(Received for publication, November 29, 1994; and in revised form, January 9, 1995)
From the
Shc protein is tyrosine phosphorylated upon B cell receptor (BCR) activation and after its phosphorylation interacts with the adaptor protein Grb2. In turn, Grb2 interacts with the guanine nucleotide exchange factor for Ras, mSOS. Several protein-tyrosine kinases (PTKs) participate in BCR signaling. However, it is not clear which PTK is involved in the phosphorylation of Shc, resulting in coupling to the Ras pathway. Tyrosine phosphorylation of Shc and its association with Grb2 were profoundly reduced in both Lyn- and Syk-deficient B cells upon BCR stimulation. Furthermore, kinase activity of these PTKs was required for phosphorylation of Shc. Shc interacted with Syk in B cells. This interaction and the requirement of Syk kinase activity for phosphorylation of Shc were also demonstrated by cotransfection in COS cells. Because Lyn is required for activation of Syk upon receptor stimulation, our results suggest that the Lyn-activated Syk phosphorylates Shc during BCR signaling.
The engagement of B cell receptor (BCR) ()leads to a
variety of biological responses ranging from programmed cell death to
cell activation and proliferation, depending on the developmental stage
of B cells. The BCR is a multisubunit complex composed of an antigen
recognition component, membrane Ig, and the associated subunits Ig
and Ig
(1) . Structures in the cytoplasmic domain of these
Ig
and Ig
chains have been shown to be critical for an
activation cascade(2, 3, 4) . These
structures, referred to as the ARH1 motif, are defined by two conserved
tyrosines 10 or 11 residues apart and by leucine or isoleucine residues
3 residues C-terminal of both tyrosine residues: XXYXX(L/I)X
YXX(L/I) (5, 6, 7) .
B cell activation by cross-linking of BCR induces intracellular signals including, at its most proximal steps, the activation of protein-tyrosine phosphorylation(8, 9) . This phosphorylation is mediated by at least two types of tyrosine kinase, Src family kinase, such as Lyn, Fyn, and Blk, and Syk kinase(10, 11, 12, 13, 14, 15, 16, 17) .
The activation of Ras, which is downstream of tyrosine kinases, appears to be a crucial early event in the intracellular signaling pathways initiated by a number of receptors(18) . Activation of tyrosine kinases increases the amount of Ras in the active GTP-bound state(19, 20, 21, 22, 23, 24, 25) , and mutational inactivation of the ras gene blocks the effects mediated by these receptors(26, 27) . Recently, several adaptor proteins have been implicated in the intermediate steps between tyrosine kinase activation and conversion of Ras to its active GTP-bound state.
First, there is the Grb2 protein that has a single Src homology 2 (SH2) domain flanked by two SH3 domains(28, 29) . The SH2 domain binds to specific phosphopeptide sequences(30) , whereas SH3 domains bind to proline-rich sequences(31) . The protein Grb2 binds to the tyrosine-phosphorylated molecule, such as autophosphorylated epidermal growth factor receptor, through its SH2 domain, and it simultaneously associates through its SH3 domains with mSOS, a guanine nucleotide exchange protein that activates Ras by inducing exchange of GDP for GTP on Ras(20, 32, 33, 34, 35, 36, 37) .
The second protein that has been implicated in the regulation of Ras is Shc. Shc contains a single SH2 domain and a glycine/proline-rich region (38) . Antisera against Shc immunoprecipitate three different Shc proteins of 48, 52, and 66 kDa. The 48- and 52-kDa proteins are both encoded by a 3.4-kilobase mRNA, whereas the 66-kDa protein is translated from a distinct transcript. Stimulation of the epidermal growth factor receptor leads to the tyrosine phosphorylation of Shc, and the Shc associates with epidermal growth factor receptor through its SH2 domain(38, 39) . The involvement of Shc in Ras activation has been demonstrated by the observation that overexpression of the shc gene induces ras-dependent neurite extensions in the PC12 pheochromocytoma neural cell line(39) . Moreover, in v-src-transformed cells, Shc is found in a complex with Grb2(40) , and a small fraction of Shc is detected in mSOS immunoprecipitates(34) . After epidermal growth factor receptor stimulation, a complex between Shc and Grb2 is also found(38, 39) .
Receptors that do not have intrinsic tyrosine kinases but signal through activation of associated tyrosine kinases also induce Ras activation. Evidence that stimulation of BCR activates Ras, leading to the up-regulation of mitogen-activated protein kinase(41, 42) , prompted us to examine the role of Shc in BCR-mediated signaling. As in T cell receptor signaling(43) , Shc is phosphorylated on tyrosine residues upon BCR stimulation, resulting in the formation of a complex containing Shc, Grb2, and mSOS(44, 45) .
Because two types of PTKs (Src family and Syk family) are associated with BCR, we reasoned whether Src family PTK or Syk family PTK is involved in phosphorylation of Shc. In this report, we demonstrate the requirement of these PTKs for BCR-induced phosphorylation of Shc and describe results relevant to the mechanism by which the BCR mediates tyrosine phosphorylation of Shc.
To examine the role of Shc upon B cell activation, we determined whether Shc is tyrosine phosphorylated upon BCR stimulation. A murine B cell line A20 expressing surface IgG was activated by anti-mouse IgG Ab. Cell lysates were immunoprecipitated by anti-Shc Ab, blotted, and analyzed by anti-phosphotyrosine mAb. Tyrosine phosphorylation of both the 48- and 52-kDa isoforms of Shc was detected within 1 min of stimulation. Shc phosphorylation peaked at about 3 min, and its phosphorylation started to diminish by 10 min (Fig. 1). Anti-Shc Ab also coimmunoprecipitated an unidentified 140-kDa tyrosine-phosphorylated protein.
Figure 1: BCR-induced tyrosine phosphorylation of Shc and its association with Grb2 in A20 cells. A20 cells were stimulated with anti-mouse IgG Ab for the indicated time, lysed, and immunoprecipitated by anti-Shc Ab. Samples were separated with 8 (upper and lowerpanels) and 12.5% SDS-polyacrylamide gels (middlepanel), transferred, and incubated with anti-phosphotyrosine mAb or anti-Grb2 Ab. The filter (8% SDS-polyacrylamide gel) was stripped and incubated with anti-Shc Ab.
Next, we assessed whether Shc would interact with Grb2 after BCR stimulation. Immunoblotting of anti-Shc immunoprecipitates with anti-Grb2 Ab revealed the coprecipitation of Grb2 with Shc after BCR stimulation (Fig. 1). These data are consistent with a previous report that Shc is tyrosine phosphorylated upon BCR stimulation, resulting in the association of Shc with Grb2 and mSOS(44, 45) . Because the expression extent of mSOS was low in A20 cells, it has been difficult to detect the association of Shc with mSOS.
BCR associates with two classes of cytoplasmic PTKs: Src family PTK and Syk family PTK. Using Lyn- and Syk-deficient DT40 B cells, we previously showed that these kinases mediate tyrosine phosphorylation of distinct molecules upon BCR stimulation(14) . Thus, we wished to examine which of these kinases are involved in phosphorylation of Shc. Lyn- and Syk-deficient DT40 cells, along with wild-type cells, were stimulated by mAb M4 that recognizes surface IgM on DT40 cells. Cell lysates were immunoprecipitated by anti-Shc, blotted, and analyzed by anti-phosphotyrosine mAb. Tyrosine phosphorylation of three isoforms of Shc was induced upon BCR stimulation in wild-type DT40 cells. Proteins corresponding to 48 and 52 kDa in mouse A20 cells showed 50- and 55-kDa forms in DT40 cells. In addition to these two isoforms, another 58-kDa form was immunoprecipitated by anti-Shc Ab. In contrast to wild-type DT40 cells, tyrosine phosphorylation of Shc was only slightly induced in both Syk- and Lyn-deficient mutants. Shc phosphorylation in Syk- and Lyn-deficient cells peaked at 1 min and 10 min, respectively (Fig. 2). These results indicate that both Syk and Lyn are required for BCR-coupled tyrosine phosphorylation of Shc. Longer exposure of this blot showed a faint tyrosine-phosphorylated 70-kDa protein only in wild-type DT40 cells after BCR stimulation. It is noteworthy that phosphorylation of the 140-kDa protein associated with Shc still occurs in Syk-deficient DT40 cells, even though the intensity is low and the time course is rapid compared with wild-type cells. This result suggests that Lyn is primarily involved in BCR-mediated tyrosine phosphorylation of this 140-kDa protein.
Figure 2: BCR-induced tyrosine phosphorylation of Shc and its association with Grb2 in various DT40 mutant cells. DT40 cells (wild-type, syk-negative, and lyn-negative) were stimulated with M4 mAb for the indicated time. Cells were lysed and immunoprecipitated by anti-Shc Ab. Samples were separated with 8 (upper and lowerpanels) and 12.5% SDS-polyacrylamide gels (middlepanel), transferred to nitrocellulose membranes, and incubated with anti-phosphotyrosine mAb, anti-Grb2 Ab, or anti-Shc Ab. The magnitudes of Shc phosphorylation (summation of three isoforms) in Syk- and Lyn-deficient DT40 cells were approximately 10% (at 1 min stimulation) and 3% (at 10 min stimulation), respectively, compared with that of wild-type cells (by densitometry).
To establish that the defect of BCR-induced tyrosine phosphorylation of Shc in Lyn- and Syk-deficient cells is due to the loss of their kinase activity, we analyzed Lyn- and Syk-deficient cells expressing kinase-negative Fyn and kinase-negative Syk, respectively. We previously demonstrated that the function of Lyn is compensated by other Src family PTKs, Fyn and Lck(14) . Indeed, transfection of wild-type fyn cDNA into Lyn-deficient cells restored BCR-induced tyrosine phosphorylation of Shc (data not shown). Although the expression extent of these kinase-negative Fyn and Syk was adequate, as judged by Western blotting (data not shown), these kinase-negative enzymes did not restore the phosphorylation of Shc upon BCR stimulation (Fig. 3). These results indicate that the kinase activity of Src-PTK and Syk is essential for the phosphorylation of Shc through BCR stimulation.
Figure 3: Requirement of kinase activity of Syk and Src PTK for BCR-induced tyrosine phosphorylation of Shc. DT40 cells (wild-type (wt) cells, syk-negative cells expressing kinase-negative Syk, and lyn-negative cells expressing kinase-negative Fyn) were stimulated with M4 for 3 min and lysed. Samples were immunoprecipitated with anti-Shc Ab, separated on 8% SDS-polyacrylamide gels, transferred, and incubated with anti-phosphotyrosine mAb or anti-Shc Ab.
As shown in Fig. 2, BCR-induced association of Grb2 with Shc in Syk-deficient cells was also profoundly reduced compared with that of wild-type cells. Longer exposure of this blot showed a faint association of Grb2 with Shc in Lyn-deficient cells at 10 min. The amount of Grb2 associated with Shc appears to correlate with the phosphorylation extent of Shc; the time course and magnitude of phosphorylation of Shc correlated well with those of associated Grb2 with Shc. These observations support the notion that Grb2 is recruited to phosphorylated Shc via its SH2 domain after BCR stimulation.
Because Shc is a cytoplasmic protein and the activation of Ras
occurs at the plasma membrane, we speculate that Shc might interact
with either the BCR complex itself or the BCR-associated proteins
during receptor stimulation. This would provide a mechanism to shuttle
Grb2 and mSOS to the membrane. Tyrosine phosphorylation of Ig and
Ig
of the BCR complex is shown to be critical for initiating a
signaling cascade(4, 49) . Particularly, Ig
is
more competent than Ig
in coupling to protein tyrosine
phosphorylation(2, 4) . Src family PTKs such as Lyn
and Fyn associate with Ig
even in the absence of ligand
stimulation(10, 11, 17, 50) ,
whereas Syk interacts with phosphorylated Ig
upon BCR
stimulation(51) . (
)Thus, we examined the
possibility that Shc associates with Ig
, Fyn, or Syk by using a
COS cell transfection system. Ig
and Fyn are expressed in the
membrane fraction through the transmembrane hydrophobic domain and the
N-terminal myristoylation site, respectively, whereas Syk is expressed
in both cytosolic and membrane fractions(53) . To minimize the
effect of subcellular localization, we made a chimeric construct
CD16/
/Syk in which intracellular Syk is fused to extracellular
CD16 and transmembrane CD3
, thereby allowing the expression of Syk
exclusively in the membrane fraction. A similar construct,
CD16/CD7/Syk, was previously demonstrated to mimic signaling events
triggered by antigen receptor stimulation(54) .
Cotransfection of Shc with CD16//Syk increased tyrosine
phosphorylation of Shc. The tyrosine phosphorylation of Shc was due to
Syk kinase activity, because the chimeric molecule bearing
kinase-negative Syk (CD16/
/Syk(K-)) did not induce this
phosphorylation (Fig. 4A). Association of Shc with
CD16/
/Syk or CD16/
/Syk(K-) was examined by
immunoblotting of anti-Shc immunoprecipitates with anti-Syk Ab. Both
CD16/
/Syk and CD16/
/Syk(K-) were associated with Shc (Fig. 4B). The quantitative difference of these
associations might be accounted for by the expression extent of
CD16/
/Syk and CD16/
/Syk(K-) (Fig. 4C).
As expected, tyrosine phosphorylation of CD16/
/Syk was observed,
whereas CD16/
/Syk(K-) yielded undetectable tyrosine
phosphorylation in COS cells (Fig. 4B). These results
demonstrate that the association of Shc with Syk is independent of the
tyrosine phosphorylation state of Syk.
Figure 4:
Interaction of Shc with CD16//Syk or
IgM/Ig
in COS cells. COS cells transfected with indicated
combinations were lysed, immunoprecipitated with anti-Shc, anti-CD16
3G8, or anti-IgM, separated on 8% SDS-polyacrylamide gels, transferred,
and incubated with anti-phosphotyrosine mAb, anti-Shc Ab, anti-Syk Ab,
or anti-human IgM Ab (A and B). Transfected COS cells
were detached and analyzed by using 3G8 or anti-IgM Ab. Nontransfected
COS cells were assayed as a negative control (C, dashedline).
To examine whether the association of Shc with Syk also occurs in B cells, coimmunoprecipitation experiments were carried out. As described above, a faint tyrosine phosphorylation of 70-kDa protein associated with Shc was observed in wild-type DT40 cells upon BCR stimulation. To identify this 70-kDa protein as Syk, we used DT40 cells overexpressing porcine Syk. As shown in Fig. 5, immunoblotting of anti-Shc immunoprecipitates with anti-Syk Ab revealed the coprecipitation of Syk with Shc. Moreover, this association was observed before receptor stimulation. Because tyrosine phosphorylation of Syk is induced by BCR stimulation(46) , this constitutive association of Shc with Syk supports the previous conclusion that this interaction is independent of the tyrosine phosphorylation state of Syk.
Figure 5: Association of Shc with Syk in B cells. Syk-deficient DT40 cells overexpressing porcine Syk were stimulated with M4 for 3 min. Cells were lysed, immunoprecipitated with anti-Shc, separated on 8% SDS-polyacrylamide gels, transferred, and incubated with anti-phosphotyrosine mAb or anti-porcine Syk Ab.
COS cells transfected
with IgM/Ig chimera and Shc showed only background tyrosine
phosphorylation of Shc. Cotransfection of Shc with IgM/Ig
and Fyn
increased tyrosine phosphorylation of IgM/Ig
(Fig. 4B), whereas tyrosine phosphorylation of Shc was
slightly increased (Fig. 4A). Although the expression
level of IgM/Ig
in the absence or presence of Fyn was roughly the
same (Fig. 4C), the amount of IgM/Ig
associated
with Shc was increased by addition of Fyn (Fig. 4B),
suggesting that Shc interacts preferentially with
tyrosine-phosphorylated Ig
probably through its SH2 domain.
Significant association of Shc with Fyn could not be detected in COS
cells (data not shown).
Stimulation of BCR results in rapid increases in tyrosine
phosphorylation on a number of proteins. This BCR-coupled tyrosine
phosphorylation is mediated by at least two types of PTKs: Src family
PTKs, including Lyn, Fyn, and Blk, and Syk kinase. Cross-linking of BCR
on syk-negative and lyn-negative DT40 cells induces a
different pattern of tyrosine phosphorylation, suggesting that Syk and
Lyn have distinct substrate specificity in BCR signaling (14) .
For instance, BCR-induced tyrosine phosphorylation of phospholipase
C-2 is mediated primarily by Syk. Thus, Syk is involved in
coupling BCR to the phosphatidylinositol pathway.
A recent report that BCR activates Ras(41) , together with the evidence that the BCR activates mitogen-activated protein kinase(42) , suggest that this pathway plays an important role in BCR signaling. Shc is tyrosine phosphorylated upon BCR stimulation and after its phosphorylation interacts with Grb2 and mSOS(44, 45) . Thus, we were interested in the mechanism(s) by which BCR mediates tyrosine phosphorylation of Shc, leading to the activation of Ras.
Consistent with previous reports(44, 45) , Shc was associated with an unidentified tyrosine-phosphorylated 140-kDa protein in both A20 and DT40 cells. It is intriguing that Lyn is primarily responsible for BCR-induced tyrosine phosphorylation of this 140-kDa protein. The association of the 140-kDa tyrosine-phosphorylated protein with Shc in B cells might evoke the speculation that Shc is recruited to this 140-kDa protein via its SH2 domain upon BCR stimulation. Immunological reagents against the 140-kDa protein will be necessary to clarify this issue.
Tyrosine phosphorylation of Shc was profoundly decreased in Lyn- and Syk-deficient DT40 cells (Fig. 2). Furthermore, transfection of kinase-negative mutants of Fyn and Syk into these deficient cells did not restore the BCR-induced tyrosine phosphorylation of Shc (Fig. 3), indicating clearly the requirement of the kinase activity of Src PTK and Syk for phosphorylation of Shc. A recent study demonstrated that Shc is tyrosine phosphorylated following T cell receptor (43) or interleukin-2 receptor stimulation(55, 56) . Because these receptors are known to associate with Src-PTKs such as Lck, Src-PTK may be one of the key molecules for coupling these receptors to tyrosine phosphorylation of Shc. The requirement of Src-PTK for phosphorylation of Shc is also exemplified by v-src-transformed fibroblasts in which Shc becomes tyrosine phosphorylated(39, 40) .
What is the underlying
mechanism that explains the requirement of Lyn and Syk for BCR-coupled
phosphorylation of Shc? Here, we showed that Shc interacts with Syk
even before BCR stimulation (Fig. 5), suggesting that this
interaction does not necessarily require tyrosine phosphorylation of
Syk. This concept is supported by the observation that Shc associates
with CD16//Syk(K-) in COS cells; tyrosine phosphorylation of
CD16/
/Syk(K-) could not be detected by anti-phosphotyrosine
mAb (Fig. 4B). Cotransfection of Shc with
CD16/
/Syk induced significant tyrosine phosphorylation of Shc in
COS cells, demonstrating the requirement of Syk kinase activity for
tyrosine phosphorylation of Shc. We previously demonstrated that
Src-PTKs, such as Lyn, phosphorylate the tyrosine residue(s) of Syk
upon BCR stimulation, enhancing the activity of Syk(46) . Taken
together, our results imply that Lyn-activated Syk induces tyrosine
phosphorylation of Shc during BCR signaling. Because Syk associates
with phosphorylated Ig
upon receptor
stimulation(51) ,
Shc complexed with Syk is thereby
recruited to the phosphorylated Ig
.
In T cell receptor
signaling, it was previously reported that Shc is directly recruited to
the phosphorylated chain upon receptor stimulation(43) .
Using the COS cell transfection system, we also confirmed the
possibility that Shc binds to the phosphorylated Ig
(Fig. 4B). If this is operative in BCR signaling, the
requirement of Lyn and Syk for BCR-coupled phosphorylation of Shc can
be explained by the following mechanism. Lyn is thought to
phosphorylate tyrosine residues of Ig
ARH1 motif upon BCR
cross-linking, allowing the recruitment of Shc via its SH2 domains to
the receptor complex. In Syk-deficient cells, this recruited Shc
appears to be phosphorylated by Lyn to some extent. Compared with
Syk-deficient cells, at least a 10-fold higher phosphorylation of Shc
was observed in wild-type cells (Fig. 2), indicating the
involvement of Syk in complete phosphorylation of Shc during BCR
signaling. Because Syk is also recruited to phosphorylated Ig
and
activated by Lyn(46, 51) ,
this activated
Syk phosphorylates Shc in wild-type B cells.
Our results with mouse A20 cells and chicken DT40 cells also indicated that Grb2 is recruited to tyrosine-phosphorylated Shc upon BCR stimulation. The time course of association of Grb2 with Shc was very similar to that of tyrosine phosphorylation of Shc upon BCR stimulation. Furthermore, glutathione S-transferase/Grb2SH2 fusion protein bound to the tyrosine-phosphorylated Shc in vitro (data not shown)(44, 45) .
Ras is a GTP-binding protein that cycles from the inactive GDP-bound state to the active GTP-bound state. It is not completely understood how Ras activity is regulated. The BCR-induced formation of complexes containing Shc, Grb2, and mSOS indicates that mSOS is involved in the activation of Ras in BCR signaling. However, GTPase-activating protein also has been postulated to be a controller of Ras, because its inactivation allows the accumulation of Ras in the GTP-bound state (57) . In fact, it was reported that stimulation of BCR decreases the GTPase-activating protein activity(58) . Moreover, Vav has been shown to increase the guanine nucleotide exchange activity in vitro, and BCR-induced tyrosine phosphorylation of Vav and its guanine nucleotide exchange activity are correlated(52) . Thus, Ras may be modulated by multiple mechanisms, with at least one that entails shuttling mSOS to membrane fraction through Lyn and Syk in BCR signaling.