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: firstname.lastname@example.org 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. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 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