Departments of *Molecular Biology, †Cardiology, and iHematology, Jichi Medical School, Kawachi-gun, Tochigi 329-0498, Japan; ¶Omiya Medical Center,
Omiya-shi, Saitama 330-8503, Japan; ‡Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892;
and §First Department of Internal Medicine, Kagawa Medical University, Kagawa 761-0793, Japan
Edited by R. L. Erikson, Harvard University, Cambridge, MA, and approved August 2, 1999 (received for review March 15, 1999)
Tec, Btk, Itk, Bmx, and Txk constitute the Tec family of protein
tyrosine kinases (PTKs), a family with the distinct feature of
containing a pleckstrin homology (PH) domain. Tec acts in signaling
pathways triggered by the B cell antigen receptor (BCR), cytokine
receptors, integrins, and receptor-type PTKs. Although upstream
regulators of Tec family kinases are relatively well characterized,
little is known of the downstream effectors of these enzymes. The
yeast two-hybrid system has identified several proteins that interact
with the kinase domain of Tec, one of which is now revealed
to be a previously unknown docking protein termed BRDG1 (BCR
downstream signaling 1). BRDG1 contains a proline-rich motif, a PH
domain, and multiple tyrosine residues that are potential target
sites for Src homology 2 domains. In 293 cells expressing recombinant
BRDG1 and various PTKs, Tec and Pyk2, but not Btk, Bmx,
Lyn, Syk, or c-Abl, induced marked phosphorylation of BRDG1 on
tyrosine residues. BRDG1 was also phosphorylated by Tec directly
in vitro. Efficient phosphorylation of BRDG1 by Tec required the PH
and SH2 domains as well as the kinase domain of the latter.
Furthermore, BRDG1 was shown to participate in a positive feedback
loop by increasing the activity of Tec. BRDG1 transcripts are
abundant in the human B cell line Ramos, and the endogenous
protein underwent tyrosine phosphorylation in response to BCR
stimulation. BRDG1 thus appears to function as a docking protein
acting downstream of Tec in BCR signaling.
he Tec protein tyrosine kinase (PTK) was initially identified
in mouse liver (1). The subsequent molecular cloning of four
Tec-related kinases, Btk (2, 3), Itk (also known as Emt or Tsk)
(4–6), Bmx (7), and Txk (or Rlk) (8, 9), revealed that these
enzymes, together with Tec, constitute a distinct subfamily of
nonreceptor PTKs. With the exception of Txk, the members of
this subfamily possess a long NH2-terminal region consisting of
a pleckstrin homology (PH) domain (10) and a Tec homology
(TH) domain (11). Because PH domains bind phosphoinositides
with high affinity, the Tec family kinases have been proposed to
act downstream of phosphatidylinositol 3-kinase (PI3-kinase) in
signaling pathways. Indeed, an increase in PI3-kinase activity
results in activation of Btk (12), and incubation of cells with
PI3-kinase inhibitors markedly reduces the activity of intracellular
Itk. Interaction of the PH domain with phosphoinositides
is probably required for targeting of Tec family kinases to the cell
membrane, given that PI3-kinase inhibitors no longer suppress
the activity of Itk when it is fused to the extracellular and
transmembrane domains of c-Kit and thereby constitutively
targeted to the membrane fraction (13).
All members of the Tec family of kinases are abundant in
hematopoietic tissues and have thus been proposed to play
important roles in blood cell development. Consistent with this
hypothesis, mutations in Btk have been shown to cause X
chromosome-linked agammaglobulinemia in humans (2, 3), indicating
that Btk is indispensable for the maturation of B
lymphocytes. Other Tec family kinases have also been shown to
act as signaling intermediaries for lymphocyte surface antigens.
For example, Tec is activated in response to engagement either
of the B cell antigen receptor (BCR) in B lymphocytes (14) or
of CD28 in T lymphocytes (15). Cross-linking of CD28 also
induces the tyrosine phosphorylation and activation of Itk (16).
The NH2-terminal region of Btk, encompassing the PH domain
and a portion of the TH domain, is able to bind directly to the
. subunit of heterotrimeric GTP-binding proteins (17), suggesting
that Tec family kinases also might act downstream of such G
proteins. Indeed, in human platelets, stimulation of the thrombin
receptor, a transmembrane receptor coupled to G proteins,
induces activation of Tec (18). Furthermore, integrin stimulation
has been shown to regulate Tec activity (18).
Whereas substantial progress has been made in identifying the
upstream regulators of Tec family kinases, much less is known of
their downstream targets. Candidates for direct substrates of
these kinases include BAP-135 (19), phospholipase C (PLC)-g2
(20), Vav (21), and Grb10 (or GrbIR) (22). Tec phosphorylation
of PLC-g2 results in the generation of inositol trisphosphate and
consequent mobilization of intracellular Ca2. from inositol
trisphosphate receptor-gated stores (23), which may play an
important role in B cell development. Tec family kinases also
contribute to the regulation of the small GTP-binding protein
Rho, which directs the formation of stress fibers as well as the
activation of serum response factor (24). In addition, Tec is a
potent activator of the promoter of the c-fos protooncogene (25).
However, the nature of the direct effectors responsible for the
transmission of these signals from Tec remains unclear. It also
remains to be determined which effectors are common to all Tec
family kinases and which ones are enzyme-specific.
To increase our understanding of the downstream signaling
mechanisms of Tec family kinases, we have used the yeast
two-hybrid system to identify Tec substrates. One of the positive
clones obtained has now been shown to encode a previously
unidentified docking protein, which we have termed BRDG1. In
a human B cell line, BRDG1 was shown to be phosphorylated on
tyrosine residues in response to stimulation of the BCR. Furthermore,
we have shown that phosphorylation of BRDG1
results in a feedback action on Tec, leading to its activation.
Materials and Methods
Cell Lines and Antibodies. UT-7 (26) was cultured in RPMI 1640
(Life Technologies, Gaithersburg, MD) supplemented with 10%
FBS and 1 ngyml human granulocyte–macrophage colonystimulating
factor. All other hematopoietic cell lines (27) were
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: PTK, protein tyrosine kinase; PH, pleckstrin homology; TH, Tec homology;
PI3-kinase, phosphatidylinositol 3-kinase; BCR, B cell antigen receptor; PLC, phospholipase
C; GST, glutathione S-transferase; SH, Src homology; DGK, diacylglycerol kinase.
Data deposition: The nucleotide sequence reported in this paper has been deposited in the
GenBankyEMBLyDDBJ databases (accession no. AB023483).
**To whom reprint requests should be addressed at: Department of Molecular Biology,
Jichi Medical School, 3311-1 Yakushiji, Kawachi-gun, Tochigi 329-0498, Japan. E-mail:
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.
§1734 solely to indicate this fact.
11976-11981 . PNAS . October 12, 1999 . vol. 96 . no. 21
maintained in RPMI 1640y10% FBS medium. For BCR stimulation,
Ramos cells (American Type Culture Collection, ATCC;
Manassas, VA) were first incubated for 12 h in Iscove’s modified
Dulbecco’s medium (IMDM; Life Technologies) containing 1%
FBS and then exposed for 5 min to anti-human IgM F(ab9)2
mgyml) (Southern Biotechnology Associates, Birmingham,
AL), as described (14). 293 cells (ATCC) were
maintained in DMEM-F12 (Life Technologies) containing 10%
FBS and 2 mM L-glutamine.
Antibodies to BRDG1 were generated in rabbits injected with
a glutathione S-transferase (GST) fusion protein containing the
COOH-terminal half (residues 172 to 295) of BRDG1. Preparation
of anti-Tec antibodies was described before (28). Antibodies
to phosphotyrosine (4G10) and the FLAG epitope tag
(M2) were obtained from Upstate Biotechnology (Lake Placid,
NY) and Eastman Kodak (New Haven, CT), respectively. Antibodies
to other PTKs were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA).
Isolation of BRDG1 cDNA and Construction of Expression Plasmids. A
cDNA corresponding to the kinase domain of human Tec (amino
acids 357 to 630) was inserted into the pGBT9 vector (CLON-
TECH), which encodes the DNA-binding domain of yeast
GAL4, thereby yielding pGAL4bd-TecKD. This plasmid was
then used in the two-hybrid screen as described (22, 29). The
TIP4 cDNA thus obtained was labeled with [a-32P]dCTP and
then used as a probe to screen a UT-7 cell cDNA library
constructed in the
lZAPII phage vector (Stratagene). After the
second round of screening, positive phage clones were converted
to pBlueScript II plasmids (Stratagene) by the in vivo excision
protocol, and the cDNA inserts were subjected to nucleotide
The coding region of BRDG1 was amplified by PCR from the
corresponding cDNA and inserted into the pcDNA3-FLAG
vector, thereby yielding pcDNA-BRDG-F, which encodes the
BRDG1 protein with a COOH-terminal FLAG epitope tag.
The BRDG1 cDNA corresponding to amino acids 1–295 or
172–295 was PCR-amplified and subcloned into pGEX2T vector
(Amersham Pharmacia Biotech) to produce the GST-fusion
protein of the full length or COOH-terminal half of BRDG1,
Transfection and Protein Analysis. 293 cells (2 . 106) were transfected
mg of each expression plasmid by the calcium
phosphate method. After 2 days of incubation, cells were solubilized
in lysis buffer [1% Nonidet P-40y50 mM TriszHCl (pH
7.4)y150 mM NaCly1mMNaFy1mMNa3VO4yaprotinin (200
unitsyml)y1 mM PMSF]. Immunoprecipitation and immunoblot
analysis were performed as described (30), and immune complexes
were detected with the enhanced chemiluminescence
For in vitro assay of kinase activity, immune complexes formed
with antibodies to PTKs were washed twice with lysis buffer and
three times with kinase buffer [20 mM TriszHCl (pH 7.4)y50 mM
NaCly10 mM MgCl2y2 mM MnCl2] and then incubated with 0.37
MBq of [g-32P]ATP. To analyze BRDG1 phosphorylation, anti-
Tec immunoprecipitates were reacted with 0.1 mM ATP plus 1
mg of GST or GST-BRDG1 fusion protein at 37°C, and the
resulting samples were subjected to immunoblot analysis with
antibodies to phosphotyrosine or GST (AMRAD, Kew, Victoria,
Introduction of pcDNA-BRDG-F with pSR. or pSRa-
TecDKD (24) into Ramos cells (5 . 106) were conducted by
electroporation as described (25). After 12 h of culture in
RPMIy10% FBS, cells were treated for1hin IMDMy1% FBS
at the concentration of 5 . 106yml. BCR of the transfected cells
were then cross-linked as described above.
Ohya et al.
Results and Discussion
Isolation of BRDG1 cDNA. With the kinase domain of human Tec
(amino acids 357-630) as a ‘‘bait,’’ we attempted to identify
substrates of Tec by yeast two-hybrid screening. From a panel of
human cDNA libraries, we identified six Tec-interacting proteins
(TIP1–TIP6) (22, 29). The TIP4 cDNA was isolated from a
cDNA library prepared from Epstein–Barr virus-transformed B
cells (CLONTECH), and was found to encode a previously
unidentified protein. To isolate a full length TIP4 cDNA, we first
attempted to identify hematopoietic cell lines in which the TIP4
transcripts are abundant.
Total RNAs were prepared from a panel of human hematopoietic
cell lines (27) including those of T cell lineage (CCRF-
CEM, Jurkat, and PEER), B cell lineage (Ramos and Raji), and
myeloid (HEL, KU812, KG1, K562, and UT-7). Northern blot
analysis of these RNAs with a probe prepared from the TIP4
cDNA obtained in the two-hybrid screen revealed the presence
of a major TIP4 transcript of 1.6 kb and a minor one of 2.2 kb
in B cells and some myeloid cells, but not in T cells (Fig. 1A). The
same membrane was rehybridized with the
b-actin cDNA to
compare the quantities of loaded RNAs (Fig. 1 A, bottom row).
On the basis of these results, a conventional cDNA library of
UT-7 cells was constructed in the
lZAPII phage vector and
screened with the TIP4 cDNA probe. A positive clone with an
insert of 1,454 bp was obtained. The 5. region of this TIP4 cDNA
was extended by 5. rapid amplification of cDNA ends (data not
shown). Nucleotide sequencing of the assembled 1,507-bp cDNA
revealed the presence of a single ORF encoding a previously
unidentified protein of 295 amino acids and with a calculated
molecular mass of 34,291 Da (Fig. 1B). Given that we have shown
that TIP4 participates in the signaling downstream of BCR (see
below), we renamed this protein BRDG1 (BCRdownstream
Database analysis revealed that the NH2-terminal half of
BRDG1 shows sequence homology to the PH domains of various
signaling proteins, including hamster diacylglycerol kinase
(DGK)-. (32% identity, 58% similarity), human DGK-. (29%
identity, 54% similarity), and a Dictyostelium discoideum homolog
of Akt (also known as Rac-PK or PKB) (20% identity,
44% similarity) (Fig. 1 B and C). In addition, BRDG1 contains
a tryptophan residue (Trp-112) at a position corresponding to
that of the hallmark tryptophan residue of PH domains. Thus,
BRDG1 is a PH domain-containing protein. The COOHterminal
half of BRDG1 is distantly related to the Src homology
(SH) 2 domain. This region of BRDG1 shares 25% and 22%
sequence identity with the SH2 domains of human PLC-g2 and
Hydra Csk, respectively. In addition, a GST fusion protein
containing the COOH-terminal half of BRDG1, but not one
containing the PH domain, interacted with a number of tyrosinephosphorylated
proteins from lysates of 293 cells, as judged by
coprecipitation experiments (data not shown). However, several
characteristic features of SH2 domains are not present in
BRDG1; the well conserved tryptophan residue in the
and the Phe-Leu-ValyIle-Arg sequence of SH2 domains (31) is
replaced by an alanine and a Met-Ile-Leu-Arg sequence, respectively.
It therefore remains unclear whether the COOH-terminal
half of BRDG1 shares the ability of genuine SH2 domains to
recognize specifically tyrosine-phosphorylated proteins. In addition
to the PH domain, BRDG1 possesses a proline-rich
sequence that constitutes a potential binding site for SH3 or WW
domains (Fig. 1 B and D). Another characteristic feature of
BRDG1 is that it is rich in tyrosine residues, many of which are
located in sequences potentially capable of binding to SH2
domains (Table 1). The overall structure of BRDG1 therefore
resembles those of docking proteins such as IRS-1 or IRS-2 (32,
33), Gab1 or Gab2 (34, 35), and Dok-1 or Dok-2 (36–38).
PNAS . October 12, 1999 . vol. 96 . no. 21 . 11977
Fig. 1. (A) Northern blot analysis of total RNA (20 mg per lane) from
CCRF-CEM (CCRF), Jurkat, PEER, Ramos, Raji, HEL, KU812, KG1, K562, and UT-7
cells with the 32P-labeled BRDG1 cDNA (top row) or b-actin cDNA (bottom
row). The positions of molecular size standards (in kilobases) are shown on the
left and the hybridizing transcripts are shown on the right. (B) The deduced
amino acid sequence of human BRDG1. The sequence is shown in single-letter
notation, with the PH domain and the putative SH2 domain indicated by solid
and broken underlines, respectively. The proline-rich motif is boxed. Residue
numbers are on the right. (C) Comparison of the amino acid sequence of
BRDG1 with those of golden hamster DGK-. (haDGK eta; GenBank accession
no. Q64398), human DGK-. (hDGK delta; accession no. D73409), and the D.
discoideum Akt homolog (dAkt; accession no. P54644). Residues identical with
those of BRDG1 are shown as asterisks; dashes represent gaps introduced to
optimize alignment. Residue numbers are shown on the left. (D) Overall
structure of BRDG1. The proline-rich motif and the PH domain are shown as
hatched and solid boxes, respectively. The positions of tyrosine residues are
indicated by arrowheads.
BRDG1 Is an
Vivo Substrate of Tec. To determine whether
BRDG1 might function as a docking protein, we investigated
whether it is phosphorylated at a high stoichiometry by PTKs in
intact cells. We subjected 293 cells to transient transfection with
an expression plasmid encoding BRDG1 with a COOH-terminal
FLAG epitope tag either alone or together with a plasmid
encoding Tec or a kinase-defective Tec mutant (TecKM in which
Lys-397 in the putative ATP binding site is replaced with Met)
(39). BRDG1 was immunoprecipitated from the various transfected
cells with antibodies to FLAG and then subjected to
immunoblot analysis with antibodies either to phosphotyrosine
or to FLAG. FLAG-tagged BRDG1 was expressed in the cells
as a protein of '37 kDa (Fig. 2A). When expressed alone,
BRDG1 showed a very low level of tyrosine phosphorylation in
293 cells. However, coexpression with Tec resulted in a marked
increase in the extent of tyrosine phosphorylation of BRDG1;
coexpression with TecKM had no such effect. When expressed
together with Tec, BRDG1 was associated with a tyrosine-
Table 1. Potential SH2-binding tyrosine in BRDG1
Flanking sequence Binding proteins
LPL Y27 FEG
RSG Y39 REY
YRE Y42 EHY Fps
YEH Y45 WTE
LFF Y57 TDK Csk
SII Y65 VDK PLC-g1,(SHP2)
TED Y168 VDV PLC-g1,(SHP2)
ACF Y180 TVS Csk
SRN Y211 SIT
IKH Y227 KVM p85, (Shc)
GQN Y236 TIE Csk
VID Y255 FVK
phosphorylated protein of '70 kDa (Fig. 2A, lane 3), the same
molecular size as that of Tec, suggestive of a physical interaction
between BRDG1 and Tec. To confirm this hypothesis, we
expressed BRDG1 in 293 cells either alone or together with Tec
or TecKM, immunoprecipitated BRDG1 with antibodies to
FLAG, and subjected the immunoprecipitates to the immunoblot
analysis with antibodies to Tec (Fig. 2B). BRDG1 was
associated with Tec protein only in the presence of Tec activity.
Reprobing of the membrane with antibodies to FLAG demonstrated
that the amount of BRDG1 immunoprecipitated from
the various transfected cells was constant. Immunoblot analysis
of total cell lysates with antibodies to Tec also confirmed that
Tec and TecKM were expressed in equivalent amounts. Therefore,
BRDG1 appears to be an efficient substrate of Tec in intact
cells, and the BRDG1–Tec interaction is phosphorylationdependent.
To determine whether BRDG1 is a specific substrate of Tec
or whether it is also phosphorylated by other PTKs, we expressed
BRDG1 in 293 cells either alone or together with representatives
of various cytoplasmic PTK subfamilies (Fig. 2C). Whereas Pyk2
induced the phosphorylation of BRDG1 to the same extent as
did Tec, BRDG1 was not phosphorylated by Lyn, Syk, or c-Abl
(Fig. 2C, top row). We further investigated the kinase specificity
of BRDG1 phosphorylation among Tec family members. Neither
Btk nor Bmx phosphorylated BRDG1 in 293 cells (Fig. 2D,
top row). These data, however, left a possibility that only Tec and
Pyk2 were highly active in 293 cells. Generally, it is difficult to
quantitatively compare the kinase activities of distinct PTKs; for
instance, Tec cannot efficiently phosphorylate poly(Glu, Tyr) or
enolase (data not shown). Therefore, to measure the kinase
activity, we conducted an in vitro kinase assay with [γ-32P]ATP
and demonstrated autophosphorylation activity of each PTK
(bottom rows Fig. 2 C and D). Although there was a diversity in
their autophosphorylation activities, it was apparent that
BRDG1 phosphorylation did not parallel the intensity of PTK
phosphorylation. These data suggest that BRDG1 receives inputs
from a highly restricted group of cytoplasmic PTKs. However,
stimulation with stem cell factor induced the tyrosine
phosphorylation of BRDG1 in 293 cells expressing c-Kit (data
not shown). It is therefore possible that BRDG1 acts downstream
of receptor-type PTKs under certain conditions.
We also verified that BRDG1 is a direct substrate of Tec in
vitro. GST or a GST-fusion protein containing full length
BRDG1 was incubated with immunoprecipitated Tec and 0.1
mM ATP, separated by SDSyPAGE, and subjected to immunoblot
analysis with antibodies to phosphotyrosine. Tec phosphorylated
tyrosine residues of GST-BRDG1 but not of GST
(Fig. 2E), indicating that BRDG1 is a direct substrate of Tec.
Although BRDG1 was initially isolated as a protein that binds
to the kinase domain of Tec in yeast cells, we investigated
11978 . www.pnas.org Ohya et al.
Fig. 2. (A) Tec-induced phosphorylation of BRDG1 in vivo. Ten micrograms
of pcDNA3-FLAG vector (V) or of pcDNA-BRDG-F (B) were introduced into 293
cells (2 . 106) by the calcium phosphate method either alone or together with
expression plasmids encoding either Tec (T) or a kinase-defective mutant of
Tec (TM). After 48 h of culture, cells were lysed and BRDG1 was immunoprecipitated
with antibodies to FLAG. The resulting precipitates were fractionated
by SDSyPAGE on a 7.5% gel and subjected to immunoblot analysis with
antibodies to either phosphotyrosine or FLAG. The positions of Tec and BRDG1
are indicated at the bottom, and positions of molecular size standards (in
kilodaltons) are on the left. The asterisk denotes the position of IgH. (B)
Physical interaction of BRDG1 with Tec in intact 293 cells. Cells transfected with
the empty vector (V) or with vectors encoding BRDG1 (B), Tec (T), or TecKM(TM)
as indicated at the top were subjected to immunoprecipitation (IP) with
antibodies to FLAG (aFLAG), and the resulting precipitates were then subjected
to immunoblot analysis either with antibodies to Tec (aTec) or FLAG.
Total cell lysates (TCL) (10 mg of protein) of each set were also subjected to
immunoblot analysis with anti-Tec antibody. (C) Effects of various PTKs on
phosphorylation of BRDG1 in 293 cells. Cells were transfected with empty
vector (Vector) or with pcDNA-BRDG-F (1BRDG) in the absence (2) or presence
of expression plasmids encoding Tec, Lyn, Syk, c-Abl, or Pyk2. BRDG1 was
immunoprecipitated from the various transfected cells with anti-FLAG antibody
and then subjected to immunoblot analysis with antibodies to phosphotyrosine
(ap-Tyr) or to FLAG (aFLAG). The PTKs were also immunoprecipitated
from the same set of cells and subjected to an in vitro kinase assay without
exogenous substrates. Autophosphorylation of each PTK (KA) is shown at the
bottom. (D) Effects of various Tec family kinases on BRDG1 phosphorylation in
293 cells. Cells were transfected with empty vector (Vector) or with pcDNA-
BRDG-F (1BRDG) in the absence (2) or presence of expression plasmids
encoding Tec, Btk, or Bmx. BRDG1 was immunoprecipitated from the transfected
cells and probed with antibodies to phosphotyrosine or FLAG. Autophosphorylation
activity of each PTK (KA) is shown at the bottom. (E) Phosphorylation
of BRDG1 by Tec in vitro. Immunoprecipitates prepared from 293
cells expressing Tec with anti-Tec antibody were washed and then incubated
at 37°C for 15 min with 0.1 mM ATP plus 1 mg of GST or GST-BRDG1 (G-BRDG),
as indicated at the top. The samples were then subjected to the immunoblot
analysis with antibodies to GST (aGST) or to phosphotyrosine (ap-Tyr).
whether other domains of Tec contribute to the interaction
between the two full length proteins. The Tec protein is composed
of five domains: a PH domain, a TH domain, an SH3
domain, an SH2 domain, and a kinase domain (Fig. 3A). We
prepared expression plasmids that encode Tec mutants lacking
each of these domains (24) and introduced them into 293 cells
together with the BRDG1 vector. Immunoblot analysis of
BRDG1 immunoprecipitates with antibodies to phosphotyrosine
revealed that deletion of the PH domain of Tec markedly
attenuated the tyrosine phosphorylation of BRDG1 (Fig. 3B,
Fig. 3. (A) Organization of the Tec protein into PH, TH, SH3, SH2, and kinase
(KD) domains. (B) Effect of various deletion mutants of Tec on BRDG1 phosphorylation
in 293 cells. Recombinant BRDG1 was immunoprecipitated with
anti-FLAG antibody from 293 cells expressing BRDG1 (B) either alone or
together with wild-type Tec (WT) or Tec mutants lacking (D) the indicated
domains. The resulting precipitates were then subjected to immunoblot analysis
with antibodies to phosphotyrosine (ap-Tyr) or FLAG (aFLAG). (C) Autophosphorylation
activity of various Tec mutants. Wild-type Tec and the various
Tec mutants used in B were immunoprecipitated from transfected 293 cells
and subjected to immunoblot analysis with either anti-phosphotyrosine antibody
or anti-Tec antibody. Autophosphorylation activity of Tec and its
mutants were shown in arbitrary units as tyrosine phosphorylation intensity
per protein amount, both measured by a densitometer.
lane 3). Deletion of the SH3 domain or TH domain did not affect
phosphorylation of BRDG1, but removal of the SH2 domain or
kinase domain completely abolished BRDG1 phosphorylation
(Fig. 3B, lanes 6 and 7). The reduced efficiency of BRDG1
phosphorylation by the PH domain and SH2 domain mutants
was not attributable to a reduction in Tec activity of these
mutants, because the autophosphorylation activity of these
mutants were not inferior to that of wild-type Tec (Fig. 3C).
Despite the high autophosphorylation activity of TecDTH, it
could phosphorylate BRDG1 to a level similar to that of
wild-type Tec. Therefore, the TH domain of Tec may also play
a role in the interaction between the two molecules.
Given that the PH domain is a binding site for phospholipids,
this domain is probably required for physical tethering of Tec
and BRDG1 to the cell membrane, resulting in an increase in the
local concentrations of and in the interaction between the two
molecules. The requirement of the Tec SH2 domain for BRDG1
phosphorylation suggests that both the SH2 and kinase domains
of Tec recognize the same target. This conclusion is consistent
with the previous observation of Songyang et al. (40) that
cytoplasmic PTKs preferentially phosphorylate peptides that are
also recognized by their own SH2 domains, providing a molecular
basis for the ‘‘processive phosphorylation’’ model (41) that
explains how hyperphosphorylation of docking proteins is
BRDG1 Regulation of Tec Activity. We next investigated whether the
interaction of BRDG1 with Tec affects Tec activity. Cell lysates
were prepared from 293 cells expressing Tec or TecKM either
alone or together with BRDG1 and were divided into two
portions. Tec was immunoprecipitated from both portions of
each lysate; one set of precipitates was then subjected to
immunoblot analysis with antibodies to phosphotyrosine, and
Ohya et al. PNAS . October 12, 1999 . vol. 96 . no. 21 . 11979
Fig. 4. (A) Activation of Tec by BRDG1. 293 cells were transfected with empty
vector (2) or with expression vectors encoding Tec (T), TecKM(TecM), or BRDG1
(B) as indicated at the top. Tec immunoprecipitates prepared from the various
transfected cells were then either subjected to immunoblot analysis with
antibodies to Tec (aTec) or to phosphotyrosine (ap-Tyr) or assayed for in vitro
kinase activity (KA) with [g-32P]ATP. (B) Ramos cells were left unstimulated (2)
or stimulated (1) with anti-human IgM F(ab9)2fragments (10 mg/ml) for 5 min.
Total cell lysates (TCL) (10 mg of protein per lane) and immunoprecipitates
prepared with antibodies to either BRDG1 (aBrdg) or Tec (aTec) were then
subjected to immunoblot analysis with antibodies to phosphotyrosine (top
lane). The positions of Tec (T) and BRDG1 (B) are indicated on the right. The
same membrane was reprobed with anti-BRDG1 antibody or anti-Tec antibody
as indicated at the bottom. (C) Ramos cells (5 . 106) were electroporated
with pcDNA-BRDG-F (10 mg) plus 15 mgofpSR. (V) or pSRa-TecDKD (DKD).
After 12 h of culture, cells were treated for1hin IMDMy1% FBS at the
concentration of 5 . 106yml and then left unstimulated (2) or stimulated (1)
with anti-IgM antibody for 10 min. From each set, FLAG-tagged BRDG1 was
immunoprecipitated and probed with either anti-phosphotyrosine antibody
or anti-FLAG antibody.
the other set was subjected to an in vitro kinase assay (Fig. 4A).
The extents of both the tyrosine phosphorylation and kinase
activity of Tec were markedly increased by coexpression with
BRDG1. Given that no phosphorylation of TecKM was apparent
by either assay (Fig. 4A, lane 4, middle and bottom rows), the
phosphorylation of the wild-type enzyme likely reflects its own
kinase activity. Reprobing of the anti-phosphotyrosine immunoblot
with antibodies to Tec revealed that the amounts of Tec
and TecKMproteins precipitated were similar (Fig. 4A, top row).
These results suggested that BRDG1 regulates the upstream
We investigated which domains of Tec are required for its
activation by BRDG1. In addition to the PH and SH2 domains
of Tec required for BRDG1 phosphorylation, the TH domain of
Tec was also necessary for the regulation of Tec activity by
BRDG1 (data not shown). A regulatory function similar to that
of BRDG1 has been described for the docking protein Sin (also
known as Efs) (42, 43). Binding of the proline-rich motif of Sin
to the SH3 domain of c-Src has been proposed to change the
conformation of the latter to an ‘‘open’’ state and thereby induce
its activation. The TH domain of Tec family kinases is thought
to contribute to the regulation of the conformation and activity
of these enzymes (44). Therefore, by analogy with the Sin-c-Src
interaction, BRDG1 may activate Tec by disrupting an intramolecular
1. Mano, H., Ishikawa, F., Nishida, J., Hirai, H. & Takaku, F. (1990) Oncogene
2. Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A., Flinter, F.,
Hammarstrom, L., Kinnon, C., Levinsky, R., Bobtoe, M., et al. (1993) Nature
BRDG1 Is Involved in the BCR Signaling. Given that the BCR is
expressed on the surface of Ramos cells and that Tec is activated
by BCR stimulation (14), we therefore investigated whether
BRDG1 participates in BCR signaling in these cells. After
culture for 12 h in 1% FBSyIMDM medium, Ramos cells were
stimulated with antibodies to IgM, lysed, and subjected to
immunoprecipitation with antibodies to BRDG1 or to Tec.
Immunoblot analysis of the resulting precipitates with antibodies
to phosphotyrosine showed that both BRDG1 and Tec became
phosphorylated on tyrosine residues in response to BCR stimulation
(Fig. 4B). Furthermore, BCR engagement induced the
binding of several phosphoproteins to BRDG1, consistent with
a role for BRDG1 as a docking protein. Two prominent phosphoproteins
(p62 and p56) also became associated with Tec in
response to BCR stimulation. The same membrane was reprobed
with the antibodies to BRDG1 or Tec to demonstrate that the
amount of precipitated each protein was constant throughout
the BCR engagement (Fig. 4B, bottom row).
However, we could not clearly observe the physical interaction
between Tec and BRDG1 in Ramos cells. This may be owing to
the low sensitivity of our anti-Tec antibody in immunoblot
analysis, because the binding was proved only weakly even in the
high expression system in 293 cells (Fig. 2B). To further support
the physiologic relevance of Tec-BRDG1 relationship, we then
tested whether a dominant-interfering mutant of Tec (TecDKD)
can suppress the BCR-driven phosphorylation of BRDG1 in
Ramos cells. A FLAG-tagged BRDG1 was introduced into
Ramos cells either alone or together with TecDKD. After 12 h
of culture, cells were stimulated with anti-IgM antibody, and the
introduced BRDG1 was immunoprecipitated. Probing the precipitates
with anti-phosphotyrosine antibody has revealed that
BCR engagement induced the phosphorylation of FLAG-tagged
BRDG1 in Ramos cells (Fig. 4C, V lanes). Coexpression of
TecDKD significantly decreased the phosphorylation level of
BRDG1 (Fig. 4C, DKD lanes), indicating that Tec is an intermediate
between BCR and BRDG1.
In this study, we describe the molecular cloning of a previously
unidentified PTK substrate, BRDG1, that exhibits a strict specificity
for upstream kinases. BRDG1 is phosphorylated on
tyrosine residues and binds to several phosphoproteins in B cells
in response to BCR stimulation. The observation that only Tec
among the Tec family kinases was able to phosphorylate BRDG1
suggests that these enzymes probably possess overlapping but
distinct substrate specificities. This conclusion is consistent with
the observation that p62Dok is an efficient substrate for Tec but
not for Itk or Btk (15) (K.Y., Y.Y., A.M., K-i.O., A.K., U.I., K.S.,
T.Y., K.O., and H.M., unpublished work). In addition, the fact
that B lymphocytes from individuals with X-linked agammaglobulinemia
express substantial amounts of Tec protein (14)
suggests that Tec cannot fully compensate for the loss of Btk
function in these cells. Together, these data indicate that the
members of the Tec family of kinases fulfill distinct roles in vivo.
Identification of substrates such as BRDG1 that are specific for
individual Tec family kinases should help to clarify these roles.
We thank M. Kawabata for the pcDNA3-FLAG vector, C. I. E. Smith for
the Btk cDNA, D. Weil for the Bmx cDNA, T. Yi for the Lyn cDNA, T.
Mustelin for Syk cDNA, Y. Maru for the c-Abl cDNA, J. Schlessinger for
the Pyk2 cDNA, and Kirin Brewery Co. (Tokyo, Japan) for cytokines.
This work was supported in part by Grants-in-Aid for Scientific Research
on Priority Areas from the Ministry of Education, Science, Sports, and
Culture of Japan. A.M. and Y.Y. are supported by Research Awards to
Jichi Medical School Graduate Students.
3. Tsukada, S., Saffran, D. C., Rawlings, D. J., Parolini, O., Allen, R. C., Klisak,
I., Sparkes, R. S., Kubagawa, H., Mohandas, T., Quan, S., Belmont, J. W.,
Cooper, M. D., Conley, M. E. & Witte, O. N. (1993) Cell 72, 279–290.
11980 . www.pnas.org Ohya et al.
4. Siliciano, J. D., Morrow, T. A. & Desiderio, S. V. (1992) Proc. Natl. Acad. Sci.
USA 89, 11194–11198.
5. Yamada, N., Kawakami, Y., Kimura, H., Fukamachi, H., Baier, G., Altman, A.,
Kato, T., Inagaki, Y. & Kawakami, T. (1993) Biochem. Biophys. Res. Commun.
6. Heyeck, S. D. & Berg, L. J. (1993) Proc. Natl. Acad. Sci. USA 90, 669–673.
7. Tamagnone, L., Lahtinen, I., Mustonen, T., Virtaneva, K., Francis, F., Muscatelli,
F., Alitalo, R., Smith, C. I. E., Larsson, C. & Alitalo, K. (1994) Oncogene
8. Haire, R. N. & Litman, G. W. (1995) Mamm. Genome 6, 476–480.
9. Hu, Q., Davidson, D., Schwartzberg, P. L., Macchiarini, F., Lenardo, M. J.,
Bluestone, J. A. & Matis, L. A. (1995) J. Biol. Chem. 270, 1928–1934.
10. Musacchio, A., Gibson, T., Rice, P., Thompson, J. & Saraste, M. (1993) Trends
Biochem. Sci. 18, 343–348.
11. Vihinen, M., Nilsson, L. & Smith, C. I. E. (1994) FEBS Lett. 350, 263–265.
12. Li, Z., Wahl, M. I., Eguinoa, A., Stephens, L. R., Hawkins, P. T. & Witte, O. N.
(1997) Proc. Natl. Acad. Sci. USA 94, 13820–13825.
13. August, A., Sadra, A., Dupont, B. & Hanafusa, H. (1997) Proc. Natl. Acad. Sci.
USA 94, 11227–11232.
14. Kitanaka, A., Mano, H., Conley, M. E. & Campana, D. (1998) Blood 91,
15. Yang, W. C., Ghiotto, M., Barbarat, B. & Olive, D. (1999) J. Biol. Chem. 274,
16. August, A., Gibson, S., Kawakami, Y., Kawakami, T., Mills, G. B. & Dupont,
B. (1994) Proc. Natl. Acad. Sci. USA 91, 9347–9351.
17. Jiang, Y., Ma, W., Wan, Y., Kozasa, T., Hattori, S. & Huang, X. Y. (1998)
Nature (London) 395, 808–813.
18. Hamazaki, Y., Kojima, H., Mano, H., Nagata, Y., Todokoro, K., Nagasawa, T.
& Abe, T. (1998) Oncogene 16, 2773–2780.
19. Yang, W. & Desiderio, S. (1997) Proc. Natl. Acad. Sci. USA 94, 604–609.
20. Takata, M. & Kurosaki, T. (1996) J. Exp. Med. 184, 31–40.
21. Machide, M., Mano, H. & Todokoro, K. (1995) Oncogene 11, 619–625.
22. Mano, H., Ohya, K., Miyazato, A., Yamashita, Y., Ogawa, W., Inazawa, J.,
Ikeda, U., Shimada, K., Hatake, K., Kasuga, M., et al. (1998) Genes Cells 3,
23. Fluckiger, A. C., Li, Z., Kato, R. M., Wahl, M. I., Ochs, H. D., Longnecker, R.,
Kinet, J. P., Witte, O. N., Scharenberg, A. M. & Rawlings, D. J. (1998) EMBO
J. 17, 1973–1985.
24. Mao, J., Xie, W., Yuan, H., Simon, M. I., Mano, H. & Wu, D. (1998) EMBO
J. 17, 5638–5646.
25. Yamashita, Y., Watanabe, S., Miyazato, A., Ohya, K., Ikeda, U., Shimada, K.,
Komatsu, N., Hatake, K., Miura, Y., Ozawa, K., et al. (1998) Blood 91,
26. Komatsu, N., Nakauchi, H., Miwa, A., Ishihara, T., Eguchi, M., Moroi, M.,
Okada, M., Sato, Y., Wada, H., Yawata, Y., et al. (1991) Cancer Res. 51,
27. Sato, K., Mano, H., Yazaki, Y. & Hirai, H. (1994) Leukemia 8, 1663–1672.
28. Yamashita, Y., Miyazato, A., Ohya, K., Ikeda, U., Shimada, K., Miura, Y.,
Ozawa, K. & Mano, H. (1996) Jpn. J. Cancer Res. 87, 1106–1110.
29. Ohya, K., Kajigaya, S., Yamashita, Y., Miyazato, A., Hatake, K., Miura, Y.,
Ikeda, U., Shimada, K., Ozawa, K. & Mano, H. (1997) J. Biol. Chem. 272,
30. Miyazato, A., Yamashita, Y., Hatake, K., Miura, Y., Ozawa, K. & Mano, H.
(1996) Cell Growth Differ. 7, 1135–1140.
31. Waksman, G., Shoelson, S. E., Pant, N., Cowburn, D. & Kuriyan, J. (1993) Cell
32. Sun, X. J., Rothenberg, P., Kahn, C. R., Backer, J. M., Araki, E., Wilden, P. A.,
Cahill, D. A., Goldstein, B. J. & White, M. F. (1991) Nature (London) 352,
33. Sun, X. J., Wang, L. M., Zhang, Y., Yenush, L., Myers, M. G., Jr., Glasheen,
E., Lane, W. S., Pierce, J. H. & White, M. F. (1995) Nature (London) 377,
34. Holgado-Madruga, M., Emlet, D. R., Moscatello, D. K., Godwin, A. K. &
Wong, A. J. (1996) Nature (London) 379, 560–564.
35. Gu, H., Pratt, J. C., Burakoff, S. J. & Neel, B. G. (1998) Mol. Cell 2, 729–740.
36. Yamanashi, Y. & Baltimore, D. (1997) Cell 88, 205–211.
37. Carpino, N., Wisniewski, D., Strife, A., Marshak, D., Kobayashi, R., Stillman,
B. & Clarkson, B. (1997) Cell 88, 197–204.
38. Di Cristofano, A., Carpino, N., Dunant, N., Friedland, G., Kobayashi, R., Strife,
A., Wisniewski, D., Clarkson, B., Pandolfi, P. P. & Resh, M. D. (1998) J. Biol.
Chem. 273, 4827–4830.
39. Mano, H., Yamashita, Y., Miyazato, A., Miura, Y. & Ozawa, K. (1996) FASEB
J. 10, 637–642.
40. Songyang, Z., Carraway, K. L., III, Eck, M. J., Harrison, S. C., Feldman, R. A.,
Mohammadi, M., Schlessinger, J., Hubbard, S. R., Smith, D. P., Eng, C., et al.
(1995) Nature (London) 373, 536–539.
41. Mayer, B. J., Hirai, H. & Sakai, R. (1995) Curr. Biol. 5, 296–305.
42. Alexandropoulos, K. & Baltimore, D. (1996) Genes Dev. 10, 1341–1355.
43. Ishino, M., Ohba, T., Sasaki, H. & Sasaki, T. (1995) Oncogene 11, 2331–2338.
44. Andreotti, A. H., Bunnell, S. C., Feng, S., Berg, L. J. & Schreiber, S. L. (1997)
Nature (London) 385, 93–97.
Ohya et al. PNAS . October 12, 1999 . vol. 96 . no. 21 . 11981