Reconstitution of the B cell antigen receptor signaling components in COS cells.

To elucidate interactions occurring between B cell protein tyrosine kinases and the signaling components of the B cell antigen receptor, we have co-transfected into COS cells individual tyrosine kinases together with chimeric cell surface receptors containing the cytoplasmic domains of Igα or Igβ. Of the tyrosine kinases transfected (Lyn, Blk, Hck, Syk, Fyn), only Blk was able to phosphorylate and subsequently associate with co-transfected Igα and Igβ chimeras in vivo. Association between Blk and the Igα and Igβ cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Igα and Igβ cytoplasmic domains phosphorylated by Blk. The enzymatic activity and membrane association of Blk were required for the observed phosphorylation of the Igα and Igβ chimeras. Sequences within the amino-terminal unique domain of Blk are responsible for recognition and subsequent phosphorylation of the Igα chimera since transfer of the unique region of Blk to Fyn results in the chimeric kinase's ability to phosphorylate the cytoplasmic domain of Igα. These findings indicate that the unique domain of Src family kinases may direct recognition of certain substrates leading to their phosphorylation.

To elucidate interactions occurring between B cell protein tyrosine kinases and the signaling components of the B cell antigen receptor, we have co-transfected into COS cells individual tyrosine kinases together with chimeric cell surface receptors containing the cytoplasmic domains of Ig␣ or Ig␤. Of the tyrosine kinases transfected (Lyn, Blk, Hck, Syk, Fyn), only Blk was able to phosphorylate and subsequently associate with cotransfected Ig␣ and Ig␤ chimeras in vivo. Association between Blk and the Ig␣ and Ig␤ cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig␣ and Ig␤ cytoplasmic domains phosphorylated by Blk. The enzymatic activity and membrane association of Blk were required for the observed phosphorylation of the Ig␣ and Ig␤ chimeras. Sequences within the amino-terminal unique domain of Blk are responsible for recognition and subsequent phosphorylation of the Ig␣ chimera since transfer of the unique region of Blk to Fyn results in the chimeric kinase's ability to phosphorylate the cytoplasmic domain of Ig␣. These findings indicate that the unique domain of Src family kinases may direct recognition of certain substrates leading to their phosphorylation.
Signal transduction through the B cell antigen receptor (BCR) 1 involves the interaction of many distinct types of signaling molecules. Cross-linking of surface immunoglobulin results in tyrosine phosphorylation of numerous proteins, including the cytoplasmic domains of the immunoglobulin-associated signaling chains Ig␣ and Ig␤, which exist as disulfide-linked heterodimers on the surface of B lymphocytes (1)(2)(3). Ig␣ and Ig␤ are necessary for signal transduction through the BCR since mutation of the surface immunoglobulin molecule such that it does not associate with the Ig␣/Ig␤ heterodimer results in B cell nonresponsiveness to antigen receptor engagement (4). Ig␣ and Ig␤ each contain one immune receptor tyrosinebased activation motif (ITAM) in their cytoplasmic domains (5). Mutation of the ITAM tyrosine residues in Ig␣ results in abrogation of B cell activation (6). The cytoplasmic domains alone of Ig␣ and Ig␤ are able to mediate signal transduction in both B and T lymphocytes (7,8). Although the signaling chains of B and T cells differ in overall sequence, the common ITAM is sufficient to mediate signaling in diverse cell types.
Tyrosine kinases expressed in B cells and implicated in induction of signaling through the BCR are Blk, Lyn, Fyn, Hck, Syk, and Btk. These kinases have been shown to be activated following BCR engagement and are activated in a sequential order (9 -12). The Src and Syk classes of tyrosine kinases have been found to be associated with the Ig receptor complex (11,(13)(14)(15)(16)(17), but elucidation of the interactions between Src kinases and the cytoplasmic domains of the receptor complex is lacking. We have therefore reconstituted the signaling components of the BCR in COS cells to define the domains of the cytoplasmic regions of Ig␣ and Ig␤ and the domains of the tyrosine kinases involved in physical and functional interactions possibly important in B cell antigen receptor-mediated signal transduction.

COS Cell Expression Constructs
Construction of platelet-derived growth factor receptor (PDGFR)-Ig␣, Ig␤, TCR-, and Fc⑀RI␤ Chimeras-DNA fragments encoding the extracellular domain of the PDGFR (amino acids 1-499) (18) or the transmembrane and cytoplasmic domain of Ig␣ (amino acid 138 -220), Ig␤ (amino acid 159 -228), TCR-chain (amino acid 31-164), and Fc⑀RI␤ (amino acid 200 -243) (19) were amplified from their respective cDNAs by polymerase chain reaction (PCR), fused at a BamHI site introduced by the amplification primers and cloned into pBluescript SKIIϩ (Stratagene). Sequencing and in vitro transcription-translation reactions showed that the fusion was in-frame and that the resulting molecule was translated into a protein of the expected size. The plasmids pSVPDGFR␣, pSVPDGFR␤, pSVPDGFR, and pSVPDGFR-Fc⑀RI␤ were generated by subcloning fusion genes into the COS expression vector pSV7C (20). Point mutations of tyrosine residues to phenylalanine were introduced into the ITAMs of Ig␣ and Ig␤ by PCR using an oligonucleotide encoding the required mutation (21).
Cloning and Mutagenesis of B Cell Protein Tyrosine Kinases-The B cell protein tyrosine kinases Blk, Lyn, Fyn, Hck, and Syk were cloned by reverse transcriptase-PCR as described elsewhere (22,23). Point mutation of the SH2 domain of Blk was performed by overlap extension PCR (24) to change the conserved arginine residue at position 145 to lysine. Using the same technique the conserved tryptophan residues at positions 88 and 89 of the Blk SH3 domain were mutated to leucine. The double mutant of Blk was made by combining the SH2 and SH3 domain mutants via a common restriction enzyme site followed by ligation. Mutation of the myristylation site and ATP binding site of Blk was performed by PCR as described (22). To construct the Blk-Fyn chimeric kinase an XhoI site was inserted by PCR at the end of the Blk unique domain (nucleotide 156 of open reading frame) and at the beginning of the Fyn SH3 domain (nucleotide 246 of open reading frame). PCRamplified products were subcloned and digested with XhoI and restriction enzymes outside the coding region to fuse the kinases together resulting in the Blk N Fyn C chimeric kinase. All cDNA constructs were subcloned into the pSV7c COS expression vector (20), sequenced to confirm mutations, and transcribed and translated in vitro to confirm that a protein of the predicted appropriate size was produced. Where appropriate, in vitro kinase assays were performed on the translated products to confirm enzymatic activity. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Transfection of COS Cells
COS cells, 1.5 ϫ 10 6 , were transfected with 1-6 g of each plasmid DNA using 15-27 l of Lipofectamine according to manufacturer's directions (Life Technologies, Inc.). Cells were harvested 24 h later.

Flow Cytometric Analysis
Transfected COS cells were removed from plates with nonenzymatic cell dissociation solution (Sigma). 1.2 ϫ 10 5 cells were stained on ice in phosphate-buffered saline containing 1% bovine serum albumin, 0.1% sodium azide, and a 1:150 dilution of normal rabbit serum or PDGFR antiserum. After washing the cells, goat anti-rabbit IgG-fluorescein isothiocyanate (Cappel) (1:50) was used for staining. Stained cells were analyzed on a Coulter EPICS flow cytometer.

Biochemical Analyses
Transfected COS cells were lysed in 1% Nonidet P-40 lysis buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, and 2 mM EDTA. Immunoprecipitation, immunoblotting, and immune complex kinase assays were performed as described previously (25,26). Exogenous substrate assays were performed by addition of 2 g of glutathione S-transferase (GST) fusion protein containing the cytoplasmic domain of Ig␤ (GST-Ig␤).

Expression of Ig␣ and Ig␤
Chimeras-Chimeric proteins containing the extracellular domain of the PDGFR coupled to the transmembrane and cytoplasmic domains of Ig␣ or Ig␤ were constructed in an SV40 based expression vector (Fig. 1). Transfection of the constructs separately into COS cells resulted in cell surface expression as determined by cell surface staining with antisera to the PDGFR and flow cytometric analyses ( Fig.  2A). Biochemical analysis of lysates from transfected COS cells revealed the size of the chimeric proteins to be approximately 97 kDa, as expected (Fig. 2B). The chimeras were recognized by antisera to both the PDGFR and Ig␣ or Ig␤ (Fig. 2B). The chimeras were expressed as monomers on the surface of COS cells as determined by two-dimensional gel electrophoresis (not shown).
Co-expression of Ig␣ or Ig␤ Chimeras with Tyrosine Kinases-To determine whether the chimeric receptors would interact with tyrosine kinases present in B cells, we co-transfected the Ig␣ chimera or Ig␤ chimera with Lyn, Blk, Hck, Syk, or Fyn (Fig. 3). The chimeras were expressed at similar levels in each of the transfectants (Fig. 3B). The tyrosine kinases were enzymatically active when expressed in COS cells as shown by their ability to autophosphorylate in an in vitro kinase assay (Fig. 3C) and by their ability to phosphorylate an exogenous substrate (GST-Ig␤) (Fig. 3D). The co-transfected kinases had similar levels of enzymatic activity. When transfected alone, the Ig␣ and Ig␤ chimeras lacked tyrosine phosphorylation. However, co-expression with the Blk tyrosine kinase resulted in phosphorylation of the Ig␣ and Ig␤ chimeras on tyrosine (Fig. 3A). No other tyrosine kinase tested, including Btk and Lck (not shown), was able to phosphorylate the Ig␣ or Ig␤ chimera. To ensure that the tyrosine phosphorylation observed was occurring within the intact transfected cells rather than occurring post lysis of the cells, Ig␣ and Ig␤ chimeras were co-transfected with Blk (Ig␣ϩBlk, Ig␤ϩBlk) or chimeras were transfected separately from Blk and cell lysates from the two transfectants were mixed and incubated (Ig␣/Blk, Ig␤/Blk) (Fig. 4). Tyrosine phosphorylation of the Ig␣ or Ig␤ chimeras was detected only in cell lysates where the chimera and Blk were co-transfected, indicating that tyrosine phosphorylation of the cytoplasmic receptor domains occurred in vivo (Fig. 4).

Co-expression of Fc⑀RI␤ and Chimeras with Tyrosine
Kinases-We tested the ability of tyrosine kinases to phosphorylate the cytoplasmic domains of the signaling proteins Fc⑀RI␤ and from the T cell receptor complex. The PDGFR extracellular domain was fused to the transmembrane and cytoplasmic domains of Fc⑀RI␤ and . When these chimeric proteins were co-expressed with tyrosine kinases in COS cells Lyn, Blk, Hck, and Fyn phosphorylated the cytoplasmic domains, while Syk did not (Fig. 5). This indicated that a unique relationship exists between the B cell specific proteins Ig␣, Ig␤, and Blk in COS cells, since only Blk phosphorylated the cytoplasmic domains of Ig␣ and Ig␤.
Mapping Tyrosine Phosphorylation Sites of Ig␣ and Ig␤ Cytoplasmic Domains-The cytoplasmic domain of Ig␣ has three potential sites for tyrosine phosphorylation, while the Ig␤ cytoplasmic domain has two such sites (Fig. 1). We mutated each of these sites individually and in combination to determine the tyrosine residues phosphorylated by Blk. No decrease in the intensity of tyrosine phosphorylation of the Ig␣ chimera was detected with mutation of the most membrane proximal tyrosine residue (Ig␣1Y Ϫ ), while phosphorylation of the 2Y and 3Y mutants was half of the wild type level (Fig. 6). Removal of the second and third tyrosine residues from the Ig␣ chimera abolished its phosphorylation by Blk (Ig␣23Y Ϫ ), indicating that only these two residues are sites for phosphorylation in vivo (Fig. 6). Mutation of each of the tyrosine residues in the Ig␤ cytoplasmic domain separately resulted in decreased phosphorylation of the chimera and mutation of both residues together yielded no phosphorylation of the chimera by Blk (Fig. 6). This indicates that both tyrosine residues in the Ig␤ cytoplasmic domain are substrates for Blk in vivo.
Physical Association of the Ig␣ and Ig␤ Chimeras with Blk-Since tyrosine kinases have been found to associate with the immunoglobulin receptor complex in B lymphocytes (11,(13)(14)(15)(16)(17) we analyzed the potential of the kinases to associate with the Ig␣ and Ig␤ chimeras. We co-transfected the Ig␣ chimera with Lyn, Blk, Hck, or Syk, lysed the cells, and immunoprecipitated the kinase. An in vitro kinase assay was then performed fol- lowed by disruption of the immune complex and reimmunoprecipitation with antiserum to Ig␣. Therefore, any kinase associated with the Ig␣ chimera would phosphorylate it in vitro with 32 P, allowing detection of the chimera upon autoradiographic analysis after SDS-polyacrylamide gel electrophoresis. Only Blk was physically associated with the Ig␣ chimera (Fig. 7A). A comparison was made between Blk's ability to phosphorylate and associate with Ig␣ and Ig␤ chimeras. As shown in Fig. 7B, Blk phosphorylates the chimeras equally and associates with them to the same extent.
Syk Binds the Ig␣ Chimera when Co-expressed with Blk-Syk contains two tandem SH2 domains in its amino terminus (27) and has been demonstrated to bind to the Ig␣/Ig␤ heterodimer in activated B lymphocytes via an SH2-phosphotyrosine dependent mechanism (9,23). Since the cytoplasmic domain of Ig␣ becomes phosphorylated on tyrosine residues when co-transfected with Blk, we sought to determine whether Syk could bind to tyrosine-phosphorylated Ig␣. We co-transfected the Ig␣ chimera, Blk, and Syk into COS cells and assessed the kinases' ability to associate with Ig␣. Upon co-transfection with Blk, Syk became associated with the Ig␣ cytoplasmic domain (Fig. 8), whereas expression of Syk alone with the Ig␣ chimera did not result in association (see Fig. 7A). Co-transfection of Lyn with Blk and the Ig␣ chimera did not result in Lyn's association with the Ig␣ chimera although Blk was associated with the chimera (Fig. 8).
Mutation of Blk's SH3 and SH2 Domains-To determine the contribution of Blk's SH3 and SH2 domains to phosphorylation of and association with the Ig␣ and Ig␤ chimeras, we made point mutations in the SH3 and SH2 domains of Blk which destroy their interaction with proline-rich regions and phosphotyrosine, respectively. These mutants retained enzymatic activity when expressed in COS cells as measured by their ability to autophosphorylate (Fig. 9C). The Blk mutants were co-expressed with the Ig␣ or Ig␤ chimeras (Fig. 9D) and were assessed for association with the chimeras (Fig. 9A). Mutation of Blk's SH2 domain alone or in combination with mutation of the SH3 domain abrogated Blk's association with the cytoplasmic domains of Ig␣ and Ig␤ (Fig. 9A). Mutation of Blk's SH3 domain had no effect on Blk's association with Ig␣ or Ig␤. Lysates were immunoprecipitated with antisera to the indicated tyrosine kinase (C and D), and immune complex kinase assays were carried out to measure autophosphorylation (D), or the exogenous substrate GST-Ig␤ was added to the assay to measure enzymatic activity (D). All immunoprecipitations were performed with equivalent protein amounts.
Phosphorylation of the Ig␣ and Ig␤ chimeras was unaffected by mutation of the SH3 and SH2 domains of Blk (Fig. 9B).
Association of Blk with Ig␣ and Ig␤ Chimeras Results from an SH2-Phosphotyrosine Interaction-Since the SH2 domain of Blk is required for association with the chimeras, we assessed the effect of mutation of the Ig␣ and Ig␤ tyrosine residues on association with wild type Blk. Mutation of the second and third cytoplasmic tyrosine residues of the Ig␣ chimera resulted in decreased association with Blk (Ig␣2Y Ϫ , Ig␣3Y Ϫ ) (Fig. 10). However, mutation of the most membrane proximal tyrosine, which we found to not be a substrate for phosphorylation by Blk, did not affect association of Blk with the Ig␣ chimera (Ig␣1Y Ϫ ) (Fig. 10). Mutation of the second and third tyrosine residues together (Ig␣23Y Ϫ ) abrogated Blk's association with the Ig␣ chimera. Mutation of the two tyrosine residues in the Ig␤ cytoplasmic domain separately resulted in decreased association with Blk (Fig. 10). No association of Blk was detected when both tyrosine residues of the Ig␤ cytoplasmic domain were mutated together (Fig. 10).
Enzymatic Activity and Membrane Association of Blk-The protein interactions mediated by the SH3 and SH2 domains of Blk are not required for recognition and phosphorylation of the Ig␣ and Ig␤ cytoplasmic domains (Fig. 9). We therefore determined if Blk's enzymatic activity was necessary for phosphorylation of the substrates by mutating the ATP binding site of the kinase which results in a catalytically inactive enzyme. Also, the Src kinase family is membrane associated due to myristylation of an amino-terminal glycine residue (28,29). We therefore mutated this glycine residue, which results in cytoplasmic expression of the kinase (30), in order to assess the importance of plasma membrane localization of Blk on Ig␣ and Ig␤ phosphorylation. The ATP binding site mutant lacks enzymatic activity as expected (Fig. 11). Enzymatic activity and membrane association of Blk are necessary for its phosphorylation of the Ig␣ and Ig␤ chimeras (Fig. 11).
The Amino-terminal Unique Domain of Blk Confers Recognition and Phosphorylation of the Ig␣ Chimera-Myristylation of Blk does not account for recognition of the Ig␣ and Ig␤ chimeras by Blk alone, since all Src family members are myristylated. We therefore replaced the amino-terminal unique domain of Fyn with that of Blk in order to localize the domain of Blk responsible for substrate recognition and phosphorylation. The chimeric kinase was enzymatically active and phosphorylated the cytoplasmic domain of the Ig␣ chimera (Fig. 12). Wild type Fyn did not phosphorylate the Ig␣ chimera. Blk's aminoterminal unique region is responsible for Ig␣ substrate recognition and tyrosine phosphorylation (Fig. 12).

DISCUSSION
Reconstitution of the B cell antigen receptor signaling components in nonlymphoid cells has allowed us to define interactions between the Ig␣ and Ig␤ cytoplasmic domains and tyrosine kinases. A unique relationship between the B cell-specific proteins Blk, Ig␣, and Ig␤ has been discovered using this system. Although in B cells many Src family tyrosine kinases have been found to be associated with the antigen receptor complex under mild cell lysis conditions (11,(13)(14)(15), only Blk is able to phosphorylate and associate with the Ig␣ and Ig␤ cytoplasmic domains in COS cells. The specificity of phosphorylation is observed only in vivo since each of the co-transfected tyrosine kinases is able to phosphorylate GST-Ig␤ in vitro (Fig. 3), indicating the inherent differences between in vivo and in vitro assays. The association detected between Blk and the Ig␣ and Ig␤ chimeras is the result of an SH2-phosphotyrosine interaction, and occurs as a consequence of phosphorylation of the receptor components (Figs. 9 and 10). The low level of association detected between Blk and individual ITAM tyrosine mutants of Ig␣ and Ig␤ may indicate that these phosphorylated tyrosine residues function in a cooperative manner to enhance kinase binding as has been suggested by in vitro studies ( Fig.  10) (31). Phosphorylation of the cytoplasmic domains of Ig␣ and Ig␤ by Blk occurs without a stable association of the proteins since the SH2 domain mutant of Blk retains its ability to phosphorylate Ig␣ and Ig␤ cytoplasmic domains yet does not form a stable complex with the proteins (Fig. 9). Interestingly, only the tyrosine residues within the ITAM of Ig␣'s cytoplasmic domain are phosphorylated by Blk. The sequence surrounding the most membrane proximal tyrosine does not conform to the consensus motif found to be phosphorylated by Src family kinases in vitro (32), whereas the sequence surrounding the ITAM tyrosine residues fits the consensus motif. Phosphorylation of the ITAM tyrosine residues by Blk supports the importance of the ITAMs in receptor mediated signal transduction. These results differ somewhat from those of Flaswinkel and Reth (6) where they identified the first tyrosine residue of the Ig␣ ITAM as the dominant tyrosine kinase phosphorylation site, without which no phosphorylation of Ig␣ was detectable. This difference may be a function of the experimental systems used; however, the results from both systems indicate the importance of both ITAM tyrosine residues in B cell signal transduction.
The roles of the Ig␣ and Ig␤ cytoplasmic domains in B cell antigen receptor signal transduction have been investigated. FIG. 4. Co-expression of the Ig␣ or Ig␤ chimera with Blk is required for phosphorylation. COS cells were co-transfected with the Ig␣ chimera or Ig␤ chimera and Blk (Ig␣ϩBlk, Ig␤ϩBlk) or COS cells were separately transfected with Ig␣ chimera, Ig␤ chimera, and Blk, and lysates from the transfectants were mixed (Ig␣/Blk, Ig␤/Blk). Cell lysates were immunoprecipitated with antisera to Ig␣ or Ig␤ (top, middle) and immunoblotting was performed with antisera to phosphotyrosine (top), or PDGFR (middle) to measure the level of chimeric receptor expressed. The enzymatic activity of Blk was assessed in an in vitro autophosphorylation assay (bottom).
FIG. 5. Fc⑀RI and TCR PDGFR chimeras are phosphorylated by several Src family tyrosine kinases. COS cells were transfected with the Fc⑀RI chimera or TCR-chimera alone, or were co-transfected with a chimera and the indicated tyrosine kinase. Cell lysates were immunoprecipitated with antibody to phosphotyrosine and immunoblotting with PDGFR antiserum was performed. Transfectants expressed equivalent levels of chimeric receptor and equivalent kinase activity (not shown).
Some findings support differing roles for the two proteins (15,33,34), whereas other results suggest that the cytoplasmic domains of these proteins are functionally equivalent in their ability to transduce signals (7,35,36). In the experimental system described here, the recognition and phosphorylation of the cytoplasmic domains of Ig␣ and Ig␤ by Blk is equivalent, as is the association of the kinase with the proteins. The interac-FIG. 6. Mapping tyrosine phosphorylation sites of Ig␣ and Ig␤ cytoplasmic domains. The Ig␣ chimera was transfected alone (Ig␣ w/o Blk) or co-transfected with Blk (Ig␣). Ig␣ chimeras with tyrosine residues mutated to phenylalanine as indicated were co-transfected with Blk into COS cells. Wild type Ig␤ chimera or Ig␤ chimeras with tyrosine residues mutated to phenylalanine as indicated were co-transfected with Blk. Cell lysates were immunoprecipitated with antisera to Ig␣ or Ig␤ and immunoblotted with antibodies to phosphotyrosine (top) or PDGFR (middle) to compare the amount of chimeric receptor expression. Equivalent enzymatic activity of co-transfected Blk was determined by in vitro autophosphorylation (bottom).

FIG. 7. Blk associates with Ig␣ and Ig␤ chimeras.
A, COS cells were transfected with the Ig␣ chimera alone, or were co-transfected with the chimera and the indicated tyrosine kinase. Association of the kinases with the Ig␣ chimera was assessed by immunoprecipitating cell lysates with antisera to the indicated tyrosine kinase, followed by an immune complex kinase assay, and subsequent disruption of the immune complex followed by immunoprecipitation with antiserum to Ig␣. Transfectants expressed equivalent levels of chimera and equivalent kinase activity (not shown). B, the Ig␣ or Ig␤ chimeras were co-transfected with Blk. Cell lysates were immunoprecipitated with the indicated antisera and immunoblotted with antiserum to the PDGFR. Equivalent levels of Blk activity were present in each immunoprecipitate (not shown).

FIG. 8. Syk binds to the Ig␣ chimera when co-expressed with
Blk. COS cells were co-transfected with the indicated cDNA constructs. Association of the kinases with the Ig␣ chimera was assessed by immunoprecipitating cell lysates with antisera to the indicated tyrosine kinase, followed by an immune complex kinase assay, and subsequent disruption of the immune complex followed by immunoprecipitation with antiserum to Ig␣ (top). Enzymatic activity of the kinases was determined by in vitro autophosphorylation (bottom).

FIG. 9. Effect of mutation of the SH3 and SH2 domains of Blk.
Wild type Blk or Blk with point mutations in the SH3 or SH2 domain or both (Blk SH23 Ϫ ) were co-transfected with the Ig␣ or Ig␤ chimera. A, association of the kinases with the chimeras was assessed by immunoprecipitating cell lysates with antiserum to Blk, followed by an immune complex kinase assay, and subsequent disruption of the immune complex followed by immunoprecipitation with antisera to Ig␣ or Ig␤. B, tyrosine phosphorylation of the chimeric receptors was assessed by immunoprecipitating cell lysates with antibody to phosphotyrosine and immunoblotting with PDGFR antiserum. C, enzymatic activity of the kinases was determined in immune-complex kinase autophosphorylation assays. D, equivalence of chimeric receptor expression was determined by immunoprecipitating cell lysates with antiserum to Ig␣ or Ig␤ and immunoblotting with antiserum to the PDGFR.
FIG . 10. Association of Blk with Ig␣ and Ig␤ cytoplasmic domains results from an SH2-phosphotyrosine interaction. The Ig␣ chimera was transfected alone (Ig␣ w/o Blk) or co-transfected with Blk (Ig␣). Ig␣ chimeras with tyrosine residues mutated to phenylalanine as indicated were co-transfected with Blk into COS cells. Wild type Ig␤ chimera or Ig␤ chimeras with tyrosine residues mutated to phenylalanine as indicated were co-transfected with Blk. To assess the mutant chimeras' association with Blk, cell lysates were immunoprecipitated with antiserum to Blk and immunoblotted with PDGFR antiserum. Transfectants expressed equivalent levels of chimeric receptor and equivalent Blk kinase activity (not shown). tion of the signaling chains with Src family kinases may therefore be equivalent at least in the initial stages of receptor mediated signal transduction.
The Syk and Zap family of protein tyrosine kinases bind to and are activated by di-phosphorylated ITAM sequences (23,37,38). Syk binds to the Ig␣ chimera when it is phosphorylated by Blk, yet Src kinases other than Blk are unable to bind revealing the specificity of SH2-phosphotyrosine interactions. In addition, binding of Syk to the Ig␣ chimera indicates that at least a population of the chimeric receptors are dually tyrosine phosphorylated since Syk requires phosphorylation of both ITAM tyrosine residues in order to bind (23). Binding of Syk specifically to the phosphorylated Ig␣ chimera supports a role for Syk post receptor engagement as has been found in B lymphocytes (9).
Src family tyrosine kinases are co-translationally myristylated at their amino terminus (39). The myristate group permits interaction with plasma membrane lipids resulting in localization of the Src kinases to the inner leaflet of the plasma membrane (40,41). Phosphorylation of the Ig␣ and Ig␤ chimeras is dependent upon localization of Blk to the plasma membrane via the myristate moiety. Localization of the enzyme to the site of Ig␣ and Ig␤ expression in the plasma membrane is important for functional interactions between the proteins.
The unique domain of Src family members is the region containing the most divergent sequences between family members. Since the SH3 and SH2 domains were not involved in recognition and phosphorylation of the Ig␣ and Ig␤ chimeras, and myristylation of Blk could not account for the specificity of Blk's phosphorylation of the cytoplasmic domains because all Src kinases are myristylated, we examined the importance of the unique domain. The ability of Blk's unique domain to transfer phosphorylation of the Ig␣ chimera to Fyn indicated that sequences necessary for recognition and phosphorylation of this substrate are contained within Blk's unique domain.
There are 51 amino acids in Blk's unique domain. A newly defined region of loose homology within the unique domain has been defined as the SH4 domain (42). This domain contains sequences important for subcellular localization of the Src kinases (43,44). Since this domain contains variations within the Src family, the region of importance in Blk for recognition and phosphorylation of Ig␣ may be within or outside this region. Association of Src family members with TCR in vivo (45) and Ig␣ in vitro (46) has been reported to involve the unique domain. The experiments described here indicate that the unique domain of Src kinases may direct recognition of certain substrates leading to their phosphorylation.
FIG . 11. Mutation of Blk's myristylation and ATP binding sites. COS cells were co-transfected with the Ig␣ or Ig␤ chimera and wild type Blk, myristylation site mutated Blk (Blk Myr Ϫ ), or ATP binding site mutated Blk (Blk ABS Ϫ ). Cell lysates were immunoprecipitated with antibodies to phosphotyrosine (top) and Ig␣ or Ig␤ (middle) and immunoblotted with PDGFR antiserum. Enzymatic activity of the kinases was measured by autophosphorylation in an in vitro kinase assay (bottom).
FIG. 12. The amino-terminal unique domain of Blk confers recognition and phosphorylation of the Ig␣ chimera. COS cells were transfected with the Ig␣ chimera alone, or were co-transfected with the indicated kinase. The Blk N Fyn C chimeric kinase contains the amino terminus of Blk up to the start of the SH3 domain together with the Fyn kinase from the SH3 domain to the carboxyl terminus of the kinase. Cell lysates were immunoprecipitated with antibody to phosphotyrosine (top) and Ig␣ (middle), and were immunoblotted with antiserum to the PDGFR. Enzymatic activity of the kinases was measured by autophosphorylation in an in vitro kinase assay (bottom).