(Received for publication, July 27, 1995; and in revised form, December 6, 1995)
From the
p21 mediates mitogenic responses and also renders
cells susceptible to apoptosis after inhibition of protein kinase C
(PKC) activity. Ras-induced apoptosis can be blocked by the
proto-oncogene bcl-2, but the biochemical or functional nature
of Bcl-2 regulation of Ras-induced apoptosis is not understood. We
demonstrate that Bcl-2 and p21
molecules can be
co-immunoprecipitated in Jurkat cells. The level of this association is
enhanced when an apoptotic stimulus (inhibition of PKC activity) is
delivered. Bcl-2/p21
association is coincident with new
phosphorylation of the Bcl-2 protein. Inhibition of this
phosphorylation prevents protection from apoptosis by Bcl-2, providing
a functional correlation to the phosphorylation event. The
Bcl-2/p21
association cannot be competed by exogenous
glutathione S-transferase-Ras fusion protein, suggesting that
the endogenous complex may be formed before cell lysis. These results
provide a possible mechanism of regulation of Ras-induced apoptosis by
Bcl-2.
p21, as key signal transducer, regulates the
proliferation or differentiation of eukaryotic
cells(1, 2) . The activation state of p21
in fibroblasts is determined by the guanine nucleotide exchange
factors such as Sos and GDP-releasing
factor(3, 4, 5, 6) . It has also
been demonstrated that p21
interacts with the
serine/threonine kinase Raf-1, which in turn regulates the activity of
a kinase cascade that includes MEK and mitogen-activated protein
kinases(7, 8, 9) . In T lymphocytes,
p21
may be involved in both PKC(
)-dependent
and PKC-independent activation pathways(1) .
In addition to
its involvement in transducing or promoting cell proliferation or
differentiation signals, p21 is also thought to be
necessary for mediating cell cycle progression, including G
to G
, and G
to M, transitions (11, 12) . Recently, it has been reported that Ras may
be involved in apoptosis mediated by FAS and tumor necrosis factor
receptors(13, 14) . Furthermore, we have demonstrated
that Ras-generated signals lead not only toward cell growth, but also
can initiate apoptosis, depending upon the state of other cellular
signaling mediators, such as Bcl-2 and PKC. This Ras-induced apoptosis
is triggered after suppression of cellular PKC activity and can be
blocked by Bcl-2, a cell survival-promoting factor(15) . A
proto-oncogene product, Bcl-2, can protect cells from apoptosis induced
by certain biological or chemical
reagents(16, 17, 18) . Bcl-2 has been
reported to bind to a human Ras-related protein,
p23
(19) , as well as to a downstream effector
protein kinase, Raf-1(20) .
Since the structure of
p23 is highly homologous to p21
(19, 20) and because p23 can bind to Bcl-2, we
have examined the interactions of these proteins in Jurkat cells
co-expressing the proto-oncogene bcl-2 and v-Ha-ras (PH1/bcl-2 cells). It is shown here that overexpressed,
activated p21
can be co-immunoprecipitated with
overexpressed Bcl-2 in PH1/bcl-2 cells, under normal growth
conditions. The association of these two molecules becomes more obvious
after delivery of an apoptotic signal (down-regulation of PKC) and may
be related to a post-translational modification of Bcl-2,
phosphorylation, which occurs simultaneously. Cell fractionation
experiments suggest that the binding of these molecules may take place
in the cell membrane. The interaction of Bcl-2 and p21
may be likely to be of physiological and biochemical relevance in
the regulation of the Ras-induced apoptosis.
Bcl-2 has been reported to interact physically with R-Ras,
which has 55% identity to p21, or with Raf, which can
bind to p21
and regulate growth
signals(19, 20) , but little is known about the
functional significance, if any, of these interactions. Since the
predicted effector domain of p23
is similar in amino
acid sequence to the corresponding region in p21
, the
human bcl-2 gene was introduced into Jurkat cells (designated
as Jurkat/bcl-2 cells) and PH1 cells (Jurkat cells containing
v-Ha-ras, designated as PH1/bcl-2 cells) to explore
possible biological and physical interactions between p21
and Bcl-2. Specific antibodies were used to determine if these
proteins could be co-immunoprecipitated, under normal growth conditions
or after delivery of a Ras-induced apoptotic signal (down-regulation of
PKC activity, by chronic treatment with the phorbol ester
PMA)(25, 26, 27, 28) . We have
confirmed elsewhere that this treatment, 24 h of exposure to a high
dose (500 nM) of PMA, down-regulates PKC activity in these
cells(15) . The proteins co-immunoprecipitating with the
anti-p21
antibody from transfected or control cell
lysates, untreated or after PMA treatment, were immunoblotted for Bcl-2
protein using an anti-human Bcl-2 antibody (Fig. 1A). A
26-kDa protein, Bcl-2, was detected in anti-p21
immunoprecipitates in lysates from PH1/bcl-2 cells, but
was not observed in lysates from cells expressing only one of the two
transfected genes, or in the control cells. In reciprocal experiments,
where the initial immunoprecipitation was carried out with an
anti-Bcl-2 antibody, immunoblotting with an anti-p21
antibody detected a protein of 21 kDa which was co-precipitated
in PH1/bcl-2 cells, both under normal growth conditions and
after down-regulation of PKC activity (Fig. 1B). The
amount of the co-precipitating protein after down-regulation of PKC
activity by PMA treatment for 24 h was increased in both cases (a
2.3-fold increase in Bcl-2 protein associating with p21
and a 1.8-fold increase in p21
associating with
Bcl-2, as measured by densitometry). As a control, an unrelated IgG
isotype mouse monoclonal antibody was used for immunoprecipitation,
followed by immunoblotting with anti-Bcl-2 or anti-Ras to exclude
nonspecific binding. There was no co-immunoprecipitation of either
Bcl-2 or p21
with this irrelevant, isotype-matched IgG
control antibody (data not shown). To further confirm the specificity
of the association of Bcl-2 and activated p21
,
immunoprecipitation with anti-human Bcl-2 antibody was performed after
[
S]methionine/cysteine metabolic labeling of
transfected and control Jurkat cells, following down-regulation of PKC
activity. 26-kDa (Bcl-2) and 21-kDa (p21
) species were
co-precipitated by the anti-Bcl-2 antibody from the lysates of
PH1/bcl-2 cells, but not from the control Jurkat cells (Fig. 1C). In contrast, only the 26-kDa Bcl-2 protein
was precipitated from lysates of Jurkat/bcl-2 cells. These
results confirmed that activated p21
and Bcl-2 are able
to associate physically in vitro and perhaps in vivo.
The quantitative increase in association of the two molecules after
down-regulation of PKC was not due to changes in the levels of the
Bcl-2 protein. The 26-kDa Bcl-2 protein was detected readily by
immunoblotting lysates from the cells transfected with the bcl-2 gene, both under normal growth conditions and after
down-regulation of PKC, and there was no significant change in Bcl-2
levels under either circumstance (Fig. 1D). In other
studies, we have found that delivery of a different signal for
apoptosis, stimulation through FAS/APO-1, also results in dramatic
enhancement of the association of Bcl-2 and p21
. (
)
Figure 1:
Association of
p21 and Bcl-2. A, co-immunoprecipitation and
detection by immunoblotting. Lysates from cells under normal growth
conditions (lanes 1-4), or after PMA (500 nM)
treatment for 24 h (lanes 5-8), were immunoprecipitated
with an anti-Ras monoclonal antibody and immunoblotted with an
anti-human-Bcl-2 antibody. Lanes 1 and 5,
PH1/bcl-2 cells; lanes 2 and 6, PH1 cells; lanes 3 and 7, Jurkat/bcl-2 cells; lanes
4 and 8, Jurkat cells. B, reciprocal
co-immunoprecipitation and detection by immunoblotting. Lanes 1 and 5, Jurkat cells; lanes 2 and 6,
Jurkat/bcl-2 cells; lanes 3 and 7, PH1
cells; lanes 4 and 8, PH1/bcl-2 cells. C, metabolic labeling and co-immunoprecipitation of
p21
and Bcl-2. After down-regulation of PKC,
S-labeled proteins were immunoprecipitated with an
anti-human Bcl-2 monoclonal antibody. An autoradiogram of the gel is
shown here. Lane 1, PH1/bcl-2 cells; lane 2,
Jurkat cells; lane 3, Jurkat/bcl-2 cells. The
different intensities of the two proteins co-precipitating from the
PH1/bcl-2 cells may reflect the different metabolic turnover
rates of the two proteins, rather than stoichiometric differences. D, immunoblot of Bcl-2 protein from cells under normal growth
conditions (lanes 1-2 and 5-6) or after
down-regulation of PKC (lanes 3-4 and 7-8). Lanes 1 and 3, PH1/bcl-2 cells; lanes 2 and 4, PH1 cells; lanes 5 and 7, Jurkat/bcl-2 cells; lanes 6 and 8, Jurkat cells.
It has been suggested that the Bcl-2 protein requires
post-translational modification, specifically phosphorylation, for
function(29, 30) . To determine whether the increased
binding of Bcl-2 to p21 following suppression of PKC
activity might be related to a modification of Bcl-2, the
phosphorylation state of Bcl-2 protein in vitro was studied.
New phosphorylation of the 26-kDa Bcl-2 protein at increased levels was
detected after down-regulation of PKC activity in PH1/bcl-2 cells which had been preloaded with [
P]ATP (Fig. 2A, lane 5). Bcl-2 was not newly
phosphorylated in Jurkat/bcl-2 cells under these same
conditions.
Figure 2:
Immunoprecipitation and kinase assays. A, cells were permeabilized under normal growth conditions (lanes 1-4), after down-regulation of PKC activity (lanes 5-8), or in the presence of staurosporine after
down-regulation of PKC (lanes 9-12), and labeled with
[P]ATP. Subsequently, the labeled proteins were
immunoprecipitated by an anti-human Bcl-2 antibody. Lanes 1, 5, and 9, PH1/bcl-2 cells; lanes 2, 6, and 10, PH1 cells; lanes 3, 7,
and 11, Jurkat/bcl-2 cells; lanes 4, 8, and 12, Jurkat cells. B, immunoblotting
of labeled proteins. After in vitro phosphorylation and
immunoprecipitation, the phosphorylated proteins from PH1/bcl-2 or PH1 cells were immunoblotted with an anti-human Bcl-2 antibody. Lanes 1 and 2, autoradiograms of immunoprecipitated
proteins; lanes 3 and 4: immunoblots of the same
membranes used for autoradiography in lanes 1 and 2. Lanes 1 and 3, PH1/bcl-2 cells; lanes 2 and 4, PH1 cells.
In normal lymphocytes and cell lines like Jurkat,
down-regulation of PKC activity can comprise a normal physiologic
signal to inhibit cell growth, rather than inducing
apoptosis(25, 26, 27, 31, 33, 34) .
Like parental Jurkat cells, Jurkat/bcl-2 cells only arrest in
the G phase of the cell cycle after down-regulation of PKC
and re-enter the cell cycle when this inhibition is
relieved(15) . Thus, phosphorylation of Bcl-2 appears to occur
only in cells for which inhibition of PKC would invoke a cell death
program (i.e. cells containing activated p21
,
PH1). In similar studies, using metabolic labeling of cells with
[
P]PO
, Bcl-2 was again found to be
phosphorylated exclusively in PH1/bcl-2 cells and only during
stimulation of Ras-induced apoptosis by inhibition of PKC
activity(15) . Immunoprecipitation using an anti-Bcl-2 antibody
after [
P]ATP labeling, followed by simultaneous
autoradiography and immunoblotting, was performed to confirm the
identity of the phosphorylated 26-kDa band as Bcl-2. The labeled 26-kDa
protein was immunoreactive with the Bcl-2 antibody (Fig. 2B, lanes 1 and 3). Another
newly kinased protein species of approximately 20 kDa, which
co-precipitated with Bcl-2, was also detected, and may possibly
represent the Bcl-2 partner, Bax, although its identity has not yet
been confirmed. Phosphorylation of both of these proteins was inhibited
by the serine/threonine kinase inhibitor, staurosporine, which
suggested the involvement of a serine/threonine kinase in Bcl-2
phosphorylation during stimulation of Ras-induced apoptosis (Fig. 2A). In contrast, the tyrosine-protein kinase
inhibitor genistein had no effect on the phosphorylation of Bcl-2 in
this permeabilized cell system (data not shown). If phosphorylation of
Bcl-2 were required for protection from Ras-induced apoptosis,
serine/threonine kinase inhibitors would be expected to abrogate this
protective effect. Inhibition of cellular PKC activity by chronic
treatment with PMA or by treatment with staurosporine led to apoptosis
in cells containing activated p21
, as expected (Table 1), but protection by Bcl-2 (PH1/bcl-2 cells), as
seen in cells treated with PMA, did not occur in the presence of
staurosporine, which also blocks the coincident phosphorylation of
Bcl-2.
It is possible that this post-translational modification of Bcl-2 (phosphorylation) occurs only when cells are facing a stress or death challenge. Whether this phosphorylation of Bcl-2 regulates its survival-promoting activity, and whether this modification is the direct result of down-regulation of PKC, or instead is the result of the activation of a rescue pathway in the face of impending apoptosis remains is not yet clear(15) . In support of a role for phosphorylation of Bcl-2 in promoting survival in these cells is our finding that, whereas overexpressed Bcl-2 protects PH1 cells from undergoing apoptosis during down-regulation of PKC, it does not protect these same cells from apoptosis during inhibition of PKC by staurosporine, a general serine/threonine kinase inhibitor. We have demonstrated herein that the serine/threonine kinase responsible for phosphorylating Bcl-2 under these conditions is sensitive to staurosporine, suggesting a requirement for this post-translational modification of Bcl-2 for its protective function. When a more specific inhibitor of PKC, chelerythrine, was utilized in parallel experiments, chelerythrine was also found to activate apoptosis in PH1 cells and with faster kinetics than did down-regulation of PKC by chronic exposure to PMA, as might be expected from the more rapid action of chelerythrine on inhibition of PKC activity. Yet, protection from Ras apoptosis by Bcl-2 was maintained, in contrast to the findings with staurosporine, and phosphorylation of Bcl-2 in response to apoptotic stimuli was not inhibited(15) .
These results suggested that
p21 and Bcl-2 can interact within cells, under conditions
where the bcl-2 gene is overexpressed, and this binding
becomes prominent in response to activation of the Ras-induced
apoptosis program. To determine whether the formation of this complex
occurred in whole cells, or after lysis of the cells, a GST-Ras fusion
protein bound to agarose was added to the cell lysates, and the
precipitates were immunoblotted with anti-human Bcl-2 and
anti-p21
antibodies. A 26-kDa Bcl-2 band was detected
only in lysates from Jurkat/bcl-2 cells (Fig. 3A). Thus, a direct interaction of p21
and Bcl-2 could be demonstrated in vitro. In the
PH1/bcl-2 cells, however, no such interaction could be
detected. This suggests that the complex of endogenous p21
and Bcl-2 is formed before lysis of the cells, and, that once
formed in vivo, the association of these two molecules could
not readily be competed by addition of exogenous GST-Ras to the cell
lysates for 2 h. In the absence of activated p21
,
inhibition of PKC activity itself does not cause apoptosis in Jurkat
cells ( Table 1and (15) ), and no new phosphorylation of
Bcl-2 protein, even when overexpressed, was detectable (Fig. 2).
The inability of GST-Ras to compete for Bcl-2 in the PH1/bcl-2 cell lysates over 2 h may therefore reflect a higher affinity of
phosphorylated Bcl-2 for p21
in general, or for activated
p21
in particular. To demonstrate that competition for
Bcl-2 could occur as a function of time or concentration of the
competitor (GST-Ras), the length of time of incubation or the
concentration of GST-Ras in the competition experiment was varied. A
26-kDa Bcl-2 band could be seen associating with the GST-Ras beads
after a 12-h competition incubation with PH1/bcl-2 cell
extracts. The quantity of bound Bcl-2 did not increase with increasing
concentrations of GST-Ras (4 µg), suggesting that the GST-Ras was
already in excess relative to the Bcl-2 protein at 2 µg (Fig. 3B). These competition results provide further
evidence that the endogenous Bcl-2/p21
complex formed in vitro is quite stable. p21
and Bcl-2 are both
known to be anchored on the cytosolic face of cytosolic membranes in
the
cell(30, 35, 36, 37, 38) .
p21
requires modifications which allow translocation to
the cellular membrane for transforming
activity(39, 40) , and, similarly, membrane
localization of Bcl-2 appears to be required for its activity in
countering a variety of apoptosis
effectors(10, 24, 32) . To determine if the
association of these two molecules or the phosphorylation of Bcl-2
might alter the subcellular localization of Bcl-2/p21
protein complex, subcellular fractions were examined after
down-regulation of PKC. Proteins from either cytosol or cytosolic
membranes were immunoprecipitated with anti-human Bcl-2 antibody and
subsequently immunoblotted with anti-p21
antibody, as
described above. A protein of 21 kDa was detected only in membrane
fractions from PH1/bcl-2 cells, with none found in the cytosol (Fig. 3C).
Figure 3:
A, affinity binding of Bcl-2 with GST-Ras
fusion protein. Cells were exposed to 500 nM PMA for 24 h and
lysed in lysis buffer containing 2 µg of GST-Ras bound to agarose
beads. The resulting complexes were immunoblotted for the presence of
Bcl-2 and GST-Ras proteins. Lane 1, Jurkat cells; lane
2, Jurkat/bcl-2 cells; lane 3, PH1 cells; lane 4, PH1/bcl-2 cells. The 50-kDa GST-Ras fusion
protein was detected in all four lanes. B, competition binding
of Bcl-2/p21 with GST-Ras fusion protein. Lysates of
PH1/bcl-2 cells were incubated with 2 µg or 4 µg of
GST-Ras beads for 2 h or 12 h. The material complexed with the beads
was separated by electrophoresis and immunoblotted with an anti-human
Bcl-2 antibody. C, membrane association of p21
and Bcl-2. After down-regulation of PKC activity, membrane (lanes 1-4) and cytosol fractions (lanes
5-8) were isolated. Each fraction was immunoprecipitated
with an anti-human Bcl-2 antibody and then immunoblotted with an
anti-Ras antibody. Lanes 1 and 5, Jurkat cells; lanes 2 and 6, Jurkat/bcl-2 cells; lanes
3 and 7, PH1 cells; lanes 4 and 8,
PH1/bcl-2 cells.
These data show that activated
p21 may physically interact with Bcl-2, and that
interactions between these two molecules may become greater when PKC
activity is suppressed. Co-precipitation of Bcl-2 with a small
GTP-binding protein p23
has been reported previously in
the yeast two-hybrid system(19) , but associations with
p21
were not observed. There are several differences
between the two experimental systems employed which may be significant
in this respect. In our studies, p21
is in a
constitutively activated (GTP-bound) state. The p21
/Bcl-2
association was most prominent under conditions favoring apoptosis in
v-ras-expressing cells. Finally, the associations we
demonstrate may require post-translational modification of Bcl-2 and
Ras(30, 39, 40) , which would not be observed
in a two-hybrid system. Membrane localization or a kinase-induced
conformational change of Bcl-2 may quantitatively or qualitatively
influence the interaction of this molecule with p21
.
p21
has been implicated as a mediator of diverse inducers
of apoptosis(13, 14, 15) . The interaction of
Bcl-2 and p21
may thus be of general physiological and
biochemical relevance in the regulation of apoptosis by Bcl-2.