©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An SH3-binding Site Conserved in Brutons Tyrosine Kinase and Related Tyrosine Kinases Mediates Specific Protein Interactions in Vitro and in Vivo(*)

(Received for publication, May 22, 1995)

Weiyi Yang Sami N. Malek Stephen Desiderio (§)

From the Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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.


MATERIALS AND METHODS

Antibodies

Polyclonal rabbit anti-Btk antibodies were raised against two synthetic peptides. Antibodies Ab1279 and Ab1280 were directed against peptide SD18, corresponding to COOH-terminal residues 636-659 of mouse Btk; antibodies Ab1300 and Ab1301 were directed against peptide SD20, corresponding to residues 81-100 of mouse Btk. Peptides were coupled to keyhole limpet hemocyanin and rabbits were immunized as described previously(27) .

Synthetic Peptides

Peptides SD26, SD27, SD28, and SD29 comprise residues 186-193 of murine Btk (KPLPPTPE) and the corresponding mutant sequences APLPPTPE, KALAPTAE and KPLAPTPE, respectively (where underlines indicate amino acid substitutions); an aspartic acid residue was added to the COOH terminus of each peptide to enhance solubility.

Construction and Isolation of Glutathione S-Transferase Fusion Proteins

DNA fragments encoding residues 1-218 of murine Btk were amplified by the polymerase chain reaction (PCR), using murine cDNA from the murine B lymphoid cell line BFO.3 as a template. The forward and reverse PCR primers incorporated BamHI and EcoRI sites, respectively, at their 5` ends. The amplified product was cloned between the BamHI and EcoRI restriction sites of the Escherichia coli expression vector pGEX-2T (Pharmacia Biotech Inc.), to produce the plasmid pGEX-2T-BtkN. DNA cassettes encoding residues 1-80 of Btk(N1) and residues 104-218 (N2) were amplified by PCR, using pGEX-2T-BtkN as a template, and cloned into pGEX-2T to yield the plasmids pGEX-2T-BtkN1 and pGEX-2T-BtkN2. DNA fragments encoding the unique region (residues 1-55) and the SH2 domain (residues 118-214) of murine Blk were similarly amplified by PCR and cloned into pGEX-2T, as were the unique region (residues 1-86) and the SH2 domain (residues 149-246) of murine Fyn(T). Cassettes specifying the SH3 domains of Blk (residues 56-115) and Fyn (residues 87-145) were amplified by PCR and cloned into pGEX-3X. All constructions were verified by nucleotide sequencing.

Plasmids encoding GST fusion proteins were introduced into E. coli strains DH5alpha 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 approx 0.6-1.0; expression of fusion proteins was then induced by addition of isopropyl-1-thio-beta-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.

Cell Lines

The murine B lymphoid cell line A20 was maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 50 µM 2-mercaptoethanol, 50 µg/ml streptomycin, and 50 units/ml penicillin. The human embryonic kidney fibroblast cell line 293 was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 µg/ml streptomycin, and 50 units/ml penicillin. All cells were grown at 37 °C in 5% CO(2).

Protein Expression in Mammalian Cells

The murine btk coding sequence was amplified by PCR from a mouse spleen cDNA library (Stratagene) and cloned into the HpaI site of the mammalian expression vector pCIS-2(28) . The murine fyn(T) coding sequence (a gift of Dr. Lawrence Samelson, National Institutes of Health) was cloned between the XhoI and NotI sites of pCIS-2. Mutagenesis was performed by the recombination PCR method(29) . Primers used to generate the Fyn W119L mutant were 5`-G GAA GGA GAC TTG TGG GAA GC-3` (forward) and 5`-GC TTC CCA CAA GTC TCC TTC C-3` (backward) corresponding to nucleotides 570-590 of fyn(T) cDNA (30) ; primers used to generate the Fyn D100N mutant were 5`-CGG ACG GAA GAT AAC CTG AG-3` (forward) and 5`-CT CAG GTT ATC TTC CGT CCG-3` (backward) corresponding to nucleotides 510-530; mutated residues are indicated by underlines. Similarly, Btk mutants K430E, Btk P189A, Btk P203A, and W251L were generated using the following primer pairs (mutated residues are marked by underlines): 5`-G GCC ATC GAG ATG ATC AGA GAA GG-3` and 5`-CC TTC TCT GAT CAT CTC GAT GGC C-3` (nucleotides 1417-1441) for K430E; 5`-G AAA AAG CCT CTT GCC CCT ACC CCA G-3` and 5`-C TGG GGT AGG GGC AAG AGG CTT TTT C-3` (nucleotides 688-713) for P189A; 5`-G AAA AAA CCG CTT GCC CCG GAG CCA-3` and 5`-TGG CTC CGG GGC AAG CGG TTT TTT C-3` (nucleotides 730-754) for P203A; and 5`-C CTA CCG TTG TGG CGA GCA CG-3` and 5`-CG TGC TCG CCA CAA CGG TAG G-3` (nucleotides 880-900) for W251L. Double mutants were made by recombining appropriate cDNA fragments. The identities of all PCR products were verified by nucleotide sequence analysis.

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).

In Vitro Binding Assays

Glutathione-agarose beads (15-20 µl of a 50% slurry containing 10-30 µg of GST fusion protein) were mixed with lysates of transfected 293 cells (300 µl, containing 200-300 µg of protein) and rocked at 4 °C for 1 h. For experiments with 293 cells expressing Btk, beads were washed three times with buffer D; for experiments with 293 cells expressing wild-type or mutant Fyn proteins, beads were washed three times with buffer N. Bound protein was eluted by boiling in SDS sample buffer for 5 min and fractionated by 7.5% or 10% SDS-PAGE. In some experiments, binding assays were carried out in the presence of competitor peptides at various concentrations (0.2 µM, 2 µM, 20 µM, 200 µM, or 2 mM).

Immunoprecipitation and Immune Complex Kinase Assays

Immunoprecipitation of Fyn was performed in buffer D (reaction volume 300 µl) containing 100 µg of cellular protein and 5 µg of anti-Fyn monoclonal antibody (Santa Cruz Biotechnology). Reactions were incubated for 2 h at 4 °C; after addition of protein A/G-Sepharose (Santa Cruz Biotechnology; 20 µl of a 50% slurry), incubation was continued for an additional hour at 4 °C with rocking.

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), 2 mM MnCl(2), 1 mM Na(3)VO(4)) 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(2), 10 mM MnCl(2), 0.1 mM Na(3)VO(4)) before initiating the kinase reaction.

Immunoblot Assays

After SDS-PAGE, protein was transferred to nitrocelluose or PVDF membranes; these were incubated with primary antibodies for 2-10 h at room temperature. The anti-phosphotyrosine antibody 4G10 (United Biotechnology) was used at 1 µg/ml. Anti-Btk antibodies were used at a 1:500 dilution. Anti-Fyn antibodies were used at 10 µg/ml. Immunoreactive proteins were detected by incubation with horseradish peroxidase-coupled goat anti-mouse or goat anti-rabbit IgG for 1 h at room temperature; immobilized horseradish peroxidase was visualized by an enhanced chemiluminescence assay (Amersham).

Pervanadate Treatment of Btk-expressing Cells

50 mM pervanadate was prepared by mixing 50 mM Na(3)VO(4) (pH 10) and 30% (8.82 M) H(2)O(2) in equimolar amounts; the mixture was allowed to stand at room temperature for 15 min and was used within 2 h. At 40-48 h after transfection, 293 cells were washed once with ice-cold PBS supplemented with 5 mM EDTA, once with serum-free Dulbecco's modified Eagle's medium, resuspended in serum-free Dulbecco's modified Eagle's medium, and incubated at 37 °C for 10 min. Pervanadate was then added to 1 mM final concentration and cells were incubated further at 37 °C for 10 min. Lysis of cells, immunoprecipitation, and immunoblotting were performed as described above. Chemiluminescence signals were digitized by high resolution optical scanning and volumetric integration of signal intensities was carried out using the ImageQuant software package (Molecular Dynamics).


RESULTS

Specific Binding of Btk to an SH3 Domain

To test the ability of Btk to bind SH3 domains in vitro, a batch adsorption method was used. The amino-terminal, SH3 and SH2 domains of Fyn(T) were expressed in E. coli as fusion proteins to glutathione S-transferase (GST) and affixed to glutathione-Sepharose beads; unfused GST was adsorbed to beads for use as a control. The resulting affinity matrices were adsorbed to a cell-free extract prepared from the mouse B lymphoid cell line A20, which expresses Btk. Retained protein was recovered and fractionated by SDS-polyacrylamide gel electrophoresis; Btk was identified by immunoblotting. A protein of apparent molecular mass 76 kDa, corresponding to the size of Btk, was readily detected among proteins eluted from the Fyn SH3 affinity matrix (Fig. 2A, laneGST-FynSH3). This species was only weakly retained by beads bearing the amino-terminal and SH2 domains of Fyn(T) (Fig. 2A, lanes GST-FynN and GST-FynSH2) and was not retained by GST alone (Fig. 2A, lane GST). These initial observations suggested that Btk is capable of binding the SH3 domain of Fyn. This was confirmed in a similar experiment performed using lysates of transfected 293 cells expressing Btk. Btk was retained by the Fyn SH3 domain (Fig. 2B, lane 7), but weakly or not at all by the SH3 domain of Blk, another member of the Src kinase family (Fig. 2B, lane 5). Neither GST alone (Fig. 2B, lane 3) nor the amino-terminal domains of Fyn or Blk (Fig. 2B, lanes 6 and 4) bound Btk to a significant extent.


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.



Identification of the SH3-binding Site Within Btk

Lysates of transfected 293 cells, expressing Fyn protein, were diluted serially and adsorbed to GST fusion proteins containing fragments of the Btk amino-terminal region. Fyn was retained by fusion proteins containing residues 1-218 (GST-BtkN) or 104-218 (GST-BtkN2) of Btk (Fig. 3, lanes 3-6 and 7-10) but not by a fusion protein containing residues 1-80 (GST-BtkN1) of Btk (Fig. 3, lanes 11-14), nor by GST alone (Fig. 3, lane 2). A binding site for Fyn was thereby localized to a 115-amino acid interval of Btk that spans the TH region and contains two consensus motifs for binding to the Src SH3 domain. In similar experiments, we observed that GST fusions to the Fyn SH3 domain were capable of also binding Itk, and, conversely, that GST fusions to the TH region of Itk were able to retain Fyn (data not shown).


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.



Mutational Analysis of the SH3-Btk Interaction

One conserved feature of SH3 domains is a hydrophobic surface that participates directly in the binding of specific peptide ligands(26, 32, 33, 34, 35) . Within this hydrophobic patch resides an invariant tryptophan residue (Trp of PI3K; Trp of Fyn) that interacts with proline at position 4 of the consensus SH3-binding motif(26, 32) . Because mutation of the corresponding proline residue in Btk abolished its SH3 binding activity, it seemed likely that mutation of Trp of Fyn would impair its interaction with the Btk TH region. To test this, wild-type Fyn and mutant Fyn proteins carrying amino acid substitutions at Trp (W119L) or another SH3 domain residue, Asp (D100N), were expressed in 293 cells and assayed for binding to the GST-BtkN2 fusion protein (Fig. 6A). Recovery of Fyn W119L from the GST-BtkN2 beads was greatly diminished (Fig. 6A, lanes 11-14) relative to wild-type Fyn (Fig. 6A, lanes 3-6) or Fyn D100N (Fig. 6A, lanes 7-10), despite similar amounts of wild-type, D100N, and W119L proteins in lysates of transfected 293 cells (Fig. 6B). Thus, the interaction of Fyn with its binding site in the Btk TH region is impaired by mutation of Trp. This impairment is not likely the result of an overall perturbation of Fyn structure, because kinase activity is unaffected by the W119L mutation (Fig. 6C). Taken together, our results are consistent with the interpretation that an interaction between the hydrophobic pocket of Fyn SH3 and Pro of Btk, corresponding to position 4 of the SH3 consensus binding site, is critical for binding.


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.

Inactivation of the SH3-binding Site at Residues 186-192 Impairs Inducible Phosphorylation of Btk in Vivo

Despite the ability of Btk to bind the Fyn SH3 domain in vitro, we were unable to detect an interaction between Btk and Fyn in vivo that required an intact SH3-binding site in the Btk TH domain (data not shown). We therefore pursued a more general approach to the question of whether the Btk TH region mediates interactions with other tyrosine kinases in vivo. This hypothesis predicts that: 1) a kinase-inactive Btk mutant should act as a tyrosine kinase substrate in vivo, and 2) tyrosine phosphorylation of kinase-inactive Btk should be diminished by a second mutation in the SH3-binding site of the Btk TH domain. To test these predictions, we assayed tyrosine phosphorylation of wild-type and mutant Btk proteins in transfected 293 cells. Tyrosine phosphorylation was induced by treatment of Btk-expressing cells with pervanadate, a tyrosine phosphatase inhibitor. In lymphocytes, pervanadate induces a series of intracellular phosphorylation events that closely resemble those induced by engagement of antigen receptors(36, 37) ; in preliminary experiments we observed that pervanadate induces tyrosine phosphorylation of endogenous Btk in the B cell lines A20/2J and M12 (data not shown).

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.

The SH3-binding Motif at Residues 186-192 Mediates Intracellular Association of Btk with a 72-kDa Phosphotyrosine-containing Protein

The experiments described above suggested that residues 186-192 of Btk mediate stable protein interactions in intact cells. To test this, we examined the P-labeled products of kinase reactions performed with anti-Btk immunoprecipitates from lysates of 293 cells expressing wild-type or mutant Btk proteins. Following fractionation of immune complex kinase products by gel electrophoresis, proteins were transferred to a PVDF membrane. After assessing the amount of filter-bound Btk protein by immunoblotting, the filter was treated with KOH and alkali-resistant radioactivity was detected by autoradiography. In immunoprecipitates from lysates of cells expressing enzymatically active forms of Btk (wild-type Btk or Btk P189A), the major P-labeled species was Btk itself, as expected; several coprecipitating proteins were also substrates for phosphorylation in vitro (Fig. 8, lanes 1 and 2). Under these conditions we could not detect significant differences between the patterns of P-labeled proteins obtained from immunoprecipitates of wild-type and P189A mutant Btk. We then asked whether differences might be uncovered by comparing the patterns of P-labeled proteins obtained from immunoprecipitates of two kinase-inactive mutants: Btk K430E and Btk K430E,P189A. Under these conditions we observed a clear difference: a 72-kDa, radiolabeled protein was detected as a major species in immunoprecipitates from cells expressing Btk K430E (Fig. 8, lane 3), but not from immunoprecipitates of cells expressing Btk K430E,P189A (Fig. 8, lane 4) or cells that did not express Btk (Fig. 8, lane 5). The loss of the 72-kDa phosphoprotein upon mutation of the Btk SH3-binding site could reflect inability of this mutant to interact with the 72-kDa phosphoprotein or with the kinase that phosphorylates it. In either event, the result implies that the Btk SH3-binding motif at residues 186-192 can mediate stable protein-protein interactions in vivo.


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.




DISCUSSION

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 XPPPXP, 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^4 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^4 resides in a pocket formed by the side chains of amino acids Tyr^14, 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^1) in the peptide library consensus motifs for phosphatidylinositol 3-kinase and Src SH3 binding. In the peptide-phosphatidylinositol 3-kinase SH3 complex, Arg^1 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^1 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 (KPLPPEP) differs from the first (KPLPPTP) 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. (^2)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.


FOOTNOTES

*
This work was supported by the Howard Hughes Medical Institute and the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-4735; Fax: 410-955-9124.

(^1)
The abbreviations used are: Btk, Bruton's tyrosine kinase; PH, pleckstrin homology; TH, Tec homology; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidine difluoride; GST, glutathione S-transferase.

(^2)
W. Yang and S. Desiderio, unpublished data.


REFERENCES

  1. Weiss, A., and Littman, D. (1994) Cell 76,263-274 [Medline] [Order article via Infotrieve]
  2. Arpaia, E., Shahar, M., Dadi, H., Cohen, A., and Roifman, C. (1994) Cell 76,947-958 [Medline] [Order article via Infotrieve]
  3. Chan, A., Kadlecek, T., Elder, M., Filipovich, A., Kuo, W., Iwashima, M., Parslow, T., and Weiss, A. (1994) Science 264,1599-1601 [Medline] [Order article via Infotrieve]
  4. Straus, D., and Weiss, A. (1992) Cell 70,585-593 [Medline] [Order article via Infotrieve]
  5. Appleby, M. W., Gross, J. A., Cooke, M. P., Levin, S. D., Qian, X., and Perlmutter, R. M. (1992) Cell 70,751-763 [Medline] [Order article via Infotrieve]
  6. Stein, P. L., Lee, H.-M., Rich, S., and Soriano, P. (1992) Cell 70,741-750 [Medline] [Order article via Infotrieve]
  7. Tsukada, S., Saffran, D. C., Rawlings, D. J., Parolini, O., Allen, R. C., Klisak, I., Sparkes, R. S., Kubagawa, H., Mohandas, T., Quan, S., Belmont, J. W., Cooper, M. D., Conley, M. E., and Witte, O. N. (1993) Cell 72,279-290 [Medline] [Order article via Infotrieve]
  8. Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A., Flinter, F., Hammarstrom, L., Kinnon, C., Levinsky, R., Bobrow, M., Smith, C. I. E., and Bentley, D. R. (1993) Nature 361,226-233 [CrossRef][Medline] [Order article via Infotrieve]
  9. Thomas, J. D., Sideras, P., Smith, C. I. E., Vorechovsky, I., Chapman, V., and Paul, W. E. (1993) Science 261,355-358 [Medline] [Order article via Infotrieve]
  10. Rawlings, D. J., Saffran, D. C., Tsukada, S., Largaespada, D. A., Grimaldi, J. C., Cohen, L., Mohr, R. N., Bazan, J. F., Howard, M., Copeland, N. G., Jenkins, N. A., and Witte, O. N. (1993) Science 261,358-361 [Medline] [Order article via Infotrieve]
  11. Rosen, F. S., Cooper, M. D., and Wedgwood, R. J. P. (1984) N. Engl. J. Med. 311,235-242 [Medline] [Order article via Infotrieve]
  12. Pearl, E., Vogler, L. B., Okos, A. J., Crist, W. M., Lawton, A. R., and Cooper, M. D. (1978) J. Immunol. 120,1169-1175 [Abstract]
  13. Scher, I. (1982) Adv. Immunol. 33,1-71 [Medline] [Order article via Infotrieve]
  14. Siliciano, J., Morrow, T. A., and Desiderio, S. V. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,11194-11198 [Abstract]
  15. Heyeck, S. D., and Berg, L. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,669-673 [Abstract]
  16. Mano, H., Ishikawa, F., Nishida, J., Hirai, H., and Takaku, F. (1990) Oncogene 5,1781-1786 [Medline] [Order article via Infotrieve]
  17. Mano, H., Mano, K., Tang, B., Koehler, M., Yi, T., Gilbert, D. J., Jenkins, N. A., Copeland, N. G., and Ihle, J. N. (1993) Oncogene 8,417-424 [Medline] [Order article via Infotrieve]
  18. Aoki, Y., Isselbacher, K., and Pillai, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,10606-10609 [Abstract/Free Full Text]
  19. de Weers, M., Brouns, G., Hinshelwood, S., Kinnon, C., Schuurman, R., Hendriks, R., and Borst, J. (1994) J. Biol. Chem. 269,23857-23860 [Abstract/Free Full Text]
  20. Kawakami, Y., Yao, L., Miura, T., Tsukada, S., Witte, O., and Kawakami, T. (1994) Mol. Cell. Biol. 14,5108-5113 [Abstract]
  21. August, A., Gibson, S., Kawakami, Y., Kawakami, T., Mills, G., and Dupont, B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,9347-9351 [Abstract/Free Full Text]
  22. Mano, H., Yamashita, Y., Sato, K., Yazaki, Y., and Hirai, H. (1995) Blood 85,343-350 [Abstract/Free Full Text]
  23. Desiderio, S., and Siliciano, J. D. (1994) Chem. Immunol. 59,191-210 [Medline] [Order article via Infotrieve]
  24. Vihinen, M., Nilsson, L., and Smith, C. I. E. (1994) FEBS Lett. 350,263-265 [CrossRef][Medline] [Order article via Infotrieve]
  25. Desiderio, S. (1993) Nature 361,202-203 [CrossRef][Medline] [Order article via Infotrieve]
  26. Yu, H., Chen, J., Feng, S., Dalgarno, D., Brawer, A., and Schreiber, S. (1994) Cell 76,933-945 [Medline] [Order article via Infotrieve]
  27. Dymecki, S. M., Zwollo, P., Zeller, K., Kuhajda, F. P., and Desiderio, S. V. (1992) J. Biol. Chem. 267,4815-4823 [Abstract/Free Full Text]
  28. Gorman, C. M., Gies, D. R., and McCray, G. (1990) DNA and Protein Eng. Tech. 2,3-10
  29. Jones, D., and Winistorfer, S. (1992) BioTechniques 12,528-530 [Medline] [Order article via Infotrieve]
  30. Cooke, M. P., and Perlmutter, R. M. (1989) New Biol. 1,66-74 [Medline] [Order article via Infotrieve]
  31. Kamps, M. P., and Sefton, B. M. (1989) Anal. Biochem. 176,22-27 [Medline] [Order article via Infotrieve]
  32. Noble, M. E. M., Musacchio, A., Saraste, M., Courtneidge, S. A., and Wierenga, R. K. (1993) EMBO J. 12,2617-2624 [Abstract]
  33. Yu, H., Rosen, M., Shin, T., Seidel-Dugan, C., Brugge, J., and Schreiber, S. (1992) Science 258,1665-1668 [Medline] [Order article via Infotrieve]
  34. Koyama, S., Yu, H., Dalgarno, D., Shin, T., Zydowsky, L., and Schreiber, S. (1993) Cell 72,945-952 [Medline] [Order article via Infotrieve]
  35. Musacchio, A., Noble, M., Pauptit, R., Wierenga, R., and Saraste, M. (1992) Nature 359,851-855 [CrossRef][Medline] [Order article via Infotrieve]
  36. Secrist, J., Burns, L., Karnitz, L., Koretzky, G., and Abraham, R. (1993) J. Biol. Chem. 268,5886-5893 [Abstract/Free Full Text]
  37. O'Shea, J., McVicar, D., Bailey, T., Burns, C., and Smyth, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,10306-10310 [Abstract]
  38. Simon, M., Dodson, G., and Rubin, G. (1993) Cell 73,169-177 [Medline] [Order article via Infotrieve]
  39. Olivier, J., Raabe, T., Henkemeyer, M., Dickson, B., Mbamalu, G., Margolis, B., Schlessinger, J., Hafen, E., and Pawson, T. (1993) Cell 73,179-191 [Medline] [Order article via Infotrieve]
  40. Buday, L., and Downward, J. (1993) Cell 73,611-620 [Medline] [Order article via Infotrieve]
  41. Cicchetti, P., Mayer, B. J., Thiel, G., and Baltimore, D. (1992) Science 257,803-806 [Medline] [Order article via Infotrieve]
  42. Ren, R., Mayer, B., Cicchetti, P., and Baltimore, D. (1993) Science 259,1157-1161 [Medline] [Order article via Infotrieve]
  43. Gout, I., Dhand, R., Hiles, I., Fry, M., Panayotou, G., Das, P., Truong, O., Totty, N., Hsuan, J., Booker, G., Campbell, I., and Waterfield, M. (1993) Cell 75,25-36 [Medline] [Order article via Infotrieve]
  44. Barfod, E., Zheng, Y., Kuang, W., Hart, M., Evans, T., Cerione, R., and Ashkenazi, A. (1993) J. Biol. Chem. 268,26059-26062 [Abstract/Free Full Text]
  45. Cheng, G., Ye, Z., and Baltimore, D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,8152-8155 [Abstract]
  46. Lin, W., Richards, F., and Fox, R. (1994) Nature 372,375-379 [CrossRef][Medline] [Order article via Infotrieve]
  47. Saouaf, S., Mahajan, S., Rowley, R., Kut, S., Fargnoli, J., Burkhardt, A., Tsukada, S., Witte, O., and Bolen, J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,9524-9528 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.