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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2357-2364
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
By
Shoji Hashimoto,
Akihiro Iwamatsu,
Masamichi Ishiai,
Katsuya Okawa,
Tomoki Yamadori,
Masato Matsushita,
Yoshihiro Baba,
Tadamitsu Kishimoto,
Tomohiro Kurosaki, and
Satoshi Tsukada
From the Department of Molecular Medicine (formerly Medicine III),
Osaka University Medical School, Osaka, Japan; the Central Laboratories
for Key Technology, Kirin Brewery Co, Ltd, Kanagawa,
Japan; and the Department of Molecular Genetics, Institute for Hepatic
Research, Kansai Medical University, Osaka, Japan.
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ABSTRACT |
Bruton's tyrosine kinase (Btk) is a critical component in the
B-cell antigen receptor (BCR)-coupled signaling pathway. Its deficiency
in B cells leads to loss or marked reduction in the BCR-induced calcium
signaling. It is known that this BCR-induced calcium signaling depends
on the activation of phospholipase C (PLC), which is mediated by
Btk and another tyrosine kinase Syk and that the SH2 and pleckstrin
homology (PH) domains of Btk play important roles in this activation
process. Although the importance of the PH domain of Btk has been
explained by its role in the membrane targeting of Btk, the functional
significance of the SH2 domain in the calcium signaling has remained
merely a matter of speculation. In this report, we identify that one of
the major Btk-SH2 domain-binding proteins in B cells is BLNK (B-cell
linker protein) and present evidences that the interaction of BLNK and the SH2 domain of Btk contributes to the complete tyrosine
phosphorylation of PLC.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
BRUTON'S TYROSINE KINASE (Btk) is a
critical cytoplasmic tyrosine kinase in B-lymphocyte development.
Mutations in the Btk gene are responsible for human X-linked
agammaglobulinemia (XLA), which usually exhibits an almost complete
block of B-cell maturation.1-3 Btk is also responsible for
murine X-linked immunodeficiency (XID) in which a point
mutation4,5 in the pleckstrin homology (PH) domain causes a
less severe block of B-cell maturation. Although the molecular
framework in which Btk participates in B-cell development is still not
fully defined, accumulating data indicate that Btk is a critical
component in B-cell antigen receptor (BCR)-coupled calcium signaling
pathway. The first evidence came from an experiment with genetic
dissection of the Btk gene in DT40 chicken B-lymphoma cells, which led
to a complete loss of BCR-coupled inositol-1,4,5-trisphosphate (IP3) production and calcium flux.6 The reduced
levels of BCR-coupled IP3 production and calcium flux seen
in B cells from XLA patients7,8 and also from XID
mice9 seem consistent with this observation and indicate
the crucial role of Btk in BCR-coupled calcium signaling.
Btk has 4 distinct domains (PH, SH3, SH2, and catalytic [SH1]) from N
to C termini).3 Although ectopic expression of wild-type Btk in Btk-deficient cells could restore the deficient BCR-coupled calcium signaling, Btk harboring mutations in its PH domain or SH2
domain could not restore it, suggesting that the functions of both the
PH and SH2 domains as well as Btk kinase activity are required for
BCR-coupled calcium signaling.6-8 The function of the PH
domain of Btk in calcium signaling has been explained by its ability to
bind phosphatidyl inositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3 ),10 a product of phosphatidyl
inositol 3 (PI3) kinase. The binding of the PH domain of Btk to
PtdIns-3,4,5-P3 seems to enhance the translocation of Btk
to the cell membrane and promote the activation of Btk by
membrane-anchoring protein kinases.11,12 The activated Btk,
in turn, activates phospholipase C (PLC), which is another
critical component of calcium signaling, by means of tyrosine
phosphorylation.6-8 This activation process of PLC has
been suggested to be concerted with the activity of another tyrosine
kinase Syk.6 The activation of PLC leads to the
production of IP3 followed by the release of internal
calcium storage through IP3 receptors, which finally
triggers extracellular calcium entry through the calcium release
activated channel by an as yet unknown mechanism.13,14 This
scheme seems to explain quite well the molecular mechanism of
BCR-coupled calcium signaling in which the function of the PH domain
and the catalytic activity of Btk are indispensable. However, for the
full understanding of the molecular framework involved in this process,
several of the molecular connections remain to be investigated. First,
although several reports have shown that the function of the Btk-SH2
domain is also indispensable for BCR-coupled calcium
signaling,6-8 the significance of this domain has remained
a matter of speculation and the binding partner of the SH2 domain of
Btk has not been clarified in any reports. Second, recent studies have
revealed that the interaction of PLC and a newly identified B-cell
linker protein (BLNK)15 (alternatively termed
SLP-6516) is also indispensable for the BCR-coupled calcium
signaling. BLNK was reported to be tyrosinephosphorylated by activated
Syk after BCR engagement and to bind to the SH2 domain of
PLC,15 which leads to the colocalization of Syk and
PLC, resulting in the activation of PLC by Syk. This observation
was confirmed by an experiment using the genetic dissection of the BLNK
gene in DT40 cells, in which BCR-coupled IP3 production and
calcium mobilization were almost completely abolished.17 Therefore, the present question is whether some molecular connection does exist between these two (Btk-PLC2 and Syk-BLNK-PLC2)
signaling pathways essential for the BCR-coupled calcium signaling.
To clarify these matters, we attempted to identify ligands for the
Btk-SH2 domain. In the present study, we report that one of the major
Btk-SH2 domain binding proteins in B cells is BLNK and present evidence
that the interaction of Btk and BLNK via the Btk-SH2 domain contributes
to the complete tyrosinephosphylation of PLC by Btk.
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MATERIALS AND METHODS |
Cell lines and antibodies.
RAMOS cells, an EBV-negative Burkitt's lymphoma cell
line,18 were obtained from the Health Science
Research Resources Bank (HSRRB, Osaka, Japan) and 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. Provided by Dr Takashi Fujita (Tokyo Metropolitan
Institute of Medical Science, Tokyo, Japan), 293 T cells19
were maintained in Dulbecco's modified Eagle's medium with 10% FCS,
100 U/mL penicillin, and 100 µg/mL streptomycin. DT40 cells (chicken
B-lymphoma cell line), which stably expressed T7-epitope tagged human
Btk,20 were cultured in RPMI medium supplemented with 10%
FCS, 1% chicken serum, 100 U/mL penicillin, 100 µg/mL streptomycin,
500 µmol/L 2-mercaptoethanol, and 2 mmol/L glutamine. Anti-Btk
monoclonal antibody (MoAb) 43-3B,21 anti-chicken BLNK
polyclonal antibody,17 anti-chicken Syk polyclonal antibody,22 and anti-chicken PLC2 polyclonal
antibody23 were described previously.
Antiphosphotyrosine MoAb 4G10, anti-T7 tag MoAb, anti-Flag
MoAb (M2), and F(ab')2 fragment of goat anti-human IgM
(µ-chain specific) were purchased from Upstate Biotechnology Inc
(Lake Placid, NY), Novagen Inc (Madison, WI), Sigma Chemical Co (St
Louis, MO), and Cappel ICN Pharmaceuticals Inc (Aurora, OH),
respectively. Anti-chicken IgM MoAb M424 was kindly
provided by Dr Max Cooper (University of Alabama, Birmingham, AL).
Expression constructs and mutagenesis.
Human wild-type and WW251LL [SH3-mutated Btk; Tryptophans 251 and 252 in the Btk-SH3 domain were replaced by Leucines] Btk cDNAs inserted
into the pEF-BOS mammalian expression vectors were described
previously.25 Human R307K [SH2-mutated Btk; Arginine 307 in the Btk-SH2 domain was replaced by Lysine] Btk cDNA was produced by
means of a polymerase chain reaction-based site directed mutagenesis
system (TaKaRa Co, Shiga, Japan) and inserted into the pEF-BOS vector.
Porcine Syk cDNA26 and rat PLC227 cDNA in
pApuro vector have been described elsewhere. The Flag-epitope tagged
BLNK expression vector was generated by in-frame insertions of an
oligonucleotide with a Flag sequence and chicken BLNK
cDNA17 into pEF-BOS vector.
Purification and identification of GST-Btk SH2 domain binding
protein.
The cDNA fragments (corresponding to amino acid residues 276 to 401) of
human wild-type (Wild) and SH2-mutated (R307K) Btk were in-frame
inserted into PGEX-2T vector (Pharmacia, Uppsala, Sweden). The GST
expression vectors were transfected into NM522 cells with a pT-Trx
(thioredoxin) expression vector.28 Purification of the GST
proteins was performed as described previously.21 Immobilization of GST-Btk-SH2 (Wild or R307K) on glutathione-Sepharose 4B beads (Pharmacia) was performed by incubating beads with 20 mmol/L
dimethyl pimelimidate dihydrochloride (Nacalai Tesque, Kyoto, Japan) in
200 mmol/L sodium borate (pH 9) for 40 minutes at room temperature,
followed by incubation in 200 mmol/L ethanolamine (pH 8) for 2 hours at
room temperature. RAMOS cells (1 × 1010) were
pelleted at 1,200 rpm for 6 minutes, resuspended in phosphate buffer
saline (PBS) at a concentration of 1 × 108 cells/mL
and stimulated with 50 µg/mL of F(ab')2 fragment of goat anti-human IgM for 3 minutes at 37°C. After stimulation, the
cells were pelleted and then lysed with the same volume of a lysis
buffer [0.2 % NP40, 10 mmol/L HEPES (pH 7), 143 mmol/L KCl, 5 mmol/L
MgCl2, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL
leupeptin, 10 µg/mL aprotinin, 1 mmol/L sodium orthovanadate]. The
cell lysate was precleared with the GST protein immobilized on
glutathione-sepharose 4B beads and then incubated with the GST-Btk-SH2
(Wild) protein immobilized on glutathione-Sepharose 4B beads for 2 hours at 4°C. Beads were washed with the lysis buffer 4 times and
eluted with elution buffer 1 [1 % sodium dodecyl sulfate (SDS), 50 mmol/L Tris pH 7.5] for 30 minutes at 100°C. The eluate was then
diluted 10-fold with the lysis buffer and incubated with
antiphosphotyrosine MoAb 4G10 immobilized on protein A-Sepharose CL4B
beads (Pharmacia) for 2 hours at 4°C. Beads were washed with the
lysis buffer 4 times and eluted with elution buffer 2 [100 mmol/L
phenyl phosphate (Nacalai Tesque), 150 mmol/L NaCl, 10 mmol/L Tris pH
7.5, 1 mmol/L sodium orthovanadate]. The eluate was concentrated by
means of Centricon-10 (Millipore Co, Bedford, MA) and then boiled with
5 × SDS sample buffer for 5 minutes. The sample was fractionated
by SDS-polyacrylamide gel electrophoresis, electrotransferred to a
polyvinylidene difluoride (PVDF) membrane (Problott;
Applied Biosystems, Foster City, CA). The membrane was stained with
Ponceau S (Nacalai Tesque) in 1% (vol/vol) acetic acid and the bands
corresponding to 68-kD and 70-kD proteins (approximately 1 µg of each
protein) were excised separately from the membrane, followed by in situ
digestions with Achromobacter protease I (a lysylendopeptidase)
as described.29 Molecular mass analysis was performed by
Matrix-assisted Laser Desorption /Ionization time-of-flight (MALDI-TOF)
mass spectrometry with a PerSeptive Biosystem Voyager-DE/RP (PE
Biosystems, Foster City, CA).30 Peptide sequencing of the 4 main digested peptide fragments was performed with a Shimadzu PPSQ-2
protein sequencer (Shimadzu, Kyoto, Japan). The proteins were
identified by searching a protein sequence database [National Center
for Biotechnology Information (NCBI; nr 10.17.98), National Institutes
of Health (NIH)].
Transfection.
The DNAs of Btk, Flag-BLNK, Syk, and PLC2 expression vectors (total
10 µg) were transfected into 293T cells with Lipofectamine (Life
Technologies Inc, Rockville, MD) and the cells were
harvested after 48 hours.
Immunoprecipitation.
For the coimmunoprecipitation assay 1 × 108 of DT40
cells (expressing the T7 epitope-tagged human Btk) were resuspended in 1 mL of PBS and preincubated for 15 minutes at 37°C. Cells were then stimulated with 4 µg/mL of the anti-chicken IgM MoAb M4 for 0, 2, 5, and 10 minutes at 37°C. Stimulation was terminated by cell
lysis with the ice-cold lysis buffer described above. To detect the
coprecipitation of BLNK with T7 epitope-tagged Btk, each cell lysate
was incubated with 5 µg of the anti-T7 tag MoAb, followed by
conjugation with protein A-Sepharose CL4B beads. The beads were washed
with the lysis buffer 4 times and then boiled with 2% SDS sample
buffer for 5 minutes. The samples were then electrophoresed and
visualized by immunoblotting with the anti-chicken BLNK polyclonal antibody.
Immunoblotting analysis.
Immunoblotting analysis was performed as described
previously.31 As primary antibodies, antiphosphotyrosine
antibody 4G10 was used at a concentration of 1 µg/mL, anti-Btk MoAb
43-3B at 3 µg/mL. The anti-chicken BLNK polyclonal antibody was used
at 1:4,000 dilution, the anti-chicken Syk polyclonal antibody at 1:1,000 dilution and the anti-chicken PLC2 polyclonal antibody at
1:2,000 dilution. Immunoreactive proteins were detected by means of the
Enhanced Chemiluminescence System (Amersham, Buckinghamshire, UK). In
some cases, the intensities of the bands were semi-quantitated by using
Scanning Imager (Amersham).
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RESULTS |
Identification of the Btk-SH2 domain binding protein as BLNK.
Stimulation of BCR on RAMOS cells by crosslinking with anti-µ
antibody rapidly induces tyrosine phosphorylation of multiple cellular
proteins. To identify the phosphoproteins, which bind to the SH2 domain
of Btk, cellular proteins were extracted from stimulated or
unstimulated RAMOS cells and incubated with the GST-SH2 (Btk) fusion
protein conjugated on glutathione S Sepharose beads. Immunoblotting
analysis with the antiphosphotyrosine antibody revealed that doublet
phosphoproteins (68 kD and 70 kD) in stimulated RAMOS cells prominently
bound to the GST-SH2 (Btk) protein, whereas the binding or tyrosine
phosphorylation of these proteins from unstimulated cells was weak
(Fig 1A, left lanes). In contrast to the
prominent binding of the 68-kD and 70-kD phosphoproteins to the GST-SH2
(Btk) (Wild) protein, no binding of these proteins was detected with a
fusion protein of GST and a mutated SH2 [Arginine 307 in Btk was
replaced by Lysin; GST-SH2 (R307K)] (Fig 1A, right lanes), indicating
that the binding of these phosphoproteins was mediated via the
conserved structure of the phosphopeptide-binding pocket in the Btk-SH2
domain. We then purified the 68-kD and 70-kD proteins by using a 2-step
affinity purification procedure based on these proteins' ability to
bind the GST-SH2 (Btk) protein and the antiphosphotyrosine antibody
(see Materials and Methods). The purified 68-kD and 70-kD proteins (Fig
1B) were separately digested by Achromobacter protease I and
the resulting peptides were subjected to MALDI mass spectrometry
analysis (Fig 1C). A search of a comprehensive peptide mass database
with the list of Achromobacter protease I-digested peptide
fragments revealed that the peptide masses derived from the 68-kD and
70-kD proteins unambiguously matched with the theoretical peptide
masses of the Achromobacter protease I-digested human BLNK-s
(shortened form of BLNK15) and BLNK respectively, which are
registered in the protein sequence database (NCBI)
(Table 1). Furthermore, the amino acid
sequences of 4 main peptides (Table 1), which were identified by
protein microsequencing, were found to completely coincide with those
of the reported BLNK-s and BLNK sequences. This result indicates that
the Btk-SH2 domain binding proteins of 68 kD and 70 kD in B cells are
human BLNK-s and BLNK, which was further confirmed by immunoblotting
analysis by using an anti-BLNK antibody (data not shown).
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| Fig 1.
Identification and purification of the Btk-SH2
domain-binding protein. (A) Lysates of RAMOS cells stimulated with
anti-µ-antibody (+) or without stimulation ( ) were incubated
with the fusion protein of GST and the wild-type Btk-SH2 [SH2(Wild)]
or mutated Btk-SH2 [SH2(R307K)] domain. Binding phosphoproteins were
detected by immunoblotting with the antiphosphotyrosine (anti-pTyr)
antibody 4G10. (B) Ponceau S staining of the purified proteins on PVDF
membrane. (C) Peptide mass map obtained by MALDI mass analysis. The
result obtained from the peptide mixture generated by
Achromobacter protease I digestion of the 68-kD protein is
representatively shown.
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Table 1.
Molecular Masses of Achromobacter Protease
I-Digested Peptides From the 68- and 70-kD Btk-SH2 Domain-Binding
Proteins
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Tyrosinephosphorylation of BLNK by Syk is responsible for the binding
of BLNK to the Btk-SH2 domain.
A recent study using tyrosine-kinase-deficient DT40 cells and a
baculovirus expression system showed that the tyrosine kinase responsible for the phosphorylation of BLNK is Syk, not Btk nor Lyn.15 Therefore, we exmained whether the tyrosine
phosphorylation of BLNK by Syk is sufficient for the binding of BLNK to
the Btk-SH2 domain (Fig 2). A Flag-epitope
tagged BLNK expression vector was transfected with or without Syk
expression vector into 293T cells. As reported previously15
and also shown in the third panel of Fig 2, BLNK was strongly
tyrosinephosphorylated when it was coexpressed with Syk in this
reconstitution system. Expressed proteins were extracted and the cell
lysates were incubated with the GST-SH2 (Wild) or the GST-SH2 (R307K)
proteins on glutathione S sepharose beads, followed by the detection of
BLNK on the beads by means of immunoblotting with the anti-Flag
antibody (Fig 2, top panel). Binding of BLNK on the GST-SH2 (Wild)
protein, not on the GST-SH2 (R307K) protein, was detected only when
BLNK was coexpressed with Syk. This result indicates that the
tyrosinephosphorylation of BLNK by Syk is actually responsible for the
binding of BLNK to the Btk-SH2 domain.
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| Fig 2.
Tyrosinephosphorylation of BLNK by Syk is responsible for
the binding of BLNK to the Btk-SH2 domain. Flag-BLNK was expressed with
or without Syk in 293T cells and the cell extracts were incubated with
the GST fusion protein of the Btk-SH2 domain. The binding of Flag-BLNK
to GST proteins was detected by immunoblotting the cellular proteins
bound to the fusion protein beads with the anti-Flag antibody (top
panel). Expression of Flag-BLNK (second) or Syk (bottom) in the lysates
was detected by immunoblotting with the anti-Flag antibody or the
anti-Syk antibody. The tyrosinephosphorylation of Flag-BLNK by Syk was
confirmed by immunoprecipitation (IP) with the anti-Flag antibody
followed by immunoblotting with the antiphosphotyrosine antibody 4G10
(third).
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In vivo interaction of BLNK with Btk.
We then examined the in vivo association of Btk and BLNK in
reconstituted cells (Fig 3A) and a B cell
line (Fig 3B). Flag-BLNK and Btk were coexpressed with or without Syk
in 293T cells. The association of Btk and BLNK was detected by
immunoprecipitation with the anti-Flag antibody followed by
immunoblotting with an anti-Btk antibody. As shown in lanes 1 and 2 of
the top panel of Fig 3A, coexpression of Syk significantly potentiated
the association of Btk and BLNK (approximately 3-fold
increased binding as quantitated by Scanning Imager), indicating that
the tyrosinephosphorylation of BLNK by Syk plays an important role in
the association of BLNK and Btk also in vivo. However, it was noted
that significant binding of Btk to BLNK was observed even when BLNK was
not phosphorylated (Fig 3A, lane 2 of the top panel),
suggesting that the association of Btk and BLNK in this reconstitution
system was partly mediated in a phosphotyrosine-independent manner.
Because of the presence of the proline-rich region in BLNK and the SH3
domain in Btk, it was presumed that this phosphotyrosine-independent
association was mediated through the Btk-SH3 domain. This was confirmed
by the fact that the SH3-mutated Btk (Tryptophans 251 and 252 in Btk
were replaced by Leucines; WW251LL) hardly bound to BLNK when it was
not tyrosinephosphorylated by Syk (Fig 3A, lane 4: the intensity of the binding is less than 10% compared with
lane 3 of Fig 3A). The association of Btk and BLNK in
these reconstituted cells was, thus, mediated through both the Btk-SH2
and SH3 domains (see Discussion).
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| Fig 3.
(A) Association of Btk and BLNK in 293T cells. Flag-BLNK
and Btk [wild-type or SH3-mutated (WW251LL)] were coexpressed with
(+) or without () Syk. Coprecipitation of Btk with Flag-BLNK was
detected by immunoprecipitation (IP) with the anti-Flag antibody
followed by immunoblotting with the anti-Btk antibody 43-3B (top
panel). (Without the expression of Flag-BLNK, Btk protein was not
detected with the same procedure; data not shown.) The equality of the
amounts of the Flag-BLNK protein in each immunoprecipitate was
confirmed by reprobing the same filter with the anti-Flag antibody
(second). Tyrosinephosphorylation of Flag-BLNK by Syk in precipitates
was confirmed by immunoblotting with the antiphosphotyrosine
(anti-pTyr) antibody 4G10 (third), and the equality of the amounts of
the Btk protein in cell lysates was confirmed by immunoblotting with
the anti-Btk antibody 43-3B (bottom). (B) Association of Btk and BLNK
in DT40 cells. After DT40 cells, in which T7-Btk was stably expressed,
were stimulated with the anti-chicken IgM antibody M4 for the indicated
time periods, the cells were lysed and immunoprecipitated with the
anti-T7 antibody. The coprecipitation of the endogenous BLNK was
detected by immunoblotting with the anti-BLNK antibody (top panel). The
equality of the amounts of T7-Btk protein in each of the
immunoprecipitates was confirmed by reprobing the same filter with the
anti-Btk antibody 43-3B (second). The tyrosinephosphorylation of BLNK
(third) or that of T7-Btk (bottom) was detected by immunoprecipitating
with the anti-BLNK antibody or the anti-T7 antibody followed by
immunoblotting with the antiphosphotyrosine antibody 4G10.
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To evaluate the contributions of the Btk-SH2 and SH3 domains to the
binding with BLNK in B cells, the association of these molecules was
investigated in DT40 cells, which stably expressed T7-epitope tagged
Btk to evaluate the association precisely. After exposure to the
anti-chicken IgM antibody M4, the cells were lysed and the cell lysates
were immunoprecipitated with the anti-T7 antibody. The coprecipitation
of the endogeneous BLNK was evaluated by means of immunoblotting with
the anti-BLNK antibody (Fig 3B, top panel). Although the association of
Btk and BLNK was observed to be weak in resting cells, BCR engagement
rapidly strengthened the association. The time course of the
association of Btk and BLNK was similar to that of the
tyrosinephosphorylation of BLNK (Fig 3B, third panel)
after BCR engagement. This finding meant that the association of Btk
and BLNK in DT40 cells is phosphorylation dependent, thus indicating
that the association is mainly mediated by the Btk-SH2 domain and
phosphotyrosine(s) in BLNK.
Interaction of the Btk-SH2 domain and phosphorylated BLNK enhances
the tyrosinephosphorylation of PLC2 by Btk.
The BCR-induced PLC2 phosphorylation is known to be mediated by both
Btk and Syk.6-8,22 Genetic dissections of Btk6 or Syk22 in DT40 cells resulted in marked reductions of the PLC2 phosphorylation. This and other7,8 observations
suggest that PLC2 serves as the in vivo substrate of Btk. However,
it is not clear whether another B-cell cytoplasmic molecule mediates the efficient PLC2 phosphorylation by Btk. A recent study using BLNK-deficient DT40 cells showed that BCR-induced PLC2
phosphorylation is almost completely eliminated by the disruption of
BLNK.17 When taken together with this report, our present
observation that Btk associates with phosphorylated BLNK suggests a
mechanism by which BLNK might mediate the efficient PLC2
phosphorylation by Btk. We tested this possibility by using a
reconstituted cell system (Fig 4). An
increase in PLC2 phosphorylation was observed when wild-type Btk was
coexpressed with PLC2 in 293T cells (Fig 4, lane 2), which was
further potentiated by the additional expression of Syk (Fig 4, lane
4). This PLC2 phosphorylation was not mediated by the Btk-SH2 domain
because a comparison of the PLC2 phosphorylation by the wild-type
Btk (Fig 4, lane 2) and by the SH2-mutated Btk (Btk [R307K], Fig 4,
lane 3) did not show any significant changes (see Discussion). Although
the additional expression of BLNK with Btk did not affect the PLC2
phosphorylation significantly (compare lanes 2 and 5, Fig 4), BLNK
markedly enhanced the PLC2 phosphorylation when it was coexpressed
with Btk and Syk (compare lanes 4 and 6, Fig 4), suggesting that the
phosphorylated BLNK contributed to the efficient PLC2
phosphorylation by Btk. In contrast to this, the additional expression
of BLNK with the SH2-mutated Btk and Syk did not enhance the PLC2
phosphorylation compared with when BLNK was not expressed (compare
lanes 7 and 8, Fig 4). These observations suggest that the interaction
of the Btk-SH2 domain and phosphotyrosine(s) in BLNK contributed to the
complete tyrosinephosphorylation of PLC2 by Btk.
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| Fig 4.
Tyrosinephosphorylation of PLC2 by Btk and Syk on
BLNK. Indicated proteins were expressed in 293T cells.
Tyrosinephosphorylation of PLC2 were detected by immunoprecipitating
(IP) with the anti-PLC2 antibody followed by immunoblotting with the
antiphosphotyrosine antibody 4G10 (top panel). The same filter was
reprobed with the anti-PLC2 antibody to confirm the equality of the
amounts of the PLC2 protein (middle). The expression of Btk in the
cell lysates is also shown (bottom).
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DISCUSSION |
In the present study, we identified one of the major Btk-SH2 domain
binding proteins in B cells as BLNK and showed that the interaction of
the Btk-SH2 domain and phosphorylated BLNK contributes to the complete
tyrosinephosphorylation of PLC2 by Btk. Our findings have identified
a new molecular connection, which may contribute to the understanding
of how each domain of Btk is involved in BCR-coupled calcium signaling.
BLNK was shown to bind the Btk-SH2 domain after its
tyrosinephosphorylation by Syk. The experiment that used reconstituted cells showed that the tyrosinephosphorylation of BLNK by Syk plays an
important role in its binding to Btk also in vivo. It should be noted,
however, that, as seen in Fig 3A, a significant binding of Btk to BLNK
was still observed even when BLNK was not phosphorylated, which
suggested that both the SH2 and SH3 domains contributed to the binding
of Btk to BLNK in the reconstitution system used in this study. The SH2
and SH3 domain-dependent binding observed here looked similar to that
reported in the binding of Grb2 to BLNK,15,16 in which Grb2
constitutively bound to BLNK in resting cells but the binding was
further enhanced by BCR stimulation15 or pervanadate
stimulation.16 However, our coprecipitation experiment with
results shown in Fig 3B indicated that the association of Btk and BLNK
was weak in the resting B cells but rapidly became enhanced after BCR
engagement, which suggested that the interaction between these
molecules is phosphorylation dependent but not constitutive in B cells
and underlined the primary importance of the Btk-SH2 domain in the
interaction with BLNK. Moreover, in the reconstituted cells, the
phosphorylated BLNK enhanced the PLC2 phosphorylation by Btk but not
by the SH2-mutated Btk, indicating that the proper binding of Btk to
BLNK via the Btk-SH2 domain is necessary for the complete
tyrosinephosphorylation of PLC2. These results may well explain the
previous observation that, although the restoration of BCR-induced
calcium flux in Btk-deficient B cells required the Btk-SH2 domain, it
was independent of the Btk-SH3 domain.7
The results obtained with the reconstitution experiment indicated that
the interaction of the Btk-SH2 domain and the phosphorylated BLNK leads
Btk to the proximity of PLC2, which also binds to the phosphorylated
BLNK,15 resulting in efficient PLC2 phosphorylation. However, (and as also described in another report7) a
certain level of PLC2 phosphorylation by Btk was observed even
without the coexpression of BLNK in our reconstitution system. This
observation does not directly mean the existence of a direct
interaction between Btk and PLC2 in B cells and, in fact, it was
observed that the BLNK-deficient DT40 cells eliminated BCR-induced
PLC2 phosphorylation.17 The direct phosphorylation of
PLC2 by Btk observed in these experiments probably reflects the
difference of the intracellular distributions of these molecules in
reconstituted cells from that in B cells or may be partly mediated by
some ubiquitously expressed adaptor molecules. It is noted that, as
apparently contradicting the finding of another report,7 no
significant difference was observed in the PLC2 phosphorylation by
wild-type Btk and the SH2-mutated Btk without the coexpression of BLNK
in our reconstituted cells, suggesting that the direct interaction of
Btk and PLC2 via the Btk-SH2 domain is unlikely.
The identification of the interaction between Btk and BLNK leads to the
emergence of a new molecular scenario in BCR-coupled calcium signaling.
BCR engagement activates Btk by means of membrane anchoring through the
interaction with PtdIns-3,4,5-P3 (the Btk-PH domain is,
thus, essential in this process)10,12 and after tyrosinephosphorylation by upstream kinases.11 Syk is also
activated after BCR engagement and phosphorylates BLNK on its
tyrosines,15 which allows the association of BLNK and Btk
as shown in this study (the Btk-SH2 domain is, thus, essential in this
process) and also the association of BLNK and PLC2.15 As
Syk also colocalizes on the phosphorylated BLNK,15 BLNK
might nucleate an activation complex, including Syk, Btk, and PLC2,
for which PLC2 is fully tyrosinephosphorylated and thereby
activated. The results of genetic dissection experiments of DT40 cells
seem to support this molecular mechanism. Syk-deficient22
or BLNK-deficient17 DT40 cells almost completely abrogated
BCR-induced PLC2 phosphorylation and Btk-deficient6 DT40
cells exhibited a lower level (almost 3 times lower than that of
wild-type cells) of the PLC2 phosphorylation. The residual
phosphorylation of PLC2 in Btk-deficient cells may be caused by the
activity of Syk, because in the above scenario Btk is not necessarily
required for the Syk-dependent PLC2 phosphorylation, whereas Syk is
required for the Btk-dependent PLC2 phosphorylation through the
phosphorylation of BLNK. However, despite the presence of residual
PLC2 phosphorylation, Btk-deficient DT40 cells showed complete loss
of the BCR-induced calcium flux,6 which raises the
possibility that Btk and Syk may mutually phosphorylate distinct tyrosines in PLC2 and that Syk-dependent phosphorylation alone may
not be sufficient to activate PLC2. Precise characterization of the
PLC2 phosphorylation sites by Btk and Syk should show the exact
activation mechanism of PLC2.
Certain reported observations still remain difficult to be explained by
the simple scenario described above. First, B cells from XID mice,
which contain a mutation in the Btk-PH domain, were reported to exhibit
only a limited (40% to 50%) reduction of BCR-induced IP3
production and calcium flux,9 which is in contrast to the
observation that the calcium signaling in Btk-deficient DT40 cells
could barely be restored by PH-mutated Btk.6 Second, it was
reported that B-cell lines established from 2 XLA patients exhibited a
detectable but markedly blunted BCR-induced calcium flux, whereas the
BCR-induced PLC2 phosphorylation in XLA B cells was not altered
compared with that in normal B cells.7 As also suggested in
previous reports,6,7 these discrepancies may reflect the
species differences in the uses of PLC isoforms or Btk-related
kinases. It should also be noted that a phosphorylation-independent activation mechanism of PLC was recently reported,32
although its biologic significance in BCR-coupled calcium signaling has not yet been determined. These complicated observations may imply the
presence of some redundancies in calcium signaling at least in some
species or alternatively may indicate that multiple pathways must
operate together to allow the IP3-gated internal calcium store to be released. Our study proposes that the molecular network including Btk and BLNK is one of the major pathways in BCR-coupled calcium signaling.
| |
ACKNOWLEDGMENT |
We thank Shigeyuki Arai and his colleagues (Fujisaki Institute,
Hayashibara Biochemical Laboratories Inc, Okayama, Japan) for the
collaboration to generate the anti-Btk MoAb 43-3B, Shunsuke Ishi
(Tsukuba Life Science Center, RIKEN, Ibaraki, Japan) for providing
pT-Trx expression vector, Hirohei Yamamura (Kobe University School of
Medicine, Kobe, Japan) for providing porcine Syk cDNA, Toshio Miyawaki
(Toyama Medical and Pharmaceutical University) for critical reading of
the manuscript, and Noriko Kameoka for preparation of the manuscript.
| |
FOOTNOTES |
Submitted March 29, 1999; accepted May 23, 1999.
Supported by Grants-in-Aid for Scientific Research (to S.T., M.I., and
T. Kurosaki), Grants-in-Aid for Scientific Research on Priority Areas
(to S.T. and T. Kurosaki) and Grants-in-Aid for COE Research (to T. Kishimoto) from the Ministry of Education, Science, Sports and Culture
of Japan, the Ministry of Health and Welfare's Research Grant for
Specific Diseases, Japan (to S.T.), Grants from Mochida Science
Foundation (to S.T.), and the Takeda Science Foundation (to T. Kurosaki).
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
Molecular Medicine (formerly Medicine III), Osaka University Medical
School, 2-2, Yamada-oka, Suita City, Osaka 565-0871, Japan; e-mail:
tsukada{at}imed3.med.osaka-u.ac.jp.
| |
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TOP
ABSTRACT
INTRODUCTION