The solution structure and intramolecular associations of the Tec kinase SRC homology 3 domain.

Tec is the prototypic member of a family of intracellular tyrosine kinases that includes Txk, Bmx, Itk, and Btk. Tec family kinases share similarities in domain structure with Src family kinases, but one of the features that differentiates them is a proline-rich region (PRR) preceding their Src homology (SH) 3 domain. Evidence that the PRR of Itk can bind in an intramolecular fashion to its SH3 domain and the lack of a regulatory tyrosine in the C terminus indicates that Tec kinases must be regulated by a different set of intramolecular interactions to the Src kinases. We have determined the solution structure of the Tec SH3 domain and have investigated interactions with its PRR, which contains two SH3-binding sites. We demonstrate that in vitro, the Tec PRR can bind in an intramolecular fashion to the SH3. However, the affinity is lower than that for dimerization via reciprocal PRR-SH3 association. Using site-directed mutagenesis we show that both sites can bind the Tec SH3 domain; site 1 (155KTLPPAP161) binds intramolecularly, while site 2 (165KRRPPPPIPP174) cannot and binds in an intermolecular fashion. These distinct roles for the SH3 binding sites in Tec family kinases could be important for protein targeting and enzyme activation.

Tec is an intracellular tyrosine kinase belonging to a family that includes Btk, Itk, Txk, and Bmx (1)(2)(3)(4). Tec family kinases are modular proteins consisting, from the N terminus, of a pleckstrin homology domain, a Tec homology domain, comprising a zinc-binding Btk motif (5) and an adjacent proline-rich region (PRR), 1 a Src homology (SH) 3 domain, a SH2 domain, and a kinase domain (6). The SH3, SH2, and kinase domains of Tec are very similar to those in the Src family kinases, while the pleckstrin homology, Btk motif, and PRR are unique to Tec family kinases (5,6). In addition, there are several other significant differences. Unlike Tec, Src kinases contain N-terminal fatty acid attachment sites responsible for localizing the proteins to the cytoplasmic membrane (7). The pleckstrin homology domains of Tec kinases could function in a similar fashion by targeting them to the membrane in response to environmental conditions (8). Src kinases also possess a Cterminal regulatory tyrosine, which binds in an intramolecular fashion to its SH2 domain in the inactive form of the kinase, and this feature is absent in Tec family proteins.
There are two major isoforms of the Tec kinase: the fulllength TecIV and a version, TecIII, generated by alternate splicing of exon 8 (9). Tec III protein lacks the C-terminal 22 amino acids of the SH3 domain, which is likely to compromise its ligand binding ability. The biological significance of this protein is unknown but it is of note that a comparable deletion in Btk causes the disease X-linked agammaglobulinemia (10). Expression of Btk and Itk is restricted to B-cells and T-cells, respectively, and Tec has been suggested to function in an equivalent manner in myeloid cells (11). However, Tec is expressed in a broad range of tissues in embryonic and adult mice (9). Tec has been shown to bind a number of proteins, including gp130 and c-Kit, implicating Tec in signaling downstream of these receptors as well as in the growth and differentiation of hematopoietic cells (12,13).
The three-dimensional crystal structures of the Src family kinases c-Src and Hck indicate that they are maintained in an inactive form via two intramolecular interactions (14 -16). Phosphorylation of the residue equivalent to Tyr 527 of avian Src allows it to form an intramolecular association with the SH2 domain. The second interaction involves the SH2 kinase linker region forming a polyproline type-II helix, which binds to the SH3 domain. While the interaction between the SH2 kinase linker and the SH3 domain may be conserved in the Tec family (17), the Tec family kinases cannot be regulated by an interaction involving a C-terminal phosphotyrosine. Studies on the Tec family member Itk (18) demonstrated an intramolecular association between the PRR and the adjacent SH3 domain. As shown in Fig. 1, all Tec family members have at least one potential class I (ϩXXPXXP) SH3 ligand consensus sequence within their PRRs. Btk has two potential class I binding sites in its PRR: 186 KPLPPTP 192 (peptides containing this sequence bind Lyn, Fyn, and Hck (19,20) and Btk SH3 (21)) and 200 KPLPPEP 206 for which surprisingly, no SH3 binding has been demonstrated. It has been proposed that an intramolecular association between the Btk SH3 domain and the PRR sequesters the Tec homology domain, restricting its availability to other cellular ligands and ensuring basal enzyme activity (21). Recently, it has been shown that the PRR of Btk mediates dimer formation, however, the question of which motif mediates self-association was not addressed (22). Murine Tec PRR contains one isolated proline-rich sequence: 155 KTLPPAP 161 (PRS-1) and two additional class I sequences: 165 KRRPPPP 171 and 167 RPPPPIP 173 which overlap with each other and an incomplete site: 170 PIPP 174 (9). We designate these overlapped sites in 165 KRRPPPPIPP 174 PRS-2.
We have determined the three-dimensional structure of the murine Tec SH3 protein by nuclear magnetic resonance (NMR) spectroscopy. NMR chemical shift perturbation studies defined the binding surface and demonstrated that a synthetic peptide corresponding to PRS-1 binds to the SH3 domain with a K d of ϳ2 mM. To further analyze interactions between the PRR and SH3 domain we produced PRR-SH3 recombinant proteins and used site-directed mutagenesis to abolish the SH3-binding sites. Analytical ultracentrifugation, surface plasmon resonance, and NMR experiments demonstrated that wild type PRR-SH3 forms dimers. Our mutational analysis indicates that PRS-1 can be used to form intramolecularly bound monomers as found for Itk (18), whereas PRS-2 preferentially directs dimer and higher order oligomer formation.

EXPERIMENTAL PROCEDURES
Plasmid Construction and Site-directed Mutagenesis-A 260-bp region encoding Tec SH3 domain, (amino acids 181-245 in mouse Tec) was PCR amplified from a cDNA library clone (a kind gift from Dr. James Ihle, St. Judes) and then cloned into the expression plasmid pGEX-4T-2 (Amersham Bioscience, Inc.). The PRR-SH3 construct, a 300-bp fragment (amino acids 151 to 245), was also PCR amplified and cloned into pGEX-4T-2. Site-directed mutagenesis was conducted on the wild type pGEX-4T-2-PRR-SH3 construct using a QuikChange site-directed mutagenesis kit (Stratagene) essentially as per the manufacturer's instructions to mutate specific Pro residues to Ala. PRR-SH3⌬1 contains a Pro-Ala mutation of residue 158 within PRS-1, PRR-SH3⌬2 contains Pro-Ala mutations of residues 168 -171 within PRS-2, and PRR-SH3⌬12 harbors both sets of mutations (Fig. 1). Correct mutagenesis and cloning was confirmed by DNA sequencing.
Protein Production-Unlabeled and 15 N-labeled samples of Tec SH3, Tec PRR-SH3wt, Tec PRR-SH3⌬1, ⌬2, and ⌬12 were prepared essentially as described for the Tec SH3 in Pursglove et al. (23). The purity of protein samples was monitored throughout the purification procedure by denaturing polyacrylamide gel electrophoresis. All proteins produced retain a Gly-Ser from the fusion partner. Mass spectrometric analysis of the SH3 samples and the PRR-SH3 proteins agreed with the expected values of M r 7,899 and M r 7,994 for the unlabeled and 15 N-labeled SH3 samples and M r 11,212, M r 11,148, M r 11,108, and M r 11,082 for PRR-SH3wt, PRR-SH3⌬1, PRR-SH3⌬2, and PRR-SH3⌬12, respectively. A high purity peptide, representing PRS-1 of the PRR of Tec kinase 155 KTLPPAP 161 (PRS-1p), was purchased from AUSPEP (Melbourne, Australia).
Nuclear Magnetic Resonance Experiments-SH3 samples were buffer exchanged into 10 mM phosphate, 0.01% NaN 3 , pH 6.0. Sample concentrations of 2.0 and 1.25 mM were used in this study for the unlabeled and 15 N-labeled samples, respectively. NMR data were recorded on Varian Inova 600 and Bruker AMX-500 and DRX-600 spectrometers. The NMR experiments used to obtain the sequence-specific chemical shift assignments for the Tec SH3 have been described elsewhere (23). PRR-SH3wt and PRR-SH3⌬2 proteins were buffer exchanged into phosphate-buffered saline, pH 6.0, and concentrated to ϳ0.5 mM. HSQC experiments were conducted as previously stated for the SH3 domain (23).
Structural Restraints-Distance restraints were derived from a twodimensional 1 H-1 H NOESY spectrum (recorded in D 2 O) and a threedimensional 1 H-15 N HSQC-NOESY spectrum, both with mixing times of 200 ms, in an identical fashion to that described for the fourth domain of the common chain in the interleukin-3, interleukin-5, and granulocyte-macrophage colony-stimulating factor receptors (24). Hydrogen bond restraints were imposed for amide groups detectable in 1 H-15 N HSQC spectra recorded more than 1 h after protein, lyophilized from H 2 O, was dissolved in D 2 O. The hydrogen-acceptor distance was restrained between 1.7 and 2.2 Å and the donor-acceptor distance was restrained between 2.7 and 3. The key residues for a class-I SH3-ligand (K/R)XXPXXP are shown as white text on black. The number of residues between the C-terminal consensus proline and the N-terminal residue of the SH3 domain are shown on the right for both PRS-1 and PRS-2. The asterisks highlight that the BMX PRS-2 sequences represent an incomplete match to the consensus and binding to SH3 domains has not been experimentally confirmed. The alignment was generated using the program MOLMOL (29). B, alignment of the N-terminal portion of the mouse Tec PRR-SH3 proteins used in this study. The N-terminal Gly-Ser are additional residues remaining after thrombin cleavage of the GST fusion partner. PRR-SH3 wt represents the wild type mouse sequence, PRR-SH3⌬1 represents a single P158A mutation disrupting the PRS-1 consensus. PRR-SH3⌬2 represents four P168A/P169A/P170A/P171A mutations disrupting the PRS-2 consensus. PRR-SH3⌬12 represents a combination of both these mutants. The mutated Ala residues are highlighted by white text on black. SH3 represents the N terminus of the protein sample used to generate the three-dimensional structure. from F1 and F2 cross-peak line widths in a 1 H-15 N HMQC-J experiment (25). Torsion angle restraints for -angles of Ϫ120 Ϯ 40°were imposed for 3 J HN-H␣ Ն 8 Hz and Ϫ60 Ϯ 30°for Յ5 Hz.
Structure Calculations-Structures were calculated in X-PLOR (version 3.851) (26) using the ARIA method (27) as implemented previously (24). An initial ensemble of 40 structures was calculated using the raw restraints, which were composed of 49 unambiguous restraints (22 intraresidue, 6 sequential, 2 medium range, and 19 long range restraints), 1566 ambiguous restraints, and 31 -angle restraints. The 10 structures with the lowest overall energy were retained. Ten ARIA iterations followed during which the assignment parameter (N p ) was reduced from 0.9999 to 0.75 and the violation tolerance (V tol ) was reduced from 2.0 to 0 Å. Hydrogen bond restraints were included for the slowly exchanging amide groups when a single acceptor was found with suitable hydrogen bonding geometry in at least 50% of the retained structures. A total of 100 structures were calculated using the final restraint list (generated with N p of 0.75 and V tol of 0 Å). These final restraints included 1228 NOE derived distance restraints. Of these, 956 were assigned unambiguously (468 intraresidue, 173 sequential, 55 medium range, and 260 long range) and 272 restraints for which the assignment remained ambiguous. The remaining restraints were 34 hydrogen bond restraints (two restraints per hydrogen bond) and 31 -angle restraints. These coordinate sets were refined in explicit solvent using the hybrid CSDX/OPLS parameter set (28). The resultant structures were all in good agreement with the experimental data, with no distance violations greater than 0.5 Å and no dihedral violations greater than 5°. Therefore, the 20 structures with the lowest overall energies were selected as the final ensemble.
Structural Analyses-Hydrogen bonding and secondary structure were analyzed using MOLMOL (29) and Ramachandran properties and angular order parameters were measured using the in-house program ANGORDER (24,30). Sixty-seven percent of all and angles have angular order parameters greater than 0.9 (Ile 182 -Thr 192 , His 195 -Lys 210 , Asp 212 -Tyr 222 , and Glu 225 -Lys 236 ). Well defined residues were identified by iterative fitting of the C ␣ atoms to define the subset with the best defined C ␣ positions. Following each iteration the C ␣ atom with the highest RMSD was excluded prior to the next fit. At each iteration the C ␣ RMSD for the retained subset was divided by the number of residues in the subset. The set of best defined residues was the subset for which this ratio was the minimum.
Ligand Titration Experiments-PRS-1p was added stepwise to a 0.75 mM 15 N-labeled SH3 sample to an 8-fold molar excess. 1 H-15 N HSQC experiments were recorded for each peptide:ligand ratio. Chemical shift change versus peptide concentration data for peaks that moved significantly upon ligand addition were fitted using nonlinear regression analysis (Prism3) to the relationship described for peptide binding to the Fyn SH3 domain (31) to obtain equilibrium dissociation constants.
BIAcore Experiments-Surface plasmon resonance experiments were performed on a BIAcore 2000 instrument. Purified Tec SH3 domain was amine-coupled to a CM5 chip according to the manufacturer's directions resulting in the deposition of ϳ1500 or 3000 response units (RU) of Tec SH3 on the chip surface. The PRR-SH3 proteins were buffer exchanged into HEPES-buffered saline and concentrations ranging from 1 to 100 M were flowed past the SH3 domain at a rate of 10 l/min. Report points were taken preinjection, postinjection, and after 50 min of continuous flow. Sensorgram analysis was carried out using the BIAevaluation (Amersham Bioscience, Inc.) software. Kinetic studies were conducted at equilibrium to determine the steady-state K d values. The concentrations used for these experiments, ranged from 1 to 85 M for PRR-SH3wt, 1-45 M for PRR-SH3⌬1, 1-25 M for PRR-SH3⌬2, and 1-60 M for PRR-SH3⌬12. These injections were conducted in duplicate and randomly to avoid incremental errors. It was determined that at a flow rate of 10 l/min there were no significant changes in response units due to bulk effects. Regeneration of the SH3-coated lanes was not possible by standard methods without destroying the SH3 domain coupled to the chip. However, it was determined that 12 injections were possible over one SH3 coupled lane before a decrease in response was observed.
Analytical Ultracentrifugation Experiments-Sedimentation equilibrium experiments to characterize the self-association of Tec PRR-SH3 wild type and mutants were carried out on a Beckman Optima XL-A analytical ultracentrifuge equipped with an An-60ti rotor. Protein samples were made up in phosphate-buffered saline, pH 7.0, at concentrations corresponding to A 280 ϭ 0.08, 0.27, and 0.8 and A 248 ϭ 0.8 and 0.27 (for a 1-cm path-length). Data were recorded at both 4 and 20°C at speeds of 20,000, 30,000, 42,000, 48,000, and 56,000 rpm. Data were collected in six-sector cells as absorbance (248 and 280 nm) versus radius scans (0.001-cm increments). Scans were collected at 4-h inter-vals and compared to determine when the sample reached equilibrium. Analysis of the data was carried out using NONLIN (32), and the best model and final parameters were determined by examination of the residuals derived from fits to monomer, monomer 7 dimer, monomer 7 trimer, and monomer 7 dimer 7 tetramer models (all ideal species models). The partial specific volumes of each domain were determined from the amino acid sequences (33), and the solvent density was taken to be 1.0066 g⅐ml Ϫ1 at 20°C.

RESULTS
Solution Structure of Tec SH3 domain-The solution structure of the Tec SH3 domain was determined using NMR spectroscopy with calculations carried out in X-PLOR using the ARIA methodology (27). The raw peak lists from the homonuclear two-dimensional NOESY and three-dimensional 1 H-15 N HSQC-NOESY experiments were interrogated to generate an initial restraint list of those NOEs able to be assigned unambiguously based purely on the chemical shift assignments. Ten ARIA iterations were completed then coordinate sets were refined in explicit solvent resulting in 20 final structures, the structural parameters of which are presented in Table I. The final ensemble of 20 structures is shown in Fig. 2. The protein backbone is well defined except for the termini and residues 190 -195 in the RT loop, as indicated by the RMSD values plotted against residue number (Fig. 3).
The SH3 domain of Tec kinase, like those found in other proteins, has two triple-stranded anti-parallel ␤-sheets arranged in a ␤-sandwich, with the two sheets at right angles to each other (Fig. 2). The second strand is shared between the two sheets (designated as B and BЈ). Strand BЈ contains a ␤-bulge due to hydrogen bonding of both HN Leu 208 and HN Glu 209 to the carbonyl O of Arg 217 . A single turn of 3 10 (Fig. 2). The 3 10 helix forms hydrogen bonds with amino acids from both of the ␤-sheets. The RT loop, between strands A and B, contains Tyr 187 which is conserved in a variety of SH3 domains including Tec family and Src family kinases. Tyr 187 is autophosphorylated in Tec, which renders these kinases fully active and, in the three-dimensional structure, is on the surface near the binding pocket (Fig. 4). The conformation of the RT loop in Tec SH3 is stabilized by the presence of a hydrogen bond between Phe 189 and Leu 199 . By comparison, the n-Src loop between ␤-strands BЈ and C, spanning amino acids Asn 211 and Leu 213 , was not found to contain any hydrogen bonds.
Peptide Binding by the SH3-A peptide corresponding to PRS-1 (PRS-1p) was titrated into a sample of 15 N-labeled SH3 domain and the change in chemical shift of resonances in the 1 H-15 N HSQC spectrum was measured as a function of ligand concentration. These data allowed us to define the interaction surface on the SH3 domain and to estimate the K d for the interaction to be 2.0 Ϯ 0.3 mM (Fig. 4) (Fig. 4C). Pro 229 lies near the center of this surface and is expected to be involved in ligand binding, however, due to its lack of a backbone NH atom it could not be detected in these experiments. Tyr 187 is located at the edge of the binding site, not integral to it, which is consistent with observations that phosphorylation of the corresponding residue in Btk has an effect on the specificity of binding but does not abolish it (34).
The SH3 domain is vital to the regulation of the tyrosine kinase activity of Src kinases due to the intramolecular contacts made in the inactive conformation (14 -16). Although it appears that regulation of the tyrosine kinase activity of Tec family members is different to that of Src, it is likely that the SH3 domain still plays an important role (18,21). Deletion of the Tec SH3 domain (35) or mutation of Tyr 187 result in a constitutively active form of the kinase (36).
Tec kinase has two main isoforms, with TecIII lacking the C-terminal 22 amino acids (223-245) of the SH3 domain rela-   tive to TecIV. This region includes the 3 10 helix and the last 2 ␤-strands and forms part of the proline-rich ligand surface. While the biological role of TecIII is not clear, the effect of SH3 truncation is expected to have a profound impact on the structure of the enzyme and its interactions with signaling partners and cellular localization.
PRR-SH3 Mutagenesis, Protein Production, and Preliminary Analysis-To determine which, if any, of the SH3-binding sites in the Tec PRR can interact with the Tec SH3 domain, we generated a series of Tec PRR and SH3 domain containing proteins that harbor mutations in the PRR. Based on the results of mutating Itk Pro 192 to Ala (18), mutation of Tec Pro 158 to Ala (PRR-SH3⌬1) should inhibit formation of a polyproline type-II helix and thus prevent PRS-1 from binding (Fig. 1). The region of protein corresponding to PRS-2 of Tec ( 165 KRRPPP-PIPP 174 ) contains four adjacent prolines (168 -171) which were mutated to Ala (PRR-SH3⌬2) to ensure that all possible consensus sequences were abolished. Finally, both sites were mutated in the same protein to act as a control (PRR-SH3⌬12).
Treatment of purified recombinant GST fusion proteins with thrombin resulted in complete digestion within 2 h, suggesting the PRR region forms a flexible tail and provides easy access for the protease. This is in stark contrast to the GST-SH3 construct, which requires much longer incubation times to achieve comparable cleavage. Size exclusion chromatography produced suboptimal separation between the GST fusion partner and the wild type PRR-SH3 protein suggesting wild type PRR-SH3 protein may have been aggregating (data not shown). Initial homonuclear NMR experiments conducted on this protein re-sulted in a broad peak shape and a lack of 1 H-15 N HSQC peaks, also indicative of protein oligomerization (data not shown). In contrast, preliminary NMR data collected from a 1 H-15 N HSQC experiment on PRR-SH3⌬2 suggested that this protein was predominately monomeric, based on peak shape and number (data not shown).
Surface Plasmon Resonance Analysis of Tec PRR-SH3 Proteins-The preliminary NMR data highlighted the possibility that the SH3 consensus binding sites within the PRR have different specificities and thus may perform different functions. These proteins were analyzed using surface plasmon resonance (BIAcore) to gain a better understanding of the specificity and strength of the interactions occurring between the SH3 domain and the different components of the Tec kinase PRR.
Tec SH3 domain was covalently linked to a Biacore chip to act as an immobilized receptor for PRR-containing proteins. Accumulation of mass, represented by increasing RU values, was therefore an indicator of intermolecular interactions between immobilized SH3 domains and PRR proteins. Sensorgrams for the various PRR proteins are shown in Fig. 5 and the observed equilibrium dissociation constants are summarized in Table II. Wild type PRR-SH3 bound to the immobilized SH3 domain surface with an equilibrium dissociation constant of 68 M, consistent with the self-aggregation observed in the preliminary NMR studies. Disruption of the PRR-1 (PRR-SH3⌬1) had no significant effect on the ability of the protein to bind intermolecularly to the SH3 domain surface (K d 64 M), indicating that PRR-1 is not critical for this interaction. Disruption of PRR-2, however, had a profound effect on the ability to bind the SH3 domain surface (K d 2200 M) indicating that this site is critical for intermolecular interactions with SH3 domains. Disruption of both PRR sites, however, results in weak binding to the immobilized SH3 domain (K d 500 M), suggesting that this protein undergoes a conformational change relative to the single mutations and is able to interact with the immobilized SH3 domain in a non-PRR directed manner.
These data, taken together with the PRS-1p binding experiments, indicate that both PRS-1 and PRS-2 have moderate affinities for the Tec SH3 domain, but that PRS-2 is sterically inhibited from binding in an intramolecular fashion to the SH3 domain. PRS-2 in Btk is the same distance from the SH3 domain as PRS-2 in Tec (Fig. 1). Therefore, it is very likely that PRS-2 in Btk will be unable to bind to its SH3 in an intramolecular fashion.
Analytical Ultracentrifugation Experiments-Analytical ultracentrifugation was used to further investigate the oligomerization properties of wild-type and mutant PRR-SH3 protein samples. Data recorded for these proteins at a range of sample concentrations and centrifugal forces were found to best-fit a monomer-dimer-tetramer model (Fig. 6), allowing calculation of equilibrium dissociation constants for these reactions (Table  II). These values were used to calculate the fraction of each species present at a range of protein concentrations (Fig. 6), providing a graphical comparison of the aggregation properties of the wild-type and mutant proteins. PRR-SH3wt was determined to have a K d of 125 (91-200) M for the monomer 7 dimer equilibrium and 0.83 (0.62-1.25) ϫ 10 12 M 3 for the monomer 7 tetramer equilibrium. PRR-SH3⌬1 has the highest proportion of tetramer at low protein concentrations, and also displays the lowest K d (50 (33-71) M) for the monomer to dimer transition. PRR-SH3⌬2 and PRR-SH3⌬12 form tetramer to a lesser extent, compared with PRR-SH3 wild type (Fig. 6) and, at all the concentrations tested (up to 0.1 mM), PRR-SH3⌬2 and PRR-SH3⌬12 samples were predominantly mono-mer. The K d values for dimerization of PRR-SH3⌬2 and PRR-SH3⌬12 were similar at 417 (312-555) M and 909 (625-1205) M, 4-and 8-fold weaker than PRR-SH3wt, respectively. As seen with the BIAcore results, these data demonstrate a degree of self-association in the absence of both PRS-1 and PRS-2, possibly of a nonspecific nature, and that the presence of an intact PRS-1 does not dramatically promote dimerization, indicating that PRS-1 prefers an intramolecular interaction with the SH3 domain. DISCUSSION In this paper we describe inter-and intramolecular interactions between the SH3 domain and SH3-binding sequences present in the PRR of Tec kinase. The three-dimensional structure of the Tec SH3 domain was determined and NMR chemical shift perturbation experiments demonstrated that a ligand representing Tec PRS-1 binds to a shallow pocket on the surface of the SH3. Using surface plasmon resonance and analytical ultracentrifugation we have shown that PRS-1 can bind intramolecularly to the SH3 domain, while PRS-2, which has a higher affinity for the Tec SH3 domain, is unable to bind intramolecularly and instead outcompetes PRS-1, resulting in dimer formation (Fig. 7).
The presence of PRR sequences is conserved within the Tec family of enzymes. Itk PRR-SH3 contains only PRS-1 and therefore displays intramolecular SH3 binding (18). Btk and Tec have both PRS-1 and PRS-2, and the Btk PRR-SH3 region was recently shown to form dimers with a dissociation constant of 60 M, similar to that shown here for the Tec PRR-SH3 (22). These results suggest that Txk, which lacks PRS-1 but retains a PRS-2 sequence, would be unable to form intramolecular PRR/SH3 interactions, but would be capable of intermolecular binding. The situation in Bmx is harder to predict. It has a PRS-1 sequence but no PRS-2, suggesting a situation more like Itk, however, the divergence in the sequence of the SH3 domain itself may mean that the Bmx SH3 domain has no affinity for its own PRR. It will be interesting to see whether this SH3-like region folds into the canonical ␤-barrel shape and binds PRR ligands in the same manner as the other Tec family members.
Recently, it has been demonstrated that a construct containing the SH3 and SH2 domains of Itk dimerizes via a novel mechanism whereby a surface on the SH2 domain, composed largely of loops from different sections of the sequence, binds to the SH3 domain in the place of more conventional polyproline helix ligands (37). The K d of this SH32 monomer-dimer equilibrium was estimated to be 25 M, which is about a factor of two tighter than the PRR-mediated dimerization of Btk and the PRS-2-mediated monomer-dimer equilibrium in Tec and it was suggested that the SH32 self-association could provide a novel mode of kinase regulation (37). Fifteen of the 33 residues of the Itk SH2 domain implicated in the dimer interface are conserved in Tec including six of nine interacting residues within the BG loop. However, given that the nonconserved residues are markedly different in character, it is difficult to predict from sequences alone whether such a mechanism may also apply to the Tec protein.
The key challenge will now be to translate these observations to the regulatory mechanisms acting in the intact enzyme. The tional change induced by phosphorylation of the kinase domain by a Src family member, or some other mechanism, could then result in a partially activated enzyme with both the SH3 and PRR becoming available for binding partners. Indeed PRS-2 in Tec, Btk, and Txk (and presumably PRS-1 in Itk and Bmx) may participate in the enzyme dimerization required for cross-phosphorylation of the regulatory tyrosine (Tyr 187 in Tec) as suggested by Hansson et al. (22) leading to full activation.
A key piece of the puzzle recently discovered by Kang et al. (38) is that phosphorylation of Btk serine 180 by the ␤ isoform of protein kinase C (PKC␤) is a potent negative regulator of Btk activity. Localization to the plasma membrane as well as phosphorylation at Tyr 551 (by Lyn) and Tyr 223 (Btk autophosphorylation) was reduced upon coexpression of Btk with increasing levels of PKC␤. A similar effect on Tec was also observed in their system. Interestingly this regulatory serine is conserved in other Tec family members (except for Bmx) and is adjacent to the PRR (4 -6 residues N-terminal to PRS-1). The mechanism for this negative regulation has yet to be determined, but introduction of a negatively charged phosphate group near PRS-1 is likely to block interactions with SH3 domain containing binding partners. Mano et al. (39) showed that the SH3  6. Sedimentation equilibrium analysis of wild type and mutant PRR-SH3 proteins. A, residuals, expressed as absorbance versus (r 2 )/2 for sedimentation equilibrium data of PRR-SH3 fitted to an ideal monomer-dimertetramer model. The residuals for three data sets corresponding to different loading concentrations are shown in different shades of gray. The fit to this model was better than to all other models tried. Similar results were obtained for the three PRR-SH3 mutants. B, plots of fraction of (q) monomer, (ϫ) dimer and (E) tetramer against total protein concentration. These plots were generated using the equilibrium constants calculated from sedimentation equilibrium data (Table II).
domain of Lyn can bind to the PRR of Tec, suggesting that one role for phosphorylation at Ser 180 (152 in Tec) may be to inhibit Lyn binding and its subsequent activation of the Tec family enzymes through tyrosine phosphorylation. Such a mechanism also suggests a role for both phosphotyrosine and phosphoserine-specific phosphatases in the regulation of Tec family enzymes.