From the Department of Pharmacology, The University of Bath, Bath BA2 7AY, United Kingdom
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ABSTRACT |
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Interleukin-3 (IL-3) acts as both a growth and
survival factor for many hemopoietic cells. IL-3 treatment of
responsive cells leads to the rapid and transient activation of Class
IA phosphoinositide-3-kinases (PI3Ks) and the
serine/threonine kinase Akt/protein kinase B (PKB) and phosphorylation
of BAD. Each of these molecules has been implicated in anti-apoptotic
signaling in a wide range of cells. Using regulated expression of
dominant-negative p85 ( Interleukin-3 (IL-3)1 is
a pleiotropic cytokine that acts as both a growth and survival factor
for a number of hemopoietic cells, including mast cells and basophils
(1, 2). The IL-3 receptor is composed of a 70-kDa specific Phosphoinositide-3 kinases (PI3Ks) are an evolutionarily conserved
family of lipid kinases (reviewed in Ref. 5). The class IA
PI3Ks can utilize phosphoinositide, phosphoinositide (4)P, and
phosphoinositide (4,5)P2 as in vitro substrates,
producing their D3-phosphorylated derivatives. The members of this
class of PI3K are composed of a regulatory/adaptor subunit, most
commonly referred to as p85, and a catalytic subunit, p110. To date,
three genes have been identified that encode 85- and 55-kDa class
IA adaptor molecules, including p85 Recently, both PI3K and PKB have been implicated in generating
anti-apoptotic signals. In neuronal cells, both PI3K (14) and PKB (15)
have been shown to be important mediators of growth factor-induced
survival. PI3K and PKB have also been shown to protect against
c-Myc-mediated apoptosis (16, 17) and anoikis (18) and are involved in
the protection from UV-B-induced apoptosis mediated by IGF-1 (19). In
addition, in a number of cytokine-dependent cells,
activation of PI3K and PKB has been linked to cell survival (20-25).
There has also been the recent suggestion that, at least in IL-2
signaling, PKB/Akt may also be important for cytokine-driven proliferation (24, 26). One of the downstream targets of PKB/Akt is the
proapoptotic member of the Bcl-2 family, BAD. In its unphosphorylated state, BAD complexes with Bcl-2 or Bcl-XL, thereby
inhibiting their function and leading to the initiation of
apoptosis. IL-3 induces phosphorylation of BAD (27, 28), which
renders it susceptible to 14-3-3 binding and hence inactivation
(28).
PI3Ks have also been implicated in controlling DNA synthesis and the
cell cycle (29). Microinjection studies demonstrated a requirement for
p110 Two relatively selective chemical inhibitors of all mammalian PI3Ks,
wortmannin (33, 34) and LY294002 (35), have been used widely in studies
examining the involvement of PI3K in anti-apoptotic signaling and
activation of PKB (15, 17-23, 25, 36). Some investigators have also
used transient expression of dominant-negative p85 molecules to probe
the function of PI3K (26, 37, 38), and others have reported stable
expression of such mutants (39), although the levels of expression
reported are very low (40). Our aim was to specifically investigate the
role of class IA PI3Ks in IL-3 signaling in BaF/3 cells,
which are absolutely dependent on IL-3 for their continued
proliferation and survival. We established regulated expression of the
p85 mutant, cDNA Constructs--
The Cell Culture--
Murine IL-3-dependent BaF/3 cells
expressing the tetracycline transactivator (tTA) from the plasmid
pUHD15-1, containing a puromycin selectable marker, were a kind gift
obtained from Dr. A. Mui, DNAX, Palo Alto, California (44). Cells were
cultured as described previously (45), with the addition of 2 µg/ml
tetracycline. 1 × 107 cells were electroporated in
0.8 ml of electroporation buffer (25 mM HEPES, pH 7.2, 140 mM KCl, 10 mM NaCl, 2 mM
MgCl2, 0.5% (w/v) Ficoll 400, filter sterilized)
containing 10 µg of linearized DNA at 960 microfarads, 450V. Cells
were plated at 5 × 105 for 48 h in the presence
of 2 µg/ml tetracycline and then plated into 96-well trays in 1 mg/ml
G418, 1 µg/ml puromycin, 2 µg/ml tetracycline. Clones of live cells
were picked after 10-14 days, expanded in the same selective medium,
and screened for inducible expression of the introduced cDNAs.
Screening for Tetracycline-regulated Expression--
G418- and
puromycin-resistant clones were expanded in the presence of 2 µg/ml
tetracycline. Cells were washed three times in 1× Hanks' buffered
saline solution containing 20 mM HEPES (Life Technologies)
and plated at 1 × 105 cells/ml in the absence or
presence of 2 µg/ml tetracycline. In order to observe reproducible,
high levels of inducible expression, we found it necessary to culture
the cells at <1 × 105 cells/ml during induction.
Failure to do this resulted in lower levels of expression, presumably
due to ineffective depletion of tetracycline when the cells are at a
higher density and not dividing as rapidly. At various time points
after the initial plating, aliquots of cells were removed, washed once
in phosphate-buffered saline, and lysed at 1 × 105
cells/10 µl in ice-cold solubilization buffer as described previously (45). 20 µg of total cell protein was used per sample for
SDS-PAGE.
Cytokine-dependent Proliferation Assays--
For
both sodium
3'-(1-[(phenylamino)-carbonyl]-3,4-tetrazolium)-bis(4-methoxy-6-nitro)benzene-sulfonic
acid hydrate (XTT) dye reduction assays and
[3H]thymidine incorporation assays, recombinant murine
IL-3 was set up in triplicate at a range of doses (0.5 pg/ml to 2 ng/ml), in serum-free AIM-V medium (Life Technologies, Inc.) in the
presence or absence of 2 µg/ml tetracycline and in flat-bottomed
96-well trays (Nunc). Transfectants were washed three times in Hanks' buffered saline solution, resuspended at 2 × 104
cells in AIM-V medium, and plated at 1000 cells/well in 100 µl of
total volume. Cells were incubated for 72 h at 37 °C. For the XTT dye reduction assays (46), 25 µl of a solution containing 1 mg/ml
XTT and 25 µM phenazine methosulfate (phenazine
methosulfate acts an electron-coupling reagent and is used to
potentiate XTT bioreduction) was added/well for the final 4 h of
incubation. The soluble formazan product was measured at 450 nm on a
Dynatech MR5000 plate reader. For thymidine incorporation assays, 0.5 µCi of [3H]thymidine (ICN) was added/well for the final
8 h of incubation. Counts were harvested using a Filtermate 196 (Packard) onto 96-unifilter GF/C plates (Packard) and dried at 50 °C
for 30 min, and 30 µl of Microscint (Packard) was added/well. Counts
were read on a Packard microplate scintillation counter.
Cell Growth Curve Analyses--
Transfectants were washed three
times in Hanks' buffered saline solution and resuspended at 2 × 104 cells in RPMI or AIM-V medium containing 5% (v/v)
conditioned medium from WEHI3B cells as a source of murine IL-3. Cells
were incubated at 37 °C and counted on a Multisizer II cell counter (Coulter) in duplicate at 24-h intervals.
Apoptosis Assays--
The Cell Death Detection enzyme-linked
immunosorbent assay (Boehringer Mannheim) was used according to the
manufacturer's recommendations. Each sample, 2 × 104
cells, was counted independently twice, and duplicate samples were
analyzed for each condition. Annexin V-fluorescein isothiocyanate and
propidium iodide dual staining was performed according to the
manufacturer's recommendations (Boehringer Mannheim). 10,000 events
were analyzed per sample using a FACS Vantage system (Becton Dickinson).
Induction of Expression in Bulk Cultures and Cell
Stimulations--
Transfectants were washed and resuspended as
described above for screening purposes, but were incubated at 37 °C
for 16-20 h to induce protein expression. Stimulation of these cells
with IL-3 was carried out as described previously (47). Unless
otherwise stated, rmIL-3 was used at a concentration of 20 ng/ml, which we had previously determined to induce maximal levels of tyrosine phosphorylation of cellular substrates. Cell pellets were lysed in
solubilization buffer at between 1 and 2 × 107
cells/ml, and clarified supernatants were used for immunoprecipitation, as described previously (45, 47, 48). The following antibodies were
used for precipitation: 5 µg of 9E10 monoclonal antibody, 1 µg of
goat polyclonal against PKB (sc-1619, Santa Cruz Biotechnology, Inc.),
4 µg of monoclonal anti-BAD antibody (B36420, Transduction Laboratories, Lexington, KY).
PKB Assays--
PKB immunocomplexes were captured on protein
G-Sepharose, 30 µl of a 50% slurry (Amersham Pharmacia Biotech) for
1 h at 4 °C with rotation. Beads were washed twice with
solubilization buffer, twice with LiCl buffer (500 mM LiCl,
100 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 7.5),
and once with kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol).
Beads were resuspended in kinase buffer containing 2.5 µg of H2B, 0.5 µM PKI, 50 µM ATP, and 3 µCi of SDS-PAGE and Immunoblotting--
SDS-PAGE and immunoblotting
were carried out as described previously (48). Primary antibodies were
used at the following concentrations: 0.1 µg/ml anti-phosphotyrosine
monoclonal antibody 4G10 (05-321, Upstate Biotechnology); 1:1000 for
the polyclonal antibodies against Akt and phosphospecific (Ser-473) Akt
(9270, New England Biolabs); 0.5 µg/ml anti-BAD antibody (sc-943,
Santa Cruz Biotechnology, Inc.); and 1:4000 for the polyclonal anti-p85 (06-195, Upstate Biotechnology) antibody. Secondary antibodies conjugated to horseradish peroxidase were used at a concentration of
0.05 µg/ml (Dako, Cambridge, UK). Immunoblots were developed using
the ECL system (Amersham Pharmacia Biotech) and Kodak X-AR 5 film.
Blots were stripped as described previously (48).
Expression of Dominant-negative p85
Given the potential role of PI3Ks in proliferation and cell survival,
we felt it likely that constitutive expression of Expression of
The effects observed on IL-3-induced proliferation could, to some
extent, be reversed in tetracycline readdition experiments. XTT assays
were set up as above, but tetracycline was added back to cells that had
been incubated for either 24 or 48 h in the absence of
tetracycline. The results for clone
As a second indicator of the effects of
In a third type of analysis, we examined the growth characteristics of
the Expression of Expression of
We also examined the effects of PI3K and its downstream target PKB have been widely implicated in
transducing survival signals in a wide variety of cells. Many of these
studies have used chemical inhibitors of PI3Ks and have implied that
the anti-apoptotic role of PI3K is its major function in growth
factor-regulated cell signaling (15, 17-22, 25). IL-3 regulates
activation of the class IA family of PI3K (6, 8); hence, in
order to address the specific involvement of these PI3Ks in
IL-3-dependent cell signaling, we have used a
dominant-negative version of p85 ( Having shown that expression of Other investigators have primarily used transient expression of
dominant-negative mutants of p85 to probe PI3K function in different
cell systems. In IL-2 signaling, PI3K has been shown to be upstream of
the transcription factor E2F, which is activated during G1,
thereby linking IL-2 and PI3K with cell cycle machinery (26) and
indicative that PI3Ks may play a role in proliferation. In 3T3-L1
adipocytes, adenovirus-mediated expression of the N-terminal src homology region 2 domain of p85 resulted in inhibition
of insulin-stimulated DNA synthesis of serum-starved cells, providing evidence that class IA PI3Ks are also important for
insulin-stimulated mitogenesis (38). Recently, inducible activation of
an active version of p110 The XTT dye reduction assay we used as one method of assessing
IL-3-induced survival is based on the reduction of XTT by
NAD/NADPH-dependent oxidoreductases (46) and is believed to
be a good indicator of cellular metabolic activity (50). It has also
been reported that these assays are a measure of glycolytic flux (56).
PI3K and PKB activity have been implicated in regulation of glucose uptake in response to insulin (39, 57-59), but the role of PI3Ks in
cytokine-mediated glucose transport is ill defined. These results suggest PI3K may also play a role in IL-3-induced glucose transport, and it will be interesting to investigate this in detail.
When characterizing proteins inducibly tyrosine-phosphorylated by
IL-3 in Using inducible expression of dominant-negative p85) in stably transfected IL-3-dependent BaF/3 cells, we have specifically
investigated the role of class IA PI3K in IL-3 signaling.
The major functional consequence of
p85 expression in these cells is
a highly reproducible, dramatic reduction in IL-3-induced
proliferation. Expression of
p85 reduces IL-3-induced PKB
phosphorylation and activation and phosphorylation of BAD dramatically,
to levels seen in unstimulated cells. Despite these reductions, the
levels of apoptosis observed in the same cells are very low and do not
account for the reduction in IL-3-dependent proliferation
we observe. These results show that
p85 inhibits both PKB activity
and BAD phosphorylation without significantly affecting levels of
apoptosis, suggesting that there are targets other than PKB and BAD
that can transmit survival signals in these cells. Our data indicate
that the prime target for PI3K action in IL-3 signaling is at the level
of regulation of proliferation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain
and a 125-kDa
chain, both of which are members of the hemopoietin
receptor superfamily lacking catalytic activity (3). However, IL-3 has been shown to induce activation of both Jak-2 and Src family tyrosine kinases, which correlate with the rapid tyrosine phosphorylation of a
number of cellular proteins and activation of intracellular signaling
cascades, including the Ras/extracellular-regulated kinase kinase
pathway, p38 and SAPKs, STAT-5, SHP2, and class IA
phosphoinositide-3 kinases (reviewed in Ref. 4).
and
. Three p110
isoforms,
,
, and
have also been described (5), with p110
expression largely restricted to leukocytes (6, 7). Both p110
and p110
have previously been shown to be coupled to IL-3 signaling (6,
8). PI3K activation has been implicated in cell survival, proliferation/mitogenesis, cell cycle regulation, membrane trafficking, glucose transport, cell metabolism, cytoskeletal rearrangement, and
membrane ruffling (5, 9). The search for the molecular intermediates
that couple PI3K to these effector systems above have identified a
number of downstream targets of PI3K activation, including protein
kinase C isoforms (10) and the serine/threonine kinase PKB (also known
as Akt) (11-13).
activity in platelet-derived growth factor-stimulated proliferation of 3T3 cells (30), and activation of an inducible version
of p110
is sufficient to induce progression of cells through
G1 and into S phase (31). Melanoma cells can be arrested in
G1 by the PI3K inhibitor LY294002 (32), and in IL-2
signaling, inhibition of PI3K results in a lack of activation of the
G1 transcription factor E2F (26).
p85, in stably transfected BaF/3 cells (see under
"Experimental Procedures" for details).
p85 lacks the p110
binding site and has been previously shown to act in a
dominant-negative manner by blocking catalytic activation of the p110
subunit (39). We show that expression of
p85 resulted in a dramatic
reduction of IL-3-induced proliferation of BaF/3 cells and effectively
abrogated the ability of IL-3 to induce PKB activation and BAD
phosphorylation. However, in stark contrast to results obtained using
PI3K inhibitors, we observed negligible changes in apoptosis when
p85 was expressed, effectively separating proliferation, PKB
activation, and BAD phosphorylation from apoptosis. These results
suggest that class IA PI3K play a complex role in integrating IL-3-induced signals to proliferative machinery and that
targets other than PKB and BAD are involved in providing survival
signals in these cells, raising the possibility that this is also the
case in other cellular systems.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
p85 form of bovine p85
(lacking amino acid residues 479-513, constituting the p110 binding
site) was a kind gift of Dr. M. Kasuga (Kobe University, Kobe, Japan)
(39). The coding sequence was amplified with Vent DNA polymerase (New
England Biolabs) according to the manufacturer's recommendations. The
following oligonucleotide primers were used: sense primer,
incorporating a BamHI site for in-frame cloning into the
Myc epitope tagging vector, pBluescript-N-Myc2,
5'-TAGGGATCCAGTGCCGAGGGGTAC-3'; antisense primer, including an
EcoRI site for cloning purposes,
5'-ATCGAATTCTCATCGCCTCTGCTG-3'. Amplification was carried out for 30 cycles in a Perkin-Elmer 9600 thermocycler, with a melting
temperature of 55 °C. Polymerase chain reaction products
were digested with BamHI and EcoRI and subcloned
into pBluescript-N-Myc2. This vector was generated by cloning duplexed
oligonucleotides incorporating the following sequence elements into
NotI-BamHI-restricted pBluescript. The oligo
contained a tandem copy of the sequence encoding the decapeptide recognized by the c-Myc monoclonal antibody 9E10 (41). Also included
were a Kozak sequence, an initiating methionine residue, and a
glycine-serine linker following the second epitope encoding sequence. A
BamHI site at the 3'-end of the oligonucleotide allows for
the in frame fusion to cDNAs of interest, generating N-terminally Myc epitope-tagged proteins. The sequence of the sense oligonucleotide was
5'-GCGGCCGCGTCGACCACCATGGAGCAGAAGCTTATCAGCGAGGAGGACCTGGGAGGAGGACAAAAGCTCATCAGCGAGGAGGACCTGATCAGCAGCGGCGGCAGCGGCGGCGGCGGATCC-3'. The sequence of the epitope tag was confirmed by dideoxy
sequencing. Epitope-tagged
p85
cDNA was subcloned into the
vector pUHD10-3neo (42, 43), which contained a neomycin resistance
gene for direct selection of transfectants. pUHD10-3neo containing
p85 or vector alone was restricted with PvuI to linearize
prior to electroporation.
-ATP
and incubated at room temperature for 30 min. Reactions were stopped by
addition of 5× SDS-PAGE sample buffer and boiling for 5 min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in
IL-3-dependent BaF/3 Cells--
The PI3K inhibitors
wortmannin and LY294002 have been widely used to study the role of
PI3Ks in a range of cell systems. However, despite the fact that these
inhibitors are relatively selective, they can inhibit all three classes
of mammalian PI3Ks and so cannot be used to clearly distinguish the
biological roles of these different families of PI3K enzymes. In order
to specifically investigate the role of class IA PI3Ks in
IL-3-induced signaling events, we have expressed a dominant-negative
version of the regulatory p85 subunit in IL-3-dependent
BaF/3 cells.
p85 lacks amino acid residues 479-513, previously been
shown to be the region of p85, which interacts with the p110
subunit
(39, 49).
p85 may prove to
be toxic to IL-3-dependent cells, hence we chose to use the
tetracycline-regulated gene expression system (42, 43). In this system,
the presence of tetracycline represses the activity of the
tetracycline-sensitive transactivator tTA. BaF/3 cells already
stably expressing tTA from the plasmid pUHD15-1 (44) were
electroporated with the response plasmid (pUHD10-3neo) encoding
N-terminally Myc epitope-tagged
p85, or vector without insert as a
control, and stable clones were selected.
p85 clones were assessed
for inducible expression of
p85 by performing tetracycline removal
time course analyses. Three representative
p85-expressing clones
(termed
p85 1A9, 1B2 and 1D8), which showed low basal expression in
the presence of tetracycline and high inducible expression when
tetracycline was absent, were selected for detailed analyses. Fig.
1A, upper panel, shows the
induction of
p85 expression upon tetracycline removal over a period
of 72 h.
p85 was specifically detected with the anti-Myc
monoclonal antibody 9E10. In all cases,
p85 expression was observed
within 7 h of tetracycline removal and was maximal 24 h after
removal of tetracycline. To compare the levels of expressed
p85 to
endogenous p85, the same immunoblots as in Fig. 1A were
reprobed with anti-p85 antibodies (see Fig. 1A, lower
panel). We consistently observed at least a 5-10-fold overexpression of the
p85 variant. Readdition of tetracycline after
an initial 24 h induction phase resulted in down-regulation of
p85 expression to basal levels within 24 h (see Fig. 1A,
upper and lower panels,
/+ lanes), and the
half-life of
p85, estimated from the decrease in levels of
expression upon tetracycline readdition, was between 4 and 6 h, as
shown in Fig. 1B.
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Fig. 1.
Inducible expression of
p85 in BaF/3 cells. A, cells were
incubated in the presence (+) of 2 µg/ml tetracycline or in its
absence (
), and aliquots of cells were removed at the indicated times
after the initiation of induction. In some cases (
/+), cells were
incubated for 24 h in the absence of tetracycline, prior to 2 µg/ml of tetracycline being added back to the cultures. Aliquots of
cells were then removed 24 and 48 h later, corresponding to 48 and
72 h after the initiation of induction. B, cells were
plated as described above, and after the initial 24 h induction
phase in the absence of tetracycline, tetracycline (tet) (2 µg/ml) was added back to the cultures and samples were removed at the
time intervals (in hours) indicated. Immunoblotting was carried out
first with 9E10, which recognizes the Myc epitope tag on the expressed
p85 protein (upper panels). The same immunoblots were
stripped and reprobed with a polyclonal antibody against p85
(lower panels). Molecular mass standards are shown in kDa,
and the positions of the expressed
p85 and endogenous p85 proteins
are indicated.
p85 Dramatically Decreases Proliferation in
Response to IL-3--
We were interested to determine what effect
expression of
p85 had on IL-3-induced survival and proliferation.
BaF/3 cells expressing
p85 were analyzed in IL-3 dose-response XTT
assays. These assays are based on the reduction of XTT by NAD/NADPH
oxidoreductases and are a measure of cells metabolic activity and
growth (46, 50). The results shown in Fig.
2 demonstrate that when
p85 was
expressed, there was a consistent and dramatic reduction in IL-3-induced proliferation, with no effect in empty vector cells. Although there was a dramatic reduction in the maximal response to IL-3
in all three
p85 clones, the ED50 values for
IL-3-responsiveness were largely unchanged at 10-20 pg/ml rmIL-3.
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Fig. 2.
Expression of p85
leads to a reduction in IL-3 responsiveness in XTT dye reduction
assays. Assays were set up as described under "Experimental
Procedures." Squares represent cells incubated in the
presence of 2 µg/ml tetracycline, and diamonds represent
cells in the absence of tetracycline. In the readdition experiment,
p85 1D8 cells were set up as above with additional samples
maintained for the first 24 or 48 h in the absence of
tetracycline, and at 24 h (circles) or 48 h
(triangles), tetracycline was added back to the cultures for
the remaining period of the 72-h total incubation time. The mean values
with S.D. are plotted for each point. In all cases, readings obtained
in the absence of IL-3 were, on average, 0.19-0.2 absorbance
units.
p85 1D8 are shown in Fig. 2, and
similar results were observed for all the
p85 clones. When
tetracycline was added back at 24 h and cells were incubated for a
further 48 h, we observed a recovery in the maximal IL-3-responsiveness to an intermediate level when compared with cells
maintained throughout the assay in the presence or absence of
tetracycline. This shows the cells can be partially rescued from the
inhibitory effects exerted by the
p85 variant, i.e. they
are not irreversibly committed.
p85 expression on
IL-3-induced proliferation, we performed assays similar to those described above, using thymidine incorporation to measure DNA synthesis. As shown in Fig. 3, a
reduction in the incorporation of [3H]thymidine in
response to IL-3 was observed when
p85 was expressed. These results
closely reflect the responses observed in the XTT assays.
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Fig. 3.
Expression of p85
leads to a reduction in IL-3-induced DNA synthesis. Cells were set
up as described in the legend to Fig. 2. Incorporation of
[3H]thymidine into DNA was determined after 72 h.
The mean values with S.D. are shown for each point. Incorporation in
the absence of any IL-3 was, on average, 525 cpm.
p85-expressing cells by performing growth curve analyses, the
results of which are shown in Fig.
4A. We observed a 20-30%
reduction in cell numbers in the absence of tetracycline at 24 h
and a 35-50% reduction in cell numbers after 48 and 72 h of
incubation. The data from these three types of assays show that
p85
perturbs the IL-3-induced signals required for proliferation of BaF/3
cells.
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Fig. 4.
The growth response to IL-3 is reduced in
the p85-expressing cells with only small
increases in apoptosis. A, growth curve analyses were
performed on the
p85 clones and the empty vector clone 2E5. Data
from at least three independent experiments were pooled, and the total
number of cells present in the absence of tetracycline (tet)
was compared with the total number of cells in its presence. Maximal
growth for each clone in the presence of tetracycline was set at 100%.
Growth in the absence of tetracycline is expressed as a percentage of
this maximal value. Mean values and S.D. are shown for each data point.
B,
p85 1D8 cells were plated at 2 × 104
cells/ml in the presence or absence of tetracycline. Aliquots of cells
were removed after 72 h and stained with Annexin V-fluorescein
isothiocyanate and propidium iodide. Cells were analyzed by flow
cytometry using a FACS Vantage system (Becton Dickinson), and 10,000 events were recorded for each sample.
p85 Results Only in Very Small Increases in
Apoptosis--
The reduction in IL-3-induced proliferation observed
above could be due to either increased apoptosis of the cells upon
p85 expression, the cells progressing through the cell cycle more slowly, or the cells becoming blocked at a certain stage of the cell
cycle and so not progressing. We wanted to assess whether apoptosis was
playing a major role in the reduction in IL-3-induced proliferation
that we observed. When we analyzed DNA fragmentation using an
enzyme-linked immunosorbent assay as a measure of apoptosis, we
observed small (1.6-1.9-fold) increases in apoptosis when cells were
cultured in the absence of tetracycline, compared with the values for
cells in the presence of tetracycline (see Table
I). However, the levels of overall
apoptosis measured in these cells were very low, compared with the
effects of LY294002, when the levels of apoptosis were 8-32-fold (data
not shown). To gain a clearer picture of the total number of cells
undergoing apoptosis in the
p85 populations, we performed dual
Annexin V/propidium iodide staining, an example of which is shown in
Fig. 4B. When
p85 was expressed, we observed only very
small increases in the number of apoptotic cells at 24, 48, and 72 h (see Table II), which were typically
from approximately 1% in the presence of tetracycline to 3% in its
absence. These results demonstrate that the decrease in IL-3
proliferation we observed when
p85 was expressed cannot be accounted
for by increased apoptosis. It appears that we have effectively
separated proliferation from apoptosis in these cells and that class
IA PI3Ks are important for regulating IL-3 growth, but are
not absolutely required for survival controlled by IL-3.
Apoptosis in p85 clones
Percentage of cells undergoing apoptosis
p85 Interacts with Tyrosine-phosphorylated Proteins in Response
to IL-3--
We have previously identified a specific set of
tyrosine-phosphorylated proteins that co-precipitate with p85 following
IL-3 treatment of murine cells. Detailed investigation of these protein interactions has led us to propose a mechanism whereby IL-3 recruits PI3K to the plasma membrane (45). Briefly, IL-3 induces tyrosine phosphorylation of the IL-3 receptor
chain, which creates a docking
site at Tyr-612 for the tyrosine phosphatase SHP2 (51). SHP2 inducibly
associates with a 100-kDa phosphotyrosine protein, and a portion of
this same p100 protein interacts with the p85 subunit of PI3K (45, 52).
This facilitates translocation of PI3K to the vicinity of the membrane
and its substrates. We were interested in determining whether the
overexpressed
p85 protein interacts with a similar profile of
tyrosine-phosphorylated proteins as endogenous p85, which would
indicate its mechanism of action in our system. In addition, we
examined the levels of overall tyrosine phosphorylation in total cell
lysates. The results for
p85 1A9 and 1D8 clones are shown in Fig.
5. In the presence of tetracycline, the
IL-3-induced profile of tyrosine phosphorylation resembled that
observed in normal BaF/3 cells (45). When
p85 was expressed, we
observed the constitutive tyrosine phosphorylation of a protein of
approximately 110 kDa. We also observed an IL-3-induced increase in the
tyrosine phosphorylation of proteins of 100 and 170 kDa. When
p85
was specifically precipitated, in addition to the expected
co-precipitation with SHP2 and p100, we observed co-precipitation with
the tyrosine-phosphorylated 170-kDa protein. Therefore, the expressed
p85 protein interacts with a similar profile of
tyrosine-phosphorylated proteins as endogenous p85, suggesting that it
exerts its dominant-negative effects by competing with endogenous p85
for binding to tyrosine-phosphorylated proteins. We unexpectedly
observed increased tyrosine phosphorylation of additional proteins in
both total cell lysates and complexed with
p85, which we are
currently investigating in greater detail.
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Fig. 5.
Association of p85
with tyrosine-phosphorylated proteins.
p85 clones 1A9 and 1D8
were incubated in the presence (+tet) or absence
(
tet) of 2 µg/ml tetracycline for 18 h prior to
being stimulated for 10 min with 20 ng/ml rmIL-3 (3), or
left untreated as controls (C). Samples of total cell
extracts were removed as the preimmunoprecipitation samples (Pre
IP). The equivalent of 5 × 106 cells were used
to perform 9E10 immunoprecipitations. Immunoblotting with 4G10 was
carried out to detect tyrosine-phosphorylated proteins
(
-PY). Molecular mass standards are shown in kDa, and the
positions of p170, p110, p100, and SHP2 are indicated.
p85 Reduces IL-3-induced Phosphorylation and
Activation of PKB and Phosphorylation of BAD--
Protein kinase B is
a downstream target of PI3K activation, and it has been proposed that
PKB plays a major role in transducing anti-apoptotic signals within
cells. Therefore, to determine the effect of
p85 expression on
IL-3-induced PKB activation, we assessed PKB phosphorylation at
Ser-473, which, together with phosphorylation of Thr-308, is required
for full activation of PKB. Expression of
p85 effectively abrogated
IL-3-induced phosphorylation of PKB at Ser 473, illustrated by the time
course analyses shown in Fig.
6A. Expression of
p85 also
reduced IL-3-induced PKB activity to the basal level observed in
unstimulated cells when measured in in vitro kinase assays
(see Fig. 6B, upper panel). Therefore, PKB activity is
effectively knocked out when
p85 is expressed. Similar results were
observed in all clones (data not shown).
View larger version (43K):
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Fig. 6.
PKB phosphorylation and activation and BAD
phosphorylation are reduced in the
p85-expressing cells. Cells were incubated in
the presence or absence of 2 µg/ml tetracycline (tet) for
18 h. A, cells were treated for the indicated periods
of time with 20 ng/ml rmIL-3 or left untreated as controls
(0). Extracts, equivalent to 4 × 105
cells, were immunoblotted with an antibody specific for phosphorylated
serine 473 of PKB (
-phosphoSer 473 PKB). B,
cells were treated for 2 min with rmIL-3 at 20 ng/ml (3) or
left untreated as controls (C). PKB immunoprecipitates were
prepared from the equivalent of 5 × 106 cells and
subjected to in vitro kinase assays using H2B as the
substrate. Blots were cut in half, and the lower part was subjected to
autoradiography, shown in the top panel. The upper halves of
the blots were probed with anti-PKB antibody to check for equivalent
loading and are shown in the lower panel. C,
extracts of
p85 1D8 were prepared as described in the legend to Fig.
5, and the equivalent of 2 × 107 cells were used per
anti-BAD precipitation. SDS-PAGE was performed through 12.5% low
bisacrylamide gels, and immunoblotting carried out with anti-BAD
antibody (sc-943). In each panel, molecular mass markers are indicated
in kDa, as are the positions of phosphorylated PKB (arrow in
A), PKB, H2B, phosphorylated BAD (pBAD) and BAD,
as appropriate.
p85 expression on BAD
phosphorylation by a combination of immunoprecipitation and
immunoblotting. This allowed us to distinguish between the faster
migrating unphosphorylated form of BAD and the slower migrating
phosphorylated form of BAD. The results are shown in Fig. 6C
for the
p85 1D8 clone. In the presence of tetracycline, addition of
IL-3 led to a clear shift of the majority of BAD to its slower
migrating phosphorylated form. However, expression of
p85 completely
inhibited this IL-3-induced shift, with all detectable BAD remaining in
its unphosphorylated form. IL-3-induced BAD phosphorylation was
unaffected in the empty vector cells by the presence or absence of
tetracycline (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
p85), which lacks the p110 binding
site and so effectively blocks PI3K activation (39, 49). We established
regulated high levels of expression of
p85 in stable transfectants
of the IL-3-dependent cell line BaF/3 using the
tetracycline-regulated system, which has not been previously reported
for
p85. Using this powerful system, we have obtained evidence that
shows that the major role played by class IA PI3Ks in IL-3
signaling is control of proliferation, because we observed a dramatic
decrease in IL-3-dependent proliferation when
p85 was
expressed. When we examined whether this decrease in proliferation was
due to increased apoptosis, we were surprised to discover that only a
small percentage of cells (2-5%) were undergoing apoptosis, and this
increased only slightly when
p85 was expressed. These results
contrast starkly to the findings of others, in which the overwhelming
effect of PI3K inhibitors is to lead to apoptosis (15, 17-19). In
IL-3-dependent MC/9 mast cells in the presence of IL-3,
50% of the cells are apoptotic following a 10-h incubation with 25 µM LY294002 (20, 21). We examined cells 24, 48, and 72 h following induction of
p85 expression and still observed only very low levels of apoptosis. One possible explanation for the
differences observed in the effects of the PI3K inhibitors versus
p85 expression is that the inhibitors target all
known mammalian PI3Ks. The class III PI3K include the human homologue of Vps34 and are important in membrane transport (53), and inhibition of this PI3K would no doubt be detrimental to cellular viability. Our
results suggest that the role of PI3Ks in proliferation can be
separated from its involvement in cell survival signaling.
p85 was not having a significant
effect on levels of apoptosis, we were interested to investigate the
underlying biochemical consequences of
p85 expression in IL-3-dependent signaling. We noted that both PKB
phosphorylation and activation were effectively abrogated upon
p85
expression, consistent with previous reports using
p85 (12) and PI3K
inhibitors (15, 17-22, 25, 36). IL-3-induced BAD phosphorylation was completely prevented upon expression of
p85, results that are consistent with reports that have shown IL-3-induced BAD
phosphorylation to be decreased in cells treated with PI3K inhibitors
(21, 23, 27). What is interesting is that despite these dramatic
effects we report on PKB activity and BAD phosphorylation, which were assessed 18-24 h after induction of
p85 expression, when we
examined apoptosis at 24, 48, and 72 h after induction of
p85, we observed only very small increases in the number of cells
undergoing apoptosis. Previous reports have made a strong argument in
favor of PKB activation and BAD phosphorylation being required to
prevent apoptosis, although the true functional significance of BAD
phosphorylation is not clear and may vary depending on cell type.
However, we have shown in this study that regulated expression of
p85 inhibits both PKB activity and BAD phosphorylation, without
significantly affecting levels of apoptosis. In an additional
study,2 we observed a lack of
correlation between PKB activation, BAD phosphorylation and the ability
of a particular cytokine to promote cell survival in three different
cytokine-dependent cells. In addition, it has been shown
that wortmannin fails to induce apoptosis of FDC-P1/Mac1 cultured in
the presence of IL-3 (25), and it has recently been suggested for
insulin-like growth factor-I that there are both
PKB-dependent and -independent survival pathways (54). In
light of these reports, there are a number of possible interpretations
of the data we have presented in this study. First, PKB activation may
be more important for IL-3-proliferative responses than anti-apoptotic
signaling; thus, when expression of
p85 reduces PKB activity,
proliferation is also reduced. A second possibility is that although we
can barely detect any activation of PKB when
p85 is expressed, we
cannot discount the formal possibility that there may a very low level
of PKB activity present, which is sufficient to provide the cells with
a survival signal. Third, the ratio of both pro- and anti-apoptotic
members of the Bcl-2 family, as well as their phosphorylation status,
is important for determining whether cells survive or apoptose, and
different family members may play dominant roles in different cell
types. Thus, although
p85 expression prevents BAD phosphorylation,
it is possible that this is not a major pathway in BaF/3 cells and that
other proapoptotic proteins, e.g. BAX, play a more critical
role. A fourth interpretation of our results is that targets other than
PKB and BAD are involved in IL-3-induced anti-apoptotic signaling, and
the adaptor molecule Shc has been suggested to play such a role (55).
Only by expressing both dominant positive and negative PKB mutants and
BAD mutants, which cannot be phosphorylated at serines 112 and 136, in
the same cell system will we be able to fully distinguish between these possibilities.
has been shown to be sufficient for cells
to progress through G1 and into S phase (31). Therefore, if
class IA PI3Ks are required for G1 progression,
then blocking their activity, as we have done in this study, may well
lead to perturbation of the cell cycle and so reduce proliferation. It
will be interesting to investigate in detail what effects
p85
expression has on regulation of the cell cycle by IL-3.
p85-expressing and nonexpressing cells, we observed increased IL-3-induced tyrosine phosphorylation of proteins of 100 and
170 kDa when
p85 was expressed. The p100 molecule is likely to be
the same protein that interacts with both PI3K and SHP2 and that we
know can be dephosphorylated by SHP2 (45). In addition, we have
identified the 170-kDa molecule as
IRS-2.3 A number of
possibilities exist that could explain these observations: 1) the
expression of
p85 leads to decreased tyrosine phosphatase activity,
and hence to increased tyrosine phosphorylation of certain substrates;
2) expression of
p85 leads to increased tyrosine kinase activity,
resulting in increased tyrosine phosphorylation of certain substrates;
and 3) expression of
p85 results in the src homology
region 2 domains having a protective effect, thus preventing
dephosphorylation of substrates. How might these effects be achieved by
p85? One possibility is that additional adaptor roles of p85, via
SH3, proline-rich, or Bcr region-mediated interactions, are important
in controlling one or more intracellular pathways involved in tyrosine
phosphorylation or dephosphorylation events. Alternatively, PI3Ks have
both protein and lipid kinase activity, although apart from p85 itself
(60) and IRS-1 (61), no substrates for PI3K protein kinase activity
have been identified. It is formally possible that downstream of PI3K
there are both lipid and protein targets that are involved in feedback
loops to protein tyrosine phosphatases and kinases. Disruption of PI3K
activity may deregulate these feedback mechanisms, leading either to a
decrease or increase in activity of the target enzyme. We are currently
devising strategies to investigate this in more detail.
p85 in stably
transfected IL-3-dependent BaF/3 cells, we have
investigated the specific role of class IA PI3K in IL-3
signaling. The major functional consequence of expression of
p85
that we observed was a dramatic reduction in IL-3-induced
proliferation, which was coupled with a reduction in PKB
phosphorylation and activation and phosphorylation of BAD. Despite
these effects, only a small percentage of the cells underwent
apoptosis. These findings contrast markedly with results reported on
the use of PI3K inhibitors, which have suggested that the major role
for PI3K is in generating anti-apoptotic signals. Our results
effectively dissociate apoptosis from proliferation, PKB activation,
and BAD phosphorylation and raise the possibility that more detailed
analyses of other systems may find this to be widely applicable.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. Mui and DNAX for the BaF/3 cells expressing tTA; Dr. S. Abbot for help with FACS analyses; and Drs. B. Vanhaesebroeck, S. Ward, and H. Wheadon for critical comments on the manuscript.
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FOOTNOTES |
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* This work was supported by a Medical Research Council project grant (to M. J. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-1225-826428;
Fax: 44-1225-826114; E-mail: M.J.Welham{at}bath.ac.uk.
2 H. J. Hinton and M. J. Welham, submitted for publication.
3 B. L. Craddock, H. K. Bone, and M. J. Welham, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: IL, interleukin; PAGE, polyacrylamide gel electrophoresis; PI3K, phosphoinositide-3 kinase; PKB, protein kinase B; XTT, sodium 3'-(1-[(phenylamino)-carbonyl]-3,4-tetrazolium)-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate; rmIL, recombinant murine interleukin; tTA, tetracycline transactivator.
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REFERENCES |
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