(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 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.
Signal transduction through the B cell antigen receptor (BCR) ()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.
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 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.
Figure 1:
PDGFR-Ig 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.
Figure 2:
Expression of the Ig 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.
Figure 3:
Only Blk phosphorylates Ig 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.
Figure 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
+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).
Figure 5:
FcRI 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).
Figure 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).
Figure 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).
Figure 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).
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 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.
Figure 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).
Figure 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
), 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).
Figure 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
Fyn
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).
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 ( 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.
The roles of the
Ig 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.
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.