(Received for publication, May 22, 1995)
From the
Mutations in Bruton's tyrosine kinase (Btk) have been associated with immunodeficiencies in man and in the mouse. Btk and two related proteins, Itk and Tec, are members of a distinct family of tyrosine kinases. These kinases are believed to function in various receptor-mediated signaling pathways, but their specific functions are as yet undefined. Btk and its homologues share extensive sequence similarity, including a conserved region, the Tec-homology (TH) domain, that has been proposed to mediate specific intermolecular or intramolecular interactions. The TH region of Btk contains a functional SH3-binding site at residues 189-192. SH3 binding is selective: Btk is retained by the SH3 domain of Fyn but not by that of Blk, another Src-type kinase. TH-SH3 binding in vitro is abolished by specific, single amino acid substitutions within the Btk TH domain or the Fyn SH3 domain. We provide two lines of evidence that the SH3-binding site in the Btk TH domain mediates protein interactions in intact cells. First, treatment of cells with pervanadate induces an increase in the phosphotyrosine content of kinase-inactive Btk; this response is substantially reduced by a mutation that inactivates the SH3-binding site in the Btk TH domain. Second, in cell lysates Btk is found in association with an as yet unidentified 72-kDa phosphotyrosine-containing protein; this interaction requires a functional SH3-binding site in the TH domain. The TH domain may therefore interact in vivo with other proteins that regulate the phosphorylation state of Btk.
The participation of protein-tyrosine kinases in lymphocyte activation and differentiation is well established. Members of the Src and Syk/ZAP-70 tyrosine kinase families have been shown to associate with antigen receptor complexes and to become activated enzymatically upon antigen receptor engagement (reviewed in (1) ). Furthermore, deficiencies in ZAP-70 (2, 3) or the Src homologues Lck (4) and Fyn (5, 6) are associated with impaired T cell receptor-mediated signaling, validating the involvement of these kinases in T cell activation.
More
recently, mutations in Bruton's tyrosine kinase (Btk) ()have been associated with a profound B cell
immunodeficiency (X-linked agammaglobulinemia or XLA) in man (7, 8) and a heritable defect of B cell function
(X-linked immunodeficiency or xid) in the CBA/N mouse
strain(9, 10) . The underlying biological defect in
XLA is an arrest of B cell development at the pre-B cell stage; as a
result, serum antibody and circulating B cells are absent or
rare(11, 12) . The defect associated with the xid mutation differs somewhat from XLA: B cells are present in reduced
numbers and responses to T cell-independent antigens are impaired, but
responses to T cell-dependent antigens are intact(13) . Several
specific B cell abnormalities are observed in xid mice,
including a failure to proliferate in response to Ig
cross-linking(13) . The XLA and xid phenotypes provide
strong genetic evidence that Btk plays critical roles in B cell
development and activation.
Btk and two related proteins, Itk (14, 15) and Tec(16, 17) , are
members of a distinct family of tyrosine kinases. Btk is preferentially
expressed in the B lymphoid and myeloid cell lineages; expression of
Itk is restricted to the T lymphoid lineage, where it is induced by
interleukin-2; Tec has a less restricted expression pattern than Btk or
Itk. All three kinases become newly phosphorylated on tyrosine and
their associated kinase activity increases in response to extracellular
stimuli: Btk upon cross-linking of surface Ig on B cells (18, 19) or FcRI receptors on myeloid
cells(20) , Itk upon ligation of the co-stimulatory molecule
CD28 in the human leukemic T cell line Jurkat (21) , and Tec
upon treatment of myeloid or B lymphoid cell lines with
interleukin-3(22) . These kinases are therefore believed to
participate in various receptor-mediated signaling pathways, but their
specific functions are as yet undefined.
Btk, Itk, and Tec resemble
Src-type kinases in the number and arrangement of Src-homology 3 (SH3),
Src-homology 2 (SH2), and catalytic domains but, unlike Src-type
kinases, they lack a regulatory tyrosine residue near the carboxyl
terminus and a consensus amino-terminal myristoylation
site(23) . The single most distinctive feature of Btk and its
homologues, however, is a region of sequence similarity extending from
the initiator methionine to the SH3 domain (Fig. 1, A and B). This interval includes a pleckstrin homology (PH)
domain and an additional conserved region, the so-called Tec-homology
or TH domain (24) that lies between the PH and SH3 domains (Fig. 1A). The conservation of amino acid sequence
(43-46% identity) in the TH region has suggested that it mediates
specific intermolecular or intramolecular interactions that are
critical for biological function(10, 25) . A striking
feature of the TH region is the presence (Fig. 1, B and C) of one or more consensus motifs (XPPPXP, where
denotes a hydrophobic residue) for binding to the Src SH3 domain, as
defined by peptide selection from a biased combinatorial
library(26) . This motif occurs once in Itk and Tec (residues
161-167 and 165-171, respectively) and twice in Btk
(residues 186-192 and 200-206); an additional site in Tec
(residues 155-161) resembles the consensus motif but lacks the
preferred proline residue at position 2. Thus, the TH regions of Btk
and related kinases may mediate specific interactions with a subset of
SH3-containing proteins; identification of such interactions may help
to define the signal transduction pathways in which Btk and its
homologues function.
Figure 1:
A,
overall organization of Itk and Btk tyrosine kinases. The amino acid
sequences of Itk and Btk are diagrammed as rectangles; the
total number of amino acid residues in each protein is indicated at the right. SH2 and SH3, Src homology regions 2 and 3; CAT, catalytic region. Numbers in italics indicate
the percent amino acid sequence identity to the corresponding region of
Itk. B, amino-terminal homology between Itk and Btk. The
amino-terminal sequences of Itk and Btk from the initiator methionine
residue to the end of the TH region are aligned. Gaps introduced to
maximize sequence identity are indicated by ellipses.
Identical residues are shaded. The pleckstrin homology region
is indicated by a dashed underline. Residues contained within
putative SH3-binding sites are indicated (±). C,
SH3-binding consensus sequences in Btk and Itk. The consensus sequence
for binding to the Src SH3 domain, as defined by selection from a
biased combinatorial peptide library (26) is indicated on the top line. The overall SH3-binding site consensus, as defined
by sequences of SH3-selected peptides sequences and a collection of
known SH3-binding sites (26) is shown on the second
line; where indicates a hydrophobic residue.
Putative SH3-binding sites in Itk and Btk are indicated on the third through fifth lines; numbers designate
the amino acid residues corresponding to the sequences at right.
In this article we characterize a functional SH3-binding site within the TH region of Btk and provide evidence that this binding site mediates protein interactions in vivo. The SH3-binding site resides at residues 189-192 of Btk. SH3 binding is selective: Btk is retained by the SH3 domain of Fyn but not by that of Blk, another Src-type kinase. TH-SH3 binding in vitro was abolished by specific, single amino acid substitutions within the Btk TH domain or the Fyn SH3 domain. We provide two lines of evidence that the SH3-binding site in the Btk TH domain mediates protein interactions in vivo. First, treatment of cells with pervanadate induces an increase in the phosphotyrosine content of kinase-inactive Btk; this response is substantially reduced by a mutation that inactivates the SH3-binding site in the Btk TH domain. Second, in cell lysates Btk is found in association with an as yet unidentified, 72-kDa phosphotyrosine-containing protein; this interaction requires a functional SH3-binding site in the Btk TH domain.
Plasmids encoding GST fusion
proteins were introduced into E. coli strains DH5 or
BL21; transformants were cultured overnight at 37 °C in 2
YT medium supplemented with carbenicillin at 25 µg/ml. Overnight
cultures were diluted 1:10 into fresh medium and grown at 37 °C to
an OD
0.6-1.0; expression of fusion proteins
was then induced by addition of
isopropyl-1-thio-
-D-galactopyranoside to 0.4 mM.
After 3.5 h, bacteria were pelleted and resuspended in ice-cold PBS
lysis buffer (150 mM NaCl, 10 mM sodium phosphate (pH
7.5), 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 1% Triton X-100, 0.5 mg/ml lysozyme, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 2.5 µg/ml pepstatin, 2.5
µg/ml antipain, and 1 µg/ml chymostatin). The mixtures were
incubated for 20 min on ice and lysis was completed by sonication (4
cycles of 10 pulses each with a Branson microtip sonifier at output
3-5, duty cycle 60%). Lysates were clarified by centrifugation at
30,000
g for 30 min at 4 °C.
A preliminary
assay was performed to determine the quantity of lysate needed to
produce affinity matrices of equivalent substituent density. Varying
amounts of lysate were mixed with 500 µl of a 50% slurry of
glutathione-agarose beads and incubated for 1 h at 4 °C. Beads were
washed once with PBS, twice with 5 PBS, and three times with
PBS; beads were then boiled in SDS sample buffer and the relative
amounts of adsorbed GST fusion proteins were estimated by SDS-PAGE and
Coomassie staining. To prepare affinity matrices for use in binding
assays, the amount of bacterial lysate used in each adsorption was
adjusted to produce beads of similar substituent density. Beads were
adsorbed to GST fusion proteins and washed as described above, then
stored on ice as a 50% slurry in PBS supplemented with 1 mM
phenylmethylsulfonyl fluoride, and 10% glycerol and used in 2 days.
Transient transfection into 293 cells was performed as
described(28) . Cells were washed at 40-48 h after
transfection with ice-cold PBS containing 5 mM EDTA. For
assays of Btk-Fyn SH3 domain interactions, cells were lysed in buffer D
(150 mM NaCl, 20 mM Tris-Cl (pH 8.3), 1 mM phenylmethylsulfonyl fluoride 0.5% digitonin, 0.5% Nonidet P-40,
10 µg/ml aprotinin, 10 µg/ml leupeptin, 2.5 µg/ml
pepstatin, 2.5 µg/ml antipain, and 1 µg/ml chymostatin) and
incubated for 20 min on ice. For preparations of wild-type and mutant
Fyn for in vitro binding assays, cells were lysed in buffer N
(150 mM NaCl, 25 mM Tris-Cl (pH 8.0), 1% Nonidet
P-40). Lysates were clarified by centrifugation at 15,000 g for 15 min at 4 °C. Concentration of protein in clarified
extracts was determined by the BCA assay (Pierce).
Immunoprecipitates were washed twice with
buffer D, twice with 500 mM LiCl, 50 mM Tris-Cl (pH
7.4), once with Fyn kinase buffer (50 mM Tris-Cl (pH 7.4), 10
mM NaCl, 10 mM MgCl, 2 mM MnCl
, 1 mM Na
VO
) and
resuspended in 100 µl of Fyn kinase buffer. The reaction was
initiated by addition of 1 µl of
[
-
P]ATP (specific activity 6000 Ci/mmol);
after 10 min at 37 °C the reaction was stopped by addition of SDS
sample buffer. Protein was fractionated by SDS-PAGE and transferred to
PVDF membranes (Millipore). Fyn was detected by immunoblotting with an
anti-Fyn rabbit polyclonal antibody (Santa Cruz Biotechnology);
membranes were then incubated in 1 M KOH for 2 h at 55 °C
to selectively dephosphorylate phosphoserine and phosphothreonine
residues(31) , and the remaining filter-bound
P
was detected by autoradiography.
Immunoprecipitation of Btk and
immune complex kinase assays were performed as described for Fyn,
except that immunoprecipitates were washed three times with buffer D
and once with Btk kinase buffer (25 mM Tris-Cl (pH 7.4), 10
mM MgCl, 10 mM MnCl
, 0.1
mM Na
VO
) before initiating the kinase
reaction.
Figure 2: Specific binding of Btk to GST-SH3 fusion proteins. A, retention of Btk from lysates of A20 B cells. A20 lysates were incubated with beads coated with GST (GST) or with GST fusions to the Fyn amino-terminal region (GST-FynN), the Fyn SH3 domain (GST-FynSH3), or the Fyn SH2 domain (GST-FynSH2). Retained protein was fractionated by SDS-PAGE and Btk was detected by immunoblotting with an anti-Btk antibody. Positions of molecular weight standards and their sizes in kilodaltons are indicated at the left. B, retention of Btk from lysates of transfected 293 cells. Btk was expressed by transient transfection in the 293 cell line and lysates of transfected cells were incubated with beads coated with GST (lane 3) or with GST fusions to the Blk amino-terminal region (lane 4), the Blk SH3 domain (lane 5), the Fyn amino-terminal region (lane 6), or the Fyn SH3 domain (lane 7). Retained protein was analyzed by immunoblotting with an anti-Btk antibody as in A. Samples of total cell lysates from 293 cells transfected with the Btk expression construct (lane 1) or with vector alone (lane 2) were analyzed in parallel. Positions of molecular weight standards and their sizes in kilodaltons are indicated at the left.
Figure 3: Localization of the SH3-binding site to the TH region of Btk. Fyn was expressed in 293 cells by transient transfection; undiluted lysate was incubated with beads coated with GST (GST; lane 2) or serially diluted and incubated with GST fusions to Btk amino acid residues 1-218 (GST-BtkN; lanes 3-6), 104-218 (GST-BtkN2; lanes 7-10), or 1-80 (GST-BtkN1; lanes 11-14). Retained protein was fractionated by SDS-PAGE and Fyn was detected by immunoblotting with an anti-Fyn antibody. Lanes 2, 3, 7, and 11, adsorptions with undiluted lysate; lanes 4, 8, and 12, lysate diluted 1:2; lanes 5, 9, and 13, lysate diluted 1:4; lanes 6, 10, and 14, lysate diluted 1:8. Positions of molecular weight standards and their sizes in kilodaltons are indicated at the left.
To localize more precisely the region of Btk that mediates binding to Fyn, a peptide competition experiment was performed. The wild-type competitor peptide, KPLPPTPED, incorporates the sequence of the putative SH3-binding site (KPLPPTP) spanning residues 186-192 of Btk; this sequence is identical to residues 161-167 of Itk. Three additional peptides, bearing single or multiple alanine substitutions, were also tested; these substitutions altered amino acid residues identified as critical for SH3 binding by peptide selection experiments(26) . A lysate of transfected 293 cells, expressing Btk, was adsorbed to the GST-FynSH3 affinity matrix in the presence of increasing concentrations of competitor peptide, and the amount of Btk retained by the matrix was assayed by immunoblotting. The wild-type peptide was seen to compete with Btk for binding to the Fyn SH3 domain (Fig. 4, lanes 2-6). Mutation of lysine at position 1 (Fig. 4, lanes 7-11), proline at position 4 (Fig. 4, lanes 17-21), or proline at positions 2, 4, and 7 (Fig. 4, lanes 12-16) abolished the ability of peptides to compete with Btk for binding to the Fyn SH3 domain at concentrations as high as 2 mM. Thus, residues 186-192 of Btk comprise a specific SH3-binding site whose structural requirements for interaction with the Fyn SH3 domain are similar to those of peptides that bind the Src SH3 domain with high affinity.
Figure 4: SH3 binding to Btk is mediated by a conserved, proline-rich motif in the TH region. Lysates of 293 cells expressing Btk by transient transfection were incubated with GST-Fyn SH3 beads in the absence of peptide competitor (lane 1), in the presence of the synthetic peptide KPLPPTPED, corresponding to residues 186-192 of wild-type Btk (lanes 2-6), or in the presence of mutant peptides APLPPTTPED (lanes 7-11), KALAPTAED (lanes 12-16), and KPLAPTPED (lanes 17-21). Peptides were introduced at concentrations of 0.2 µM (lanes 2, 7, 12, and 17), 2 µM (lanes 3, 8, 13, and 18); 20 µM (lanes 4, 9, 14, and 19); 200 µM (lanes 5, 10, 15, and 20), and 2 mM (lanes 6, 11, 16, and 21). Retained protein was fractionated by SDS-PAGE and Btk was detected by immuoblotting with an anti-Btk antibody.
Residues 200-206 of Btk (KPLPPEP) resemble the SH3-binding
motif at residues 186-192. We used site-directed mutagenesis to
assess the relative contributions of these two regions to SH3 binding.
The wild-type GST-BtkN2 fusion protein and a mutant fusion protein
carrying a proline to alanine substitution at residue 203 (GST-BtkN2
P203A) were similarly able to bind Fyn in vitro (Fig. 5A, lanes 1-4 and 9-12); in contrast, fusion proteins bearing a proline to
alanine substitution at residue 189 (GST-BtkN2 P189A) or alanine
substitutions at both Pro and Pro
(GST
BtkN2 P189A, P203A) showed similar reductions in binding to Fyn (Fig. 5A, lanes 5-8 and 13-16). To
extend these observations to the binding of full-length Btk, wild-type
or mutant Btk proteins were expressed in 293 cells and tested for their
ability to bind to GST-FynSH3. Wild-type Btk and Btk P203A were both
retained on GST-FynSH3 beads (Fig. 5B, lanes 1-4 and 9-12). SH3 binding was unaffected by mutation
of a conserved tryptophan residue in the Btk SH3 domain (W251L),
indicating that under conditions of this assay the Btk SH3 domain does
not mask accessibility of the Btk SH3-binding site (Fig. 5B,
lanes 17-20). In contrast, the binding of Btk P189A and Btk
P189A,P203A to GST-FynSH3 was substantially reduced (Fig. 5B, lanes 5-8 and 13-16).
Taken together, these observations indicate that intervals spanning
residues 186-192 mediates binding of Btk to the SH3 domain of
Fyn.
Figure 5: Residues 189-192 of Btk comprise the principal SH3-binding site. A, binding of Fyn to GST fusion proteins containing amino acid residues 104-218 of wild-type Btk (GST-BtkN2), to similar fusion proteins carrying mutations in the first (GST-BtkN2 P189A) or second (GST-BtkN2 P203A) putative SH3-binding motif, or to fusion proteins carrying mutations in both motifs (GST-BtkN2 P189A, P203A). Lysates of 293 cells expressing Fyn were serially diluted 2-fold and incubated with beads coated with GST fusion proteins; retained protein was fractionated by SDS-PAGE and Fyn was detected by immunoblotting with an anti-Fyn antibody. Lanes1-4, binding to wild-type GST-BtkN2; lanes 5-8, binding to GST-BtkN2 P189A; lanes 9-12, binding to GST-BtkN2 P203A; lanes 13-16, binding to GST-BtkN2 P189A, P203A. Lanes 1, 5, 9, and 13, adsorptions with undiluted lysate; lanes 2, 6, 10, and 14, lysate diluted 1:2; lanes 3, 7, 11, and 15, lysate diluted 1:4; lanes 4, 8, 12, and 16, lysate diluted 1:8. Positions of molecular weight standards and their sizes in kilodaltons are indicated at the left; positions of Fyn and GST-BtkN2 fusion proteins are indicated by arrows at the right. B, binding of wild-type and mutant Btk proteins to the Fyn SH3 domain. Lysates of 293 cells expressing wild-type Btk (lanes 1-4), Btk P189A (lanes 5-8), Btk P203A (lanes 9-12), Btk P189A,P203A (lanes 13-16), or Btk W251L (lanes 17-20) were serially diluted 2-fold and incubated with GST-Fyn SH3 beads. Retained protein was fractionated by SDS-PAGE and Btk was detected by immunoblotting with an anti-Btk antibody. The position of Btk is indicated by the arrow at the right.
Figure 6:
Binding of Fyn to the Btk TH region is
selectively abolished by mutation of Trp in the Fyn SH3
domain. A, binding of wild-type and mutant Fyn proteins to the
Btk TH region. Lysates of 293 cells expressing wild-type Fyn, Fyn
D100N, or Fyn W119L were serially diluted 2-fold and assayed for
binding to the GST-BtkN2 fusion protein. Retained protein was
fractionated by SDS-PAGE and Fyn was detected by immunoblotting with an
anti-Fyn antibody. Lanes 1 and 3-6, Fyn
wild-type lysate; lanes 7-10, Fyn D100N lysate; lanes 11-14, Fyn W119L lysate. Lane 2, lysate
of 293 cells transfected with vector alone. Lanes 1, 2, 3, 7,
and 11, undiluted lysates; lanes 4, 8, and 12, lysates diluted 1:2; lanes 5, 9, and 13,
lysates diluted 1:4; lanes 6, 10, and 14, lysates
diluted 1:8. Lane 1, binding to GST alone; lanes
2-14, binding to GST-BtkN2. Positions of molecular weight
standards and their sizes in kilodaltons are indicated at the left.
B, assay for wild-type and mutant Fyn proteins in lysates of
transfected 293 cells. Equivalent amounts (20 µg of protein) of
lysate from 293 cells transfected with vector alone (lane 1),
or cells expressing Fyn (lane 2), Fyn W119L (lane 3),
or Fyn D100N (lane 4) were fractionated by SDS-PAGE and
assayed for Fyn by immunoblotting with an anti-Fyn antibody. Positions
of molecular mass standards and their sizes in kilodaltons are
indicated at the left. C, wild-type Fyn and the Fyn W119L
mutant exhibit similar autophosphorylation activities in
vitro. Fyn was immunoprecipitated, using a mouse anti-Fyn
monoclonal antibody, from equivalent amounts of lysate from 293 cells
transfected with vector alone (lanes 1 and 7) or from
cells expressing Btk (lanes 2 and 8), wild-type Fyn (lanes 3 and 9), Fyn D100N (lanes 4 and 10), Fyn W119L (lanes 5 and 11), or
kinase-inactive Fyn K296E (lanes 6 and 12). Immune
complex kinase reactions were performed as described under
``Materials and Methods''; products were fractionated by
SDS-PAGE and
P-labeled proteins were detected by
autoradiography (lanes 1-6). Fyn was detected by
immunoblotting with a rabbit anti-Fyn antibody (lanes
7-12). Positions of molecular mass standards and their sizes
in kilodaltons are indicated at the left.
Although the residue corresponding to
Asp of Fyn is highly conserved among SH3 domains, the
D100N mutation had little or no effect on binding of Btk by Fyn SH3. In
the SH3 domain of PI3K the analogous residue, Asp
, appears
to interact with the amino-terminal arginine of a high-affinity peptide
ligand(26) . The SH3-binding site at residues 186-192 of
Btk has an initial lysine (Lys
) in place of arginine; the
peptide competition data presented above indicate that Lys
contributes significantly to Fyn SH3 binding. The behavior of the
Fyn D100N mutant, however, suggests that binding does not involve a
direct interaction between Lys
of Btk and Asp
of Fyn.
In the experiment shown in Fig. 7, cells were treated for 10 min with pervanadate; Btk was then immunoprecipitated and phosphotyrosine was detected by immunoblotting. Pervanadate treatment was accompanied by an increase in the phosphotyrosine content of Btk (Fig. 7A, upper panel, lanes 2 and 7) and a concomitant shift of Btk to lower mobility (Fig. 7A, lower panel, lanes 2 and 7), presumably as a consequence of increased phosphorylation. Tyrosine phosphorylation of a kinase-inactive Btk mutant (K430E) was undetectable in untreated cells; tyrosine phosphorylation of Btk K430E was clearly detectable, however, after pervanadate treatment. The P189A mutation, which abolishes SH3 binding by Btk, had no detectable effect on basal or pervanadate-inducible tyrosine phosphorylation of kinase-active Btk (Fig. 7A, lanes 3 and 8), but resulted in a substantial reduction in the pervanadate-inducible tyrosine phosphorylation of enzymatically inactive Btk (Fig. 7A, lanes 9 and 10, and Fig. 7B).
Figure 7: Induction of Btk trans-phosphorylation by pervanadate is impaired by inactivation of the SH3-binding site at residues 186-192. A, tyrosine phosphorylation of Btk in vivo in the presence or absence of pervanadate. 293 cells expressing wild-type Btk (lanes 2 and 7), Btk P189A (lanes 3 and 8), Btk K430E (lanes 4 and 9), or Btk P189A,K430E (lanes 5 and 10), or cells transfected with vector alone (lanes 1 and 6) were left untreated (lanes 1-5) or incubated for 10 min in the presence of 1 mM pervanadate (lanes 6-10). Cells were lysed and Btk was collected by immunoprecipitation; immunoprecipitates were fractionated by SDS-PAGE and phosphotyrosine was detected by immunoblotting with an anti-phosphotyrosine antibody (upper). The same filter was subsequently stripped and Btk was detected by immunoblotting with an anti-Btk antibody (lower). Positions of molecular mass standards and their sizes in kilodaltons are indicated at the left. B and C, quantitation of pervanadate-inducible, tyrosine phosphorylation of Btk K430E and Btk K430E,P189A. 293 cells expressing Btk K430E or Btk K430E,P189A were treated with pervanadate and Btk was immunoprecipitated as in A. Cell lysates were serially diluted 2-fold and fractionated by SDS-PAGE. Btk (B, upper) and phosphotyrosine (B, lower) were detected by immunoblotting. Chemiluminescent signals were digitized by optical scanning and intensities were quantitated using ImageQuant software. At each dilution, the phosphotyrosine signal amount of Btk protein; the phosphotyrosine signal the ratio of these normalized intensities are expressed on the ordinate as the ratio of levels of phosphotyrosine in Btk K430E,P189A were plotted as the percent of phosphotyrosine in Btk K430E after normalization of Btk protein. At each dilution, the phosphotyrosine signals associated with Btk K430E,P189A and Btk K430E were normalized to the corresponding amount of Btk protein. In C, the amount of phosphotyrosine detected in Btk P189A,K430E, relative to the amount detected in Btk K430E (P-Tyr (P189A, K430E)/P-Tyr (K430E)), is indicated for each dilution assayed.
This
reduction was quantitated by densitometry of anti-phosphotyrosine and
anti-Btk chemiluminescent signals. To ensure that our measurements were
not distorted by non-linearity of the enhanced chemiluminescence assay
or of the densitometric analysis, we analyzed varying amounts of
immunoprecipitated protein. Btk and phosphotyrosine by immunoblotting
of serially diluted, anti-Btk immunoprecipitates from
pervanadate-treated cells expressing Btk K430E or Btk P189A,K430E (Fig. 7B). At each dilution, the density of the
anti-phosphotyrosine signal was normalized to the density of the
anti-Btk signal. The normalized anti-phosphotyrosine signals obtained
for Btk P189A,K430E and Btk K430E were then expressed as a ratio;
mutation of Pro clearly resulted in a 3-4-fold
reduction in the pervanadate-induced phosphorylation of Btk (Fig. 7C). We conclude that under the conditions of
this experiment: 1) most basal and pervanadate-inducible tyrosine
phosphorylation of wild-type Btk is apparently the result of
autophosphorylation; 2) Btk autokinase activity is not affected by
inactivation of the SH3-binding site in the TH domain; 3) Btk is a
substrate for tyrosine phosphorylation by one or more additional
kinases; and 4) interactions mediated by the SH3-binding site at
186-192 facilitates phosphorylation of Btk by other tyrosine
kinases.
Figure 8:
Association of Btk with a 72-kDa,
phosphotyrosine-containing protein is impaired by inactivation of the
SH3-binding site in the TH domain. 293 cells expressing wild-type Btk (lane 1), Btk P189A (lane 2), Btk K430E (lane
3), Btk K430E,P189A (lane 4), or cells transfected with
vector alone (lane 5) were lysed and lysates were subjected to
immunoprecipitation with an anti-Btk antibody. Immune complex kinase
assays were performed as described under ``Materials and
Methods''; products were fractionated by SDS-PAGE and transferred
to a PVDF membrane. The membrane was treated with KOH and
alkali-resistant P was detected by autoradiography. Lanes 1 and 2 were exposed for 2 h; lanes
3-5 were exposed for 24 h. Positions of molecular mass
standards and their sizes in kilodaltons are indicated at the left.
Genetic evidence implicates Btk as essential for development of B cells in humans and for normal B cell responsiveness in mice, but its function remains unknown. Tyrosine kinases, in general, contain discrete structural domains that mediate assembly of multicomponent signaling complexes. Btk and its homologues share two such domains, SH2 and SH3, with members of the Src family. In addition, Btk and its homologues each contain a PH domain at the extreme amino terminus and an additional conserved region (the TH region) between the PH and SH3 domains. As one approach to defining the signaling pathways in which Btk and related kinases act, we have pursued the following strategy: 1) to identify specific ligands for the Btk TH region in vitro; 2) to construct specific Btk mutations that abolish TH-mediated protein binding; and 3) to determine whether such mutations affect interactions between Btk and other proteins within the cell. Here we have presented evidence that the TH region of Btk mediates protein-protein interactions in vitro and in vivo, and that these interactions involve specific binding to SH3 domains.
SH3 domains
are found in proteins that carry out such diverse functions as signal
transduction, cytoskeletal assembly, and the phagocytic oxidative
burst. All of these proteins function as parts of multicomponent arrays
whose assembly may involve specific SH3-mediated interactions. Similar
consensus binding motifs for SH3 domains of phosphatidylinositol
3-kinase and Src have been defined by selection from a biased
combinatorial peptide library: RXLPPRPXX for
phosphatidylinositol 3-kinase and RXLPPLPR (where
denotes a hydrophobic residue) for Src(26) . A number of
SH3-binding proteins have been identified, including the Ras nucleotide
exchange factor Sos1, which binds the SH2- and SH3-containing adaptor
protein Grb-2(38, 39, 40) ; 3BP1 and 3BP2,
proteins of unknown function that bind the SH3 domain of the c-Abl
tyrosine kinase(41, 42) ; dynamin, which binds a
variety of SH3 domains (43) ; and the GTPase activating protein
CDC42, which binds the SH3 domains of Src and phosphatidylinositol
3-kinase(44) . Taken together, the SH3-binding motifs of these
proteins and the motifs obtained by peptide selection define the core
consensus sequence XP
PPXP, in which the
underlined proline residues at positions 4 and 7 are
invariant(26) . Of these residues, the proline at position 4 is
most critical for SH3 binding.
The amino acid residues that mediate
binding of Btk to the Fyn SH3 domain, KPLPPTP, conform to
the SH3 binding consensus as defined by peptide selection. A point
mutation at Pro
of Btk (corresponding to Pro
of the core binding consensus sequence) was sufficient to abolish
binding. In the solution structure of a high-affinity peptide bound to
the phosphatidylinositol 3-kinase SH3 domain, Pro
resides
in a pocket formed by the side chains of amino acids Tyr
,
Trp
, Pro
, and Tyr
(26) .
As this structure predicted, mutation of Fyn at Trp
(corresponding to Trp
of the phosphatidylinositol
3-kinase SH3 domain) abolished binding to Btk. Lys
of Btk
also appears to participate in binding to SH3, and this residue is
conserved in Itk and Tec. Arginine, however, is found at the
corresponding position (Arg
) in the peptide library
consensus motifs for phosphatidylinositol 3-kinase and Src SH3 binding.
In the peptide-phosphatidylinositol 3-kinase SH3 complex, Arg
of the bound peptide forms a salt bridge with Asp
in
the phosphatidylinositol 3-kinase SH3 domain and mutation of Asp
impairs peptide binding(26) . In contrast, mutation of
the corresponding residue in the Fyn SH3 domain (Asp
) did
not affect the binding of Btk. This may reflect differences in the
pattern of side chain contacts made by Lys
of Btk with
the Fyn SH3 domain, in comparison with those made by Arg
of
the core consensus binding motif with the SH3 domain of
phosphatidylinositol 3-kinase.
While this work was in progress,
others documented the ability of Btk and Itk to bind SH3 domains of Fyn
and two other Src-type kinases, Lyn, and Hck, in
vitro(45) . The in vitro binding experiments we
report here are in general agreement but permit a more detailed
assessment of the TH-SH3 interaction. A single point mutation at
Pro, in the first putative SH3-binding site of Btk, was
sufficient to abolish specific SH3-binding by intact Btk; an analogous
mutation at Pro
, in the second putative binding site, had
no detectable effect on retention of full-length Btk by an SH3 matrix.
While the effects of single point mutations on the binding of intact
Btk were not assessed in the previous study, the observation that GST
fusions containing the first proline-rich site adsorb soluble SH3 more
avidly than fusions to the second site in a filter-binding assay (45) are consistent with our results. The second proline-rich
site (K
PLPPEP) differs from the first
(K
PLPPTP) at position Glu
and at the two
positions immediately carboxyl-terminal (Thr
and
Ala
); these differences may account for preferential SH3
binding to the first site, as non-proline residues in SH3-binding
peptides are likely to contribute to the specificity of high-affinity
interactions(26, 46) .
We have proceeded to obtain
evidence that the SH3-binding site in the TH domain mediates protein
interactions in vivo. Sodium pervanadate, a potent inhibitor
of protein tyrosine phosphatases, induces increases in the
phosphotyrosine content of endogenous Btk in mouse B cells (data not
shown) and transfected Btk in 293 cells. Tyrosine phosphorylation of
the kinase-inactive Btk K430E mutant is also increased by pervanadate
treatment; this increase is largely abrogated by a second mutation
(P189A) that inactivates the SH3-binding site in the Btk TH domain. The
P189A mutation has no detectable effect on the intracellular
distribution of Btk protein. ()These observations suggest
that the increase in tyrosine phosphorylation of kinase-inactive Btk
observed upon pervanadate treatment requires interactions mediated by
the SH3-binding site at residues 186-192. Although putative
physiologic targets for binding by the Btk TH domain have not yet been
defined, our results indicate that Btk associates stably in vivo with an as yet unidentified 72-kDa phosphotyrosine-containing
protein, and that this association is abolished by the P189A mutation.
The 72-kDa protein becomes phosphorylated on tyrosine in
immunoprecipitates of kinase-inactive Btk; whether it is itself a
kinase or simply a substrate for a coprecipitating kinase is not yet
clear.
Taken together, observations presented here suggest that Btk
is a substrate for one or more other protein tyrosine kinases in
vivo, and that its ability to act as a substrate is impaired by
inactivation of the SH3-binding site in the TH domain. Tyrosine
phosphorylation may regulate Btk activity or its accessibility to
downstream targets. Btk is newly phosphorylated on tyrosine and
activated upon cross-linking of surface Ig on B cells (18, 19) or FcRI receptors on myeloid
cells(20) . Maximal tyrosine phosphorylation and activation of
Btk occurs by about 5 min after ligation of surface Ig, in contrast
with members of the Src family, whose phosphorylation and activation
are maximal within seconds after receptor cross-linking(47) .
These kinetics would be consistent with models in which Btk is
activated, directly or indirectly, through an interaction with one or
more other tyrosine kinases.