(Received for publication, December 3, 1996, and in revised form, January 30, 1997)
From the Department of Pharmacology and Cancer Center, University of Virginia, Charlottesville, Virginia 22908
Two tyrosine phosphoproteins in phorbol ester-sensitive EL4 (S-EL4) mouse thymoma cells have been identified as the p120 c-Cbl protooncogene product and the p85 subunit of phosphatidylinositol 3-kinase. Tyrosine phosphorylation of p120 and p85 increased rapidly after phorbol ester stimulation. Phorbol ester-resistant EL4 (R-EL4) cells expressed comparable amounts of c-Cbl and phosphatidylinositol 3-kinase protein but greatly diminished tyrosine phosphorylation. Co-immunoprecipitation experiments revealed complexes of c-Cbl with p85, and of p85 with the tyrosine kinase Lck in phorbol ester-stimulated S-EL4 but not in unstimulated S-EL4 or in R-EL4 cells. In vitro binding of c-Cbl with Lck SH2 or SH3 domains was detected in both S-EL4 and R-EL4 cells, suggesting that c-Cbl, p85, and Lck may form a ternary complex. In vitro kinase assays revealed phosphorylation of p85 by Lck only in phorbol ester-stimulated S-EL4 cells. Collectively, these results suggest that Cbl-p85 and Lck-p85 complexes may form in unstimulated S-EL4 and R-EL4 cells but were not detected due to absence of tyrosine phosphorylation of p85. Greatly decreased tyrosine phosphorylation of c-Cbl and p85 in the complexes may contribute to the failure of R-EL4 cells to respond to phorbol ester.
T lymphocyte activation is triggered by interaction between the T
cell antigen receptor (TCR)1 and its
cognate antigen. One of the earliest signaling events following TCR
stimulation is the rapid increase in tyrosine phosphorylation of a
number of proteins (reviewed in Refs. 1 and 2). Unlike growth factor
receptors that have intrinsic tyrosine kinase activity (reviewed in
Ref. 3), the TCR components transduce their signals through
noncovalently associated cytoplasmic tyrosine kinases (1, 2). Three
tyrosine kinases, Lck, Fyn, and ZAP-70, have been implicated in the
function of the TCR. Identification of substrates for these tyrosine
kinases has been an area of intense investigation. Several tyrosine
kinase substrates in T cells have been identified in the past few
years, including phospholipase C- (4, 5), the guanine nucleotide
exchange factor Vav (6-8), an oligomeric ATPase valosin-containing
protein (9, 10), the membrane-cytoskeleton linker protein ezrin (11,
12), and the
subunit of TCR (13). Recently, c-Cbl, a protooncogene protein of 120 kDa has been identified as another prominent tyrosine kinase substrate in T cells (14). Although it has been demonstrated that c-Cbl becomes rapidly tyrosine-phosphorylated upon TCR stimulation (14) and forms complexes with several signaling molecules (15-19), the
function of c-Cbl in T cells is not clear.
T cell activation can be mimicked by the addition of tumor-promoting
phorbol esters plus calcium ionophores, suggesting important roles for
protein kinase C (PKC) and calcium (20). The role of PKC in T cell
activation is further emphasized by studies showing that induction of
the transcription factors AP-1, NF-B, and nuclear factor of
activated T cell for cytokine gene expression is regulated by PKC (21).
PKC is a family of phospholipid-dependent serine/threonine kinases, most of which can be stimulated directly by phorbol ester treatment (reviewed in Refs. 22 and 23). Although a functional role for
PKC in T cell activation is demonstrated, the signaling events mediated
by PKC are not clear. Stimulation of T lymphocytes with phorbol esters
induces a marked phosphorylation of Lck at N-terminal serine residues
(24, 25). Whether this phorbol ester-stimulated serine phosphorylation
of Lck has any functional significance is not well understood.
Stimulation of some T cells with phorbol ester also induces
accumulation of a Ras-GTP complex (26), suggesting that PKC might
mediate Ras activation. Ras has been shown to interact directly with
Raf-1 kinase in vitro and in vivo (27, 28). The
evidence that PKC can phosphorylate and activate Raf-1 suggests that
PKC may be an upstream regulator of this kinase (29, 30). Raf-1 can
trigger a kinase cascade by phosphorylating and activating
mitogen-activated protein (MAP) kinase kinase (MEK), which subsequently
phosphorylates and activates MAP kinase (reviewed in Ref. 31). The
demonstration that MAP kinase can translocate to the nucleus (32),
where it can directly modulate transcriptional factors (33, 34), might
provide a mechanism for PKC regulation of cytokine gene expression.
To study the PKC-mediated signaling events, we have employed an EL4 mouse thymoma cell line that requires only phorbol ester treatment to stimulate interleukin-2 (IL-2) production, growth inhibition, and adherence to the substrate (35, 36). Comparison with an EL4 variant that lacks all of these responses (36, 37) has allowed us to investigate signaling molecules that may be essential for these responses. Previous work has revealed deficient induction of c-Jun and Fra transcription factors (38) and deficient activation of MEK in phorbol ester-resistant EL4 (R-EL4) cells (39), suggesting potential functional roles for these signaling molecules. In addition, treatment of phorbol ester-sensitive EL4 (S-EL4) cells with tyrosine kinase inhibitors (genistein and herbimycin) blocks phorbol ester-stimulated IL-2 mRNA production (40), which raised the possibility that tyrosine phosphorylation may be important for phorbol ester-stimulated IL-2 production. To examine the tyrosine phosphorylation events potentially important for phorbol ester-stimulated IL-2 production and other responses, we have compared tyrosine phsophoproteins between S-EL4 and R-EL4 cells. A tyrosine phosphoprotein, p85, was detected in S-EL4 but not R-EL4 cells, and phorbol ester stimulation enhanced its tyrosine phosphorylation only in sensitive cells (41). Here we report an additional tyrosine phosphoprotein, p120, that has a tyrosine phosphorylation response similar to that of p85. We have identified this p120 protein as the product of the c-Cbl protooncogene and further investigated its associated signaling proteins. Our studies have revealed greatly decreased tyrosine phosphorylation of c-Cbl in R-EL4 cells and some deficiencies in its associated signaling molecules in these cells.
Anti-phosphotyrosine (anti-Tyr(P)) and anti-PI 3-kinase antibodies were obtained from Upstate Biotechnology Inc. (Lake Placid, NY). All other antibodies, glutathione S-transferase (GST) fusion proteins, and glutathione-agarose beads were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phorbol dibutyrate (PDB), aprotinin, sodium pyrophosphate, sodium orthovanadate, phenylmethylsulfonyl fluoride, and HEPES were purchased from Sigma. Protein A-agarose beads and enhanced chemiluminescence (ECL) reagents were supplied by Amersham Corp. Phorbol ester-sensitive and -resistant EL4 cells were purchased from ATCC (Rockville, MD), and fetal calf serum was obtained from Life Technologies, Inc. Luminescent stickers for marking and aligning autoradiographs were obtained from Stratagene (La Jolla, CA).
Cell Culture and Phorbol Ester TreatmentEL4 cells were grown in RPMI 1640 medium supplemented with 5% heat-inactivated fetal calf serum and 2 mM glutamine to a density of approximately 1-2 × 106/ml. Cells were treated with 150 mM PDB for the times indicated.
ImmunoprecipitationCells (2 × 107) were lysed in 1 ml of lysis buffer (50 mM HEPES, pH 7.5, 150 nM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA) supplemented with protease and phosphatase inhibitors (1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µM sodium pyrophosphate). Particulate material was removed by centrifugation at 14,000 × g for 15 min, and supernatants were incubated with 2-5 µg of the indicated antibodies at 4 °C for 3 h, followed by incubation with 15 µl of protein A-agarose beads for 1 h. The immunoprecipitates were washed with lysis buffer five times and treated with 1 × Laemmli sample buffer and then subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting with the indicated antibodies.
Binding of Cellular Proteins to GST Fusion ProteinsCell lysates from 1.5 × 107 cells were precleared with 40 µl of glutathione-agarose beads overnight and then incubated with 10 µg of purified GST proteins noncovalently coupled to 15 µl of glutathione-agarose beads for 3 h at 4 °C. The bead-bound proteins were washed five times with lysis buffer, suspended in 1 × Laemmli sample buffer, and subjected to SDS-PAGE and immunoblotting with the indicated antibodies.
Western BlottingProteins from 2 × 105 cells were separated by electrophoresis through an 8% polyacrylamide gel and transferred to nitrocellulose. Immunoblots were blocked by incubation (30 min) with either 1% bovine serum albumin in Tris-buffered saline-Tween (10 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) for Tyr(P) blots or 5% nonfat dried milk in Tris-buffered saline-Tween, followed by incubation for 1 h with the indicated primary antibodies at concentrations of 0.5 µg/ml. Blots were then washed with Tris-buffered saline-Tween, and horseradish peroxidase-linked secondary antibodies were added for a further 30 min. Blots were washed, and proteins were visualized by the addition of enhanced chemiluminescence reagents as described by the manufacturers. For two-dimensional gel electrophoresis, proteins from 2.5 × 106 cells were separated on isoelectric focusing tube gels with pH 3.5-10 ampholines followed by SDS-PAGE and immunoblotting with Tyr(P) antibody, and then the blots were stripped and reprobed with the antibody to the p85 subunit of PI 3-kinase or c-Cbl. Luminescent stickers were used during exposure of the immunoblots to film to help align the films with the immunoblots and to mark the positions of molecular weight markers. The positions of tyrosine-phosphorylated p85 and the p85 subunit of PI 3-kinase and of p120 and c-Cbl on two-dimensional gel could be compared unambiguously by overlaying and aligning the two films.
Lck Kinase AssayCell lysates from 2 × 107 cells were precleared with 30 µl of protein A-agarose
beads for about 3 h. Precleared cell lysates were incubated
overnight with 5 µg of Lck antibodies, followed by incubation with 15 µl of protein A-agarose beads for 1 h. The beads were washed
four times with lysis buffer and one time with 25 mM HEPES
and incubated at 30 °C for 5 min in 50 µl of kinase reaction
buffer (25 mM HEPES, pH 7.5, 5 mM
MgCl2, 5 mM MnCl2, 1 mM
sodium vanadate, 50 µM ATP, 10 µCi of
[-32P]ATP). The kinase reactions were stopped with
3 × Laemmli sample buffer. Samples were then subjected to
SDS-PAGE and transferred to nitrocellulose. The nitrocellulose was
exposed to Kodak film at
70 °C overnight and then immunoblotted
with the indicated antibodies.
Previous studies
revealed a tyrosine phosphoprotein, p85, in phorbol ester-sensitive but
not -resistant EL4 mouse thymoma cells and found that phorbol ester
stimulation enhanced its tyrosine phosphorylation only in sensitive
cells (41). As shown in Fig. 1A,
antiphosphotyrosine immunoblotting reveals an additional protein, p120,
that exhibits a similar pattern. It could not be discerned from this
experiment whether the proteins were deficient in the resistant cells or merely the tyrosine phosphorylation.
In attempt to identify these proteins, blots were probed with antisera
to known tyrosine phosphoproteins of similar molecular weight that
could have a role in T cell signaling. Probing with antibodies to
c-Cbl, a prominent tyrosine kinase substrate of 120 kDa recently
identified in T cells (14), revealed expression of c-Cbl in S-EL4 cells
and equal or greater expression in R-EL4 cells (Fig. 1B).
Identification of p120 as c-Cbl would suggest that tyrosine
phosphorylation of c-Cbl was much higher in S- than in R-EL4 cells. To
test this possibility, c-Cbl proteins were immunoprecipitated from both
S- and R-EL4 cells, and the tyrosine phosphorylation of c-Cbl was
determined by anti-Tyr(P) immunoblotting. C-Cbl did exhibit much
greater tyrosine phosphorylation in S- than in R-EL4 cells (Fig.
2), consistent with the possibility that c-Cbl was p120.
To further address the possible identity of p120 as c-Cbl, attempts to
separate p120 from c-Cbl were made using two-dimensional gel
electrophoresis. Lysates from cells that were stimulated with 150 nM PDB for 5 min were electrophoresed through isoelectric
focusing gels followed by SDS-PAGE gels and then subjected to
anti-Tyr(P) immunoblotting. The tyrosine phosphoproteins were resolved
into a number of distinct spots or arrays of spots, corresponding to
major tyrosine phosphoproteins observed in one-dimensional gel analysis
(Fig. 1A). A series of 120-kDa tyrosine phosphoproteins of
varying isoelectric points was detected in S- but not in R-EL4 cells
(Fig. 3, left panels). When the anti-Tyr(P)
immunoblots were stripped and reprobed with anti-Cbl antibody, a set of
anti-Cbl reactive proteins, also exhibiting several pI forms, was
detected in both cell lines (Fig. 3, right panels).
Overlaying the two films revealed that the p120 signal exactly
overlapped with the c-Cbl signal, arguing strongly that p120 was
c-Cbl.
Since phorbol ester stimulation of S-EL4 cells enhanced the tyrosine
phosphorylation of p120, the time course of tyrosine phosphorylation
after phorbol ester stimulation was examined. Lysates from cells
stimulated with 150 nM PDB for various times up to 60 min
were analyzed by anti-Tyr(P) immunoblotting. The tyrosine
phosphorylation of p120 in S-EL4 cells began to increase about 1.5 min
after PDB stimulation, remained elevated for 20 min, and decreased by
about 30 min (Fig. 4A). Although resistant cells exhibited a faint tyrosine phosphorylation of p85, no obvious p120 tyrosine phosphorylation was observed in R-EL4 cells (Fig. 4B). The blots were stripped and reprobed with anti-Cbl
antibody to confirm that the amount of c-Cbl proteins did not change
over the assay period (Fig. 4).
c-Cbl, p85, and Lck Complex Formation in Phorbol Ester-stimulated Sensitive EL4 Cells
Since it has been reported that c-Cbl could
form complexes with several signaling molecules (15-19), the
possibility that c-Cbl may form a complex with tyrosine phosphoproteins
in EL4 cells was investigated. c-Cbl proteins were immunoprecipitated
from lysates of cells that had been unstimulated or treated with PDB for 2 min, and Cbl-associated tyrosine phosphoproteins were detected with an anti-Tyr(P) antibody. An 85-kDa tyrosine phosphoprotein was
observed in phorbol ester-stimulated S-EL4 cells but not in unstimulated S-EL4 or in R-EL4 cells despite comparable
immunoprecipitation of c-Cbl in both cells (Fig. 5). In
addition to the similar pattern of expression, this 85-kDa tyrosine
phosphoprotein co-migrated with the previously observed p85 detected by
direct immunoblotting of lysates (data not shown), suggesting that they
are the same protein. To our surprise, we did not observe enhanced
tyrosine phosphorylation of c-Cbl in phorbol ester-stimulated S-EL4
cells. We suspect that some phosphotyrosines of c-Cbl in phorbol
ester-stimulated S-EL4 cells may be lost during the longer time
required for immunoprecipitation as opposed to direct immunoblotting of
lysates.
Although p85 was detected by anti-Tyr(P) antibody in Cbl
immunoprecipitates after phorbol ester stimulation of S-EL4 cells, it
was not clear whether phorbol ester stimulation induced more tyrosine-phosphorylated p85 to be associated with c-Cbl in S-EL4 cells
or whether phorbol ester stimulation induced the tyrosine phosphorylation of p85 that associated with c-Cbl in either
phosphorylation state. To explore these possible mechanisms, we first
tried to identify potential tyrosine kinases for p85. The lymphocyte
tyrosine kinases Lck and Fyn were immunoprecipitated from lysates
prepared after a 10-min treatment of cells with 150 nM PDB
to determine whether p85 can be co-immunoprecipitated with these
tyrosine kinases. Monoclonal antibodies to c-Fos and cyclin E were used
as negative controls. The tyrosine kinase-associated tyrosine
phosphoproteins were detected by anti-Tyr(P) antibody. A strong p85
signal was detected in Lck immunoprecipitates from S-EL4, not R-EL4,
cells (Fig. 6A). A weak p85 signal also could
be detected in Fyn immunoprecipitates from S-EL4 cells; however, this
signal did not exceed that detected in control immunoprecipitates (Fig.
6A). Since p85 was observed to co-immunoprecipitate with Lck
in phorbol ester-stimulated S-EL4 cells, we next tried to determine
whether phorbol ester stimulation had any effect on the
co-immunoprecipitation of p85 with Lck. Lysates from S-EL4 cells that
were unstimulated or treated with 150 nM PDB for 10 min
were immunoprecipitated with anti-Lck antibody, and p85 was detected by
anti-Tyr(P) antibody. A prominent p85 band was detected in Lck
immunoprecipitates from stimulated S-EL4 cells, but signals in
immunoprecipitates from unstimulated S-EL4 cells did not exceed those
from control immunoprecipitates (Fig. 6B). The appearance of
p85 in Lck immunoprecipitates after phorbol ester stimulation of S-EL4
cells was very rapid, being detectable after only 2 min of phorbol
ester stimulation (data not shown).
To investigate which domains of Lck could bind p85, lysates from cells
that were unstimulated or were treated with 150 nM PDB were
incubated with GST fusion proteins containing the SH2 or SH3 domains of
Lck, and the tyrosine phosphoproteins that bound to these GST fusion
proteins were detected by anti-Tyr(P) antibody. An 85-kDa tyrosine
phosphoprotein was observed to bind the Lck SH2 (Fig.
7A) and the Lck SH3 domains (Fig.
7B). This 85-kDa tyrosine phosphoprotein co-migrated with
the p85 detected in whole cell lysates and was detected only in S-EL4
cells (Fig. 7). The binding of p85 with the SH2 or SH3 domains of Lck
was enhanced after phorbol ester stimulation of S-EL4 cells (Fig. 7).
Since the Lck SH2 domain should bind only tyrosine-phosphorylated p85,
whereas the SH3 domain would be expected to bind the 85-kDa protein
independent of its phosphorylation state, it is not surprising that
Western blotting with anti-Tyr(P) antibody provides a stronger p85
signal with the SH2 GST fusion protein than with the SH3. Collectively, these results suggested that Lck was a potential tyrosine kinase for
p85.
Since we observed p85 in both c-Cbl and Lck immunoprecipitates after
phorbol ester stimulation of S-EL4 cells, we tried to test whether
c-Cbl could bind Lck and form a ternary complex with p85 in EL4 cells.
When Lck proteins were immunoprecipitated from sensitive or resistant
EL4 cells that had been unstimulated or treated with PDB, no obvious
c-Cbl proteins were detected in Lck immunoprecipitates (data not
shown). Since it was reported that c-Cbl could bind the SH2 and SH3
domains of Lck in vitro in Jurkat cells (14), we next
examined whether this was the case for EL4 cells. Lysates from
sensitive and resistant EL4 cells that were unstimulated or treated
with 150 nM PDB for 5 min were incubated with GST fusion
proteins containing Lck SH3 or Lck SH2 domains. c-Cbl proteins were
observed to constitutively bind GST fusion proteins containing Lck SH3
(Fig. 8A) or Lck SH2 domains (Fig. 8B) in both S- and R-EL4 cells but not to control GST fusion
proteins. The binding of c-Cbl to Lck SH2 domains was better in S- than in R-EL4 cells. This was not surprising, since SH2 domains serve as the
binding sites for phosphotyrosine during protein-protein interaction
(42, 43) and c-Cbl exhibited significantly greater tyrosine
phosphorylation in sensitive than in resistant EL4 cells (Fig. 2). The
co-immunoprecipitation of p85 with c-Cbl and with Lck after phorbol
ester stimulation of S-EL4 cells and the in vitro binding of
Lck SH2 or SH3 domains with c-Cbl suggested that c-Cbl, Lck, and p85
might form a ternary complex in phorbol ester-stimulated S-EL4 cells.
The failure to detect an Lck-Cbl complex via co-immunoprecipitation may
be due to weak or transient complex formation.
Phorbol Ester-stimulated Phosphorylation of p85 by Lck Only in S-EL4 Cells
Although Cbl-p85 and Lck-p85 complexes were observed
only in phorbol ester-stimulated S-EL4 cells, we could not exclude the possibility that a Cbl-p85 or Lck-p85 complex did form in unstimulated S-EL4 or in R-EL4 cells but was not detected due to absence of tyrosine
phosphorylation of p85. We therefore tested whether p85 could be
phosphorylated by Lck in vitro after phorbol ester
stimulation of the cells. Lck proteins were immunoprecipitated from
lysates prepared from unstimulated cells or from cells treated with 150 nM PDB for 5 min and subjected to an in vitro
kinase assay to detect 32P transfer to any
co-immunoprecipitated proteins. A 32P-containing band of 56 kDa, probably reflecting autophosphorylation of Lck, was detected in
all Lck immunoprecipitates, but an 85-kDa protein was phosphorylated in
Lck immune complex kinase assays only in phorbol ester-stimulated S-EL4
cells, not in unstimulated S-EL4 cells, in R-EL4 cells, or in control
immune complexes (Fig. 9, upper panel).
Anti-Tyr(P) immunoblotting demonstrated its co-migration with p85 (Fig.
9, lower panel). The phosphorylation of p85 in Lck immune
complex was very rapid, being detectable after only 2 min of phorbol
ester stimulation (data not shown). These results supported the
possibility that detection of p85 in Cbl-p85 and Lck-p85 complexes in
phorbol ester-stimulated S-EL4 cells was due to the tyrosine
phosphorylation of p85. But without identification of p85, we could not
exclude the possibility that the detection of p85 in these complexes in
phorbol ester-stimulated S-EL4 cells was due to association of more
tyrosine-phosphorylated p85 with c-Cbl or Lck.
Identification of p85 as the p85 Subunit of PI 3-Kinase
Since
it was reported that the p85 subunit of PI 3-kinase associated with
c-Cbl (15, 16, 17) and bound the SH3 domains of Lck (42, 43), we
speculated that p85 might be the p85 subunit of PI 3-kinase. To test
this possibility, lysates from cells that were stimulated with 150 nM PDB for 5 min were subjected to two-dimensional gel
electrophoresis, and immunoblots were probed successively with
anti-Tyr(P) and anti-PI 3-kinase p85 antibodies. While a series of
tyrosine-phosphorylated p85 spots was detected in S- but not in
R-EL4 cells (Fig. 10, left panels), a set of
spots reactive with the antibodies to the p85 subunit of PI 3-kinase
was observed in both S- and R-EL4 cells (Fig. 10, right
panels). These PI 3-kinase spots exactly overlapped with the
Tyr(P)-p85 spots in S-EL4 cells when the films were superimposed,
arguing that p85 was the p85 subunit of PI 3-kinase.
To further confirm that p85 was the p85 subunit of PI 3-kinase, we
examined whether Cbl- and Lck-associated p85 was recognized by the
antibody to the p85 subunit of PI 3-kinase. c-Cbl proteins were
immunoprecipitated from lysates prepared from cells that were
unstimulated or treated with PDB and subjected to SDS-PAGE followed by
anti-PI 3-kinase immunoblotting. In phorbol ester-stimulated S-EL4
cells, Cbl-associated p85 was recognized by the antibody to PI 3-kinase
p85 subunit (Fig. 11A). However, we also
observed the co-immunoprecipitation of PI 3-kinase with c-Cbl in
unstimulated S-EL4 or in R-EL4 cells (Fig. 11A), whereas we
did not detect the co-immunoprecipitation of tyrosine-phosphorylated
p85 with c-Cbl in these cells (Fig. 5). This may indicate that p85 does
associate with c-Cbl in both cell lines but is not detected with Tyr(P) antibodies due to the lack of tyrosine phosphorylation of p85. This
notion was supported by our observations that p85 was phosphorylated in vitro in Lck immunoprecipitates only in phorbol
ester-stimulated S-EL4 cells but not in unstimulated S-EL4 or in R-EL4
cells (Fig. 9). Furthermore, PI 3-kinase, like p85, also could be
co-immunoprecipitated with Lck in phorbol ester-stimulated S-EL4 cells.
In addition, binding between PI 3-kinase and Lck was observed in
unstimulated S-EL4 and in R-EL4 cells, and phorbol ester stimulation
slightly enhanced the association of PI 3-kinase with Lck in both cell lines (Fig. 11B). Compared with the strong
tyrosine-phosphorylated p85 signal in Lck immunoprecipitates from
phorbol ester-stimulated S-EL4 cells, the amount of PI 3-kinase p85
that associated with Lck seemed quite small. A potential explanation
for this difference could be heavy tyrosine phosphorylation of the few
Lck-associated PI 3-kinase molecules.
Two potentially important tyrosine phosphoproteins, p120 and p85, have been identified in phorbol ester-sensitive EL4 cells. These two proteins were detected by anti-phosphotyrosine antibody in S-EL4 but not in R-EL4 cells, and phorbol ester stimulation enhanced their tyrosine phosphorylation only in sensitive cells (Fig. 1). P120 was identified as the c-Cbl protooncogene product by anti-Cbl immunoblotting, by comparison of c-Cbl tyrosine phosphorylation between S- and R-EL4 cells, and by two-dimensional gel analysis (Figs. 1, 2, 3). Co-migration of p85 with the p85 subunit of PI 3-kinase on two-dimensional gel analysis and reactivity of Cbl- and Lck-associated p85 with the antibody to the p85 subunit of PI 3-kinase (Figs. 10 and 11) strongly argue that p85 is the PI 3-kinase p85 subunit.
Association of c-Cbl with some signaling molecules has been observed in several cell lines (15-19) and also occurred in EL4 cells. Co-immunoprecipitation experiments revealed formation of complexes of c-Cbl with the p85 subunit of PI 3-kinase and of the p85 subunit of PI 3-kinase with Lck in both S-EL4 and R-EL4 cells (Fig. 11, A and B). Co-immunoprecipitation of c-Cbl with Lck was not observed. However, in vitro binding of c-Cbl with Lck SH2 or SH3 domains was detected in both cell lines (Fig. 8), suggesting that c-Cbl, Lck, and the p85 subunit of PI 3-kinase may form a ternary complex. Failure to detect association of c-Cbl with Lck in immune complex may be due to weak or transient binding. Alternatively, c-Cbl may associate with Lck SH2 or SH3 domains indirectly via their association first with p85. While this might explain the lack of co-immunoprecipitation of Lck with c-Cbl and the weak Cbl-Lck SH2 association detected in vitro, the strong signal detected with the Lck-SH3 domain suggests some direct Cbl-Lck SH3 interaction.
Complexes of Cbl-PI 3-kinase p85 and of Lck-PI 3-kinase p85 have been observed also in other cells (16, 17). In Jurkat T cells, the p85 subunit of PI 3-kinase and PI 3-kinase activity were observed in c-Cbl immunoprecipitates. After TCR stimulation, the association of the p85 subunit of PI 3-kinase with c-Cbl and the PI 3-kinase activity in c-Cbl immunoprecipitates was further enhanced. In addition to binding to c-Cbl, the p85 subunit of PI 3-kinase also was observed in Lck immunoprecipitates in IL-2-dependent helper and cytolytic T cell clones (46). IL-2 stimulation not only enhanced the association of the p85 subunit of PI 3-kinase with Lck but also induced the tyrosine phosphorylation and activation of Lck-associated PI 3-kinase (46). The p85 subunit of PI 3-kinase can bind to Lck SH2 or SH3 domains dependent on the cell lines examined (44, 45). While the binding of PI 3-kinase p85 to Lck SH2 domains is largely phosphotyrosine-dependent, PI 3-kinase p85 binds to Lck SH3 domains through a proline-rich motif independent of phosphotyrosine (43). We also observed binding of the p85 subunit of PI 3-kinase to Lck SH2 or SH3 domains in EL4 cells (data not shown) in addition to the binding of tyrosine-phosphorylated p85 to these domains (Fig. 7).
Although S-EL4 and R-EL4 cells have comparable amounts of c-Cbl, PI 3-kinase, and Lck proteins and form complexes of Cbl-PI 3-kinase p85 and Lck-PI 3-kinase p85, the p85 subunit of PI 3-kinase in these complexes becomes tyrosine-phosphorylated only in phorbol ester-stimulated S-EL4 cells but not in unstimulated S-EL4 or in R-EL4 cells (Figs. 5 and 6), and tyrosine phosphorylation of c-Cbl is greatly diminished in R-EL4 cells (Fig. 2). Since phosphotyrosine residues can bind SH2 domains, promoting protein-protein interaction (42, 43), tyrosine phosphorylation of c-Cbl and of the PI 3-kinase p85 subunit that associates with c-Cbl and Lck may promote the downstream signal transduction that contributes to the phorbol ester-stimulated responses in S-EL4 cells. Since this tyrosine phosphorylation happens rapidly after phorbol ester stimulation of S-EL4 cells (2 min), it may contribute to rapid phorbol ester-stimulated responses such as adherence to the substrate, which occurs 30 min after phorbol ester stimulation, as well as to late responses such as IL-2 production or growth inhibition. Although treatment of osteoclast-like cells with c-Cbl antisense did not inhibit cell adherence (47), potential roles for c-Cbl in lymphocyte adherence or other responses remain to be tested.
Greatly diminished tyrosine phosphorylation of c-Cbl and the p85 subunit of PI 3-kinase that associates with c-Cbl or Lck in R-EL4 cells suggests that the tyrosine kinases for these proteins may be defective in R-EL4 cells. While the tyrosine kinases for c-Cbl have not been well examined, the fact that Lck can phosphorylate p85 in vitro only in phorbol ester-stimulated S-EL4 cells (Fig. 9) suggests that Lck may be responsible for the tyrosine phosphorylation of Cbl- and Lck-associated PI 3-kinase p85. However, we cannot exclude the possible involvement of other tyrosine kinases. Lck is expressed in both S-EL4 and R-EL4 cells and undergoes autophosphorylation in both cell lines (Fig. 9), suggesting that it is not inactive in R-EL4 cells. Determination of whether mutation of some tyrosine phosphorylation sites in c-Cbl or PI 3-kinase p85 contributes to the greatly decreased tyrosine phosphorylation of these proteins will require further investigation.
In addition to possible defects in tyrosine kinases or tyrosine
phosphorylation sites, it is possible that some upstream regulators for
c-Cbl, PI 3-kinase p85, or Lck are defective in R-EL4 cells, accounting
for the greatly decreased tyrosine phosphorylation of these proteins.
Several PKC isoforms (PKC-, -
, and -
) exhibit greatly
decreased expression in R-EL4 cells (48, 49), raising the possibility
that these PKC isoforms may contribute to the regulation of c-Cbl, PI
3-kinase p85, or Lck by serine or threonine phosphorylation in S-EL4
cells and facilitate phorbol ester-stimulated tyrosine phosphorylation
of these proteins.
In conclusion, these studies have identified two potentially important phosphotyrosine proteins in EL4 cells, p120 and p85, as the c-Cbl protooncogene product and the p85 subunit of PI 3-kinase. C-Cbl, Lck, and PI 3-kinase p85 proteins and complexes of Cbl-PI 3-kinase p85 and of Lck-PI 3-kinase p85 were observed in S-EL4 and R-EL4 cells; however, tyrosine phosphorylation of PI 3-kinase p85 in these complexes was detected only in phorbol ester-stimulated S-EL4 cells, and tyrosine phosphorylation of c-Cbl was much higher in S-EL4 than in R-EL4 cells, raising the possibility that these complexes and tyrosine phosphorylation of the proteins may be important for phorbol ester-stimulated responses in S-EL4 cells. Understanding how PKC regulates these signaling molecules and which PKC isoforms may be involved will be of special interest in the future.
We thank Moira Resnick for helpful advice and discussion throughout this work and Xuqiong Wu from the laboratories of A. P. and A. V. Somlyo at the University of Virginia for technical help with the two-dimensional gel analysis.