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(Received for publication, May 25,
1995; and in revised form, August 11, 1995) From the
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
Signal transduction through the B cell antigen receptor (BCR) ( 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 Ig
Figure 1:
PDGFR-Ig
Figure 2:
Expression of the Ig
Figure 3:
Only Blk phosphorylates Ig
Figure 4:
Co-expression of the Ig
Figure 5:
Fc
Figure 6:
Mapping tyrosine phosphorylation sites of
Ig
Figure 7:
Blk associates with Ig
Figure 8:
Syk
binds to the Ig
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
Figure 10:
Association of Blk with Ig
Figure 11:
Mutation of Blk's myristylation and
ATP binding sites. COS cells were co-transfected with the Ig
Figure 12:
The amino-terminal unique domain of Blk
confers recognition and phosphorylation of the Ig
Reconstitution of the B cell antigen receptor signaling
components in nonlymphoid cells has allowed us to define interactions
between the Ig The roles of the
Ig 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 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 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
Volume 270,
Number 45,
Issue of November 10, 1995 pp. 27072-27078
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
or Ig
. Of the tyrosine kinases
transfected (Lyn, Blk, Hck, Syk, Fyn), only Blk was able to
phosphorylate and subsequently associate with co-transfected 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.
)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 tyrosine-based
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. 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
DNA fragments encoding the extracellular domain of the
PDGFR (amino acids 1-499) (18) or the transmembrane and
cytoplasmic domain of Ig, Ig, TCR-, and Fc
RI
Chimeras (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 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 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).
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
Fyn
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 
10
, 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
10
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 Ig
(GST-Ig).
Expression of Ig
Chimeric proteins containing the extracellular domain
of the PDGFR coupled to the transmembrane and cytoplasmic domains of
Ig and Ig
Chimeras 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).
and Ig chimeras. The
extracellular domain of the PDGFR (amino acids 1-499) was fused
to the transmembrane and cytoplasmic domains of Ig (amino acids
138-220) and Ig (amino acids 159-228). The numbered
tyrosine residues correspond to potential sites of
phosphorylation.
and Ig
chimeras. A, flow cytometric analysis of Ig and Ig
chimera transfected COS cells. Mock-, Ig chimera-, and Ig
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 Ig or Ig.
The Ig and Ig chimeras have an apparent molecular mass of
approximately 97 kDa.
Co-expression of Ig
To determine whether the chimeric receptors
would interact with tyrosine kinases present in B cells, we
co-transfected the Ig or Ig Chimeras with
Tyrosine Kinases 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+Blk, Ig+Blk) 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).
and
Ig chimeras in vivo. COS cells were transfected with the
Ig chimera or Ig chimera alone, or were co-transfected with a
chimera and the indicated tyrosine kinase. Cells lysates were
immunoprecipitated with antibodies to phosphotyrosine (A) and
Ig or Ig (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-Ig was added to the assay to measure
enzymatic activity (D). All immunoprecipitations were
performed with equivalent protein amounts.
or Ig
chimera with Blk is required for phosphorylation. COS cells were
co-transfected with the Ig chimera or Ig chimera and Blk (Ig+Blk, Ig+Blk) 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).
Co-expression of Fc
We tested the ability of tyrosine kinases to
phosphorylate the cytoplasmic domains of the signaling proteins
FcRI
and Chimeras with
Tyrosine Kinases
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.
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).
Mapping Tyrosine Phosphorylation Sites of Ig
The cytoplasmic domain of Ig and
Ig Cytoplasmic Domains
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 (Ig1Y), 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 (Ig23Y), 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.
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).
Physical Association of the Ig
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
with Blk 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 followed 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 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.
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).
Syk Binds the Ig
Syk contains two tandem SH2 domains in its amino terminus (27) and has been demonstrated to bind to the Ig Chimera when Co-expressed with
Blk/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).
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).
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.
Phosphorylation of the Ig and Ig chimeras was unaffected by
mutation of the SH3 and SH2 domains of Blk (Fig. 9B).
) 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.
Association of Blk with Ig
Since the SH2
domain of Blk is required for association with the chimeras, we
assessed the effect of mutation of the Ig and Ig Chimeras
Results from an SH2-Phosphotyrosine Interaction 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 (Ig2Y, Ig
3Y) (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 (Ig1Y) (Fig. 10). Mutation of the second and third tyrosine residues
together (Ig
23Y) 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).
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).
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).
or
Ig 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 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).
The Amino-terminal Unique Domain of Blk Confers
Recognition and Phosphorylation of the Ig
Myristylation of Blk does not account for recognition of
the Ig
Chimera 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
amino-terminal unique region is responsible for Ig substrate
recognition and tyrosine phosphorylation (Fig. 12).
chimera. COS
cells were transfected with the Ig chimera alone, or were
co-transfected with the indicated kinase. The BlkFyn 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).
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 ( Fig. 9and 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. and Ig 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 Ig and Ig 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. 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) . 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. 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.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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L. I. Pao, S. J. Famiglietti, and J. C. Cambier Asymmetrical Phosphorylation and Function of Immunoreceptor Tyrosine-Based Activation Motif Tyrosines in B Cell Antigen Receptor Signal Transduction J. Immunol., April 1, 1998; 160(7): 3305 - 3314. [Abstract] [Full Text] [PDF]
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L. K. Verkoczy, B.-a. Guinn, and N. L. Berinstein Characterization of the Human B Cell RAG-associated Gene, hBRAG, as a B Cell Receptor Signal-enhancing Glycoprotein Dimer That Associates with Phosphorylated Proteins in Resting B Cells J. Biol. Chem., July 7, 2000; 275(28): 20967 - 20979. [Abstract] [Full Text] [PDF]
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ABSTRACT
