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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 2003-2012
By
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.
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.
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 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.
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.
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
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
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.
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.
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.
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).
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.
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.
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.
1.
Tsukada S, Saffran DC, Rawlings DJ, Parolini O, Allen RC, Klisak I, Sparkes RS, Kubagawa H, Mohandas T, Quan S, Belmont JW, Cooper MD, Conley ME, Witte ON:
Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia.
Cell
72:279, 1993[Medline]
[Order article via Infotrieve]
2.
Vetrie D, Vorechovsky I, Sideras P, Holland J, Davies A, Flinter F, Hammarström L, Kinnon C, Levinsky R, Bobrow M, Smith CIE, Bentley DR:
The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases.
Nature
361:226, 1993[Medline]
[Order article via Infotrieve]
3.
Thomas JD, Sideras P, Smith CIE, Vorechovsky I, Chapman V, Paul WE:
Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes.
Science
261:355, 1993
4.
Rawlings DJ, Saffran DC, Tsukada S, Largaespada DA, Grimaldi JC, Cohen L, Mohr RN, Bazan JF, Howard M, Copeland NG, Jenkins NA, Witte ON:
Mutation of unique region of Bruton's tyrosine kinase in immunodeficient XID mice.
Science
261:358, 1993
5.
Saouaf SJ, Mahajan S, Rowley RB, Kut SA, Fargnoli J, Burkhardt AL, Tsukada S, Witte ON, Bolen JB:
Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.
Proc Natl Acad Sci USA
91:9524, 1994
6.
de Weers M, Brouns GS, Hinshelwood S, Kinnon C, Schuurman RK, Hendriks RW, Borst J:
B-cell antigen receptor stimulation activates the human Bruton's tyrosine kinase, which is deficient in X-linked agammaglobulinemia.
J Biol Chem
269:23857, 1994
7.
Aoki Y, Isselbacher KJ, Pillai S:
Bruton tyrosine kinase is tyrosine phosphorylated and activated in pre-B lymphocytes and receptor-ligated B cells.
Proc Natl Acad Sci USA
91:10606, 1994
8.
Takata M, Kurosaki T:
A role for Bruton's tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-
9.
Fluckiger AC, Li Z, Kato RM, Wahl MI, Ochs HD, Longnecker R, Kinet JP, Witte ON, Scharenberg AM, Rawling DJ:
Btk/Tec kinase regulate sustained increase s in intracellular Ca2+ following B-cell receptor activation.
EMBO J
17:1973, 1998[Medline]
[Order article via Infotrieve]
10.
Kawakami Y, Yao L, Miura T, Tsukada S, Witte ON, Kawakami T:
Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon Fc
11.
Sato S, Katagiri T, Takaki S, Kikuchi Y, Hitoshi Y, Yonehara S, Tsukada S, Kitamura D, Watanabe T, Witte ON, Takatsu K:
IL-5 receptor-mediated tyrosine phosphorylation of SH2/SH3-containing proteins and activation of Bruton's tyrosine and Janus 2 kinases.
J Exp Med
180:2101, 1994
12.
Kikuchi Y, Yasue T, Miyake K, Kimoto M, Takatsu K:
CD38 ligation induces tyrosine phosphorylation of Bruton tyrosine kinase and enhanced expression of interleukin 5-receptor
13.
Tsukada S, Rawlings DJ, Witte ON:
Role of Bruton's tyrosine kinase in immunodeficiency.
Curr Opin Immunol
6:623, 1994[Medline]
[Order article via Infotrieve]
14.
Cheng G, Ye ZS, Baltimore D:
Binding of Bruton's tyrosine kinase to Fyn, Lyn, or Hck through a Src homology 3 domain-mediated interaction.
Proc Natl Acad Sci USA
91:8152, 1994
15.
Yao L, Kawakami Y, Kawakami T:
The pleckstrin homology domain of Bruton tyrosine kinase interacts with protein kinase C.
Proc Natl Acad Sci USA
91:9175, 1994
16.
Tsukada S, Simon MI, Witte ON, Katz A:
Binding of
17.
Yang W, Malek SN, Desiderio S:
An SH3-binding site conserved in Bruton's tyrosine kinase and related tyrosine kinases mediates specific protein interactions in vitro and in vivo.
J Biol Chem
270:20832, 1995
18.
Yang W, Desiderio S:
BAP-135, a target for Bruton's tyrosine kinase in response to B cell receptor engagement.
Proc Natl Acad Sci USA
94:604, 1997
19.
Matsushita M, Yamadori T, Kato S, Takemoto Y, Inazawa J, Baba Y, Hashimoto S, Sekine S, Arai S, Kunikata T, Kurimoto M, Kishimoto T, Tsukada S:
Identification and characterization of a novel SH3-domain binding protein, Sab, which preferentially associates with Bruton's tyrosine kinase (Btk).
Biochem Biophys Res Commun
245:337, 1998[Medline]
[Order article via Infotrieve]
20.
Aldrich RA, Steinberg AG, Campbell DC:
Pedigree demonstrating a sex-linked recessive condition characterized by draining ears, eczematoid dermatitis and bloody diarrhea.
Pediatrics
13:133, 1954
21.
Wolff JA:
Wiskott-Aldrich syndrome: Clinical, immunologic, and pathologic observations.
J Pediatr
70:221, 1967[Medline]
[Order article via Infotrieve]
22.
Derry JMJ, Ochs HD, Francke U:
Isolation of a novel gene mutated in Wiskott-Aldrich syndrome.
Cell
78:635, 1994[Medline]
[Order article via Infotrieve]
23.
Kolluri R, Tolias KF, Carpenter CL, Rosen FS, Kirchhausen T:
Direct interaction of the Wiskott-Aldrich syndrome protein with the GTPase Cdc42.
Proc Natl Acad Sci USA
93:5615, 1996
24.
Symons M, Derry JMJ, Karlak B, Jiang S, Lemahieu V, McCormick F, Francke U, Abo A:
Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization.
Cell
84:723, 1996[Medline]
[Order article via Infotrieve]
25.
Lamarche N, Tapon N, Stowers L, Burbelo PD, Aspenström P, Bridges T, Chant J, Hall A:
Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade.
Cell
87:519, 1996[Medline]
[Order article via Infotrieve]
26.
Miki H, Sasaki T, Takai Y, Takenawa T:
Induction of filopodium formaition by a WASP-related actin-depolymerizing protein N-WASP.
Nature
391:93, 1998[Medline]
[Order article via Infotrieve]
27.
Wengler G, Gorlin JB, Williamson JM, Rosen FS, Bing DH:
Nonrandom inactivation of the X chromosome in early lineage hematopoietic cells in carriers of Wiskott-Aldrich syndrome.
Blood
85:2471, 1995
28.
Ochs HD, Slichter SJ, Harker LA, Von Behrens WE, Clark RA, Wedgwood RJ:
The Wiskott-Aldrich syndrome: Studies of lymphocytes, granulocytes, and platelets.
Blood
55:243, 1980
29.
Blaese RM, Strober W, Brown RS, Waldmann TA:
The Wiskott-Aldrich syndrome. A disorder with a possible defect in antigen processing or recognition.
Lancet
1:1056, 1968[Medline]
[Order article via Infotrieve]
30.
Simon HU, Mills GB, Hashimoto S, Siminovitch KA:
Evidence for defective transmembrane signaling in B cells from patients with Wiskott-Aldrich syndrome.
J Clin Invest
90:1396, 1992
31.
Rivero-Lezcano OM, Marcilla A, Sameshima JH, Robbins KC:
Wiskott-Aldrich syndrome protein physically associates with Nck through Src homology 3 domains.
Mol Cell Biol
15:5725, 1995[Abstract]
32.
Bunnell SC, Henry PA, Kolluri R, Kirchhausen T, Rickles RJ, Berg LJ:
Identification of Itk/Tsk Src homology 3 domain ligands.
J Biol Chem
271:25646, 1996
33.
Cory GOC, MacCarthy Morrogh L, Banin S, Gout I, Brickell PM, Levinsky RJ, Kinnon C, Lovering RC:
Evidence that the Wiskott-Aldrich syndrome protein may be involved in lymphoid cell signaling pathways.
J Immunol
157:3791, 1996[Abstract]
34.
Banin S, Truong O, Katz DR, Waterfield MD, Brickell PM, Gout I:
Wiskott-Aldrich syndrome protein (WASp) is a binding partner for c-Src family protein-tyrosine kinases.
Curr Biol
6:981, 1996[Medline]
[Order article via Infotrieve]
35.
Miki H, Nonoyama S, Zhu Q, Aruffo A, Ochs HD, Takenawa T:
Tyrosine kinase signaling regulates Wiskott-Aldrich syndrome protein function, which is essential for megakaryocyte differentiation.
Cell Growth Differ
8:195, 1997[Abstract]
36.
Wu Y, Spencer SD, Lasky LA:
Tyrosine phosphorylation regulates the SH3-mediated binding of the Wiskott-Aldrich syndrome protein to PSTPIP, a cytoskeletal-associated protein.
J Biol Chem
273:5765, 1998
37.
Klein G, Giovanella B, Westman A, Stehlin JS, Mumford D:
An EBV-genome-negative cell line established from an American Burkitt lymphoma; receptor characteristics. EBV infectibility and permanent conversion into EBV-positive sublines by in vitro infection.
Intervirology
5:319, 1975[Medline]
[Order article via Infotrieve]
38.
Hurwitz R, Hozier J, LeBien T, Minowada J, Gajl Peczalska K, Kubonishi I, Kersey J:
Characterization of a leukemic cell line of the pre-B phenotype.
Int J Cancer
23:174, 1979[Medline]
[Order article via Infotrieve]
39.
Kuwahara K, Kawai T, Mitsuyoshi S, Matsuo Y, Kikuchi H, Imajoh Ohmi S, Hashimoto E, Inui S, Cooper MD, Sakaguchi N:
Cross-linking of B cell antigen receptor-related structure of pre-B cell lines induces tyrosine phosphorylation of p85 and p110 subunits and activation of phosphatidylinositol 3-kinase.
Int Immunol
8:1273, 1996
40.
Kohno S, Minowada J, Sandberg AA:
Chromosome evolution of near-haploid clones in an established human acute lymphoblastic leukemia cell line (NALM-16).
J Natl Cancer Inst
64:485, 1980
41.
Pear WS, Nolan GP, Scott ML, Baltimore D:
Production of high titer helper-free retroviruses by transient transfection.
Proc Natl Acad Sci USA
90:8392, 1993
42.
Zhu Q, Watanabe C, Liu T, Hollenbaugh D, Blaese RM, Kanner SB, Aruffo A, Ochs HD:
Wiskott-Aldrich syndrome/X-linked thrombocytopenia: WASP gene mutations, protein expressi |
TOP
ABSTRACT
INTRODUCTION
RI),
) 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.
2.
J Exp Med
184:31, 1996
chain: Synergistic effects with interleukin 5.
Proc Natl Acad Sci USA
92:11814, 1995