(Received for publication, July 12, 1995; and in revised form, September 12, 1995)
From the
A 120-kDa protein that is tyrosine-phosphorylated upon antigen
receptor ligation in B lymphocytes has been identified as the product
of the c-cbl protooncogene. Tyrosine phosphorylation of Cbl
depends on the efficient association of membrane immunoglobulin heavy
chains with the Ig/
heterodimer but is unimpaired in splenic
B cells from the Xid mouse. Cross-linking of membrane IgM and membrane
IgG, but not of CD40, leads to the tyrosine phosphorylation of Cbl. In
receptor-ligated B lymphocytes, p120
associates
with an 85-kDa protein that has been identified as the 85-kDa subunit
of phosphatidylinositol 3-kinase.
The antigen receptor on B lymphocytes is made up of membrane
immunoglobulins associated with the Ig/
heterodimer(1, 2) . This heterodimer serves to link
the receptor with associated Src family kinases such as Blk, Lyn, Fyn,
and Fgr(3, 4, 5, 6) , and also with
the Syk tyrosine kinase(7, 8) . Tyrosine
phosphorylation appears to be an obligatory event (9) in the
initiation of signal transduction pathways that promote B lymphocyte
proliferation and differentiation. A number of signaling events
downstream of this receptor depend on the interaction of membrane
immunoglobulin heavy chains with the associated Ig
/
heterodimer(10, 11, 12) , while some are
initiated independently of these associated glycoproteins(13) .
Activated protein tyrosine kinases are presumed to participate in
triggering the Ras pathway, in activating PI
3-kinase, and
in initiating the hydrolysis of phosphatidylinositol 4,5-bisphosphate
by specific isoforms of phosphatidylinositol-specific phospholipase C.
Following B cell receptor ligation, cytoplasmic tyrosines in the
ITAMs (immunoreceptor tyrosine-associated motifs) of Ig and
Ig
are tyrosine-phosphorylated, presumably by activated Src family
kinases. The subsequent recruitment of Syk to phosphorylated
ITAMs(14, 15) , and possibly an interaction between
Src family kinases and Syk(16, 17, 18) ,
leads to the activation of the latter. Another protein tyrosine kinase
that is activated following receptor ligation and which plays a role in
antigen receptor mediated signaling is
Btk(19, 20, 21) . Splenic B cells in Xid
mice, which carry a point mutation in the PH domain of Btk, appear to
be identical to gene-targeted mice in which no Btk protein is
synthesized(22) . Btk appears to be critical for the entry into
S phase of B lymphocytes that have been stimulated by antigen receptor
cross-linking(22, 23) .
An alternative pathway by
which antigen-selected B lymphocytes may be induced to proliferate is
mediated by T lymphocyte-derived cytokines and the triggering of CD40
on B cells by its ligand on activated T
cells(24, 25, 26) . Cross-linking of CD40 can
lead to the activation of tyrosine and serine/threonine kinases, the
activation of PI 3-kinase, the activation of phospholipase C
isoforms, and the nuclear translocation of the NF
B transcription
factor. While the molecular consequences of activating B lymphocytes
via the antigen receptor and by CD40-mediated signaling are similar,
the mechanisms by which these individual pathways are activated by
these different ligands may well be distinct.
Cbl is the cellular
homolog of the v-Cbl oncoprotein (27, 28) and is a
predominantly cytosolic protein, which contains 17 proline-rich motifs
potentially capable of binding a range of SH3 domains. Tyrosine
phosphorylation of Cbl has been described in response to receptor
occupancy of a number of receptors including the antigen receptor on T
cells, the erythropoietin receptor, the granulocyte/macrophage
colony-stimulating factor receptor, the Fc receptor, the
colony-stimulating factor-1 receptor, and the epidermal growth factor
receptor(29, 30, 31, 32) . Tyrosine
phosphorylation of Cbl has also been observed in v-src- and
v-abl-transformed cells (32, 33) , and this
modification may be a critical event in mitogenic signaling.
We
describe here the identification of Cbl as a prominent substrate for
tyrosine phosphorylation in antigen-receptor-ligated B lymphocytes.
This phosphorylation event requires the efficient interaction of
membrane immunoglobulin heavy chains with the Ig/
heterodimer, and is unimpaired in splenic B cells from the Xid mouse.
Following receptor ligation, Cbl is seen to associate with an 85-kDa
phosphoprotein, identified as the 85-kDa subunit of PI 3-kinase. Our
studies suggest that a major role of Cbl in B cells may be the
recruitment and activation of PI 3-kinase following antigen receptor
ligation.
Immunoprecipitations, anti-phosphotyrosine immunoblots, and in vitro kinase assays were performed as described previously(17, 34) .
Figure 1: Cbl is the prominent 120-kDa protein that is tyrosine-phosphorylated following anti-IgM and anti-IgG cross-linking. Upper panel, anti-phosphotyrosine immunoblot. Lower panel, anti-Cbl immunoblot. WEHI 231 (IgM) B cells and the 4JJ subclone of A20.25 (IgG; A20/4JJ) were either not stimulated (N) or stimulated (S) with F(ab)2 anti-mouse IgM (WEHI 231)or anti-mouse IgG (A20/4JJ) for 5 min. Lysates were either not precleared(-) or were precleared with anti-Cbl (Cbl) or with a control rabbit IgG (IgG). Total lysate was then separated on SDS/PAGE transferred to an Immobilon-P (Millipore) membrane and reacted sequentially with an anti-phosphotyrosine monoclonal antibody and a peroxidase conjugated rabbit anti-mouse antibody. The ECL system (Amersham Corp.) was used for detection. Positions of molecular weight markers are indicated on the left of each panel.
In Fig. 2A, Cbl tyrosine phosphorylation is apparent from the 30-s time point. In a slightly longer exposure, phosphorylation was apparent from the 10-s time point. Tyrosine-phosphorylated proteins with a slightly slower mobility than the major Cbl band probably represent Cbl species phosphorylated on multiple sites. These bands were depleted by anti-Cbl as seen in Fig. 1and Fig. 2A. Similar bands were observed on anti-Cbl immunoblots of lysates from activated B cells (data not shown). The tyrosine-phosphorylated 70-kDa species seen both in Fig. 1and 2A, exactly comigrates with, and probably represents, tyrosine-phosphorylated Syk. Phosphorylation of Cbl is observed soon after the cross-linking of membrane immunoglobulins but is not observed after cross-linking CD40 (Fig. 2B), although both stimuli can independently contribute to the initiation of similar signaling pathways.
Figure 2: A, time course of tyrosine phosphorylation following receptor ligation of BAL 17 B cells. Lysates were prepared 10 s (10``), 30 s (30''), 1 min (1`), 3 min (3`), 5 min (5`), and 15 min (15`) after activation. Additional lysates at the 5-min time point were precleared either with anti-Cbl or with preimmune IgG. Details as in legend to Fig. 1. B, Cbl is tyrosine-phosphorylated after antigen receptor ligation but not following CD40 cross-linking. BAL 17 B cells were either not stimulated (N) or were stimulated with anti-IgM for 5 min (S1), anti-CD40 for 30 s (S2), or anti-CD40 for 5 min (S3). In leftmost four lanes, total lysates were separated on SDS-PAGE. In rightmost four lanes, anti-Cbl immunoprecipitates were separated. Proteins were visualized by an anti-phosphotyrosine immunoblot.
Figure 3:
Cross-linking of a transmembrane IgM
mutant that associates poorly with the Ig/
heterodimer
compromises the tyrosine phosphorylation of Cbl. A20.25 cells
expressing transfected wild-type human IgM (A20 WT) or a
transmembrane YS/VV mutant (A20/4J) were individually either
not stimulated(-) or cross-linked with anti-human IgM
(
µ) or anti-mouse IgG (
). Lysates were
immunoprecipitated with anti-Cbl and were separated on SDS-PAGE,
followed by an immunoblot analysis using anti-phosphotyrosine antibody (upper panel). Immunoprecipitated Cbl was quantified (lower panel) by reprobing with
anti-Cbl.
Figure 4: Cbl is efficiently tyrosine-phosphorylated following receptor ligation in Xid splenic B cells. Splenic B cells were purified from CBA/CaHN-xid/J mice and were either not stimulated (N) or cross-linked with anti-IgM (S). Total lysates and anti-Cbl immunoprecipitates were separated on SDS/PAGE and tyrosine-phosphorylated proteins revealed by an immunoblot assay.
We wished to ascertain whether the
formation of a complex between Shc, Grb2, and Cbl could be demonstrated
in activated B lymphocytes. We were able to confirm, as described
previously(41) , the formation of Shc-Grb2 complexes after B
cell activation (Fig. 5A) and also the association of
fusion proteins containing an intact N-terminal SH3 domain of Grb2 with
Cbl from B cell lysates (Fig. 5B). While these data
suggest that Shc-Grb2-Cbl complexes may well be formed in activated B
cells, in our hands we were able to observe an in vivo association between Grb2 and Cbl only in three murine pre-B cell
lines. Although we could detect phosphorylated Shc species
in anti-Cbl immunoprecipitates from lysates of activated B cells (data
not shown), we were unable, in any of the three activated B cell lines
tested, to demonstrate an interaction in vivo between Grb2 and
Cbl. However, Shc-Grb2-Cbl complexes are readily observed in activated
human B cell lines.
Figure 5:
Grb2
complexes with Shc in activated B cells. BAL 17 cells were either not
stimulated (N) or stimulated with anti-IgM (S) and
immunoprecipitated with anti-Grb2 or anti-Shc. Transferred proteins
were revealed on an immunoblot using anti-Grb2 (upper panel)
or anti-phosphotyrosine (lower panel) antibodies. B,
Cbl binds to the N-terminal SH3 domain of Grb2. Lysates from
non-stimulated (N) and anti-IgG-stimulated (S) A20.25
B cells were bound to a control glutathione S-transferase
affinity matrix (GST), to a wild type GST-Grb2 matrix (G2), or to a mutant GSTGrb2 matrix (K36; Trp
Lys mutation in codon 36, which abrogates binding in the N-terminal SH3
domain). Bound proteins were separated in parallel with proteins from
original lysate (Total). Presence of Cbl was monitored by an
immunoblot assay.
The band that has been labeled as Shc in the lower panel of Fig. 5A is inferred to be the phosphorylated form of p52 Shc. Anti-Shc immunoblots detect the p52 and p46 species of Shc in B cells but fail to reveal p66. Although the p46 form of Shc is also phosphorylated in activated B cells, the band representing it is probably obscured in Fig. 5A by rabbit IgG detected by the second antibody.
Figure 6:
An 85-kDa protein is associated with Cbl
in activated B cells. WEHI 231 B cells were either not stimulated (N) or stimulated with anti-IgM (S),
immunoprecipitated with preimmune serum or anti-Cbl, and subjected to
an in vitro kinase assay using
[-
P]ATP.
Figure 7:
A, the p85 subunit of PI 3-kinase
associates with Cbl in receptor-ligated B cells. BAL 17 B cells were
either not stimulated (N) or cross-linked with anti-mouse IgM (S) and immunoprecipitated with anti-Cbl. The presence of
associated p85 was detected using an immunoblot assay. B,
Anti-Cbl-associated PI 3-kinase activity in receptor-ligated B cells.
BAL 17 B cells were either not stimulated (N) or cross-linked
with anti-IgM (S) and immunoprecipitated with preimmune serum,
anti-Cbl, or anti-phosphotyrosine antibodies. The presence of
associated PI 3-kinase activity was detected by incubation with
micellar PI and [-
P]ATP and analysis on
thin layer chromatography.
Since B lymphocyte antigen receptor cross-linking leads to an increase in PI 3-kinase activity(42, 43) , we were keen to establish whether there was an increase in PI 3- kinase activity associated with Cbl following anti-IgM exposure. As seen in Fig. 7B, a dramatic increase in Cbl-associated phosphatidylinositol 3-kinase kinase activity was observed in activated B cell lysates.
Three different cell lines representing distinct stages of differentiation were used in all the studies described above (other than those examining membrane IgM mutants). Most experiments were performed with all three cell lines, although in some only two of the three lines were used. No functional differences between these cell lines was observed in any of the above studies.
Since the tyrosine phosphorylation of Cbl is an early event downstream of the engagement of a number of mitogenic receptors, we sought to identify proteins that specifically associate with Cbl after the initiation of signal transduction. A number of proteins associate constitutively with Cbl usually via SH3 domain-mediated interactions. These proteins include Src family kinases, Grb2, and Nck(29, 31, 44) . We have demonstrated here that an 85-kDa protein specifically associates with Cbl following B cell receptor ligation and that this 85-kDa protein is the p85 subunit of PI 3-kinase. In addition we have also demonstrated a convincing increase in PI 3-kinase activity associated with Cbl in activated B cells, suggesting that both subunits of this enzyme associate with Cbl and implying that Cbl might play a role in the activation of PI 3-kinase. It is quite likely that the major functional role of Cbl in activated B cells is to serve as a docking/activation site for PI 3-kinase.
PI 3-kinase is known to be activated when one or both of
the SH2 domains in the p85 subunit of this enzyme bind to
tyrosine-phosphorylated proteins that contain Y-X-X-M
motifs(45) . The activation of PI-3 kinase in response to
antigen receptor ligation in B cells has been described previously (42, 43) and association of PI-3 kinase with CD19
(which contains a Y-X-X-M motif in its cytoplasmic
tail) has also been demonstrated(46) . Given the ease with
which a Cbl-PI 3-kinase complex can be detected in vivo in
activated B cells, and the dramatic increase in Cbl-associated PI-3
kinase catalytic activity following antigen receptor ligation, we
postulate that the major functional consequence of Cbl tyrosine
phosphorylation is the catalytic activation of PI 3-kinase. The 110-kDa
catalytic subunit of this enzyme possesses both a lipid kinase activity
and a serine/threonine protein kinase activity. The lipid kinase
activity may, via PI intermediates phosphorylated in the 3-position of
inositol, contribute to the activation of the isoform of protein
kinase C(47) . Activation of Cbl presumably initiates a cascade
of events including the activation of S6 kinase and the induction of
proliferation(48, 49) . While this report was being
prepared, the association of PI 3-kinase with Cbl was reported in
receptor-ligated T cells(50) , and we are aware of similar to
be reported results from a second group(51) . It is likely that
the activation of PI-3 kinase by Cbl will prove to be a recurring theme
in mitogenic signal transduction.
The tyrosine phosphorylation of
Cbl in response to antigen receptor cross-linking requires the
associated Ig/
complex and does not require functional Btk.
While CD40 cross-linking can also lead to the activation of PI-3 kinase
in B cells, this apparently occurs via a Cbl-independent mechanism. It
is unclear as to whether there is a functional significance to the
association of Cbl with Grb2 and the formation of complexes between
Shc, Grb2, and Cbl following B cell activation. While such a complex
could in theory promote the association of Cbl with the antigen
receptor and therefore with cellular membranes, given the apparently
low stoichiometry with which such complexes are generated, it is
unclear whether such an event is necessary for the activation of PI
3-kinase activity. It is worth noting that previous attempts to
demonstrate PI 3-kinase association in vivo with the
Ig
/
heterodimer have proved unsuccessful(44) ,
suggesting that recruitment of PI 3-kinase via Shc, Grb2 and Cbl to the
antigen receptor does not occur readily. However it has also been
suggested, albeit based on indirect evidence, that membrane association
of PI 3-kinase might be necessary for catalytic
activation(52) . The association of Cbl with Golgi membranes
has been demonstrated in activated macrophages(32) . This
phenomenon, as well as the activation of PI 3-kinase, might be linked
to the formation of Grb2-Cbl or Shc-Grb2-Cbl complexes in cells that
have received a mitogenic stimulus.