(Received for publication, April 13, 1995)
From the
Phosphatidylinositol-3 kinase (PI-3 kinase) has been implicated
in cellular events such as mitogenic signaling, actin organization, and
receptor sorting. The p85 subunit of PI-3 kinase contains multiple
domains capable of protein-protein interactions that may contribute to
mediate the multiple physiological functions of this enzyme. Here, we
demonstrate that antibodies raised against the p85 subunit of PI-3
kinase immunoprecipitate a single tyrosine-phosphorylated protein of
120 kDa (pp120) from lysates of activated Jurkat T cells and A20 B
cells. This protein is the only significant phosphotyrosine-containing
protein in p85 immunoprecipitates from these cells, and it cannot be
detected in immunoprecipitates of other signaling proteins such as
PLC
Phosphatidylinositol 3-kinase is a lipid kinase that
phosphorylates PIns, ( The multiplicity of potential functions of PI-3 kinase
activity is paralleled by the complexity of the PI-3 kinase itself. The
mammalian kinase is composed of two subunits, an 85-kDa regulatory
subunit (p85) and a 110-kDa catalytic subunit (p110), of which two
different respective isoforms have been characterized, though many
isoforms are likely to exist(6) . The p85 subunit contains two
SH2 domains, one SH3 domain, a Bcr homology domain, and two different
proline-rich regions, which represent potential SH3 domain binding
sites(7) . Thus, the p85 subunit has the potential for forming
oligomers or heteroligomers with proteins that also contain SH3 or
poly-proline domains. The signaling or sorting functions of PI-3 kinase
may involve its direct interaction with these other cellular
components. We have set out to identify proteins that stably
interact with PI-3 kinase, which may constitute possible targets or
effectors of PI-3 kinase function. We initiated these studies using T
and B lymphoid cell lines, which, when activated through the TCR/CD3
complex or through cross-linked surface immunoglobulins, display
massive increases in tyrosine phosphorylation of cellular proteins and
rapid changes in cellular functions ranging from increased adhesion to
receptor up-regulation. PI-3 kinase does not directly associate with
the TCR/CD3 complex or with surface immunoglobulins. It only interacts
directly with the membrane receptors CD28 in T cells and CD19 in B
cells(10, 11) . It has also been reported to interact
weakly with members of the Src family of tyrosine
kinases(8, 9) . Thus, cells stimulated through the
TCR/CD3 or immunoglobulin complexes are ideally suited for
investigating new potential high affinity interactions between PI-3
kinase and cellular components elicited upon activation. In this paper,
we report that PI-3 kinase rapidly and stably associates with a 120-kDa
protein that is tyrosine phosphorylated in cells activated through the
TCR/CD3 or immunoglobulin complexes. This protein appears to correspond
to the proto-oncogene c-cbl. In addition, our results suggest
that, in vivo, c-cbl specifically associates with the
Figure 6:
The SH2 domain of p85 associates with cbl. Jurkat T cells were incubated at 37 °C in the
presence of soluble anti-CD3 and secondary cross-linking antibody for 4
min. Cells were then lysed, and extracts were incubated with the
indicated concentration of a GST fusion protein comprising the two SH2
domains of p85
Immunoprecipitates obtained using monoclonal antibodies to p85
contained two major bands that migrated with molecular weights of
approximately 85 and 110 kDa (Fig. 1A). These two bands
probably represent the p85 and catalytic subunits of PI-3 kinase,
respectively. Minor polypeptides were also observed, the most prominent
of which migrated slightly above the 110-kDa band (Fig. 1, leftlane). In contrast, immunoprecipitates of
activated cell extracts obtained with monoclonal antibodies to
phosphotyrosine contained a large number of bands, many of which were
clustered around 100-120 kDa. Immunoblotting of the p85
immunoprecipitates with antibodies against phosphotyrosine (Fig. 1B) revealed only a single band of 120 kDa. This
phosphoprotein comigrates with the methionine-labeled band detected
above the p110 subunit of PI-3 kinase. It was striking in these
experiments that pp120 was the only major tyrosine phosphoprotein
detected in p85 immunoprecipitates, despite the massive increase in the
number of tyrosine-containing proteins in cell extracts after
activation. The coprecipitation of a single phosphotyrosine protein
with p85 in T lymphocytes is in marked contrast to that observed in
non-lymphoid cells. For example, immunoprecipitation of p85 from
fibroblastic cells stimulated with PDGF coprecipitates many
phosphotyrosine-containing proteins including the PDGF receptor and
other proteins associated with the receptor complex, such as PLC
Figure 1:
A tyrosine-phosphorylated protein is
coprecipitated with PI-3 kinase. Jurkat T cells were labeled with
[
Immunoprecipitation of p85 at various time points after activation
revealed some pp120 in immunoprecipitates from non-stimulated Jurkat T
cells. However, the level of pp120 phosphorylation significantly
increased over this constitutive basal level after 1 min of stimulation (Fig. 2A). In addition, a decrease in mobility of pp120
could be observed. Phosphorlyation of pp120 decreased subsequently,
approximating background levels after 15 min of stimulation. This
increase in the phosphotyrosine signal is not due to changes in the
amount of p85 present in the immunoprecipitates (Fig. 2B).
Figure 2:
Time course of tyrosine phosphorylation of
the PI-3 kinase-associated pp120. Jurkat T cells were incubated at 37
°C in the presence of soluble anti-CD3 and secondary cross-linking
antibody for the times indicated above each lane.
Cells were then lysed, and extracts were immunoprecipitated with
polyclonal antisera to p85. Immunoprecipitates were then resolved on
7.5% SDS-PAGE and transferred to nitrocellulose. A,
phosphotyrosine immunoblot; B, P85 immunoblot. Arrows point to pp120 (A) and p85 (B). The intense band
at 105 kDa is a contaminant in the polyclonal anti-p85 antiserum, which
is detected by the secondary antibody.
We next investigated whether the
association of p85 with pp120 was restricted to T lymphocytes. For
these experiments, we employed the murine B cell lymphoma, A20, which
expresses IgG on the cell surface and is efficient at presenting
antigen to T cells. Resting A20 B cells display very low levels of
tyrosine phosphorylation, and stimulation results in a massive increase
in tyrosine phosphorylation of numerous bands (not shown).
Immunoprecipitates of p85 from resting A20 B cells were devoid any
coprecipitating tyrosine phosphoprotein, but p85 immunoprecipitates
from activated A20 cells contained three prominent
tyrosine-phosphorylated bands at 95-100, 120, and 150 kDa (Fig. 3). Thus, the association of a tyrosine-phosphorylated,
120-kDa protein with p85 also appears to occur in B cell lines. A
potential candidate for the 95-100-kDa band is the surface
molecule CD19, known to be tyrosine phosphorylated and associate with
PI-3 kinase after stimulation(11) . The phosphotyrosine band at
150 kDa was not consistently observed in p85 precipitates and was
occasionally observed in the preimmune precipitates.
Figure 3:
Tyrosine-phosphorylated bands associated
with p85 in A20 B cells. A20 B cells were incubated at 37 °C with
anti-Ig for the times indicated above each lane.
Cells were then lysed, and extracts were immunoprecipitated with
polyclonal antisera to p85. Immunoprecipitates were then resolved on
7.5% SDS-PAGE, transferred to nitrocellulose, and immunoblotted for
phosphotyrosine.
Figure 4:
c-cbl is associated with p85. A20
B cells were incubated at 37 °C with anti-Ig for the times
indicated above each lane. Cells were then lysed, and
extracts were divided into four aliquots. Aliquots were
immunoprecipitated with polyclonal antisera to p85, non-immune rabbit
serum (P.I.), polyclonal antisera against c-cbl (cbl), or boiled in sample buffer (TCL).
Immunoprecipitates and total cell lysate were resolved on 7.5% SDS-PAGE
and transferred to nitrocellulose. The nitrocellulose blot was cut, and
the regions above and below the 100-kDa marker were blotted with
anti-phosphotyrosine (A) or anti-p85 antibodies (B),
respectively. The position of prestained molecular weight markers is
indicated.
The significance of the p85-c-cbl interaction cannot
be determined from these studies because the functional cellular role
of c-cbl is unknown. The v-cbl oncogene was
originally identified as expressed by the transforming retrovirus Cas
NS-1 (14, 15, 16) and was later found to be a
truncated form of an endogenous cellular protein. The truncated form,
v-cbl, can be found in the nucleus and has been shown to have
the ability to bind DNA. In contrast, the endogenous c-cbl exists as a 120-kDa protein that does not appear to reside in the
nucleus and does not bind DNA. It has not been possible to detect
truncated forms of c-cbl, though a smaller mRNA transcript has
been shown to be expressed by some cells. The mechanism whereby
v-cbl is transforming and the physiological function of the
larger c-cbl have yet to be determined.
Figure 5:
c-cbl is specifically associated
with p85
Work by Songyang and co-workers (19) to
determine the consensus binding sites for p85 SH2 domains failed to
show any differences in selectivity between the SH2 domains of the
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
. Furthermore, antibodies specific for the
isoform of p85
but not antibodies specific for the
isoform immunoprecipitate
this tyrosine-phosphorylated protein. pp120 completely comigrates with
the proto-oncogene c-cbl, which is a 120 kDa protein product
abundant in lymphoid cells. Furthermore, immunoblots of p85
immunoprecipitates using antibodies raised against c-cbl detect a band at exactly the position of pp120. In addition, p85
can be detected in immunoblots of c-cbl immunoprecipitates.
Thus, pp120 appears to correspond to c-cbl. A direct
association between c-cbl and p85 can be observed in vitro using a fusion protein comprising the Src homology 2 (SH2) domains
of p85, and this binding is abolished by phenyl phosphate, suggesting
that the interaction is mediated through phosphotyrosine-SH2 domain
interactions. Thus, these results show important functional differences
between the
and
isoforms of p85 in vivo and point
to c-cbl as a potentially important mediator of some of the
functions of PI-3 kinase in intact cells.
)PIns(4)P, and PIns(4,5)P2 on the D3
position of the inositol ring (PI-3 kinase). PI-3 kinase was first
identified as a lipid kinase activity associated with the middle T
antigen in polyoma virus (SV40) transformed cells. It is now known to
associate with several different receptor tyrosine kinases, including
the receptors for PDGF, epidermal growth factor, insulin/IRS-1, and
with non-receptor tyrosine kinases, such as
p60
(1, 2, 3) . The
association with multiple tyrosine kinases has suggested that PI-3
kinase plays an important role in signaling pathways leading to growth
and proliferation. Interestingly, cloning of the catalytic subunit of
PI-3 kinase revealed a high degree of homology with a yeast protein,
vps34p, which plays a fundamental role in the delivery of newly
synthesized proteins to the yeast vacuole, indicating an important role
for PI-3 kinase activity in protein sorting(4) . Recent studies
from our laboratory strongly suggest that PI-3 kinase activity may play
an important role in the intracellular sorting and down-regulation of
the PDGF receptor(5) . Thus, PI-3 kinase may coordinate or
regulate diverse functions of receptor tyrosine kinases in mammalian
cells.
isoform of the p85 subunit of PI-3 kinase, suggesting important
physiological differences between these kinase isoforms.
Antibodies
Monoclonal antibodies
directed to the N-terminal SH2 domain (UB 93-3, 05-217) or
to the SH3 domain (05-212) of p85, as well as rabbit polyclonal
antisera to rat PI-3 kinase (06-195) were obtained from Upstate
Biotechnology Inc. Monoclonal antibodies were always used in
combination to maximize the recovery of p85 molecules that might be
complexed through SH2 or SH3 domains. Isoform-specific rabbit antisera
were raised against peptides based on the extreme C-terminal 15 amino
acids of each isoform as described (12) . Unlabeled goat
anti-Ig (IgM, IgG, IgA) used for stimulating A20 cells and
cross-linking OKT3, was obtained from Cappel. Affinity-purified
c-cbl antisera was purchased from Santa Cruz Biotechnology.
The OKT3 and OKT4 hybridomas were purchased from ATCC (CRL8001,
CRL8002). Antibody was concentrated from cell supernatants using ABx
bakerbond (J. T. Baker). Monoclonal antibody raised against
phosphotyrosine (4G10) was obtained from UBI. The fusion protein was
constructed using a portion of the human cDNA for p85, consisting
of the two SH2 domains and the intervening sequences (nucleotides
986-2313). The DNA was inserted into the pGEX2T vector and expressed in Escherichia coli strain, XA90. The GST-SH2 fusion protein was
adsorbed onto glutathione-agarose beads and used directly for
adsorption studies.
Cells
Jurkat T cells and A20 murine B
lymphoma cells were obtained from ATCC. All cells were grown in
complete RPMI media supplemented with 10% fetal calf serum. T cells
were aliquoted (3-5 10
cells/ml) into
microfuge tubes and stimulated using 50 µg/ml ABx-purified OKT3
supplemented with excess goat anti-mouse IgG. At the time points
indicated in each experiment, cells were pelleted in a microfuge for 5
s, placed on ice, and resuspended in cold lysis buffer (see below). B
cells were aliquoted at 5
10
cells/ml and
stimulated with 20 µg of purified anti-IgG. For metabolic labeling
experiments, cells were incubated in serum-free, methionine-free RPMI
with 200 µCi/ml [
S]methionine at 37 °C
for 3 h. Cells were then transferred to serum-free RPMI for an
additional hour before stimulation.
Immunoprecipitation and
Immunoblotting
Cells were lysed in 1 ml of an ice-cold
buffer composed of 1% Triton X-100, 20 mM Tris, 150 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 1
mM benzamidine, 1 mM 1,10-phenanthroline, 1 mM sodium vanadate, and 50 mM sodium fluoride. Lysates were
clarified by centrifugation at 14,000 g for 15 min and
precleared by incubation with Protein A-Sepharose prior to addition of
specific antibodies. After 120 min of incubation at 5 °C, protein
A-Sepharose was added, and incubations continued for a further 60 min.
Protein A beads were then washed three to five times in a wash buffer
composed of 20 mM Tris, 150 mM sodium chloride, 0.2%
Triton X-100, and 0.1% SDS. Immunoprecipitates were resolved by
SDS-PAGE and transferred to nitrocellulose for immunoblotting. Primary
antibodies were detected by chemiluminescence (Amersham Corp.). For
adsorption to the agarose-immobilized SH2-p85 fusion protein, lysates
were clarified as above and incubated with the concentrations of fusion
protein indicated in Fig. 6. After 60 min of incubation at 5
°C, the agarose beads were washed as described above. Beads were
then boiled in SDS-sample buffer, and adsorbed proteins were resolved
by SDS-PAGE.
(A) or the indicated concentration of GST
alone (B). C, p85 was immunoprecipitated from the
supernatants of the lanes in B using the polyclonal
antisera (UBI). Precipitates were resolved on 7.5% SDS-PAGE,
transferred to nitrocellulose, and probed with anti-phosphotyrosine
antibodies.
A Single Tyrosine-phosphorylated Protein Associates
with PI-3 Kinase in Activated Lymphocytes
The complex
structure of the p85 subunit of PI-3 kinase can potentially give rise
to numerous protein-protein interactions in cells stimulated by
mitogenic factors. At least a subset of interacting proteins might be
expected to interact stably and to coimmunoprecipitate with kinase
isolated from cell extracts. To identify such components, we analyzed
the polypeptide composition of p85 immunoprecipitates from stimulated
Jurkat T cells labeled to equilibrium with
[S]methionine. Polypeptides in such
immunoprecipitates were separated by polyacrylamide gel electrophoresis
and transferred onto nitrocellulose paper for immunoblotting.
1,
GAP, and Syp. These results suggest that pp120 may be a major target or
effector of PI-3 kinase function in lymphoid cells.
S]methionine as described under
``Materials and Methods'' and then stimulated with soluble
anti-CD3 for 4 min at 37 °C. Total cell lysates were
immunoprecipitated with a combination of two monoclonal antibodies
raised against the SH2 and SH3 domains of p85 or with a monoclonal
antibody raised against pTyr. Immunoprecipitates were then resolved on
7.5% SDS-PAGE and transferred to nitrocellulose. A, an
autoradiogram of the nitrocellulose blot. Arrows indicate
major bands immunoprecipitated specifically by the antibodies. B, phosphotyrosine immunoblot of lanes shown in A. The primary antibody was detected using horseradish
peroxidase-coupled secondary antibodies and ECL. An arrow points to the single phosphotyrosine band, pp120. The intense
lower band at approximately 40 kDa corresponds to the precipitating
immunoglobulin, detected by the anti-mouse secondary
antibodies.
pp120 Is the Proto-oncogene c-cbl
The
identity of some of the numerous proteins that are tyrosine
phosphorylated in response to stimulation through the T cell receptor
has been determined. Two of the proteins in the molecular range of
100-120 kDa have been shown to correspond to the mammalian
protein VCP (13) and to the proto-oncogene
c-cbl(14) . Antibodies to VCP and to c-cbl were used to determine whether these polypeptides corresponded to
the 120-kDa tyrosine-phosphorylated polypeptide associated with p85.
Antibodies against c-cbl but not antibodies against VCP
immunoprecipitated a tyrosine-phosphorylated protein that comigrated
exactly with the p120 band associated with p85 in lysates of A20 B
cells (Fig. 4A). In addition, immunoblotting with
anti-p85 antibodies revealed the presence of p85 in c-cbl immunoprecipitates of extracts from stimulated but not resting B
cells (Fig. 4B). Although the amount of p85 in
c-cbl immunoprecipitates represented a small fraction of the
total cellular p85 pool, it represented a significant amount
(approximately 50%) of the p85 protein associated with phosphotyrosine
immunoprecipitates (not shown). These results suggest that c-cbl is the major tyrosine phosphoprotein associated with p85 in these
cells.
c-cbl Specifically Interacts with
p85
Jurkat T cells have been shown to contain two of
the known isoforms of p85 ( and
), which display high
sequence similarity. The functional differences between the two p85
isoforms are not known, though there is some evidence that p85 isoforms
regulate the catalytic activity of PI-3 kinase differentially (13) and that in lymphocytes they are differentially
phosphorylated in response to stimulation(17) . We sought to
determine the specificity of the interaction of c-cbl with the
or
isoforms of p85. These two characterized isoforms of p85
display slightly different mobilities on SDS-PAGE and can be separated
by immunoprecipitation with isoform-specific antisera (Fig. 5A, toppanel). Despite the
presence of large amounts of p85
in immunoprecipitates obtained
with
-isoform-specific antiserum, c-cbl could not be
detected by immunoblotting these precipitates with c-cbl antiserum (Fig. 5A, middlepanel). Furthermore, p85
immunoprecipitates from
activated Jurkat T cells were devoid of coprecipitating tyrosine
phosphoproteins. These results are consistent with the findings of Ward etal.(18) , which concluded that PI-3 kinase
is not a substrate for tyrosine kinases in T cells and does not
interact with any tyrosine-phosphorylated proteins. These investigators
employed anti-p85 antibodies specific for p85
. In contrast,
p85
immunoprecipitates contained a single
phosphotyrosine-containing protein at 120 kDa (Fig. 5A, bottom), which was also detected with anti-c-cbl antibodies (Fig. 5A, middle). A time
course shows that this isoform-specific interaction occurs rapidly
after activation and declines over 15 min (Fig. 5B).
This time course is identical to that observed using polyclonal
antisera against p85 which does not distinguish among different
isoforms (Fig. 2).
. A, Jurkat T cells (2.5
10
)
were stimulated for 2 min at 37 °C by cross-linking CD3 and CD4,
lysed, and immunoprecipitated with isoform-specific antisera raised
against p85
and p85
. Immunoprecipitates were resolved on 7.5%
SDS-PAGE, blotted onto nitrocellulose, and probed with anti-p85
antiserum, anti-phosphotyrosine, and anti-c-cbl. Toppanel illustrates the region of the blot containing p85,
and middle and bottom panels are the region above the
105-kDa marker, which contained the only tyrosine phosphoprotein in p85
immunoprecipitates. B, Jurkat T cells were incubated at 37
°C in the presence of soluble anti-CD3 and secondary cross-linking
antibody for the times indicated above each lane.
Cells were then lysed, and extracts were immunoprecipitated with
isoform-specific anti-p85 antisera as indicated. Immunoprecipitates
were then resolved on 7.5% SDS-PAGE, transferred to nitrocellulose, and
probed with anti-phosphotyrosine
antibodies.
p85 Can Associate with c-cbl through Its SH2
Domains
An analysis of the human c-cbl sequence
shows a C-terminal proline-rich region as well as multiple tyrosine
residues(15) , including one within the context of a consensus
p85 binding site (YEXM, tyrosine 731), as determined by
Songyang et al.(19) . The proline-rich region has
recently been reported to bind the SH3 domains of the adaptor molecule
Grb-2 in vitro(14) and was previously shown to bind
p47 Nck(20) . To determine the nature of the interactions
between p85 and c-cbl, we tested the ability of a p85 fusion
protein to adsorb to c-cbl from cell extracts. A GST-fusion
protein comprising the two SH2 domains of p85 readily adsorbed a
tyrosine-phosphorylated protein of 120 kDa from Jurkat cells, as well
as many other bands not seen in polyclonal or isoform-specific p85
precipitates (Fig. 6). Both the fusion protein and polyclonal
p85 antisera fail to coprecipitate this band when 40 mM phenyl
phosphate was added to the lysis buffer (data not shown). These results
suggest that the interaction between p85 and c-cbl occurs
through SH2 domains of p85 and phosphotyrosine residues in
c-cbl.
or
isoforms(19) . Thus, it is unlikely that
different affinities for c-cbl can explain the observed
differential interaction between the
and
isoforms of p85
and c-cbl that appear to occur in intact cells. Other
potential factors that may influence in vivo interactions and
that may not be apparent from in vitro peptide affinity
studies are additional protein-protein interactions or cellular
localization. These factors may also explain our failure to detect an
interaction between PLC
1 and c-cbl in immunoprecipitates
of c-cbl or PLC
1 obtained from cell extracts (not shown
and (21) ), despite the fact that an interaction between the
SH2 domains of PLC
1 and c-cbl can be readily observed in vitro(14) . We believe that the observed in
vitro interaction between the p85 fusion protein and c-cbl probably reflects the mechanism of interaction between p85
and c-cbl in intact cells. The basis for the isoform-specific
interaction in vivo between p85
and c-cbl cannot
be determined from the data presented here, but understanding the
nature and significance of this selective interaction is an important
goal for future studies.
We acknowledge Drs. L. Samelson and A. B. Reynolds
for the generous gifts of reagents.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.