(Received for publication, July 21, 1995; and in revised form, October 11, 1995)
From the
We and others have demonstrated that the c-cbl proto-oncogene product is one of the earliest targets of tyrosine
phosphorylation upon T cell receptor stimulation. Given the
similarities in the B and T lymphocyte antigen receptors, and the
induction of pre-B leukemias in mice by the v-cbl oncogene, we
examined the potential involvement of Cbl in B cell receptor signaling.
We demonstrate prominent and early tyrosine phosphorylation of Cbl upon
stimulation of human B cell lines through surface IgM. Cbl was
associated in vivo with Fyn and, to a lesser extent, other Src
family kinases. B cell activation also induced a prominent association
of Cbl with Syk tyrosine kinase. A substantial fraction of Cbl was
constitutively associated with Grb2 and this interaction was mediated
by Grb2 SH3 domains. Tyrosine-phosphorylated Shc, which prominently
associated with Grb2, was detected in association with Cbl in activated
B cells. Thus, Grb2 and Shc adaptors, which associate with
immunoreceptor tyrosine based activation motifs, may link Cbl to the B
cell receptor. B cell activation also induced a prominent association
between Cbl and the p85 subunit of phosphatidylinositol (PI) 3-kinase
resulting in the association of a substantial fraction of PI 3-kinase
activity with Cbl. Thus, Cbl is likely to play an important role to
couple the B cell receptor to the PI 3-kinase pathway. Our results
strongly suggest a role for p120 in signaling
downstream of the B cell receptor and support the idea that Cbl
participates in a general signal transduction function downstream of
the immune cell surface receptors.
Recognition of antigens by antigen receptors (T cell receptor
(TCR) ()or B cell receptor (BCR)) leads to a cascade of
biochemical events that culminates in lymphocyte activation (1) . The T and B cell antigen receptors signal through
associated CD3/TCR
/
and the Ig
/
chains,
respectively(1, 2) . The cytoplasmic tails of these
receptor-associated polypeptides contain conserved sequence motifs, the
immunoreceptor tyrosine-based activation motifs (I-TAMs), which are
necessary and sufficient for coupling to downstream signaling
machinery(1, 2) . An early and an obligatory step in
lymphocyte activation is the tyrosine phosphorylation of the I-TAMs and
a number of cellular substrates(1, 2) . However,
lymphocyte antigen receptors and their associated chains lack intrinsic
tyrosine kinase domains, indicating the role of non-receptor protein
tyrosine kinases (PTKs). Indeed, the CD3/
chains of the TCR and
the CD4/8 co-receptors associate with the Src-family PTKs Fyn and Lck,
respectively(3, 4, 5, 6) .
Similarly, Ig
/Ig
chains interact with the Src family kinases
Lyn, Blk, Fyn, and Lck in B cells(7, 8, 9) .
Furthermore, upon tyrosine phosphorylation by the Src family kinases,
the I-TAMs recruit the Syk/ZAP-70 PTKs in an activation-dependent
manner(10, 11, 12, 13, 14) .
Genetic and biochemical data have clearly shown that Src family and
ZAP-70/Syk PTKs are crucial for lymphocyte
activation(15, 16, 17, 18, 19, 20, 21, 22) .
Therefore, identification and characterization of the early targets of
receptor-associated PTKs has become a major focus of studies aimed at
elucidating the mechanisms of lymphocyte signaling.
Many of the
identified effectors that function downstream of the PTKs in T and B
cells are shared; these include phospholipase C-1,
phosphatidylinositol (PI) 3-kinase, adaptor proteins Shc and Grb2 that
couple receptors to Ras pathway, and the proto-oncogene product
Vav(1, 2) . Thus, additional effectors are also likely
to be shared between T and B cells.
Recently, we identified a Fyn/Lck SH3 domain-binding protein, p120, which was present as preformed complexes with Fyn and Lck in unstimulated T cells and was rapidly tyrosine phosphorylated upon TCR stimulation(23, 24) . Subsequently, we and others have demonstrated this polypeptide to be the product of the c-cbl proto-oncogene(25, 26) . c-cbl is the cellular homolog of v-cbl, the oncogene of the Cas NS-1 murine retrovirus that induces pre-B and myeloid leukemias in neonatal mice(27, 28, 29, 30) . Furthermore, c-cbl resides on human chromosome 11-q23 near a translocation breakpoint found in about 10% of leukemias; although a direct involvement of Cbl in the pathogenesis of these leukemias remains to be shown, it is interesting to note that a large proportion of these are of B or mixed cell lineage(31) .
The Cbl protein possesses a
long proline-rich region, which could mediate its interactions with the
SH3 domain-containing proteins, and a number of tyrosine residues whose
phosphorylation could recruit SH2 domains of signaling
proteins(28) . Indeed, we and others have demonstrated in
vivo complexes of Cbl with several widely expressed signaling
proteins. p120 associates with Grb2 by binding
to its SH3 domains, and this fraction of Cbl is rapidly
tyrosine-phosphorylated in activated T cells(25, 32) .
A p120
-Grb2 complex was also observed in a human
leukemic cell line UT-7(33) . In addition,
tyrosine-phosphorylated p120
associates with the
p85 subunit of PI 3-kinase in T cells, primarily in a SH2-dependent
manner, resulting in the association of PI 3-kinase activity with
Cbl(25, 32) . Recently, we have shown that
p120
forms a TCR activation-dependent complex
with Crk proteins by binding to their SH2 domains(34) .
Finally, p120
also associates with Nck adaptor
protein by binding to its SH3 domains(35) . These studies
suggested that p120
may link the components of
multiple signaling pathways coupled to non-receptor PTKs. This
possibility is supported by reported tyrosine phosphorylation of Cbl
upon stimulation through Fc
, granulocyte macrophage colony
stimulating factor, and erythropoietin receptors, which also utilize
non-receptor PTKs(33, 36, 37) . However, Cbl
phosphorylation was not observed upon Fc
R triggering(36) ,
indicating that the involvement of Cbl in signaling through additional
receptors cannot be assumed. The close resemblance between the TCR and
BCR signaling mechanisms, and the propensity of v-cbl to
induce B-cell leukemias, prompted us to examine the potential
involvement of Cbl in tyrosine kinase-mediated signaling downstream of
the BCR.
Here, we demonstrate that Cbl is a major and early substrate of tyrosine phosphorylation in B cells stimulated through surface IgM. Moreover, we show that Cbl, which is constitutively associated with Grb2, is recruited into PTK signaling machinery and forms activation-dependent complexes with Shc, PI 3-kinase, and B-cell PTKs Syk and Fyn. These results suggest a role for Cbl in B cell activation and raise the possibility that subversion of its signaling functions may contribute to B cell leukemogenesis by v-cbl, a truncated protein that lacks the C-terminal two-thirds of Cbl including the proline-rich SH3 domain-binding region(28) .
Figure 6:
BCR activation does not induce detectable
tyrosine phosphorylation of the p85 and p110 subunits of PI 3-kinase.
Anti-PI 3-kinase p85 immunoprecipitations from 2 10
unstimulated Ramos cells(-) or from cells stimulated with
anti-IgM for 2 min (+) were subjected to serial immunoblotting
with antibodies shown on top. Arrows point to p120
(Cbl), the p110 and p85 subunits of PI 3-kinase,
and Ig heavy chain (Ig). Overlay of autoradiograms confirmed
that p85 and p110 did not correspond to any of the major phosphotyrosyl
polypeptides.
Relatively low levels of
tyrosine phosphorylation on various polypeptides were seen in
unstimulated cell lysates harvested at either 2 or 30 min of incubation
at 37 °C (Fig. 1, lanes 1 and 2). Anti-IgM
stimulation led to a time-dependent increase in tyrosine
phosphorylation of a number of cellular polypeptides, including a
prominent 120-kDa phosphoprotein. Phosphorylation of this polypeptide
was observed at the earliest time point tested (10 s), reached a
maximum by 1-2 min, and declined slightly thereafter with
substantial phosphorylation still detectable at 60 min (Fig. 1, lanes 3-9, and data not shown). Anti-pY immunoblotting
of anti-Cbl immunoprecipitates showed that the 120-kDa pY polypeptide
observed in lysates comigrated with Cbl (Fig. 1, right
panel, lanes 10-18). Low basal phosphorylation of Cbl was
observed in the absence of anti-IgM stimulation (lanes 10 and 11). Notably, directly immunoprecipitated Cbl was rapidly and
prominently tyrosine phosphorylated with a kinetics identical to that
observed for the p120 band in cell lysates (compare lanes
12-18 with lanes 3-9). Anti-Cbl reprobing of
immunoblots (lower panels) confirmed that equal amounts of
lysates or immunoprecipitates were resolved in all lanes. Thus,
p120 is a major early substrate of tyrosine
phosphorylation upon stimulation of Ramos B cells through their antigen
receptor.
Figure 1:
Cross-linking of B cell antigen
receptor on the Ramos human B cell line induces rapid and sustained
tyrosine phosphorylation of p120. Cells were
washed and resuspended in serum-free medium, warmed to 37 °C, and
then incubated for the indicated times (seconds or minutes) either in
the absence(-) or in the presence (+) of 20 µg/ml rabbit
anti-human IgM. Cells were lysed in Triton X-100 lysis buffer, and
precleared lysates were resolved on SDS-9% PAGE either as such (Lysate, left panel; 10
cells per lane) or after
anti-Cbl immunoprecipitation (
-cbl i.p., right
panel; 5
10
cells/lane). Resolved polypeptides
were transferred to PVDF membrane and incubated with anti-pY antibody
followed by Protein A-horseradish peroxidase (upper panels).
Blots were developed using the ECL method. Filters were then stripped
and re-immunoblotted with anti-Cbl antibody to demonstrate equal
loading in all lanes (lower panels). Cbl and Ig (heavy chain
of the immunoprecipitating antibody) are indicated on the left. All
lanes within each blot are from a single
exposure.
In addition to Ramos, we also examined anti-IgM-induced Cbl phosphorylation in two other B cell lines, Daudi and Raji. In each case, anti-pY immunoblotting of the immunoprecipitated Cbl showed prominent tyrosine phosphorylation when examined at 2 min after stimulation (Fig. 2, lanes 3 and 4 in each panel). Similarly, anti-Cbl immunoblotting showed that Cbl was prominently immunoprecipitated by anti-pY antibody from lysates of the anti-IgM-activated but not the unstimulated cells (Fig. 2, lanes 5 and 6, in each panel). These results demonstrate that Cbl tyrosine phosphorylation is not restricted to Ramos B cells, but is a general feature of B cells that carry a surface IgM receptor.
Figure 2:
Tyrosine phosphorylation of Cbl by BCR
cross-linking in Daudi and Raji human B cell lines.
Immunoprecipitations were carried out with the indicated antibodies (I.P. Abs) from Triton X-100 lysates of 5 10
unstimulated(-) or anti-IgM-stimulated (+) Daudi (left) or Raji cells (right). Immunoprecipitates were
resolved on SDS-9% PAGE, transferred to PVDF membrane, and subjected to
serial immunoblotting with anti-pY (top) and anti-Cbl (bottom) antibodies. NRS, normal rabbit serum, used
as a negative control. A constitutively phosphorylated polypeptide
co-migrating with Cbl is immunoprecipitated by anti-pY antibody from
unstimulated Daudi cells; however, it is unreactive with anti-cbl
antibody. All lanes within each blot are from a single
exposure.
A small fraction of Cbl was readily detectable in association with Fyn prior to anti-IgM activation of Ramos cells and this association increased considerably following stimulation (Fig. 3, anti-Cbl blot, lanes 9 and 10). Anti-pY immunoblotting showed that Fyn-associated Cbl underwent an activation-dependent tyrosine phosphorylation. A much smaller amount of Cbl was observed in anti-Blk immunoprecipitates from activated Ramos cells (anti-Cbl blot, lanes 5 and 6), and activation-dependent tyrosine phosphorylation of Blk-associated Cbl was also observed upon longer exposures. Much smaller amounts of Cbl were detected in association with Lyn and Lck and could be visualized primarily in anti-pY immunoblot upon longer exposure (not shown). Thus, Cbl associates detectably with some B cell Src-family PTKs (Fyn and Blk), but poorly with others (Lyn and Lck). Under the conditions of the experiment, all antibodies were able to immunoprecipitate their respective polypeptides, as determined by direct immunoblotting (data not shown); these polypeptides are visible in anti-pY immunoblotting of anti-Lyn, anti-Lck, and anti-Fyn immunoprecipitates just above the Ig heavy chain bands (Lck resolves as a doublet) (Fig. 3, lanes 3, 4, and 7-10). Low stoichiometry of association, together with comigrating Ig heavy chains made it difficult to use immunoblotting with antibodies to Src family PTKs to demonstrate their association with Cbl (data not shown).
Figure 3:
In vivo association of Cbl with B
cell tyrosine kinases. Immunoprecipitations were carried out with the
indicated antibodies (I.P. Abs, shown on top) from Triton
X-100 lysates of 5 10
unstimulated(-) or
anti-igM-stimulated (+) Ramos cells. Immunoprecipitates or whole
cell lysates (10
cells) were resolved on SDS-9% PAGE,
transferred to PVDF membrane, and subjected to anti-pY immunoblotting (top panel). The membrane was stripped and reprobed with
anti-cbl antibody (lower panel). Immunoprecipitated species
and Ig are indicated on the left. Bands corresponding to Lyn,
Lck (a doublet), and Fyn are seen immediately above the Ig band. Blk
bands co-migrate with Ig. In lane 6 of the anti-pY immunoblot
(anti-Blk), Cbl migrates immediately above a major background band
which is seen in lanes 5 and 6. All lanes within each
blot are from a single exposure.
Anti-pY immunoblotting of anti-Cbl immunoprecipitates showed several associated polypeptides migrating between Ig and Cbl bands as well as other higher molecular weight species (Fig. 3, lanes 11 and 12). A Cbl-associated 70 kDa polypeptide comigrated with Syk (compare lanes 11 and 12 with 13 and 14) and anti-Syk immunoblotting confirmed its identity (data not shown). Anti-Syk immunoprecipitates revealed a prominent 120-kDa phosphotyrosyl polypeptide comigrating with Cbl; the phosphotyrosine content of this band showed an activation-dependent increase (Fig. 3, lanes 13 and 14). Anti-Cbl immunoblot confirmed that Cbl was associated with Syk in an activation-dependent manner. Thus, Syk and Cbl show a prominent activation-dependent association in B cells. A 70-kDa tyrosyl phosphoprotein comigrating with Syk was also observed to associate with Src kinases, in particular with Lck, in an activation-dependent manner (Fig. 3, lanes 3-10); additional unidentified pY polypeptides were also noted in anti-Lck and anti-Blk immunoprecipitates (lanes 5-8).
As expected from the above studies, anti-pY immunoblotting showed activation-dependent tyrosine phosphorylation of Cbl (Fig. 4, lanes 3 and 4, both bands of the doublet are specific), whereas anti-Cbl immunoblotting revealed activation-dependent immunoprecipitation of Cbl by anti-pY antibody (lanes 11 and 12). Anti-Cbl immunoblotting revealed that a substantial fraction of this polypeptide co-immunoprecipitated with Grb2 when lysates of unstimulated Ramos cells were analyzed (Fig. 4, lane 5). This was confirmed by anti-Grb2 immunoblotting of anti-Cbl immunoprecipitate (lane 3). Cbl-Grb2 association was relatively unaltered after B cell activation (Fig. 4, lanes 3-6); in some experiments a modest increase in association was noted (data not shown). In keeping with the association of a substantial fraction of Cbl with Grb2, anti-Cbl antibody coimmunoprecipitated small amounts of a number of phosphotyrosyl polypeptides which represented major Grb2-associated phosphotyrosyl proteins in activated Ramos cells (Fig. 4, anti-pY blot, compare lanes 4 and 6). These included a 52-kDa polypeptide comigrating with Shc (lane 8), a 145-kDa polypeptide previously observed in association with Shc in B cells (40, 41) (see lane 8), and several others migrating at 70-100 kDa. Association between Shc and Cbl was directly confirmed by anti-Shc blotting which revealed Shc in anti-Cbl immunoprecipitate of activated cells (Fig. 4, lane 4); anti-Cbl antibody also detected a small amount of Cbl in anti-Shc immunoprecipitate upon longer exposure of the blot (not shown). Concurrently, a tyrosine-phosphorylated 120-kDa species comigrating with Cbl was observed in anti-Shc immunoprecipitate (lane 8). As expected, a prominent association between Grb2 and Shc was detected by anti-Grb2 immunoblotting of anti-Shc immunoprecipitates (lanes 7 and 8) as well as by anti-Shc and anti-pY immunoblotting of anti-Grb2 immunoprecipitates (lanes 5 and 6).
Figure 4:
In vivo association of
p120with Grb2, Shc, and p85 subunit of the PI
3-kinase in Ramos B cells. Immunoprecipitations from Triton X-100
lysates of 5
10
unstimulated(-) or
anti-IgM-stimulated (+) Ramos cells were carried out with the
indicated antibodies (I.P. Abs., shown on top).
Immunoprecipitates or whole cell lysates (from 10
cells)
were resolved on SDS-9% PAGE, transferred to PVDF membrane, and
subjected to serial immunoblotting with antibodies indicated on the right. Immunoprecipitated species are indicated by arrows or brackets; Ig, immunoglobulin heavy chain.
Each immunoblot represents a reprobing of the appropriate parts of the
filter shown on top. All lanes within each blot are from a single
exposure.
Anti-pY immunoblotting of anti-PI 3-kinase p85 immunoprecipitates (Fig. 4, lanes 9 and 10) revealed the activation-dependent appearance of major pY polypeptides migrating at 120 and 100 kDa. The 100-kDa polypeptide is likely to be the CD19, previously shown to be tyrosine phosphorylated upon B cell activation and to interact with the PI 3-kinase p85(42) . The 120-kDa pY polypeptide comigrated with Cbl. Anti-Cbl immunoblotting of PI 3-kinase p85 immunoprecipitates (Fig. 4, lanes 9 and 10) and anti-PI 3-kinase p85 blotting of anti-Cbl immunoprecipitates (Fig. 4, lanes 3 and 4) demonstrated that a substantial fraction of Cbl became associated with PI 3-kinase p85 after anti-IgM activation of Ramos cells, whereas the association was undetectable in unstimulated cells. The amount of PI 3-kinase p85 polypeptide detected in anti-Cbl immunoprecipitates was nearly as much as in anti-pY immunoprecipitates (Fig. 4, compare lanes 4 and 12). Concurrently, small amounts of Grb2 and Shc also became associated with PI 3-kinase p85, suggesting that larger order complexes of Grb2, Cbl, Shc, and PI 3-kinase p85 are induced by B cell activation. Consistent with this suggestion, all of these polypeptides were detected in anti-pY immunoprecipitates from activated cells (lane 12), even though Grb2 and PI 3-kinase p85 were not tyrosine phosphorylated ( Fig. 6and data not shown).
Figure 5:
Binding of p120 to
GST fusion proteins of Grb2 is exclusively mediated through Grb2 SH3
domains. Cell lysates from 1.5
10
unstimulated
cells(-) or cells stimulated with anti-IgM antibody for 2 min at
37 °C (+) were incubated for 1 h at 4 °C with GST fusion
proteins noncovalently immobilized on glutathione-Sepharose beads (5
µl of packed beads; 10 µg of fusion protein; total volume 1
ml), and bound proteins were solubilized in sample buffer. Binding
reactions or whole cell lysate (from 10
cells) were
resolved by SDS-PAGE and subjected to anti-Cbl immunoblotting (upper panel) using Protein A-horseradish peroxidase conjugate
and ECL detection. The filter was stripped and immunoblotted with
anti-pY antibody (lower panel). w.t., wild type Grb2. 3-2-3 refers to NH
-terminal SH3, SH2, and
C-terminal SH3 domains of Grb2. Asterisks denote mutated
domains. Mutated residues were: NH
-terminal SH3, P49L; SH2,
R86K; COOH-terminal SH3, P206L. Cbl and Shc are indicated on the left.
cbl i.p. (lanes 9-10),
anti-Cbl immunoprecipitation from the same amount of cell lysate as
used for binding reactions.
Anti-pY reprobing of the blot
showed the expected increase in tyrosine phosphorylation of cellular
polypeptides upon anti-IgM stimulation (lanes 9 and 10). Notably, Cbl itself was tyrosine phosphorylated upon
anti-IgM stimulation and associated with various pY polypeptides (Fig. 5, lower panel, lanes 9 and 10).
Furthermore, the Grb2 SH2 mutant showed essentially undetectable
binding to pY polypeptides aside from Cbl (lanes 9 and 10), whereas the Grb2 SH3 domain mutant was fully able to bind
to most pY polypeptides but showed little binding to Cbl (lanes 11 and 12). Anti-Shc reprobing confirmed the identity of the
band designated as Shc in Fig. 5, and showed that the SH3 mutant
but not the SH2 mutant of Grb2 could bind to Shc in lysates of
activated cells (data not shown). These data demonstrate that anti-IgM
stimulation of Ramos cells did not induce a Grb2 SH2 domain-binding
site on Cbl, indicating that the p120-Grb2 interaction in
B cells is exclusively Grb2 SH3 domain-mediated, similar to our
findings in T cells(25) .
Having established that the PI 3-kinase subunits are not tyrosine phosphorylated, we examined the relative role of Cbl in recruiting PI 3-kinase activity into phosphotyrosyl signaling complexes. For this purpose, we measured the lipid kinase activity associated with anti-Cbl immunoprecipitates from unstimulated and anti-IgM-stimulated Ramos cells. As shown in Fig. 7(upper left panel), a substantial fraction of PI-kinase activity was observed in anti-Cbl immunoprecipitates of activated Ramos cells when compared to the activity present in anti-PI 3-kinase p85 immunoprecipitates. Further analysis of the Cbl-associated lipid kinase activity revealed that it contained specific PI 3-kinase activity (Fig. 7, upper right panel). Significantly, the level of PI-kinase activity in anti-Cbl and anti-pY immunoprecipitates was comparable (Fig. 7, upper left panel, lane 4 versus 6), suggesting that Cbl may represent a predominant mechanism to recruit PI 3-kinase activity into phosphotyrosyl complexes. In each experiment, anti-PI 3-kinase p85 immunoblots of the same samples showed that the level of lipid kinase activity correlated well with the amount of p85 PI 3-kinase protein (Fig. 6, bottom panels).
Figure 7:
A large fraction of PI 3-kinase activity
is associated with Cbl in anti-IgM-stimulated Ramos B cells.
Immunoprecipitations carried out with the indicated antibodies (I.P. Abs., shown on top) from lysates of 5 10
unstimulated(-) or anti-IgM-stimulated (+) Ramos cell
were subjected to lipid kinase assays using phosphatidylinositol (left panel) or phosphatidylinositol 4,5-bisphosphate (right panel) as substrate, as described under
``Materials and Methods.'' The reaction products were
subjected to TLC to resolve phosphatidylinositol phosphates (PIP; left panel) or phosphatidylinositol
3,4,5-trisphosphate (PIP
; right panel) and
visualized by autoradiography. Following the kinase reaction,
bead-bound proteins were resolved by SDS-PAGE and immunoblotted with
anti-PI 3-kinase p85 antibody (bottom). Right and left panels are separate experiments. All lanes within each
panel are from a single exposure. The apparently unequal migration of
PIP3 in the right panel reflects transposition of lanes from
different parts of a TLC plate which ran with a
slant.
The potential signal transduction role of the c-cbl proto-oncogene product was revealed by its cloning as an NCK
SH3-domain-binding protein(35) , a prominent tyrosine
phosphorylation of Cbl polypeptide upon triggering through the
TCR(26) , and identification of the Fyn/Lck SH3 domain-binding
T cell phosphoprotein p120 as
Cbl(23, 24, 25) . Given the strong
similarities between T and BCR signaling, it appeared likely that Cbl
may also participate in signaling downstream of the BCR. Here, we used
several human B cell lines that have been widely used to study BCR
signaling to demonstrate that Cbl is a prominent and early substrate of
tyrosine phosphorylation upon triggering through the B cell antigen
receptor. Recently, Cbl was also shown to undergo rapid tyrosine
phosphorylation upon triggering through Fc receptor (36, 37) as well as the granulocyte macrophage colony
stimulating factor and erythropoietin receptors(33) , which
also signal through non-receptor PTKs. Thus, it is likely that Cbl
plays a general role in signaling through hematopoietic cell surface
receptors. However, Cbl phosphorylation was not observed upon
triggering through Fc
R, which also utilizes Src family kinases and
Syk(36) , suggesting that the role of Cbl downstream of the
cell surface receptors is somewhat selective.
Cbl phosphorylation
upon BCR stimulation was rapid and sustained. Moreover, Cbl was one of
the major tyrosine-phosphorylated proteins in activated B cell lysates.
These results indicate that a relatively large fraction of Cbl is
accessible to BCR-associated PTKs. The Src family PTKs are thought to
mediate the early phosphorylation events upon BCR
triggering(7, 8, 9, 22) . Consistent
with Cbl being a substrate for Src kinases, a Fyn-Cbl complex was
detectable prior to activation and increased further upon activation.
In fact, Cbl represented the major Fyn-associated phosphotyrosyl
polypeptide in activated Ramos cells. Smaller amounts of Cbl were also
detected in association with Blk, Lck, and Lyn. However, the fraction
of Cbl associated with Src family kinases was substantially smaller
than the total tyrosine phosphorylated pool of Cbl (Fig. 4),
suggesting that either Cbl is transiently recruited to Src family
kinases and quickly released, or that additional cytoplasmic PTKs are
involved. Since the SH3 and SH2 domains of Src family kinases can
concurrently bind to phosphorylated Cbl and thus provide a high
affinity interaction(23, 24) , the second possibility
is more likely. Consistent with this idea, a substantial fraction of
Cbl became physically associated with Syk following B cell activation.
Thus, Syk may represent one of the additional PTKs that phosphorylate
Cbl. By analogy, a similar role of Syk/ZAP-70 in Cbl phosphorylation
can be postulated in T cells particularly since a large
non-Fyn/Lck-associated pool of phosphorylated Cbl is also found in
activated T cells(23, 25) . The possible involvement
of other B cell PTKs, such as the Bruton's tyrosine kinase (44, 45) remains to be investigated. Interestingly,
Bruton's tyrosine kinase SH3 domain can also bind to Cbl in
vitro(46) ; however, this association has proven difficult
to demonstrate in vivo. ()
The differential association between Cbl and various Src family kinases was not due to inefficient immunoprecipitation of Lyn and Lck, as shown by immunoblotting with immunoprecipitating antibodies (data not shown), and by co-immunoprecipitation of several phosphotyrosyl polypeptides (Fig. 3). A lower association of Cbl with Lck compared to Fyn was also observed in our analyses of T cells(23, 25) . The structural basis for differential interaction is not clear at present, but may include relative abundance of Src family proteins in cells, their relative affinities for Cbl versus other substrates, and the respective affinities of their SH3 and SH2 domains for Cbl. For example, a stronger in vitro binding to Cbl is observed with Fyn SH3 compared to Lck SH3(23) . Similarly, Lyn SH3 was able to interact with Cbl (36) whereas Blk SH3 was not(26) . All analyzed Src family SH2 domains show some binding to tyrosine-phosphorylated Cbl in vitro, although this has not been carefully quantified (23, 25, 26) . Regardless of the mechanism, however, differential association of Cbl with members of the Src family is consistent with distinct signaling roles of these kinases in B cell activation(47, 48) .
Association of a large fraction of cellular Cbl with Grb2 before anti-IgM-stimulation of B cells (Fig. 4) is consistent with our observation in T cells where Grb2-Cbl interaction was demonstrated to be entirely mediated by Grb2 SH3 domains(25) . As in T cells(25, 32) , Grb2-associated fraction of Cbl was a prominent target of tyrosine phosphorylation upon BCR activation with a kinetics similar to that observed in lysates and anti-Cbl immunoprecipitates (data not shown). Recently, increased Grb2-Cbl association was noted in T cells concurrently stimulated through TCR and CD4, and it was suggested that the Grb2 SH2 domain may mediate this enhanced association(32) . However, Cbl derived from unstimulated or anti-IgM-stimulated B cells was unable to bind in vitro to a Grb2 fusion protein containing mutations in ligand-binding pouches of both SH3 domains (Fig. 5), or to isolated Grb2-SH2 (data not shown). These results clearly demonstrate that anti-IgM stimulation of B cells does not create Grb2 SH2 domain-binding sites on Cbl. Use of Grb2 fusion protein with a mutation in the phosphopeptide-binding pocket of the SH2 domain further established that Grb2-Cbl interaction in B cells was exclusively SH3 domain-mediated.
Interaction of Cbl with the SH3 domains of Grb2 is
likely to mediate its indirect association with other signaling
proteins that bind to the Grb2 SH2 domain. Consistent with this idea,
Cbl was found to associate with Shc, which binds to Grb2 SH2 domain
when tyrosine phosphorylated (49, 50) . Our findings
that a smaller amount of Shc was associated with Cbl, compared to a
more abundant Shc-Grb2 complex, support the role of Grb2 as a bridge
between Shc and Cbl. This scheme is also favored by the inability of
Shc SH2 fusion protein to bind to phosphorylated Cbl in vitro (data not shown). Since Shc is capable of binding to cytoplasmic
tails of TCR and Ig
/
chains, apparently through a direct
interaction between Shc SH2 domain and the I-TAM motifs (50, 51, 52) , we suggest that Shc may
recruit Grb2-associated Cbl to antigen receptors. As such, Shc-Grb2-Cbl
represents a pathway distinct from Shc-Grb2-Sos that has been
previously defined(50, 53) . Further analyses will be
required to directly test this idea.
The recruitment of PI 3-kinase activity into BCR signaling complexes has been well documented(34, 42, 54, 55, 56) , yet the PI 3-kinase subunits themselves do not appear to undergo significant tyrosine phosphorylation ( Fig. 6and data not shown). Thus, PI 3-kinase recruitment into phosphotyrosyl complexes is likely to involve its interactions with other polypeptides. We show here that Cbl represents one such polypeptide. Cbl became prominently associated with PI 3-kinase p85 and co-immunoprecipitated a substantial level of PI 3-kinase activity. The activation dependence of these associations is consistent with our earlier observations that binding between PI 3-kinase p85 and Cbl in T cells was primarily mediated by PI 3-kinase p85 SH2 domains binding to pY motifs on Cbl(25) . Prior analyses have demonstrated that BCR activation induces tyrosine phosphorylation of CD19 creating a PI 3-kinase p85 SH2 domain-binding motif which allows PI 3-kinase recruitment(42) . In addition, the SH3 domains of Src family kinases can interact with proline-rich sequences of PI 3-kinase p85 protein in vitro(47, 48) , although the relative contribution of this mechanism to recruit PI 3-kinase to BCR in vivo remains to be clarified. In fact, relatively little PI 3-kinase activity co-immunoprecipitated with Fyn in T cells, whereas a large fraction of activity was associated with Cbl(25) . Given the extent of Cbl-PI 3-kinase association in B cells, it is likely that this represents a major mechanism of activation-dependent recruitment of PI 3-kinase activity into phosphotyrosyl complexes, complementing the role of CD19 and Src family kinase SH3-mediated interactions with PI 3-kinase p85(42, 47, 48) .
In conclusion, we demonstrate that Cbl is a major substrate of tyrosine phosphorylation upon B cell antigen receptor activation, and represents a major in vivo associate for Grb2/Shc adaptors, the PI 3-kinase and the B cell PTKs Fyn and Syk. These findings strongly suggest the involvement of Cbl as a cytoplasmic signaling protein in tyrosine kinase-dependent activation of B lymphocytes through the B cell antigen receptor. Given the propensity of oncogenic Cbl to induce B cell leukemias(27) , our results raise the possibility that Cbl may be involved in signaling cell proliferation and/or survival in B lymphocytes.