (RIP) in the case of CD95; and TNF receptor-1-associated death domain protein (TRADD) and RIP in the case of TNF receptor-1 (3, 38, 45). FADD is the point of convergence between the Fas/CD95- and TNF receptor-1-linked apoptotic signal transduction pathways. Whereas Fas/CD95 directly recruits FADD, TNF receptor-1 binds TRADD, which then acts as an adaptor protein to recruit FADD. The formation of CD95-FADD or TNF receptor-1-TRADD-FADD complexes after ligand binding are important for the induction of apoptosis. The assembly of a pro-apoptotic DISC is completed by the recruitment and concomitant activation of the cytosolic caspase FLICE, a member of the ICE protease family (3, 38-45). Recently, a number of proteins have been identified as inhibitors of Fas- as well as TNF receptor-1-induced apoptosis (3, 39-41). These proteins interact directly with FADD or FLICE, thereby interfering with DISC assembly and function. Notably, the death domain of Fas contains a conserved YXXL motif similar to the immunoreceptor tyrosine-based activation motif sequences as a potential binding site for SH2-containing proteins, and Fas has recently been shown to associate with Fyn and Lck kinases as pro-apoptotic regulators that are required for induction of Fas-mediated apoptosis (30, 31). We therefore postulated that BTK could interact with Fas and prevent the assembly of a pro-apoptotic DISC after Fas ligation.
We first investigated if BTK is capable of a physical association with Fas and other members of DISC by examining the Fas, FLICE, FADD, and TRADD immune complexes from the Nonidet P-40 lysates of untreated DT-40 cells for the presence of BTK. BTK was detected by Western blot analysis in Fas (but not the other) immune complexes by anti-BTK immunoblotting (Fig. 5A). Similarly, Fas was detected by anti-Fas immunoblotting in BTK immune complexes from wild-type DT-40 cells as well as BTK-deficient DT-40 cells reconstituted with wild-type human btk gene (Fig. 5B). The constitutive association of BTK with Fas protein was also found in the human B-cell precursor leukemia cell line NALM-6-UM1 (Fig. 5C). Taken together, these results demonstrated that BTK is capable of associating with Fas protein, and this physical association does not require prior engagement of the Fas receptor. As shown in Fig. 5D, Fas is associated with FADD in BTK-deficient DT-40 cells, as evidenced by detection of Fas in FADD immune complexes, and this physical interaction was enhanced markedly after Fas ligation. In Fas-activated BTK-deficient DT-40 cells, Fas-associated FADD molecules could be detected by anti-FADD immunoblotting (Fig. 5D). In contrast to BTK-deficient DT-40 cells, very little Fas-FADD association was found in untreated or anti-Fas-treated BTK-deficient DT-40 cells reconstituted with wild-type human BTK (Fig. 5B). Similarly, Fas ligation failed to enhance the Fas-FADD association in human NALM-6-UM1 leukemia cells (Fig. 5C). Thus, BTK associates with Fas and impairs its interaction with FADD, a protein that is essential for the recruitment and activation of FLICE by Fas during the apoptotic signal. Although these results do not exclude the possibility that BTK may alter the fate of the apoptotic signal triggered by Fas ligation by multiple mechanisms including modulation of the function of positive or negative regulators of apoptotic signal transduction, they do provide at least one plausible explanation for the observed anti-apoptotic function of BTK.
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To elucidate further the physiologic significance of the observed BTK-Fas association in human leukemic B-cell precursors, we compared the sensitivities of BTK-positive NALM-6-UM1 human pre-B leukemia cell line and BTK-deficient RAMOS-1 human B-cell leukemia cell line to Fas-mediated apoptosis. As shown in Fig. 6A, these two cell lines express similar levels of Fas protein. BTK-deficient RAMOS-1 cells underwent apoptosis after Fas ligation with the agonistic anti-Fas antibody, but BTK-positive NALM-6-UM1 cells did not (Fig. 6B). We next examined the effects of the leflunomide metabolite analog LFM-A13, a potent inhibitor of BTK, on BTK-Fas association and resistance to Fas-mediated apoptosis in NALM-6-UM1 cells. Anti-BTK and anti-Fas Western blot analyses of whole cell lysates from LFM-A13-treated NALM-6-UM1 cells showed no reduction in BTK (Fig. 6C1) or Fas protein (Fig. 6C2) expression levels. There was substantially less Fas protein in the BTK immune complexes, providing direct evidence that inhibition of BTK by LFM-A13 abrogates the BTK-Fas association (Fig. 6C3). Notably, a 4-h treatment with LFM-A13 did not induce apoptosis in NALM-6-UM1 cells but rendered these highly resistant human leukemia cells sensitive to Fas-mediated apoptosis (Fig. 6D). These results provided further support for our hypothesis that BTK is a physiologically important negative regulator of Fas-mediated apoptosis.
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The ability of the BTK inhibitor LFM-A13 to abrogate the BTK-Fas
association provided circumstantial evidence that the kinase activity
of BTK plays an important role for the formation of the BTK-Fas
complexes. To establish further whether or not the association of BTK
with Fas is dependent on its kinase activity, we next examined BTK-Fas
interactions in BTK-deficient DT-40 cells reconstituted with either
wild-type human BTK (BTK, rBTK[WT]) or kinase inactive
mutant (Arg525
Gln) human BTK (BTK
,
rBTK[K
]). As shown in Fig.
7A, Western blot analysis of
whole cell lysates from these two cell lines with anti-BTK or anti-Fas
antibodies did not reveal any substantial differences (i.e.
in both of two independent experiments, we observed slightly higher BTK
and Fas expression levels in BTK
, rBTK[K
]
cells). Fas immune complexes from lysates of BTK
,
rBTK[WT] cells contained BTK protein, and this BTK-Fas association was enhanced further by treatment of cells with the anti-Fas antibody (Fig. 7B, first two lanes). In
contrast, no BTK protein was detectable in Fas immune complexes from
BTK
, rBTK[K
] cells regardless of
treatment with the anti-Fas antibody (Fig. 7B,
third and fourth lanes). These results
provide corroborating evidence that the association of BTK with Fas is
dependent on the kinase activity of BTK.
|
We next performed binding experiments with full-length MBP-BTK and truncated MBP-BTK and GST-BTK fusion proteins corresponding to various domains of BTK (Fig. 8, A-C) to elucidate the structural requirements for BTK association with Fas. MBP-BTK 1-659 (full-length BTK) as well as MBP-BTK 408-659 (BTK kinase domain) and MBP-BTK 2-137 (BTK PH domain) were able to bind and pull down Fas from lysates of BTK-deficient DT-40 cells (Fig. 8D), human NALM-6 pre-B leukemia cells (Fig. 8E), and KL2 human Epstein-Barr virus-transformed B-lymphoblastoid cells (Fig. 8F). However, Fas did not bind to the control MBP-BTK 519-567 fusion protein corresponding to a truncated kinase domain containing the Tyr551 transphosphorylation site (C5 in Fig. 7D), GST-BTK 219-377 containing the SH3 plus SH2 domains, GST-BTK 219-268 corresponding to the SH3 domain, or GST-BTK 281-377 corresponding to SH2 domain (the last two were used only with lysates from BTK-deficient DT-40 cells) (Fig. 8, D-F).
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Although the crystal structure of full-length BTK has not been
reported, the recently published structures of the PH domain and BTK
motif (46) provide useful information applicable to the binding
capability of BTK and its PH domain. FADD has been reported to interact
with the cytoplasmic domain of Fas, which is largely composed of a
death domain consisting of six antiparallel -helices assembled from
residues 230-314 (47). The YXXL sequence of the Fas death
domain has been speculated to resemble ITAMs and be recognized by an
SH2 domain of a PTK upon tyrosine phosphorylation or by other
mechanisms (30, 31). An analysis of the conformation of this
YXXL sequence shows that it is located in the middle of an
-helix, and unless a substantial conformational change of that
-helix would occur to make the tyrosine residue more accessible, it
may be too rigid for interaction with a PTK. Thus, the structural geometry of the YXXL sequence would likely prevent Fas and
BTK from adopting a binding mode such as that of CD3-
ITAM/ZAP-70 as
was suggested (31). The inability of the BTK SH2 domain to pull down
Fas from whole cell lysates further supports this notion. How then does
BTK associate with Fas? BTK and Fas may associate via complementary
electrostatic attractions and hydrogen bond interactions, which could
involve the previously reported charged residues on the surfaces of the
-helices of the Fas death domain. This association could be mediated
by a third protein that forms an interface between Fas and BTK. The
importance of the SH2 and kinase domains of BTK for its anti-apoptotic
function prompts the hypothesis that a tyrosine-phosphorylated
substrate of BTK may provide such an interface. Further studies will be
required to elucidate the exact structural basis for the BTK-Fas interactions.
The ability of BTK to inhibit the pro-apoptotic effects of Fas ligation prompts the hypothesis that apoptosis of developing B-cell precursors during normal human B-cell ontogeny may be regulated reciprocally by Fas and BTK. The absence of BTK or mutations in its kinase, PH, and SH2 domains could lead to inappropriate apoptotic cell death of pre-B-cells, leading to the phenotype of X-linked agammaglobulinemia (13, 15, 16). Inappropriate apoptosis may underlie the pathogenesis as well as drug resistance of human leukemias and lymphomas, which makes control of apoptosis an important potential target for therapeutic intervention. The fate of leukemia/lymphoma cells exposed to chemotherapeutic agents such as vincristine and daunorubicin may reside in the balance between the opposing pro-apoptotic effects of caspases activated by DISC and an upstream negative regulatory mechanism involving BTK and/or its substrates. Therefore, inhibitors of BTK are likely to enhance the drug sensitivity of B-lineage leukemia/lymphoma cells.
Our findings provide unique biochemical evidence to link a death
receptor physically to an anti-apoptotic tyrosine kinase and
corroborate the growing evidence that there are multiple
counterregulatory mechanisms in B-cell precursors which operate to
preserve cell survival and growth, thereby ensuring their orderly
development and differentiation (4, 6, 7). BTK is the first tyrosine kinase to be identified as a dual function regulator of apoptosis which
promotes radiation-induced apoptosis but inhibits Fas-activated apoptosis. We have reported previously that BTK but not LYN is required
for radiation-induced apoptosis (12). Here, we showed that targeted
disruption of lyn gene does not abrogate the anti-apoptotic activity of BTK. Thus, the pro- as well as anti-apoptotic functions of
BTK do not depend on LYN kinase, which is thought to act upstream of
BTK in B-cell antigen receptor-linked mitogenic signaling (14, 22).
Indeed, our findings taken together with previously published role of
LYN as a promoter of Fas-activated apoptosis (32) suggest that these
two tyrosine kinases may play opposite roles in regulation of
Fas-linked death signaling. We believe that the identification of BTK
as a dual function regulator of apoptosis will significantly increase
our understanding of both the biological processes involved in
programmed cell death and diseases associated with dysregulation of apoptosis.
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ACKNOWLEDGEMENTS |
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We thank C. Mao and E. Sudbeck for helpful discussions, T. Dong for technical assistance, and A. Geegan for typing the manuscript.
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FOOTNOTES |
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* This work was supported in part by research grants from the Parker Hughes Trust and by NCI, National Institutes of Health, Grant U01-CA-72157.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The first two authors contributed equally to this work.
Stohlman Scholar of the Leukemia Society of America and a Parker
Hughes Chair in Oncology. To whom requests for reprints should be
addressed: Hughes Institute, 2665 Long Lake Rd., Suite 330, St. Paul,
MN 55113. Tel.: 612-697-9228; Fax: 612-697-1042; E-mail: fatih_uckun{at}mercury.ih.org.
The abbreviations used are: TNF, tumor necrosis factor; BTK, Bruton's tyrosine kinase; PTK, protein tyrosine kinase(s); DICS, death-inducing signaling complex; PH domain, pleckstrin homology domain; SH2 and SH3 domains, Src homology 2 and Src homology 3 domains, respectively; GST, glutathione S-transferase; MBP, maltose-binding protein; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PI, propidium iodide.
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REFERENCES |
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