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PROTEINS: Structure, Function, and Genetics 29:545–552 (1997)
SH3 Domain of Bruton’s Tyrosine Kinase Can Bind
to Proline-Rich Peptides of TH Domain
of the Kinase and p120cbl
Himatkumar V. Patel,1 Shiou-Ru Tzeng,1 Chen-Yee Liao,1 Shi-Han Chen,2 and Jya-Wei Cheng1*
1Division of Structural Biology and Biomedical Science, Department of Life Science, National Tsing Hua University,
Hsinchu, Taiwan
2Department of Pediatrics, University of Washington, Seattle, Washington
ABSTRACT
X-linked agammaglobulinemia (XLA), an inherited disease, is caused by
mutations in the Bruton’s tyrosine kinase
(BTK). The absence of functional BTK leads to
failure of B-cell differentiation; this incapacitates antibody production in XLA patients,
who suffer from recurrent, sometimes lethal,
bacterial infections. BTK plays an important
role in B-cell development; it interacts with
several proteins in the context of signal transduction. Point mutation in the BTK gene that
leads to deletion of C-terminal 14 aa residues of
BTK SH3 domain was found in a patient family.
To understand the role of BTK, we studied
binding of BTK SH3 domain (aa 216–273, 58
residues) and truncated SH3 domain (216–259,
44 residues) with proline-rich peptides; the
first peptide constitutes the SH3 domain of
BTK, while the latter peptide lacks 14 amino
acid residues of the C terminal. Proline-rich
peptides selected from TH domain of BTK and
p120cbl were studied. It is known that BTK TH
domain binds to SH3 domains of various proteins. We found that BTK SH3 domain binds to
peptides of BTK TH domain. This suggests that
BTK SH3 and TH domains may associate in
inter- or intramolecular fashion, which raises
the possibility that the kinase may be regulating its own activity by restricting the availability of both its ligand-binding modules. We also
found that truncated SH3 domain binds to BTK
TH domain peptide less avidly than does normal SH3 domain. Also, we show that the SH3
and truncated SH3 domains bind to peptide of
p120cbl, but the latter domain binds weakly. It
is likely that the truncated SH3 domain fails to
present to the ligand the crucial residues in
the correct context, hence the weaker binding.
These results delineate the importance of Cterminal in binding of SH3 domains and indicate also that improper folding and the altered
binding behavior of mutant BTK SH3 domain
likely leads to XLA. Proteins 29:545–552,
1997. r 1997 Wiley-Liss, Inc.
r 1997 WILEY-LISS, INC.
Key words: BTK; XLA; SH3 domain; TH domain; proline-rich peptide; p120cbl;
peptide binding
INTRODUCTION
X-Linked agammaglobulinemia (XLA) patients present markedly reduced or absent serum immunoglobulins of all isotypes and fail to produce antigen-specific
antibodies.1 They suffer from recurrent bacterial
infections, some of which can be life-threatening.2
This gene defect is intrinsic to B-cell lineage: T
cell-dependent immunity is normal in XLA patients.3
The number of pre-B cells in the patient’s bone
marrow is normal; the defect thus resides in the
development pathway of B cells.3-5 The gene responsible for this disease encodes a cytoplasmic tyrosine
kinase, Bruton’s tyrosine kinase (BTK).5 This kinase
is expressed in early and mature human B-cell lines
but is absent in terminally differentiated plasma cell
lines. This distribution indicates that BTK protein,
like other nonreceptor tyrosine kinases, is required
for normal B-cell differentiation. The kinase is expressed in most hematopoietic cells, but is selectively downregulated in plasma cells and T lymphocytes. This explains the normal T-cell immunity in
XLA patients. Mutations or deletions in the BTK
gene were detected in unrelated XLA patients,4,6
suggesting strongly that the kinase is directly involved in the disease, and therefore, in the process of
B-cell development.
The signal transduction pathways that regulate
cell growth and differentiation are characterized by
a cascade of specific protein–protein recognition
events that occur intracellularly upon extracellular
stimulation.7–10 Cytoplasmic kinases, which translate the signal into the final biological effect incorporate protein modules that mediate the recognition
events.
Contract grant support: National Science Council, ROC;
Contract grant support: March of Dimes Birth Defects Foundation.
*Correspondence to: Dr. Jya-Wei Cheng, Division of Structural Biology and Biomedical Science, Department of Life
Science, National Tsing Hua University, Hsinchu 300, Taiwan.
E-mail: lscjw@life.nthu.edu.tw
Received 10 February 1997; Accepted 13 May 1997
546
H. V. PATEL ET AL.
Fig. 1. Domains present in BTK shown from N to C terminal. A:
Amino acid sequence of proline-rich segment of TH domain
chosen for this study, residues 181–194, is shown. Amino acid
sequence of SH3 domain, labeled as SH3-58 corresponds to
residues 216–273 of BTK. Truncated SH3 domain (SH3-44), the
factor that causes XLA, corresponds to amino acids 216–259 of
BTK, and lacks 14 aa residues of C-terminal. Residues within SH3
domain implicated in binding to proline-rich peptides are indicated
by filled circles. B: Established SH3 binding consensus; X represents any residue and F represents a hydrophobic residue.
Peptide P1 corresponds to the proline-rich segment 185–193 of
BTK TH domain, while peptide P4 corresponds to residues
181–192 of BTK TH domain.
The latter render possible the selective phosphorylation–activation of the member immediately downstream in the process, out of a myriad of possible substrates. How the given the cell achieves the exquisite
specificity inherent to signal transmission is intriguing.7,8 But the progress in uncovering the mechanism has made great strides in the recent past so
that glimpses of what may be essential steps in the
whole scenario are now seen. Kinases are endowed
with, in addition to the catalytic domain, several
protein modules that recognize and bind to their
target, which, in turn, presents specific motifs in the
context of the signal. The result of such specificity is
manifested as the faultless proliferation of the cell,
and, eventually, as the smooth maintenance of the
organism. The reductionistic approach of studying
individual domains has been successful as well as
informative.7,8,11 Nonetheless, details of interaction
between the domains still remain unclear. Study of
the interaction of inter- and intramolecular organization of the domains is thus a significant objective.
BTK, along with Tec, Ltk, and Atk, belongs to a
small family of tyrosine kinases (the Tec family) that
share common structural features.2,7–9 In vivo and in
vitro studies have shown that BTK can bind to the
protein product of the c-cbl protooncogene, the bg
subunits of heterotrimeric G proteins, Fyn, Lyn,
Hck, and protein kinase C,12–16 but little is known of
its biological role. Like many other cytoplasmic
tyrosine kinases in signaling pathways, BTK contains an N-terminal pleckstrin homology (PH) domain, a proline-rich Tec homology (TH) domain, a
Src homology 2 (SH2) domain, a Src homology 3
(SH3) domain and a catalytic tyrosine kinase domain (Fig. 1A).7-10 The exact function of PH domain
in signaling proteins is still unknown, but it is
thought to effect protein–protein interactions and/or
membrane localization.7–9
SH3 and SH2 domains are small protein modules
that mediate protein–protein interactions and occur
in many proteins involved in intracellular signal
transduction.7–10 They participate in a diverse array
of cellular events. SH3 domains bind to proline-rich
sequences and SH2 domains bind to phosphotyrosine sequences on the receptor protein tyrosine
kinases.11,17,18 The structural basis for interaction
between peptide ligands and SH3 domains is now
well understood.19,20 Two classes, I and II, bind with
opposite amide bond directionality. Ligands of both
classes contain a PXXP core sequence that anchors
the ligand to the receptor in the polyproline type II
(PPII) helical conformation. Nearby flanking sequences provide binding specificity.21 Although the
core sequences of ligands of different SH3 domains
are conserved, the flanking sequences are unique to
individual SH3 domains and bind to a specific
pocket.22–24 The structures of these SH3 domains,
about 60 amino acid residues in length and lacking
disulfide bonds, are highly homologous, forming
mainly a b-barrel type structure.11 The structures of
several SH3 domains from various signaling proteins and their complexes with proline-rich peptides
have been solved by x-ray crystallography25–30 and
NMR spectroscopy.31-38
547
BTK SH3 DOMAIN BINDING TO TH DOMAIN
Study of the association of the various domains
with their ligands can help define consensus motifs
and their modes of binding.39 These rules should lay
the foundation for design of peptide inhibitors or
effectors of proteins that block their interaction with
other proteins. They would also enable study and
control of signaling processes. We report herein that
SH3 domain of BTK binds to a proline-rich peptide of
TH domain of the same kinase. This binding, interor intramolecular, may represent (one of the) ground
state(s) of the kinase. Whether the complex participates in signal transduction remains to be elucidated.
SH3 domain of BTK has been shown to bind to the
protein product of c-cbl oncogene (p120cbl)12. We
quantitated the affinity of BTK SH3 domain for an
SH3 binding peptide of p120cbl. Point mutation,
resulting in the deletion of 14 amino acids constituting the C–terminal of BTK SH3 domain has been
identified as a cause of XLA in a patient family
studied.1 We therefore studied a 58-residue peptide
corresponding to SH3 domain of BTK and a truncated peptide of 44 residues lacking the C-terminal
14 aa residues of BTK SH3 domain. Studies on the
latter peptide can shed light on the direct factor that
causes XLA. We found that the truncated peptide
binds considerably weakly to the BTK TH domain
peptide. Furthermore, the association of the truncated SH3 domain with proline-rich peptide of p120cbl
was weaker than with intact SH3 domain. We have
demonstrated earlier that the mutation (loss of the
C-terminal 14 aa residues) results in transformation
of the b-barrel structure of SH3 domain to random
coil conformation.40 Herein we show that the mutation-truncation that causes XLA saps the SH3 domain of its ability to bind to its target, due probably
to the aberrant folding. The altered binding behavior
likely renders the kinase abnormal.
MATERIALS AND METHODS
Synthesis of the 216,273 and 216,259 BTK
SH3 Domain and Proline-Rich Peptides
The SH3 domains corresponding to residues
216,273 (58 aa residues), 216,259 (44 aa residues)
of BTK and proline-rich peptides, HRKTKKPLPPTPYQ and SLHKDKPLPVPPYQ were synthesized on
an Applied Biosystems 431A peptide synthesizer by
using standard solid phase methods. After synthesis,
the peptides were deprotected at side chains and
cleaved from the resin by treating with a mixture of
trifluoroacetic acid and water containing phenol/1,2ethandithiol/thioanisole (reagent ‘‘K’’). The fluorescein carbonyl-HRKTKKPLPPTPYQ was prepared
by alkylation of the resin linked peptide with dichlorotriazinylaminofluorescein (DTAF, dihydrochloride,
Calbiochem) and triethylamine in dimethylformamide, followed by side chain deprotection and
resin cleavage with reagent ‘‘K’’. Purification was
performed on a reversed phase HPLC preparative
column (Vydac, RP-18 column).41,42 Purity of these
synthetic peptides was confirmed by HPLC and
Mass spectroscopy. Other peptides were purchased
(Genemed Biotechnologies, Inc., USA) and used without further purification.
Fluorescence Measurements
Intrinsic fluorescence intensity of Trp of SH3
domain was monitored to determine dissociation
constant of the domain with the peptides. The excitation wavelength was set at 297 nm with 4-nm slit
and emission wavelength, at 345–349 nm, with 8-nm
slit by using SLM 4800 fluorescence spectrometer.
Fluorescence spectra were taken at 400–300 nm by
using a 1-nm step size, 5 averaging scans in a 1 cm 3
1 cm cell. Fluorescence emission spectra of FITClinked peptide were measured with excitation at 494
nm and emission at 518 nm. Fluorescence anisotropy
was also monitored to study binding. T-format measurement of fluorescence anisotropy (r) of SH3 domain upon peptide binding was performed by macroprogram to obtain the fluorescence intensity (I) and
gain (G) in the parallel and perpendicular modes.
The fluorescence anisotropy values were then calculated by using the following equation:
r5
Rv 2 RH
Rv 1 RH
(1)
where RH 5 G00I00/G2I2 and RV 5 G00/G2
Determination of the Kd by Fluorescence
The dissociation constant, Kd, was determined by
assuming one-to-one binding of the BTK SH3 domain with peptides. In these experiments, protein
concentration was kept constant at 1.5 µM for SH3-58
and at 3 µM for SH3-44 in phosphate-buffered saline
(PBS), pH 7.4 at room temperature. Peptide concentration was increased gradually and the fluorescence
intensity measured for each addition. In the case of
tyrosine-containing peptides, excitation was set at
297 nm. Under this condition, peptides alone still
gave a small, linear, and concentration-dependent
fluorescence signal. To obtain actual fluorescence
change upon binding, emission intensities were corrected for this component. The dissociation constant,
Kd, was determined by nonlinear least-squares fitting of data by monitoring the change in the fluorescence emission with Equations (2) and (3):
F 5 F0 1
(Fb 2 F0 )[pep]
(Kd 1 [pep])
(2)
where F0 is fluorescence intensity of the free SH3
domain, that is, in the absence of peptide, and Fb is
fluorescence intensity of the complex at saturation.
Complete (100%) binding was assumed to occur
when the fluorescence intensity assumed a plateau.
548
H. V. PATEL ET AL.
TABLE I. Interaction of SH3 Domain (SH3-58) and
Truncated SH3 Domain (SH3-44) of BTK With
Proline-Rich Peptides*
No.
Peptide sequence
SH3-58
Kd (µM)
SH3-44
Kd (µM)
P1
P2
P3
P4
P5
P6
P7
TKKPLPPTPE
TKRALPPLPE
TKRALAPLPE
HRKTKKPLPPTPYQ
FITC-HRKTKKPLPPTPYQ
AAPPLPPRKT
SLHKDKPLPVPPYQ
54.8
38.4
No binding
14.9
3.2†
239
34.5
76.5
112.2
*Kd values were determined by measuring change in fluorescence intensity of the respective SH3 domain upon addition of
the peptides.
†Change in fluorescence of FITC-peptide was monitored for
different concentrations of SH3 domain, for peptide concentration constant (see Materials and Methods section). Errors are
estimated to be less than 610%.
RESULTS
Binding of BTK SH3 With Peptide of
BTK TH Domain
Fig. 2. Change in fluorescence emission spectra upon binding
of SH3 domain to the indicated peptide. A: Representative
emission spectra of intrinsic Trp fluorescence of BTK SH3 domain
in the absence (0 µM) and presence (500 µM) of peptide. B:
Binding isotherms of binding of SH3 domain with the indicated
BTK-TH derived peptides. Concentration of SH3 domain was kept
constant (1.5 µM). Relative change in fluorescence intensity is
plotted against peptide/SH3 molar ratio; solid line represents the
line of best fit, data were fitted by nonlinear least-squares analysis.
Concentration of free peptide is denoted by [pep] and
that of free SH3 domain and complex of peptide–SH3
domain, by [SH3] and [Comp], respectively, at equilibrium.
Kd 5
[SH3][pep]
[Comp]
(3)
Concentration of the free peptide can be calculated
by subtracting the fluorescence-estimated concentration of the complex from the concentration of added
peptide. In the case of FITC–peptide, the concentration of peptide was kept constant (50 nM), which was
titrated against SH3 domain. Anisotropy measurement was performed under similar conditions, and
Kd was calculated by using Equations (2) and (3) in
monitoring fluorescence change of peptide instead of
SH3.
TH domain of BTK binds to SH3 domains of Lyn,
Fyn, and Hck.14 Binding is shown to control the
biological activity. Here we sought to test the ability
of TH domain of BTK to bind to SH3 domain of BTK;
the association state between the two can reflect the
organization of the enzyme (Fig. 1). There is evidence suggesting that the sequence KPLPPEP of
BTK TH domain (residues 200–206) does not bind to
SH3 domains of Lyn, Fyn, and Hck.43 But sequence
KPLPPTP of BTK TH domain (residues 186–192)
may be involved in binding to these SH3 domains.43
We therefore studied the binding of BTK SH3 domain with a 10 aa peptide TKKPLPPTPE (P1) of
BTK TH domain (residues 184–193) (Fig. 1B) by
monitoring the intrinsic fluorescence intensity and
anisotropy of Trp residue of SH3 domain. An increase in fluorescence intensity accompanied the
binding process. The emission spectra of SH3 domain, alone and in the presence of 500 µM peptide,
are shown in Figure 2A. The fluorescence intensity
assumed a plateau for higher concentrations of the
ligand (Fig 2B).
Hydrophobic interactions are known to dominate
the binding of SH3 domains, but electrostatic interaction undoubtedly plays a crucial role. The rise in
intensity can thus be explained as the shielding from
solvent quenching of the aromatic residues upon
peptide binding. The dissociation constant of BTK
SH3–TH peptide (P1) was found to be 54.8 µM (Table
I). The binding of BTK TH domain peptide to its own
SH3 domain thus falls in the range of the binding of
BTK TH domain peptides with Fyn and Lck SH3
domains.14,43 Contained within the BTK SH3 domains is, therefore, comparable propensity to bind to
TH domains of the relevant protein. It is likely that
BTK SH3 DOMAIN BINDING TO TH DOMAIN
549
the kinase exploits the binding of its SH3 domain
with its own TH domain to maintain the structural
integrity and to regulate its activity.
Importance of Distinct Residues
To delineate the contribution of the residues of
BTK TH domain peptide involved in binding, we
studied mutated peptide P1. The mutated peptide
TKRALPPLPE (P2), chosen from class I peptide19
binds to BTK SH3 domain, with Kd 5 38.4 µM (Table
I). The binding isotherm is shown in Figure 2B.
Substitution of Arg-3 for Lys-3 potentiates binding:
Kd for Arg-3 containing peptide P2 increased to 38.4
µM from 54.8 µM for Lys-3 containing peptide P1.
Increased binding of peptide P2 suggests that Arg-3
is preferred to Lys-3. Increased binding of peptide P2
relative to peptide P1, despite the Pro-4 to Ala-4
mutation in peptide P2, suggests also that Pro-4 is
silent in this binding. Pro-6, on the other hand, is
crucial to binding: we found that peptide P3
TKRALAPLPE, wherein Pro-6 is substituted by
Ala-6, fails to bind altogether to BTK SH3 domain
(Table I). Feng and colleagues19 concluded similarly
that replacement of Pro-6 by other residue abolishes
SH3 binding.
Basic Residues Assist in Binding
To investigate the importance of basic residues in
binding, we constructed peptide HRKTKKPLPPTPYQ (P4) of BTK TH domain (Fig. 1). This peptide
contains three additional residues, HRK, N-terminal
to peptide P1. We carried out similar titration of
peptide P4 with BTK SH3 domain. Peptide P4 is the
only ligand for which a steady decrease in fluorescence intensity accompanied the binding, followed by
saturation; we found that Kd 5 14.9 µM. The binding
isotherm is depicted in Figure 4A. The stronger
binding of peptide P4, relative to peptide P1 (Kd 5
54.8 µM), can be attributed to the three basic residues that peptide P4 incorporates. Basic residues of
ligands participate in binding to SH3 domains by
stabilizing the anionic moieties of the SH3 domain;
the latter would otherwise be unfavored in the
rather hydrophobic environment encircling the binding region.19–21 It has been observed that distinct
residues potentiate the binding of SH3 domains with
its ligands.22 Charged residues surrounding the hydrophobic region effect ligand binding to the SH3
domain of Sem-5.44
FITC Fluorophore Assists in Binding
We incorporated FITC in peptide P4 to serve as an
intrinsic probe (peptide P5, Table I). We found that
Kd dropped from 14.9 µM without FITC for the same
peptide to 3.2 µM with peptide incorporating FITC
(Table I). Figure 3 depicts the binding isotherm. It
has been observed that ligands incorporating small
aromatic moieties bind potently to SH3 domains of
Fig. 3. Binding isotherms of binding of SH3 domain with the
indicated BTK-TH derived FITC-peptide. Concentration of FITCpeptide was kept constant (50 nM). Relative change in fluorescence intensity (filled circles), and fluorescence anisotropy (filled
squares) of FITC fluorophore (see Materials and Methods section)
is plotted against SH3/peptide molar ratio; solid line represents the
line of best fit.
Src and other proteins.45 The observation that nonpeptide tails attached to peptide ligands containing
the PXXP core motif potentiate binding is important
in drug design. We studied the binding also by
monitoring change in fluorescence anisotropy, which
increased as ligand concentration was increased and
later assumed a plateau for higher ligand concentrations (Fig. 3). The dissociation constant was deduced
by applying similar nonlinear least-squares fitting
method.
Class II peptides bind to SH3 domains with opposite amide backbone directionality relative to class I
peptides.19,20 They bind with a consensus sequence
XPPLPXR. To determine if BTK SH3 domain binds
to class II peptides, we deduced Kd of class II peptide
AAPPLPPRKT (P6). The dissociation constant was
found to be 239 µM. The higher dissociation constant
for peptide P6 suggests that class II binding in this
case may be less feasible.
Binding of BTK SH3 Domain With
Proline-Rich Peptide of Protein Product c-cbl
Oncogene
The protein product of c-cbl oncogene (p120cbl) is
present in early B lineage and myeloid cells. SH3
domains of numerous proteins, including Fyn, Grb2,
Lck, Fgr, Nck, and PLCg1 have been shown to bind
to p120cbl. p120cbl is also expressed in T cells and is
rapidly phosphorylated on stimulation of T-cell receptor. BTK SH3 domain binds to p120cbl, and consequently it is thought to be involved in the signaling
pathways.12 We extend this finding herein by quantitating the affinity. The dissociation constant of BTK
SH3 domain with a proline-rich peptide (P7) segment SLHKDKPLPVPPYQ of p120cbl was found to
550
H. V. PATEL ET AL.
be 34.5 µM (Fig. 4B shows the binding curve; see also
Table I).
Truncated BTK SH3 Domain Binds Weakly
to Peptides of BTK TH Domain and p120cbl
Point mutation in the BTK gene that leads to
deletion of the C-terminal 14 aa residues in SH3
domain of BTK protein has been found to cause XLA
in a patient family studied.1 To shed light on the
molecular details of the cause of XLA, we studied the
binding of BTK SH3 domain lacking the C-terminal
14 aa residues (referred to as truncated SH3 domain), the direct factor that causes XLA.
We found that truncated SH3 domain binds to
peptide P4, an SH3 binding segment of BTK TH
domain, only weakly; Kd increased to 76.5 µM from
14.9 µM for that with normal SH3 domain (Table I
and Fig. 4A). We reported that the truncated SH3
domain fails to fold as a b barrel, but adopts, instead,
random coil conformations.40 The binding of peptide
with truncated SH3 domain, albeit weak, occurs,
despite loss of structure of truncated SH3 domain,
probably because of induced fit. Also, binding is
weaker because energy may be expended in folding
the domain appropriately to effect binding. Truncation in our case and mutation in vivo thus weakens
the binding of BTK SH3 domain with its own TH
domain. We also deduced Kd of truncated SH3 domain with peptide of p120cbl, P7, to be 112.2 µM, in
contrast to 34.5 µM for normal SH3 domain. The
deletion of the 14 aa thus weakens the affinity of
truncated SH3 domain with its ligands.
DISCUSSION
Bruton’s tyrosine kinase is crucial for the development of B cells: dysfunction in this protein’s activity,
even when catalytic activity is retained, causes the
inheritable genetic disorder, XLA.3–5 Despite the
importance of BTK in signaling in normal cells and
in XLA, its function remains unknown. The presence
in this kinase of SH3, SH2, TH, and PH domains, in
addition to the catalytic domain, implies that the
kinase must link some of the many steps involved in
the differentiation signal. BTK resides largely in the
cytoplasm.
TH domain of BTK binds to SH3 domains of Fyn,
Lyn, and Hck.14,43 But the contribution of binding to
the biological activity is unclear. Unknown also is
the influence of the various domains it comprises of
on each other. We demonstrated that BTK TH domain peptide binds to BTK SH3 domain with affinity
comparable to that of other SH3 domains implicated
in signaling cascade.14,43 BTK SH3 and TH domains
may, therefore, associate in vivo in an inter- or
intramolecular fashion. This raises the possibility
that BTK may associate in an inter- or intramolecular fashion to control signaling pathways.
Also conceivable is that BTK SH3 domain sequesters the BTK TH domain in the resting stage,
Fig. 4. Binding of SH3 domain (filled circles) and truncated
SH3 domain (filled squares) with peptide derived from BTK-TH
domain, peptide P4 (A) and with peptide derived from p120cbl,
peptide P7 (B). Relative change in intrinsic Trp fluorescence
intensity of BTK SH3 domain is plotted against peptide/SH3 molar
ratio; solid lines represent the line of best fit. The isotherms show
that SH3 domain binds to both peptides more strongly than does
truncated SH3 domain.
restricting, thereby, the availability of TH domain to
its ligands and ensuring that basal level activity of
the kinase and its partners be maintained. Upon
inception of the signal, however, change in BTK
protein may loosen the binding of BTK SH3–TH
domains (inter- or intramolecular). This dissociation
can expose both the domains and make them available to bind to their cognate ligands. The latter
process can allow propagation of the signal further.
The kinase might thus make use of the strategically
positioned domains to ensure timeliness, efficiency
and precision in its activity.
Cory and coworkers demonstrated that BTK SH3
domain binds to p120cbl12. This oncogenic protein
from B-cell receptor stimulated cell lysates bound
more strongly than did the protein from unactivated
cell lysates. We extended their observation and
quantitated the affinity. This suggests that p120cbl
BTK SH3 DOMAIN BINDING TO TH DOMAIN
may indeed participate in the cascade through which
BTK protein transmits the differentiation signal.
The most important aspect of our work is the
demonstration that truncated SH3 domain binds
only weakly to the ligands studied. The weak binding of BTK SH3–TH domains suggests that the
mutation-deletion makes for greater availability of
both the domains even in the unactivated state—an
event that would for normal BTK, occur upon arrival
of the signal. This disruption may impair the signaling cascade, resulting, eventually, in XLA. It is
difficult to define the exact consequence of the weakened binding observed because whether BTK regulates differentiation positively or negatively is unknown.
The truncated SH3 domain also exhibits weaker
affinity for p120cbl Cory and coworkers12 have demonstrated that binding of BTK SH3 domain with this
protein contains physiological significance. Taken
together, our results suggest that mutated BTK SH3
domain defies the binding behavior of wild-type SH3
domain and, by analogy, of the BTK protein. We have
shown earlier that the mutated SH3 domain loses its
stability and structure due to deletion of C-terminal
14 aa residues.40 It is likely that the truncated SH3
domain fails to present to the ligand the crucial
residues in the correct context, hence the weaker
binding.
In conclusion, we have shown that BTK SH3
domain can bind to proline-rich peptides of BTK TH
domain. While the dissociation constant falls in the
µM range, the affinity is sufficiently high, nevertheless, to hold together the two in an inter- or intramolecular complex in the absence of external stimulation. The kinase may thus be regulating its own
activity by restricting the availability of both its
ligand binding modules. Point mutation in the BTK
gene causes deletion of 14 aa residues in the C-terminal of BTK SH3 domain. We show that the truncated SH3 domain binds weakly to peptides of BTK
TH domain and p120cbl This loss of function might
render the kinase abnormal.
While the present paper was under review, three
papers appeared, which described also internal regulation in Src,46 Hck,47 and Itk48 kinases.
ACKNOWLEDGMENT
We thank Dr. Wen-Guey Wu, Dr. Ping-Chiang Lyu,
and Dr. Woei-Jer Chuang for fruitful discussions.
Peptides were synthesized at the Regional Instrumental Center at Hsinchu, National Science Council, Taiwan, ROC. This work is supported in part by
grants from the National Science Council,ROC (JWC)
and by a clinical research grant 6-463 from the
March of Dimes Birth Defects fundation (SHC).
2.
3.
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