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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 2003-2012
Involvement of Wiskott-Aldrich Syndrome Protein in B-Cell Cytoplasmic
Tyrosine Kinase Pathway
By
Yoshihiro Baba,
Shigeaki Nonoyama,
Masato Matsushita,
Tomoki Yamadori,
Shoji Hashimoto,
Kohsuke Imai,
Shigeyuki Arai,
Toshio Kunikata,
Masashi Kurimoto,
Tomohiro Kurosaki,
Hans D. Ochs,
Jun-ichi Yata,
Tadamitsu Kishimoto, and
Satoshi Tsukada
From the Department of Medicine III, Osaka University Medical School,
Osaka, Japan; the Department of Pediatrics, School of Medicine, Tokyo
Medical and Dental University, Tokyo, Japan; the Fujisaki Institute,
Hayashibara Biochemical Laboratories Inc, Okayama, Japan; the
Department of Molecular Genetics, Institute for Hepatic Research,
Kansai Medical University, Osaka, Japan; and the Department of
Pediatrics, School of Medicine, University of Washington, Seattle, WA.
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ABSTRACT |
Bruton's tyrosine kinase (Btk) has been shown to play a role in
normal B-lymphocyte development. Defective expression of Btk leads to
human and murine immunodeficiencies. However, the exact role of Btk in
the cytoplasmic signal transduction in B cells is still unclear. This
study represents a search for the substrate for Btk in vivo. We
identified one of the major phosphoproteins associated with Btk in the
preB cell line NALM6 as the Wiskott-Aldrich syndrome protein (WASP),
the gene product responsible for Wiskott-Aldrich syndrome, which is
another hereditary immunodeficiency with distinct abnormalities in
hematopoietic cells. We demonstrated that WASP was transiently
tyrosine-phosphorylated after B-cell antigen receptor cross-linking on
B cells, suggesting that WASP is located downstream of cytoplasmic
tyrosine kinases. An in vivo reconstitution system demonstrated that
WASP is physically associated with Btk and can serve as the substrate
for Btk. A protein binding assay suggested that the
tyrosine-phosphorylation of WASP alters the association between WASP
and a cellular protein. Furthermore, identification of the
phosphorylation site of WASP in reconstituted cells allowed us to
evaluate the catalytic specificity of Btk, the exact nature of which is
still unknown.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
BRUTON'S TYROSINE kinase (Btk) is a
cytoplasmic tyrosine kinase that is involved in the pathogenesis of
human and murine B-cell deficiencies (human X-linked agammaglobulinemia
[XLA]1,2 and murine X-linked immunodeficiency
[XID]3,4). Since the identification of this tyrosine
kinase, several reports have demonstrated the involvement of Btk in
cytoplasmic signal transductions through the B-cell antigen receptor
(BCR),5-9 the high-affinity IgE receptor (Fc RI),10 the interleukin-5 (IL-5)
receptor,11 and CD38.12 Btk, a member of the
Btk/Tec family (Itk, Tec, Bmx, Txk), is composed of several domains,
including a PH (pleckstrin homology), TH (tec homology), SH (src
homology) 3, SH2, and SH1 (=kinase) domain from the N to C
termini.13 Several molecules that interact with these
domains of Btk have been described,14-19 although the
unique role of Btk in B-cell development has remained unclear.
The Wiskott-Aldrich syndrome (WAS)20,21 is another X-linked
hereditary immunodeficiency, characterized by thrombocytopenia, eczema,
and abnormal humoral and cell-mediated immunity. The gene responsible
for WAS has been identified and termed WAS protein (WASP).22 Recent studies have shown that WASP specifically
associates with the activated form of CDC42, suggesting that WASP is
involved in regulating cytoskeletal architecture.23,24 This
finding may explain the abnormalities seen in the cytoskeleton of
hematopoietic cells in WAS patients, although arguments can be put
forward against such a role for WASP.25,26 X-chromosome
inactivation studies in obligate carriers for WAS have demonstrated a
nonrandom inactivation pattern in most hematopoietic cell lineages,
including T cells, B cells and CD34+ cells, 27
suggesting the requirement of WASP for the normal differentiation and
growth of hematopoietic cells. A variety of morphologic and functional
abnormalities affecting T and B lymphocytes, neutrophils, and platelets
have been identified. WAS patients consistently fail to mount an
antibody response to polysaccharides and often respond poorly to
protein antigens.28,29 In addition, transmembrane signaling
in B cells of WAS patients has been reported to be
defective.30 Exon 10 of WASP contains several polyproline
stretches, which represent several potential SH3 domain binding motifs.
Binding studies using glutathione S-transferase (GST)-SH3 fusion
proteins have suggested that the proline-rich region of WASP binds, at least in vitro, a variety (>10) of SH3 containing proteins, which include those of the Btk/Tec family kinases.31-35 However,
it seems doubtful that WASP is involved in the signaling pathways of
all these SH3 domain containing molecules and, in fact, only a few of
them (Nck,31 Fyn, Fgr,34 Grb2,35
and PSTPIP, the recently described cytoskeletal association
protein36) have been demonstrated to bind WASP in vivo.
Although it was reported that PSTPIP is involved in the control of the
cytoskeleton together with WASP and that tyrosine-phosphorylation of
PSTPIP regulates the SH3-mediated binding of WASP to PSTPIP, the
significance of the potential binding of WASP to the tyrosine kinases
has not been demonstrated. In addition, no studies have evaluated WASP
as a possible substrate of these tyrosine kinases.
We report in this study that WASP can serve as a participant in the
tyrosine kinase pathway in B-lineage cells. WASP was found to be
constitutively tyrosine-phosphorylated in the pre-B-cell line NALM6;
furthermore, we showed that WASP is transiently
tyrosine-phoshphorylated after BCR cross-linking on B cells. A
reconstitution cell system allowed us to observe the association of
WASP and Btk in vivo and to identify WASP as the substrate for Btk.
Protein binding assay demonstrated that the phosphorylation of WASP
dramatically alters the association between WASP and an unidentified
220-kD cellular protein. The phosphotyrosine motif of WASP by Btk was determined, which may give a new insight to the still-unknown substrate
specificity of Btk.
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MATERIALS AND METHODS |
Cell lines and antibodies.
RAMOS cells,37 an Epstein-Barr virus (EBV)-negative
Burkitt's lymphoma cell line, were obtained from the Health Science Research Resources Bank (HSRRB, Osaka, Japan). NALM6
cells, a human pre-B-cell line that has been described
earlier,38,39 and NALM16 cells,40 a human
pro-B-cell line, were obtained from Fujisaki Cell Center Hayashibara
(FCCH) Biochemical Laboratories, Inc (Okayama, Japan). These cells were
cultured in RPMI 1640 medium supplemented with 10% fetal calf serum
(FCS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 500 µmol/L
2-mercaptoethanol. 293T cells41 were provided by Dr Takashi
Fujita (The Tokyo Metropolitan Institute of Medical Science, Tokyo,
Japan). Anti-WASP polyclonal antibody 503, generated against amino acid
residues 209 to 226 of human WASP, has been described
previously.42 Anti-Btk monoclonal antibody 43-3B was
generated by immunizing mice with a fusion protein consisting of GST
and the unique region (amino acid residues 1 to 186) of the human Btk.
Hybridomas were generated as previously described43 and
were screened by enzyme-linked immunosorbent assay using the GST-Btk
unique region fusion protein or GST alone. The antibody from clone
43-3B binds the GST-Btk unique region, but not GST alone. This antibody
could detect a 77-kD immunoblot band in the lysate of PA317 cells that
stably expressed Btk protein by transfection of Btk cDNA,1
but not in the lysate of nontransfected PA317 cells. The same 77-kD
band was detected in the lysate of B cells, but not in that of T cells
(data not shown). Antibody 43-3B was shown to recognize human as well
as mouse Btk, whereas another anti-Btk monoclonal antibody, 48-2H,
which was generated against the SH3 domain of Btk and previously
described by us,43,44 only recognizes human Btk. Mouse
antiphosphotyrosine monoclonal antibody 4G10, purified mouse myeloma
IgG2b (isotype-matched control for 43-3B), the anti-T7 tag monoclonal
antibody, and F(ab')2 fragment of goat antihuman IgM
(µ chain specific) were purchased from Upstate Biotechnology Inc
(Lake Placid, NY), Zymed Laboratories Inc (SanFrancisco, CA), Novagen,
Inc (Madison, WI), and Cappel ICN Pharmaceuticals Inc (Aurora, OH), respectively.
Constructs and mutagenesis.
The structure of human Btk cDNA was previously described 1
and the cDNA was inserted into the pEF-BOS mammalian expression
vector.45 Human WASP cDNA (nucleotides 2-1708 of the WASP
gene)46 was generated by reverse transcriptase-polymerase
chain reaction (RT-PCR) from normal peripheral blood
mononuclear cells with the aid of synthetic oligonucleotide primers and
inserted into the pcDNA3 (Invitrogen Co, San Diego, CA) mammalian
expression vector or the pRc/CMV mammalian expression vector
(Invitrogen) in which two T7 sequences (MASMTGGQQMG) had been inserted
in tandem.47 Point mutations were introduced by a T7 DNA
polymerase-based site directed mutagenesis system (Stratagene, La
Jolla, CA), and confirmed by limited nucleotide sequencing.
Cell stimulation.
For BCR stimulation, 1 × 108 RAMOS cells were
preincubated for 30 minutes in 1 mL of serum-free RPMI 1640 medium at
37°C and subsequently incubated for the indicated time at 37°C
with 100 µg of a F(ab')2 fragment of goat antihuman
IgM (µ chain specific). Stimulation was terminated by cell lysis with
the ice-cold Triton X-100 lysis buffer described below.
Transfection.
The Btk or WASP expression vectors (total, 10 µg) were transfected
into 293T cells with Lipofectamine (GIBCO/BRL, Rockville, MD) and the
cells were harvested 48 hours later. For the phosphorylation assay of
WASP, cells were lysed in a Triton X-100 lysis buffer described below.
For the coimmunoprecipitation experiments, cells were lysed in the
digitonin lysis buffer described below.
Cell lysis, immunoprecipitation, and GST-SH3 binding assay.
For the coimmunoprecipitation experiments, we used a digitonin lysis
buffer containing 1% digitonin, 10 mmol/L triethanolamine (pH 7.5),
150 mmol/L NaCl, 10 mmol/L iodoacetoamide, 1 mmol/L EDTA, and protease
inhibitors (1 mmol/L phenylmethylsulfonyl fluoride [PMSF], 10 µg/mL
leupeptin, and 10 µg/mL aprotinin). The GST-SH3 binding assay
used a NP-40 lysis buffer containing 0.2% NP-40, 10 mmol/L HEPES
(pH 7.0), 143 mmol/L KCl, 5 mmol/L MgCl2, the same protease
inhibitors as for the digitonin lysis buffer, and 1 mmol/L sodium
orthovanadate. To perform the transfections and the cell stimulation
experiments, we selected a Triton X-100 lysis buffer containing 1%
Triton X-100, 0.05% sodium dodecyl sulfate (SDS), 10 mmol/L
NaH2PO4/Na2HPO4 (pH
7.0), 150 mmol/L NaCl, the same protease inhibitors as for the other
lysis buffer, and 1 mmol/L sodium orthovanadate. To detect the
tyrosine-phosphorylation of WASP after BCR cross-linking of RAMOS
cells, we used 0.5 mmol/L pervanadate instead of 1 mmol/L sodium
orthovanadate as the phosphotyrosine phosphatase (PTPase) inhibitor by
the reason described in the Discussion. Pervanadate stock solution (10 mmol/L) was prepared by mixing equal volumes of 20 mmol/L sodium
orthovanadate (pH 10) and 20 mmol/L of H2O2.
The mixture was allowed to stand at room temperature for 15 minutes and
then added to the Triton X-100 lysis buffer for a final concentration
of 0.5 mmol/L.
For the immunoprecipitation experiments, the cell lysate was
centrifuged for 15 minutes at 14,000 rpm at 4°C and the supernatant was precleared for 30 minutes at 4°C with an excess amount of protein-A Sepharose CL4B beads (Pharmacia, Uppsala, Sweden). The precleared lysate was incubated for 60 minutes at 4°C with
the appropriate antibodies, followed by conjugation with
protein-A Sepharose CL4B beads for 60 minutes at 4°C, and then
washed four times with a suitable buffer. For the GST-SH3 binding
assay, the cell lysate was centrifuged for 15 minutes at 14,000 rpm at
4°C and the supernatant was precleared for 30 minutes at 4°C
with glutathione-Sepharose 4B beads (Pharmacia). The precleared lysate was incubated for 120 minutes at 4°C with 8 µg of GST or GST-SH3 (corresponding to amino acid residues 212 to 275 of human Btk) bound to
glutathione-Sepharose 4B beads. The beads were then washed four times
with NP-40 lysis buffer and boiled for 5 minutes with a 2× SDS
loading buffer. Proteins were fractionated by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE; 5% to 15% gradient).
Protein analysis.
Western analysis was performed as described previously.43
As primary antibodies, the antiphosphotyrosine antibody 4G10 was used
at 1 µg/mL and the anti-Btk monoclonal antibody 43-3B was used at 3 µg/mL. The anti-WASP polyclonal antibody 503 was used at 1:3,000
dilution. Immunoreactive proteins were detected by the Enhanced
Chemiluminescence System (Amersham, Bucks, UK). The in vitro kinase
assay was performed as described previously,43,48 except
for the addition of denatured enolase (5 µg) as a
transphosphorylation substrate. 35S-metabolic labeling was
performed as also described previously.1,48
| |
RESULTS |
WASP is a major phosphoprotein associated with the Btk-SH3 domain in
the pre-B-cell line NALM6.
To identify the possible substrate(s) for Btk, we searched for
molecules that could physically associate with Btk and, in addition,
could be tyrosine-phosphorylated in B-lineage cells. Lysates of NALM16
or NALM6 cells were incubated with a GST-SH3 (Btk) fusion protein
conjugated to beads and the precipitated phosphoproteins were detected
by immunoblotting with the antiphosphotyrosine antibody 4G10. A
phosphorylated 62-kD protein was detected in the precipitate from the
NALM6 cell lysate but not in that of the NALM16 cells or in the
precipitate by GST alone (Fig 1A). In
addition to the 62-kD protein, a 120-kD tyrosine-phosphorylated protein
associated with GST-SH3 (Btk) was identified (the asterisk in Fig 1A)
in the cell lysates of both cell lines, although the band representing
the phosphorylation of this protein in NALM6 cells was weak compared
with that of the NALM16 cells. The 120-kD phosphoprotein was
subsequently identified to be Cbl (data not shown), which was
previously described as a SH3 domain binding protein.49
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| Fig 1.
Constitutive tyrosine-phosphorylation of WASP in NALM6
cells. (A) Serum-starved 5 × 107 cells (NALM16 and NALM6)
were lysed with the NP-40 lysis buffer and, after preclearance, lysates
were incubated with GST alone or BtkSH3-GST fusion protein bound to
glutathione-Sepharose 4B beads at 4°C for 2 hours. After washing,
the beads were boiled with 2× SDS loading buffer and samples were
fractionated by SDS-PAGE (5% to 15% gradient gel) and immunoblotted
(IB) with the antiphosphotyrosine (APT) antibody 4G10. The asterisk
indicates Cbl (120 kD). Molecular mass standards are shown in
kilodaltons. (B) The same membrane was reprobed with the anti-WASP
antibody 503 (top). Total cell lysates (TCL) of NALM16 and NALM6 cells
were loaded and immunoblotted with the anti-WASP antibody 503 (bottom).
(C) NALM16 and NALM6 cells (1 × 108) were lysed with the
NP-40 lysis buffer and, after preclearance, lysates were
immunoprecipitated (IP) with the anti-WASP antibody 503 (lanes 3 and 4)
or preimmune rabbit IgG as a control (lanes 1 and 2).
Immunoprecipitates were fractionated by SDS-PAGE and immunoblotted with
the antiphosphotyrosine (APT) antibody 4G10 (top), followed by
reprobing with the anti-WASP antibody 503 (bottom).
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Because of a recent report suggesting that the GST-SH3 (Btk) fusion
protein binds to the polyproline region of WASP33 and because the reported molecular size of WASP on SDS-PAGE42
was similar to that of the 62-kD protein that was constitutively
tyrosine-phosphorylated in NALM6 cells and bound to the Btk-SH3 domain,
we investigated whether this phosphoprotein could be WASP. The
reprobing of the membrane with the anti-WASP antibody 503 (Fig 1B, top
panel) showed that the two bands completely coincided, strongly
suggesting that this 62-kD phosphoprotein is indeed WASP. The amounts
of WASP recovered from the GST-SH3 (Btk) fusion protein coated beads, as well as the total amounts of WASP in the cell lysates (Fig 1B,
bottom panel), were similar between these cell lines, suggesting that
the binding affinity of WASP with the Btk-SH3 domain is independent on
the phosphorylation of WASP. To confirm the phosphorylation of WASP,
the lysate of NALM6 cells was immunoprecipitated with the anti-WASP
antibody 503 and immunoblotted with the antiphosphotyrosine antibody
4G10. As shown in Fig 1C, the tyrosine-phosphorylation of WASP was
readily detectable in this cells, demonstrating that WASP is
constitutively tyrosine-phosphorylated in NALM6 cells. This observation
suggests that WASP, one of the Btk-SH3 domain binding proteins, could
serve as the substrate for tyrosine kinase(s) in B-lineage cells at
least under certain intracellular conditions.
WASP is tyrosine-phosphorylated after BCR cross-linking on B cells.
The identification of WASP as the tyrosine-phosphorylated protein in a
B-lineage cell line prompted us to examine the possibility that WASP
would also become tyrosine-phosphorylated in B cells after some
physiological stimulation such as BCR cross-linking. After exposure to
the anti-µ antibody, RAMOS cells were lysed in Trition X-100 lysis
buffer containing pervanadate at different time points
(Fig 2). WASP was immunoprecipitated from
the lysates using the anti-WASP antibody 503, and the
tyrosine-phosphorylation of WASP was evaluated by immunoblotting with
the antiphosphotyrosine antibody 4G10 (Fig 2A, top panel). At the same
time points, the tyrosine-phosphorylation of Btk was assessed as a
control experiment by immunoprecipitating the lysates using the
anti-Btk antibody 48-2H. As previously reported,5,6 rapid
tyrosine-phosphorylation of Btk became apparent after the addition of
the anti-µ antibody, reached a maximum at 0.5 minutes, and decreased
within 30 minutes in our experiment (Fig 2B, top panel). As shown in
Fig 2A, WASP was also transiently tyrosine-phosphorylated, reaching a
peak at 3 minutes after stimulation and having decreased to an
undetectable level (no band visible even after long exposure of the
immunoblot) by 60 minutes. This increase in tyrosine-phosphorylation
after BCR cross-linking was not due to a change in the amount of WASP proteins in the cell lysates (Fig 2A, bottom panel). The
observation that WASP is tyrosine-phosphorylated after BCR
cross-linking suggests that WASP is an active participant in the
cytoplasmic tyrosine kinase pathway triggered by BCR cross-linking on B
cells.
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| Fig 2.
BCR cross-linking induces tyrosine-phosphorylations of
Btk and WASP. Serum-starved 1 × 108 RAMOS cells were
stimulated with 100 µg/mL F(ab')2 fragment of goat
antihuman IgM antibody for the indicated times (0, 0.5, 3, 15, 30, and
60 minutes). Cells were lysed with Triton X-100 lysis buffer and, after
preclearance, lysates were immunoprecipitated with the anti-WASP
antibody 503 (A) or the anti-Btk antibody 48-2H (B). Immunoprecipitates
were fractionated by SDS-PAGE and immunoblotted with the
antiphosphotyrosine (APT) antibody 4G10 (A and B; top). These membranes
were reprobed with the anti-WASP antibody 503 (A; bottom) or the
anti-Btk antibody 43-3B (B; bottom).
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Tyrosine-phosphorylation of WASP by Btk in an in vivo reconstitution
system.
Because of the findings that WASP can be tyrosine-phosphorylated in
vivo and associates with the SH3 domain of Btk, we decided to assess
the possibility of WASP being actually tyrosine-phosphorylated by Btk
in vivo. Human WASP cDNA inserted into the pcDNA3 expression vector was
transfected with or without Btk cDNA into 293T cells. After lysis with
a Triton X-100 lysis buffer, the lysates were immunoprecipitated by
using the anti-WASP antibody 503. Tyrosine-phosphorylation of the
precipitated WASP was evaluated by immunoblotting with the
antiphosphotyrosine antibody 4G10. As shown in
Fig 3A, tyrosine-phosphorylation of WASP
was not observed in 293T cells when WASP alone was expressed (lane 1; a
faint band was detected by very long exposure of the immunoblot; data
not shown). In contrast, prominent tyrosine-phosphorylation of WASP was
detected when WASP was coexpressed with Btk (lane 2) despite the
approximately equal amounts of precipitated WASP from cell lysates (Fig
3A, middle panel), indicating that the presence of Btk was requirement
for the phosphorylation of WASP on its tyrosine. To further investigate
the role of Btk in the tyrosine-phosphorylation of WASP, we selected
two mutant Btk proteins for coexpression with WASP in 293T cells. It
has been previously shown that the substitution of lysine by arginine
at amino acid residue 430 (K430R mutant) generates a kinase-inactive
Btk.50 This mutation completely abolished the kinase
activity of Btk in the 293T cell reconstitution system (Fig 3B, lane
3). In the second mutant Btk protein, the two conserved tryptophans at
codons 251 and 252 were replaced by two leucines (WW252LL mutant); this substitution has been shown to greatly diminish the interaction of SH3
domains with their ligands (Fig 3C, lane 4 of top panel and previous
studies19,51-55). However, the kinase activities of both
wild and WW252LL mutant Btk, when transfected into 293T cells, were at
a similar level (Fig 3B, lanes 1 and 2). No significant tyrosine-phosphorylation of WASP was observed when WASP and K430R mutant Btk were coexpressed in 293T cells (Fig 3A, lane 3),
whereas cotransfection of WASP and WW251LL mutant Btk resulted in a
much lower level of tyrosine-phosphorylation (Fig 3A, lane 4)
when compared with that induced by wild-type Btk (lane 2), despite the
fact that the protein levels expressed by the transfections were
identical (Fig 3A, middle panel for WASP and bottom panel for Btk).
These results demonstrate that WASP, under this experimental condition,
can be tyrosine-phosphorylated by the kinase activity of Btk and that
the direct interaction between WASP and the SH3 domain of Btk is
required for this phosphorylation to occur.
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| Fig 3.
(A) Phosphorylation of WASP by Btk in a reconstitution
system. Cotransfection of pcDNA3/WASP with pEF-BOS vector alone (lane
1), pEF-BOS/wild-type (WT) Btk (lane 2), pEF-BOS/Btk (K430R) (lane 3)
or pEF-BOS/Btk (WW251LL) (lane 4) into 293T cells was performed with
lipofectamine. Cells were harvested after 48 hours and lysed with
Triton X-100 lysis buffer. After preclearance, lysates were
immunoprecipitated with the anti-WASP antibody 503 and immunoblotted
with the antiphosphotyrosine (APT) antibody 4G10 (top). The same
membrane was reprobed with the anti-WASP antibody 503 (middle). To
detect the expression of Btk constructs, the total cell lysates (TCL)
were also loaded and immunoblotted with the anti-Btk antibody 43-3B
(bottom). (B) In vitro kinase assays of wild and mutant Btk. The
expression vectors of Btk (WT) (lane 1), Btk (WW251LL) (lane 2), or Btk
(K430R) (lane 3) were transfected into 293T cells. Cells were harvested
after 48 hours, and the lysates were immunoprecipitated with the
anti-Btk antibody 48-2H. The in vitro kinase assay was performed with
denatured enolase as a substrate. The bottom panel demonstrates the
expression of Btk protein in the lysates by immunoblotting with the
anti-Btk antibody 43-3B. (C) Association of WASP with wild-type or
mutant Btk. Transfection of pEF-BOS/wild type (WT) Btk (lane 1) or
cotransfection of T7 epitope-tagged WASP with pEF-BOS/wild-type (WT)
Btk (lane 2), pEF-BOS/Btk (K430R) (lane 3), or pEF-BOS/Btk (WW251LL)
(lane 4) into 293T cells was performed with lipofectamine. Cells were
harvested after 48 hours and lysed with digitonin lysis buffer. After
preclearance, lysates were immunoprecipitated with the anti-T7 tag
antibody and immunoblotted with the anti-Btk antibody 43-3B (top) or
with the antiphosphotyrosine (APT) antibody 4G10 (second), followed by
reprobing with the anti-WASP antibody 503 (third). To detect the
expression of Btk proteins, the total cell lysates (TCL) were also
loaded and immunoblotted with the anti-Btk antibody 43-3B (bottom).
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To further examine the interaction of these molecules, we next compared
the association of WASP with wild-type Btk with that with mutant Btks.
To evaluate the association of these molecules more precisely, T7
epitope-tagged WASP was transiently coexpressed with wild-type or
mutant Btk in 293T cells. The cell lysates (1% digitonin) were
immunoprecipitated by the anti-T7 tag antibody, and the coprecipitated
Btk was detected by immunoblotting with the anti-Btk antibody 43-3B
(Fig 3C, top panel). Simultaneously, the tyrosine-phosphorylation of
the precipitated T7-WASP and Btk was evaluated by immunoblotting with
the antiphosphotyrosine antibody 4G10 (Fig 3C, second panel). As a
result of using the digitonin lysis buffer, which is less stringent
than the Triton X-100 lysis buffer (1% Triton X-100 plus 0.05% SDS),
the coprecipitation of T7-WASP and wild-type Btk could be readily
observed (Fig 3C, lane 2 of top and second panels). The amount of Btk
coprecipitated with T7-WASP was almost identical regardless of the
activation of Btk or the phosphorylation of WASP (lanes 2 and 3 of top
and second panels), suggesting that the association of Btk with WASP is
constitutive and does not depend on the activation or phosphorylation of Btk or WASP.
Tyrosine-phosphorylation of WASP alters the association of a 220-kD
cellular protein with WASP.
To investigate the functional significance of the
tyrosine-phosphorylation of WASP, we examined possible changes in the
association between WASP and cellular proteins. T7-WASP was transiently
coexpressed in 293T cells either with wild-type Btk or K430R mutant Btk
(phosphorylated or unphosphorylated WASP, respectively). Expressed WASP
proteins were then immunopurified by incubation with the anti-T7 tag
antibody followed by conjugation with protein A beads and washing with the Triton X-100 lysis buffer. Silver staining of the
immunoprecipitates demonstrated that purified WASP proteins were
essentially free of proteins other than Ig used for purification (data
not shown). Purified WASP proteins were then incubated with the RAMOS
cell lysate that was metabolically labeled with
35S-methionine (see Materials and Methods). After washing,
labeled proteins bound to the beads were visualized by autoradiograpy. As shown in Fig 4, binding of a 220-kD
cellular protein was detected in the presence of unphosphorylated WASP
(lane 1), whereas such binding of this 220-kD protein was greatly
reduced in the presence of phosphorylated WASP (lane 2). There were no
other significant differences between the binding of the proteins to
phosphorylated and unphosphorylated WASP. This result suggests that
WASP bound to an unidentified 220-kD cellular protein under these
experimental conditions and that this association was greatly reduced
by the tyrosine-phosphorylation of WASP.
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| Fig 4.
Tyrosine-phosphorylation of WASP alters the association
of a 220-kD cellular protein with WASP. WASP was expressed by
cotransfection with Btk (K430R) (lane 1) or wild-type Btk (lane 2).
Unphosphorylated ( ) or phosphorylated (+) WASP proteins were
immunopurified and then incubated with 35S-metabolically
labeled RAMOS cell lysate. Labeled proteins bound to the beads were
detected by autoradiography.
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The phosphotyrosine motif of WASP is similar to the
autophosphorylation site of Btk.
As demonstrated above, WASP can serve as the substrate for Btk at least
in the in vivo reconstitution system used by us. Because only a few
molecules have been reported to date to serve as Btk substrate, the
observed in vivo phosphorylation of WASP by Btk provided a unique
opportunity to study the in vivo substrate specificity of Btk. WASP has
seven tyrosines located at amino acid residues 51, 83, 88, 102, 107, 212, and 291 (Fig 5A). The
flanking amino acid residues of each of the tyrosines are shown in Fig
5B. To identify the phosphorylation site of WASP by Btk, we generated seven constructs in which each of the seven tyrosines of WASP was
replaced by a phenylalanine (designated as Y51F, Y83F, Y88F, Y102F,
Y107F, Y212F, and Y291F mutant). Each of these mutant constructs was
then cotransfected with Btk cDNA into 293T cells and the
tyrosine-phosphorylation of the mutant WASP was evaluated. As shown in
Fig 5C, Y51F, Y83F, Y88F, Y102F, Y107F, and Y212F mutant proteins were
phosphorylated at a level similar to that of wild-type WASP (lanes 1 through 7). However, in the case of Y291F mutant protein, the major
62-kD band representing tyrosine-phosphorylation of WASP by Btk was absent and only a very weak band in the high molecular range was observed. As shown in Fig 5D, the introduction of Y291F mutation into
WASP did not alter the association of WASP with Btk, meaning that the
absence of phosphorylation does not imply a weaker association of Btk
with the Y291F mutant than that with wild-type WASP. These results
indicate that Btk phosphorylates WASP on its tyrosine 291 in the
reconstituted cells and, furthermore, indicate that the phosphorylation
of WASP does not alter the association with Btk. The very weak band
seen in Y291F mutant transfected 293T cells (Fig 5C, lane 8 of top
panel) may represent a weak phosphorylation of other tyrosine residues
of WASP, possibly due to the hyperexpressions of Btk and WASP in the
reconstitution system. As will be discussed later, the flanking amino
acid residues of tyrosine 291 of WASP exhibit a distinct similarity to
those of tyrosine 223, the autophosphorylation site of
Btk55 (Fig 5B).
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| Fig 5.
Identification of the phosphorylation site of
WASP by Btk. (A) A schematic representation of the structure of WASP
and its functional domains (pleckstrin homology domain [PH], the
GTPase binding domain [GBD], and proline rich regions [Poly-Pro])
and the distribution of the tyrosine residues. The positions and amino
acid numbers of tyrosine residues are indicated (arrows). (B) The
flanking amino acid sequences of each tyrosine residue in WASP and of
tyrosine 223 (the autophosphorylation site) of Btk are listed. (C)
Coexpression of Btk and mutant WASP constructs. Cotransfection of
pEF-BOS/wild-type Btk with pcDNA3/wild-type WASP (lane 1), pcDNA3/Y51F
(lane 2), pcDNA3/Y83F (lane 3), pcDNA3/Y88F (lane 4), pcDNA3/Y102F
(lane 5), pcDNA3/Y107F (lane 6), pcDNA3/Y212F (lane 7), or pcDNA3/Y291F
mutant WASP (lane 8) was performed with lipofectamine. Cells were
harvested after 48 hours and lysed with Triton X-100 lysis buffer.
After preclearance, lysates were immunoprecipitated with the anti-WASP
antibody 503 and immunoblotted with the antiphosphotyrosine (APT)
antibody 4G10 (top). The same membrane was reprobed with the anti-WASP
antibody 503 (middle). To detect the expression of Btk proteins, the
total cell lysates (TCL) were loaded and immunoblotted with the
anti-Btk antibody 43-3B (bottom). (D) Association of wild-type or Y291F
mutant WASP with Btk. Transfection of pEF-BOS/wild-type Btk as a
control (lane 1) or cotransfection of pEF-BOS/wild-type Btk with T7
epitope-tagged WASP (lane 2) or T7 epitope-tagged Y291F (lane 3) into
293T cells was performed to transiently express the proteins. Cells
were harvested after 48 hours and lysed with digitonin lysis buffer.
After preclearance, lysates were immunoprecipitated with the anti-T7
tag antibody and immunoblotted with the anti-Btk antibody 43-3B (top),
followed by reprobing with the anti-WASP antibody 503 (middle). To
detect the expression of Btk proteins, the total cell lysates (TCL)
were also loaded and immunoblotted with the anti-Btk antibody 43-3B
(bottom).
|
|
| |
DISCUSSION |
The recent observation that mutations within Btk and WASP cause
characteristic immunodeficiency disorders clearly demonstrates the
importance of these proteins for normal lymphocyte function. In the
present study, we demonstrated that WASP, which has been reported to be
one of the Btk-SH3 domain binding proteins, is a participant in the
cytoplasmic tyrosine kinase pathway in B-lineage cells and may actually
serve as the substrate of Btk in vivo.
In contrast to our finding presented here, one report claimed that the
tyrosine-phosphorylation of WASP was not observed after BCR
cross-linking.33 This discrepancy may be due to the
addition of pervanadate to the cell lysis buffer that we used in all of our procedures. We initially observed that the tyrosine-phosphorylation of WASP was barely visible when a lysis buffer was used that contained orthovanadate only. Because of residual PTPase activities observed even
at high concentrations of orthovanadate and the fact that its
inhibitory effect is reversible,56 we decided to add
pervanadate to our buffer. Pervanadate, consisting of a variety of
complexes formed between orthovanadate and hydrogen peroxide, is much
more potent than orthovanadate as a PTPase inhibitor and its inhibitory effect is irreversible.56 The observation that the addition of pervanadate to the lysis buffer was a prerequisite for the detection
of tyrosine-phosphorylation of WASP suggests that phosphorylated WASP
is very sensitive to cellular PTPases. Although exposure of cells to
pervanadate has been shown to mimic limited receptor signalings under
certain conditions,57-59 the time course of phosphorylation after BCR cross-linking (Fig 2A) demonstrates that the increased tyrosine-phosphorylation of WASP we observed depends on the signaling through the BCR and was not simply an artifact of pervanadate. Several
cytoplasmic tyrosine kinases, including Btk, Syk, and such Src-family
kinases as Lyn, have been reported to be involved in the cytoplasmic
signal transduction via BCR. The rapid tyrosine-phosphorylation of WASP
after BCR cross-linking thus suggests an involvement of WASP in the
cytoplasmic tyrosine kinase pathway in B-lineage cells. Simultaneously,
in another report, we have recently demonstrated that WASP is
transiently tyrosine-phosphorylated in normal human platelets after
stimulation with thrombopoietin (Imai et al, manuscript submitted). Putting these observations together suggests
the involvement of WASP in tyrosine kinase pathways in a variety of
hematopoietic cell lineages.
The in vivo reconstitution experiments used in this study have provided
evidence that WASP associates with Btk and can serve as its substrate
in vivo. These observations raise the possibility that Btk may play a
significant role in the phosphorylation of WASP in B cells as well,
although the data presented here do not allow us to determine if under
physiologic conditons, eg, BCR cross-linking, Btk is the dominant
kinase that contributes to the tyrosine phosphorylation of WASP.
Several molecules have been reported to associate with the SH3 domain
of Btk at least in vitro.33,49,60 However, no evaluations
of these molecules as possible substrates of Btk have been reported.
Recently, we reported a novel protein Sab, which also binds the SH3
domain of Btk.19 Although in vivo association of Btk and
Sab was observed,19 the tyrosine-phosphorylation of Sab by
Btk could not be detected when using a reconstitution system similar to
the one described in this report (our unpublished data).
Therefore, the observed tyrosine-phosphorylation of WASP by Btk cannot
be extrapolated to other Btk-SH3 domain binding molecules and they may
play different roles in the Btk signaling pathway.
The precise biological significance of the phosphorylation of WASP is
currently unknown. However, in this study, we demonstrated that
tyrosine-phosphorylation of WASP regulates the association of a 220-kD
protein with WASP. It seems likely that some conformational change is
induced in WASP by the tyrosine-phosphorylation. The significance of
this association is currently being investigated. Our observation also
suggests the possibility that the tyrosine-phosphorylation may also
alter the interaction between WASP and its other binding partners.
Furthermore, one must certainly include the possibility that the
tyrosine-phosphorylation of WASP may create a docking site for SH2 or
PTB-domain containing protein(s), although the results of our
experiment shown in Fig 4 failed to detect any increase in the binding
of cellular proteins to the phosphorylated WASP.
With the aid of mutant constructs in which one of the seven tyrosines
was systematically replaced with phenylalanine, the major
phosphorylation site of WASP by Btk was determined as tyrosine 291. By
using a chemical peptide library61 or a phage display approach,62 it has been shown that each of the tyrosine
kinases or its family displays distinct substrate specificities that
are mainly determined by the residues immediately flanking the
phosphorylated tyrosine residue. Several cytoplasmic tyrosine kinases
(Fps, Abl, Src family kinases) seem to have a preference for acidic
residues (glutamic acid or aspartic acid) at the 4 to 2
positions (especially the 3 position) from the targeted
tyrosine, hydrophobic residues (isoleucine, leucine or valine) at the
1 position, acidic residues at the +1 and +2 positions, and
hydrophobic residues that tend to dominate at the +3 position. In
contrast, the cytoplasmic tyrosine kinase Syk commands acidic residues
at the 1 position. Although the general substrate specificity of
Btk has not yet been determined, it is of interest that the flanking
amino acid residues of tyrosine 291 (SKLIYDFI) of WASP have a distinct
similarity to those of tyrosine 223, the autophosphorylation site of
Btk, which is located in its SH3 domain55 and has the
flanking sequence VVALYDYM (Fig 5B). Comparing the flanking sequences
of these two tyrosines, the following common characteristics were
noted: 4 to 2 (nonacidic), 1 (hydrophobic), +1
(acidic), +2 (aromatic), and +3 (hydrophobic). These considerations may
also explain why tyrosine 551 of Btk, which is located in its catalytic
domain and corresponds to the autophosphorylation sites of other
cytoplasmic tyrosine kinases,63 serves as a
transphosphorylation site for Src family kinases (mainly Lyn)55 rather than as a site for autophosphorylation by Btk itself. The 3 and 2 positions from tyrosine 551 of Btk
are occupied by acidic residues (the flanking sequence of tyrosine 551 is LDDEYTSS). It appears, therefore, that the flanking sequence of
tyrosine 551 is preferred by Src family kinases rather than Btk itself.
As mentioned in the introduction, several observations have suggested
that the B cells of WAS patients contain some intrinsic defects.
However, in contrast to the clearly identified defect in the B cells of
XLA patients or XID mice, these developmental or functional defects in
WAS-B cells have still not been precisely defined. Clarification of the
significance of the tyrsoine-phosphorylation of WASP, which is mediated
by cytoplasmic tyrosine kinase as described in this report, may further
clarify the exact roles of the immunodeficiency-causing molecules in
cytoplasmic signal transduction as well as the exact pathogenesis of
these diseases.
| |
ACKNOWLEDGMENT |
The authors thank Dr Hajime Karasuyama (The Tokyo Metropolitan
Institute of Medical Science) and Drs Nobuo Sakaguchi and Kazuhiko Kuwahara (Kumamoto University, Kumamoto, Japan) for their valuable comments and thank Dr Shigekazu Nagata (Osaka University Medical School, Osaka, Japan) and Dr Yoshihiro Takemoto (Nippon Glaxo Limited,
Tsukuba Research Laboratory, Ibaraki, Japan) for providing materials.
| |
FOOTNOTES |
Submitted June 3, 1998; accepted November 3, 1998.
Supported by grants from the Ministry of Education, Science and Culture
of Japan (to T.K. and S.T.), from the Ministry of Health and Welfare of
Japan (to S.T.), from the National Institutes of Health (HD17427), and
from the March of Dimes Birth Defects Foundation (6-FY96-0330; to
H.D.O.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Satoshi Tsukada, MD, PhD, Department of
Medicine III, Osaka University Medical School, 2-2, Yamada-oka
Suita City, Osaka 565, Japan; e-mail:
tsukada{at}imed3.med.osaka-u.ac.jp.
| |
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S. Matsuda, Y. Mikami, M. Ohtani, M. Fujiwara, Y. Hirata, A. Minowa, Y. Terauchi, T. Kadowaki, and S. Koyasu
Critical role of class IA PI3K for c-Rel expression in B lymphocytes
Blood,
January 29, 2009;
113(5):
1037 - 1044.
[Abstract]
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S. Sharma, G. Orlowski, and W. Song
Btk Regulates B Cell Receptor-Mediated Antigen Processing and Presentation by Controlling Actin Cytoskeleton Dynamics in B Cells
J. Immunol.,
January 1, 2009;
182(1):
329 - 339.
[Abstract]
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H. Niu, D. T. M. Leung, C. H. Ma, E. C. Y. Law, F. C. H. Tam, and P.-L. Lim
Cells That Produce Deleterious Autoreactive Antibodies Are Vulnerable to Suicide
J. Immunol.,
August 1, 2008;
181(3):
2246 - 2257.
[Abstract]
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J. Jongstra-Bilen, A. Puig Cano, M. Hasija, H. Xiao, C. I. E. Smith, and M. I. Cybulsky
Dual Functions of Bruton's Tyrosine Kinase and Tec Kinase during Fc{gamma} Receptor-Induced Signaling and Phagocytosis
J. Immunol.,
July 1, 2008;
181(1):
288 - 298.
[Abstract]
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S. Tsuboi and J. Meerloo
Wiskott-Aldrich Syndrome Protein Is a Key Regulator of the Phagocytic Cup Formation in Macrophages
J. Biol. Chem.,
November 23, 2007;
282(47):
34194 - 34203.
[Abstract]
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D. Caracino, C. Jones, M. Compton, and C. L. Saxe III
The N-Terminus of Dictyostelium Scar Interacts with Abi and HSPC300 and Is Essential for Proper Regulation and Function
Mol. Biol. Cell,
May 1, 2007;
18(5):
1609 - 1620.
[Abstract]
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S. Tsuboi
A Complex of Wiskott-Aldrich Syndrome Protein with Mammalian Verprolins Plays an Important Role in Monocyte Chemotaxis.
J. Immunol.,
June 1, 2006;
176(11):
6576 - 6585.
[Abstract]
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E. Torres and M. K. Rosen
Protein-tyrosine Kinase and GTPase Signals Cooperate to Phosphorylate and Activate Wiskott-Aldrich Syndrome Protein (WASP)/Neuronal WASP
J. Biol. Chem.,
February 10, 2006;
281(6):
3513 - 3520.
[Abstract]
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N. Yokoyama, J. Lougheed, and W. T. Miller
Phosphorylation of WASP by the Cdc42-associated Kinase ACK1: DUAL HYDROXYAMINO ACID SPECIFICITY IN A TYROSINE KINASE
J. Biol. Chem.,
December 23, 2005;
280(51):
42219 - 42226.
[Abstract]
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E. A. Burton, T. N. Oliver, and A. M. Pendergast
Abl Kinases Regulate Actin Comet Tail Elongation via an N-WASP-Dependent Pathway
Mol. Cell. Biol.,
October 15, 2005;
25(20):
8834 - 8843.
[Abstract]
[Full Text]
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V. Chandrasekaran and S. K. Beckendorf
Tec29 controls actin remodeling and endoreplication during invagination of the Drosophila embryonic salivary glands
Development,
August 1, 2005;
132(15):
3515 - 3524.
[Abstract]
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S. Burns, G. O. Cory, W. Vainchenker, and A. J. Thrasher
Mechanisms of WASp-mediated hematologic and immunologic disease
Blood,
December 1, 2004;
104(12):
3454 - 3462.
[Abstract]
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A E Turco and L M Bambara
Pregnancy, microchimerism and autoimmunity: an update
Lupus,
September 1, 2004;
13(9):
659 - 660.
[Abstract]
[PDF]
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A. Gismondi, L. Cifaldi, C. Mazza, S. Giliani, S. Parolini, S. Morrone, J. Jacobelli, E. Bandiera, L. Notarangelo, and A. Santoni
Impaired natural and CD16-mediated NK cell cytotoxicity in patients with WAS and XLT: ability of IL-2 to correct NK cell functional defect
Blood,
July 15, 2004;
104(2):
436 - 443.
[Abstract]
[Full Text]
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X. Wu, S. Suetsugu, L. A. Cooper, T. Takenawa, and J.-L. Guan
Focal Adhesion Kinase Regulation of N-WASP Subcellular Localization and Function
J. Biol. Chem.,
March 5, 2004;
279(10):
9565 - 9576.
[Abstract]
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K. Badour, J. Zhang, F. Shi, Y. Leng, M. Collins, and K. A. Siminovitch
Fyn and PTP-PEST-mediated Regulation of Wiskott-Aldrich Syndrome Protein (WASp) Tyrosine Phosphorylation Is Required for Coupling T Cell Antigen Receptor Engagement to WASp Effector Function and T Cell Activation
J. Exp. Med.,
January 5, 2004;
199(1):
99 - 112.
[Abstract]
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C. A. Jefferies, S. Doyle, C. Brunner, A. Dunne, E. Brint, C. Wietek, E. Walch, T. Wirth, and L. A. J. O'Neill
Bruton's Tyrosine Kinase Is a Toll/Interleukin-1 Receptor Domain-binding Protein That Participates in Nuclear Factor {kappa}B Activation by Toll-like Receptor 4
J. Biol. Chem.,
July 3, 2003;
278(28):
26258 - 26264.
[Abstract]
[Full Text]
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G. O. C. Cory, R. Garg, R. Cramer, and A. J. Ridley
Phosphorylation of Tyrosine 291 Enhances the Ability of WASp to Stimulate Actin Polymerization and Filopodium Formation
J. Biol. Chem.,
November 15, 2002;
277(47):
45115 - 45121.
[Abstract]
[Full Text]
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A. Takesono, L. D. Finkelstein, and P. L. Schwartzberg
Beyond calcium: new signaling pathways for Tec family kinases
J. Cell Sci.,
January 8, 2002;
115(15):
3039 - 3048.
[Abstract]
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D. Watanabe, S. Hashimoto, M. Ishiai, M. Matsushita, Y. Baba, T. Kishimoto, T. Kurosaki, and S. Tsukada
Four Tyrosine Residues in Phospholipase C-gamma 2, Identified as Btk-dependent Phosphorylation Sites, Are Required for B Cell Antigen Receptor-coupled Calcium Signaling
J. Biol. Chem.,
October 12, 2001;
276(42):
38595 - 38601.
[Abstract]
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A. Oda, H. D. Ochs, L. A. Lasky, S. Spencer, K. Ozaki, M. Fujihara, M. Handa, K. Ikebuchi, and H. Ikeda
CrkL is an adapter for Wiskott-Aldrich syndrome protein and Syk
Blood,
May 1, 2001;
97(9):
2633 - 2639.
[Abstract]
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Y. Baba, S. Hashimoto, M. Matsushita, D. Watanabe, T. Kishimoto, T. Kurosaki, and S. Tsukada
BLNK mediates Syk-dependent Btk activation
PNAS,
February 8, 2001;
(2001)
51626198.
[Abstract]
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U. D. Bajpai, K. Zhang, M. Teutsch, R. Sen, and H. H. Wortis
Bruton's Tyrosine Kinase Links the B Cell Receptor to Nuclear Factor {kappa}B Activation
J. Exp. Med.,
May 15, 2000;
191(10):
1735 - 1744.
[Abstract]
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A. Oda, Y. Ikeda, H. D. Ochs, B. J. Druker, K. Ozaki, M. Handa, T. Ariga, Y. Sakiyama, O. N. Witte, and M. I. Wahl
Rapid tyrosine phosphorylation and activation of Bruton's tyrosine/Tec kinases in platelets induced by collagen binding or CD32 cross-linking
Blood,
March 1, 2000;
95(5):
1663 - 1670.
[Abstract]
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B. S. Gross, J. I. Wilde, L. Quek, H. Chapel, D. L. Nelson, and S. P. Watson
Regulation and Function of WASp in Platelets by the Collagen Receptor, Glycoprotein VI
Blood,
December 15, 1999;
94(12):
4166 - 4176.
[Abstract]
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H. N. Higgs and T. D. Pollard
Regulation of Actin Polymerization by Arp2/3 Complex and WASp/Scar Proteins
J. Biol. Chem.,
November 12, 1999;
274(46):
32531 - 32534.
[Full Text]
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S. Hashimoto, A. Iwamatsu, M. Ishiai, K. Okawa, T. Yamadori, M. Matsushita, Y. Baba, T. Kishimoto, T. Kurosaki, and S. Tsukada
Identification of the SH2 Domain Binding Protein of Bruton's Tyrosine Kinase as BLNK---Functional Significance of Btk-SH2 Domain in B-Cell Antigen Receptor-Coupled Calcium Signaling
Blood,
October 1, 1999;
94(7):
2357 - 2364.
[Abstract]
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T. Yamadori, Y. Baba, M. Matsushita, S. Hashimoto, M. Kurosaki, T. Kurosaki, T. Kishimoto, and S. Tsukada
Bruton's tyrosine kinase activity is negatively regulated by Sab, the Btk-SH3 domain-binding protein
PNAS,
May 25, 1999;
96(11):
6341 - 6346.
[Abstract]
[Full Text]
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A. M. Egloff and S. Desiderio
Identification of Phosphorylation Sites for Bruton's Tyrosine Kinase within the Transcriptional Regulator BAP/TFII-I
J. Biol. Chem.,
July 20, 2001;
276(30):
27806 - 27815.
[Abstract]
[Full Text]
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J.-F. Cote, P. L. Chung, J.-F. Theberge, M. Halle, S. Spencer, L. A. Lasky, and M. L. Tremblay
PSTPIP Is a Substrate of PTP-PEST and Serves as a Scaffold Guiding PTP-PEST Toward a Specific Dephosphorylation of WASP
J. Biol. Chem.,
January 18, 2002;
277(4):
2973 - 2986.
[Abstract]
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K. Yoshida, Y. Yamashita, A. Miyazato, K.-i. Ohya, A. Kitanaka, U. Ikeda, K. Shimada, T. Yamanaka, K. Ozawa, and H. Mano
Mediation by the Protein-tyrosine Kinase Tec of Signaling between the B Cell Antigen Receptor and Dok-1
J. Biol. Chem.,
August 4, 2000;
275(32):
24945 - 24952.
[Abstract]
[Full Text]
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Y. Baba, S. Hashimoto, M. Matsushita, D. Watanabe, T. Kishimoto, T. Kurosaki, and S. Tsukada
BLNK mediates Syk-dependent Btk activation
PNAS,
February 27, 2001;
98(5):
2582 - 2586.
[Abstract]
[Full Text]
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TOP
ABSTRACT
INTRODUCTION