©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reconstitution of the B Cell Antigen Receptor Signaling Components in COS Cells (*)

(Received for publication, May 25, 1995; and in revised form, August 11, 1995)

Sandra J. Saouaf (§) Stephanie A. Kut Joseph Fargnoli R. Bruce Rowley Joseph B. Bolen Sandeep Mahajan

From the Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 Igalpha or Igbeta. Of the tyrosine kinases transfected (Lyn, Blk, Hck, Syk, Fyn), only Blk was able to phosphorylate and subsequently associate with co-transfected Igalpha and Igbeta chimeras in vivo. Association between Blk and the Igalpha and Igbeta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Igalpha and Igbeta cytoplasmic domains phosphorylated by Blk. The enzymatic activity and membrane association of Blk were required for the observed phosphorylation of the Igalpha and Igbeta chimeras. Sequences within the amino-terminal unique domain of Blk are responsible for recognition and subsequent phosphorylation of the Igalpha chimera since transfer of the unique region of Blk to Fyn results in the chimeric kinase's ability to phosphorylate the cytoplasmic domain of Igalpha. These findings indicate that the unique domain of Src family kinases may direct recognition of certain substrates leading to their phosphorylation.


INTRODUCTION

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 Igalpha and Igbeta, which exist as disulfide-linked heterodimers on the surface of B lymphocytes (1, 2, 3) . Igalpha and Igbeta are necessary for signal transduction through the BCR since mutation of the surface immunoglobulin molecule such that it does not associate with the Igalpha/Igbeta heterodimer results in B cell nonresponsiveness to antigen receptor engagement(4) . Igalpha and Igbeta each contain one immune receptor tyrosine-based activation motif (ITAM) in their cytoplasmic domains(5) . Mutation of the ITAM tyrosine residues in Igalpha results in abrogation of B cell activation(6) . The cytoplasmic domains alone of Igalpha and Igbeta 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, 10, 11, 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 Igalpha and Igbeta and the domains of the tyrosine kinases involved in physical and functional interactions possibly important in B cell antigen receptor-mediated signal transduction.


MATERIALS AND METHODS

COS Cell Expression Constructs

Construction of platelet-derived growth factor receptor (PDGFR)-Igalpha, Igbeta, TCR-, and FcRIbeta Chimeras

DNA fragments encoding the extracellular domain of the PDGFR (amino acids 1-499) (18) or the transmembrane and cytoplasmic domain of Igalpha (amino acid 138-220), Igbeta (amino acid 159-228), TCR- chain (amino acid 31-164), and FcRIbeta (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 SKII+ (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 pSVPDGFRalpha, pSVPDGFRbeta, pSVPDGFR, and pSVPDGFR-FcRIbeta 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 Igalpha and Igbeta 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). PCR-amplified 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.

Transfection of COS Cells

COS cells, 1.5 times 10^6, were transfected with 1-6 µg of each plasmid DNA using 15-27 µl 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 times 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 µg of glutathione S-transferase (GST) fusion protein containing the cytoplasmic domain of Igbeta (GST-Igbeta).


RESULTS

Expression of Igalpha and Igbeta Chimeras

Chimeric proteins containing the extracellular domain of the PDGFR coupled to the transmembrane and cytoplasmic domains of Igalpha or Igbeta 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 Igalpha or Igbeta (Fig. 2B). The chimeras were expressed as monomers on the surface of COS cells as determined by two-dimensional gel electrophoresis (not shown).


Figure 1: PDGFR-Igalpha and Igbeta chimeras. The extracellular domain of the PDGFR (amino acids 1-499) was fused to the transmembrane and cytoplasmic domains of Igalpha (amino acids 138-220) and Igbeta (amino acids 159-228). The numbered tyrosine residues correspond to potential sites of phosphorylation.




Figure 2: Expression of the Igalpha and Igbeta chimeras. A, flow cytometric analysis of Igalpha and Igbeta chimera transfected COS cells. Mock-, Igalpha chimera-, and Igbeta chimera-transfected COS cells were stained with antibodies to PDGFR (solid lines) or normal rabbit serum (dotted lines), followed by goat anti-rabbit IgG-fluorescein isothiocyanate-labeled secondary antibody and analyzed on a Coulter EPICS flow cytometer. B, chimera-transfected cells and WEHI231 B cells were lysed and lysates were immunoprecipitated with the indicated antisera and fixed Staphylococcus aureus (Staph). SDS-PAGE was carried out on the immunoprecipitates, proteins were transferred to nitrocellulose, and immunoblotted with antisera to Igalpha or Igbeta. The Igalpha and Igbeta chimeras have an apparent molecular mass of approximately 97 kDa.



Co-expression of Igalpha or Igbeta Chimeras with Tyrosine Kinases

To determine whether the chimeric receptors would interact with tyrosine kinases present in B cells, we co-transfected the Igalpha chimera or Igbeta 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-Igbeta) (Fig. 3D). The co-transfected kinases had similar levels of enzymatic activity. When transfected alone, the Igalpha and Igbeta chimeras lacked tyrosine phosphorylation. However, co-expression with the Blk tyrosine kinase resulted in phosphorylation of the Igalpha and Igbeta chimeras on tyrosine (Fig. 3A). No other tyrosine kinase tested, including Btk and Lck (not shown), was able to phosphorylate the Igalpha or Igbeta chimera. To ensure that the tyrosine phosphorylation observed was occurring within the intact transfected cells rather than occurring post lysis of the cells, Igalpha and Igbeta chimeras were co-transfected with Blk (Igalpha+Blk, Igbeta+Blk) or chimeras were transfected separately from Blk and cell lysates from the two transfectants were mixed and incubated (Igalpha/Blk, Igbeta/Blk) (Fig. 4). Tyrosine phosphorylation of the Igalpha or Igbeta 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).


Figure 3: Only Blk phosphorylates Igalpha and Igbeta chimeras in vivo. COS cells were transfected with the Igalpha chimera or Igbeta chimera alone, or were co-transfected with a chimera and the indicated tyrosine kinase. Cells lysates were immunoprecipitated with antibodies to phosphotyrosine (A) and Igalpha or Igbeta (B) in order to measure the level of chimeric receptor expression. Immunoblotting with antiserum to the PDGFR was performed (A and B). 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-Igbeta was added to the assay to measure enzymatic activity (D). All immunoprecipitations were performed with equivalent protein amounts.




Figure 4: Co-expression of the Igalpha or Igbeta chimera with Blk is required for phosphorylation. COS cells were co-transfected with the Igalpha chimera or Igbeta chimera and Blk (Igalpha+Blk, Igbeta+Blk) or COS cells were separately transfected with Igalpha chimera, Igbeta chimera, and Blk, and lysates from the transfectants were mixed (Igalpha/Blk, Igbeta/Blk). Cell lysates were immunoprecipitated with antisera to Igalpha or Igbeta (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).



Co-expression of FcRIbeta and Chimeras with Tyrosine Kinases

We tested the ability of tyrosine kinases to phosphorylate the cytoplasmic domains of the signaling proteins FcRIbeta and from the T cell receptor complex. The PDGFR extracellular domain was fused to the transmembrane and cytoplasmic domains of FcRIbeta 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 Igalpha, Igbeta, and Blk in COS cells, since only Blk phosphorylated the cytoplasmic domains of Igalpha and Igbeta.


Figure 5: FcRI and TCR PDGFR chimeras are phosphorylated by several Src family tyrosine kinases. COS cells were transfected with the FcRI 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).



Mapping Tyrosine Phosphorylation Sites of Igalpha and Igbeta Cytoplasmic Domains

The cytoplasmic domain of Igalpha has three potential sites for tyrosine phosphorylation, while the Igbeta 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 Igalpha chimera was detected with mutation of the most membrane proximal tyrosine residue (Igalpha1Y), 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 Igalpha chimera abolished its phosphorylation by Blk (Igalpha23Y), indicating that only these two residues are sites for phosphorylation in vivo (Fig. 6). Mutation of each of the tyrosine residues in the Igbeta 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 Igbeta cytoplasmic domain are substrates for Blk in vivo.


Figure 6: Mapping tyrosine phosphorylation sites of Igalpha and Igbeta cytoplasmic domains. The Igalpha chimera was transfected alone (Igalpha w/o Blk) or co-transfected with Blk (Igalpha). Igalpha chimeras with tyrosine residues mutated to phenylalanine as indicated were co-transfected with Blk into COS cells. Wild type Igbeta chimera or Igbeta chimeras with tyrosine residues mutated to phenylalanine as indicated were co-transfected with Blk. Cell lysates were immunoprecipitated with antisera to Igalpha or Igbeta 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).



Physical Association of the Igalpha and Igbeta 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 Igalpha and Igbeta chimeras. We co-transfected the Igalpha chimera with Lyn, Blk, Hck, or Syk, lysed the cells, and immunoprecipitated the kinase. An in vitro kinase assay was then performed followed by disruption of the immune complex and reimmunoprecipitation with antiserum to Igalpha. Therefore, any kinase associated with the Igalpha chimera would phosphorylate it in vitro with P, allowing detection of the chimera upon autoradiographic analysis after SDS-polyacrylamide gel electrophoresis. Only Blk was physically associated with the Igalpha chimera (Fig. 7A). A comparison was made between Blk's ability to phosphorylate and associate with Igalpha and Igbeta chimeras. As shown in Fig. 7B, Blk phosphorylates the chimeras equally and associates with them to the same extent.


Figure 7: Blk associates with Igalpha and Igbeta chimeras. A, COS cells were transfected with the Igalpha chimera alone, or were co-transfected with the chimera and the indicated tyrosine kinase. Association of the kinases with the Igalpha 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 Igalpha. Transfectants expressed equivalent levels of chimera and equivalent kinase activity (not shown). B, the Igalpha or Igbeta 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).



Syk Binds the Igalpha 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 Igalpha/Igbeta heterodimer in activated B lymphocytes via an SH2-phosphotyrosine dependent mechanism(9, 23) . Since the cytoplasmic domain of Igalpha becomes phosphorylated on tyrosine residues when co-transfected with Blk, we sought to determine whether Syk could bind to tyrosine-phosphorylated Igalpha. We co-transfected the Igalpha chimera, Blk, and Syk into COS cells and assessed the kinases' ability to associate with Igalpha. Upon co-transfection with Blk, Syk became associated with the Igalpha cytoplasmic domain (Fig. 8), whereas expression of Syk alone with the Igalpha chimera did not result in association (see Fig. 7A). Co-transfection of Lyn with Blk and the Igalpha chimera did not result in Lyn's association with the Igalpha chimera although Blk was associated with the chimera (Fig. 8).


Figure 8: Syk binds to the Igalpha chimera when co-expressed with Blk. COS cells were co-transfected with the indicated cDNA constructs. Association of the kinases with the Igalpha 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 Igalpha (top). Enzymatic activity of the kinases was determined by in vitro autophosphorylation (bottom).



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 Igalpha and Igbeta 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 Igalpha or Igbeta 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 Igalpha and Igbeta (Fig. 9A). Mutation of Blk's SH3 domain had no effect on Blk's association with Igalpha or Igbeta. Phosphorylation of the Igalpha and Igbeta chimeras was unaffected by mutation of the SH3 and SH2 domains of Blk (Fig. 9B).


Figure 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) were co-transfected with the Igalpha or Igbeta 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 Igalpha or Igbeta. 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 Igalpha or Igbeta and immunoblotting with antiserum to the PDGFR.



Association of Blk with Igalpha and Igbeta 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 Igalpha and Igbeta tyrosine residues on association with wild type Blk. Mutation of the second and third cytoplasmic tyrosine residues of the Igalpha chimera resulted in decreased association with Blk (Igalpha2Y, Igalpha3Y) (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 Igalpha chimera (Igalpha1Y) (Fig. 10). Mutation of the second and third tyrosine residues together (Igalpha23Y) abrogated Blk's association with the Igalpha chimera. Mutation of the two tyrosine residues in the Igbeta cytoplasmic domain separately resulted in decreased association with Blk (Fig. 10). No association of Blk was detected when both tyrosine residues of the Igbeta cytoplasmic domain were mutated together (Fig. 10).


Figure 10: Association of Blk with Igalpha and Igbeta cytoplasmic domains results from an SH2-phosphotyrosine interaction. The Igalpha chimera was transfected alone (Igalpha w/o Blk) or co-transfected with Blk (Igalpha). Igalpha chimeras with tyrosine residues mutated to phenylalanine as indicated were co-transfected with Blk into COS cells. Wild type Igbeta chimera or Igbeta 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).



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 Igalpha and Igbeta 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 Igalpha and Igbeta 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 Igalpha and Igbeta chimeras (Fig. 11).


Figure 11: Mutation of Blk's myristylation and ATP binding sites. COS cells were co-transfected with the Igalpha or Igbeta chimera and wild type Blk, myristylation site mutated Blk (Blk Myr), or ATP binding site mutated Blk (Blk ABS). Cell lysates were immunoprecipitated with antibodies to phosphotyrosine (top) and Igalpha or Igbeta (middle) and immunoblotted with PDGFR antiserum. Enzymatic activity of the kinases was measured by autophosphorylation in an in vitro kinase assay (bottom).



The Amino-terminal Unique Domain of Blk Confers Recognition and Phosphorylation of the Igalpha Chimera

Myristylation of Blk does not account for recognition of the Igalpha and Igbeta 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 Igalpha chimera (Fig. 12). Wild type Fyn did not phosphorylate the Igalpha chimera. Blk's amino-terminal unique region is responsible for Igalpha substrate recognition and tyrosine phosphorylation (Fig. 12).


Figure 12: The amino-terminal unique domain of Blk confers recognition and phosphorylation of the Igalpha chimera. COS cells were transfected with the Igalpha 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 Igalpha (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).




DISCUSSION

Reconstitution of the B cell antigen receptor signaling components in nonlymphoid cells has allowed us to define interactions between the Igalpha and Igbeta cytoplasmic domains and tyrosine kinases. A unique relationship between the B cell-specific proteins Blk, Igalpha, and Igbeta 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 Igalpha and Igbeta 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-Igbeta in vitro (Fig. 3), indicating the inherent differences between in vivo and in vitro assays. The association detected between Blk and the Igalpha and Igbeta chimeras is the result of an SH2-phosphotyrosine interaction, and occurs as a consequence of phosphorylation of the receptor components ( Fig. 9and 10). The low level of association detected between Blk and individual ITAM tyrosine mutants of Igalpha and Igbeta 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 Igalpha and Igbeta by Blk occurs without a stable association of the proteins since the SH2 domain mutant of Blk retains its ability to phosphorylate Igalpha and Igbeta cytoplasmic domains yet does not form a stable complex with the proteins (Fig. 9). Interestingly, only the tyrosine residues within the ITAM of Igalpha'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 Igalpha ITAM as the dominant tyrosine kinase phosphorylation site, without which no phosphorylation of Igalpha 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 Igalpha and Igbeta cytoplasmic domains in B cell antigen receptor signal transduction have been investigated. 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 Igalpha and Igbeta by Blk is equivalent, as is the association of the kinase with the proteins. The interaction 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 Igalpha 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 Igalpha 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 Igalpha 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 Igalpha and Igbeta chimeras is dependent upon localization of Blk to the plasma membrane via the myristate moiety. Localization of the enzyme to the site of Igalpha and Igbeta 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 Igalpha and Igbeta 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 Igalpha 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 Igalpha may be within or outside this region. Association of Src family members with TCR in vivo(45) and Igalpha 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.


FOOTNOTES

*
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 Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, H24-03, P.O. Box 4000, Princeton, NJ 08543-4000. Tel.: 609-252-5279; Fax: 609-252-6051.

(^1)
The abbreviations used are: BCR, B cell antigen receptor; ITAM, immune receptor tyrosine-based activation motif; PDGFR, platelet-derived growth factor receptor; PCR, polymerase chain reaction; GST, glutathione S-transferase; TCR, T cell receptor.


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