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Department of Pharmacology, University of Bath, Bath, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The widespread use of two chemical inhibitors of PI3K, wortmannin and LY294002, have led to the suggestion that PI3K activation is important for providing a survival signal to many different cell types, since treatment with these inhibitors results in increased apoptosis (6, 7, 8, 9, 10, 11). A key downstream target of PI3K in mediating this survival signal has been identified as the serine/threonine kinase, protein kinase B (PKB; also known as Akt; 12). PKB was first identified due to its homology to PKA and PKC and as the cellular homologue of the oncogene v-Akt (13, 14). It is activated in response to a number of growth factors, including platelet-derived growth factor (15), insulin (16, 17), epidermal growth factor, basic fibroblast growth factor (18), IL-3 (11, 19, 20, 21), IL-4, stem cell factor (SCF), and GM-CSF (11). Activation is brought about by a combination of phosphorylation and targeting to the cellular membrane via an N-terminal PH domain (22, 23, 24). Active PKB is phosphorylated on two C-terminal sites, Thr308 and Ser473 (25), the former is phosphorylated by the PI3K-dependent kinase, PDK1 (26). The only direct downstream substrates of PKB identified to date are the glycogen synthesis up-regulator glycogen synthase kinase-3 (16) and the proapoptotic Bcl-2 family member Bad (19, 20).
The Bcl-2 proteins are intracellular regulators of apoptosis. Overexpression of the antiapoptotic members (e.g., Bcl-2, Bcl-XL) prevent cell death, while overexpression of the proapoptotic members (e.g., Bax, Bak, Bad) promote cell death (reviewed in Refs. 27, 28). Bcl-2 family members are able to form homo- and heterodimers through the interactions of their Bcl-homology (BH3) domains (27). These interactions and the relative protein levels are thought to determine the fate of the cell (27, 28). An additional level of regulation is the modification of Bcl-2 family members by survival factor-induced phosphorylation, altering the binding properties of family members. For example, Bad forms heterodimers with both Bcl-2 and Bcl-XL, neutralizing their antiapoptotic affects and thus promoting cell death (29, 30, 31). However, in the presence of IL-3, Bad becomes phosphorylated on Ser112 and Ser136, resulting in its association with the protein adaptor 14-3-3. This abrogates its interaction with Bcl-XL, allowing Bcl-XL to promote survival of the cell (31).
There is some conflicting evidence as to the requirement of PI3K and
PKB in survival signaling in hemopoietic cells. The use of PI3K
inhibitors and overexpression of PKB mutants have led to the suggestion
that PI3K, PKB, and Bad are essential elements in antiapoptotic
signaling (19, 20, 21). In contrast, it has been shown that GM-CSF can
provide survival signals independently of PI3K activity, PKB
activation, and Bad phosphorylation (11) and that, in the presence of
IL-3, wortmannin does not induce apoptosis of factor-dependent
(FD)-P1/Mac1 cells (9). In addition, we have recently shown, using
regulated expression of dominant-negative PI3K in stably transfected
BaF/3 cells, that expression of 
p85 dramatically reduces
IL-3-induced PKB activation and phosphorylation of Bad, without having
a significant effect on levels of apoptosis (32). Thus, PI3K and PKB
activation may not be absolutely required, or indeed not be sufficient,
for survival signaling. The aim of this study was to address this issue
by examining the relationships between cytokine-induced activation of
PKB, phosphorylation of Bad, and cell survival. We show that there is
not an absolute correlation between PKB activation, Bad
phosphorylation, and cell survival, and have identified cell
type-specific responses to IL-4. Thus, it appears that PKB activation
alone is not sufficient to promote cell survival, and may indeed not be
necessary, suggesting that other pathways must also be important for
cytokine-induced survival of hemopoietic cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells were maintained at 37°C, 5% (v/v) CO2 in a humidified incubator in RPMI 1640 medium supplemented with 10% (v/v) FBS (Life Technologies, Paisley, Scotland), 20 µM 2-ME, 100 U penicillin/streptomycin, and 2 mM glutamine (RPMI media). BaF/3 and MC/9 cells were cultured with the addition of 5% (v/v) conditioned media from WEHI3B cells as a source of murine IL-3. FD-6 cells were cultured with the addition of 5% (v/v) conditioned media from X63omIL-4 as a source of murine IL-4 (33).
Cell stimulations
Cytokine stimulations were conducted as previously described (34) using concentrations of factor previously shown to produce maximal tyrosine phosphorylation: IL-3, 20 ng/ml; SCF, 50 ng/ml (murine recombinant; R&D Systems, Minneapolis, MN); IL-4, 20 µg/ml; GM-CSF, 5 µg/ml (synthetic; gifts from Dr. I. Clark-Lewis, Biomedical Research Centre, Vancouver, Canada); insulin, 5 µg/ml (Sigma, St. Louis, MO). Cell pellets were lysed in solublization buffer (50 mM Tris-HCl (pH 7.5), 10% (v/v) glycerol, 1% (v/v) Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 1 mM sodium orthovanadate, 1 mM sodium molybdate, 10 mM sodium flouride, 40 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, 0.7 µg/ml pepstatin) as previously described (34).
XTT bioreduction assays
Recombinant cytokines were set up in triplicate at a range of doses in RPMI media (see above) and in flat-bottom 96-well trays (Nunc, Naperville, IL). Cells were washed three times in HBSS containing 20 mM HEPES, resuspended at 1 x 105 cells/ml in RPMI media, and plated at 5000 cells/well in 100 µl total volume. Cells were incubated for 72 h at 37°C. A total of 25 µl of a solution containing 1 mg/ml XTT (sodium 3'-(1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate) and 25 µM phenazine methosulfate (PMS acts as an electron-coupling reagent and is used to potentiate XTT bioreduction) were added per well for the final 4 h of incubation. The soluble formazan product was measured at 450 nm on a Dynatech (Chantilly, VA) MR5000 plate reader. When LY294002 was used, cells were incubated with the appropriate concentration of inhibitor, or vehicle (DMSO) alone before plating out.
Viability assays
Cells were washed as above and resuspended at 5 x 104/ml in RPMI media. Cells were plated in the absence or presence of cytokine (as indicated in figure legends). Duplicate samples were set up for each treatment, and, at 24 h intervals, each sample was double counted and the number of dead vs live cells determined on the basis of trypan blue exclusion.
Immunoprecipitations
Cells were stimulated and solublized as described and immunoprecipitations conducted either with 1 µg anti-PKB Ab (sc-1619; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4°C or with 4 µg of anti-Bad mAb (B36420; Transduction Laboratories, Lexington, KY) overnight at 4°C. Immunocomplexes were captured with 30 µl of protein-G Sepharose beads at 4°C with rotation for 1 h. For Bad precipitations, beads were washed three times in solublization buffer, resuspended in 1x SDS-PAGE sample buffer, and boiled.
SDS-PAGE and immunoblotting
SDS-PAGE and immunoblotting were conducted as previously described (34). PKB samples were fractionated through 7.5% polyacrylamide gels with an acrylamide:bisacrylamide ratio of 37.5:1. Bad immunoprecipitates were fractionated through 13.5% polyacrylamide gels with an acrylamide:bisacrylamide ratio of 118:1 (low bis). Abs were used for immunoblotting at the following concentrations: 1:1000 dilution of polyclonal Abs against PKB/AKT, phosphospecific (Ser473) Akt (9270; New England Biolabs, Beverly, MA) or phosphospecific (Ser112) Bad (9290; New England Biolabs); 0.5 µg/ml anti-Bad (sc-943; Santa Cruz Biotechnology); 0.5 µg/ml 4G10 anti-phosphotyrosine mAb (Upstate Biotechnology, Lake Placid, NY). Secondary Abs conjugated to HRP were used at a concentration of 0.05 µg/ml (Dako, Cambridge, U.K.). ECL or ECLplus (Amersham Pharmacia Biotech, Piscataway, NJ) were used for development of the immunoblots, according to the manufacturer’s instructions. Blots were stripped and reprobed as previously described (35).
In vitro kinase assays
PKB immunoprecipitates 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 DTT). Beads were resuspended in kinase buffer
containing 2.5 µg H2B, 0.5 µM PKI, 50 µM ATP, and 3 µCi 
-ATP
and incubated at room temperature for 30 min. Reactions were stopped by
addition of 5x SDS-PAGE sample buffer and boiling for 5 min. Samples
were fractionated through a 15% polyacrylamide gel with an
acrylamide:bisacrylamide ratio of 37.5:1 and transferred to
nitrocellulose. Membranes were cut in half, the upper part was
immunoblotted for PKB, and the lower part was subjected to
autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been widely reported that PKB is a common intermediate in antiapoptotic signaling in many cell systems. However, to date, little information is available on whether this represents a universal phenomenon, or if there may be cell-type-specific differences in survival signaling. It is also not clear whether activation of PKB is sufficient to provide a survival signal, or whether it is a necessary event in survival of hemopoietic cells. To investigate these issues, we investigated whether activation of PKB correlated with the ability to induce cell survival in a number of cytokine-dependent cells, which we have used extensively for studying mechanisms of cytokine signal transduction. MC/9 and BaF/3 cells are both dependent on IL-3 for their continued proliferation (36, 37), whereas FD-6 cells are dependent on IL-4 for their proliferation (35). In addition to the cytokines upon which they depend for proliferation and survival, MC/9 also respond to SCF, GM-CSF, IL-4, and insulin; BaF/3 will respond to IL-4 and insulin and FD-6 to IL-3, GM-CSF, and insulin.
Although activation of PKB has been previously reported for a number of
cytokines, this has been limited to FL5.12 (19), 32D (21), and MC/9
(11) for IL-3 and only MC/9 for SCF, IL-4, and GM-CSF (11). In
addition, only short-term cytokine stimulations were performed in each
case. Therefore, we first decided to examine cytokine-induced PKB
activation in our three different cells, using extensive time course
analyses, which we also compared with the kinetics of total cellular
tyrosine phosphorylation induced by the individual cytokines. The
activation of PKB was assessed by examining the phosphorylation state
of Ser473, one of two sites on PKB phosphorylated in its
activated form (25). In the cell lines tested, all the cytokines
induced phosphorylation of PKB at Ser473 (see Figs. 1
,
2A, middle panels,
and 2B, upper panels). The kinetics of this
phosphorylation varied according to the cytokine and cell line
examined, but in each case mimicked the kinetics of total cellular
tyrosine phosphorylation (see Figs. 1 and 2A, upper
panels). SCF-induced phosphorylation of PKB was very rapid, seen
after 10 s of treatment, it reached a peak at 2 min and was back
to basal levels by 30 min (Fig. 1A). IL-3-induced
phosphorylation of Ser473 of PKB reached a maximum after
2–5 min in MC/9 (Fig. 1B) and BaF/3 (Fig. 2A)
and after 10 min in FD-6 cells (Fig. 2B).
Phosphorylation of PKB induced by IL-4 reached a maximum after
2–5 min in MC/9 cells, which was then sustained for 30 min (Fig. 1C), and after 10 min IL-4 treatment in FD-6 cell (see Fig. 2B).
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A, in MC/9 cells, IL-3, IL-4,
GM-CSF, and insulin all induce phosphorylation of PKB to similar
levels, while SCF induced a much greater level of phosphorylation. In
FD-6 cells, the insulin-induced phosphorylation of PKB was far greater
than that induced by the other cytokines, while relatively speaking,
IL-4 induced little phosphorylation (see Fig. 3B). Both IL-3
and IL-4 induced the phosphorylation of PKB in BaF/3 cells, although
IL-4 to a very low level (see Fig. 3C).
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To determine whether the cytokine-induced phosphorylation of PKB
is mediated through PI3K, as suggested in other cell types, cells were
preincubated with the PI3K inhibitor LY294002 30 min before
stimulation. With the exception of insulin-treated FD-6 cells, 10 µM
LY294002 completely abrogated the cytokine-induced phosphorylation of
PKB in all cell lines tested (see Fig. 3
). Importantly, levels of
cytokine-induced tyrosine phopshorylation were not affected by LY294002
preincubation. Increasing the concentration of LY294002 to 30 µM in
FD-6 cells completely blocked the insulin-induced phosphorylation of
PKB-Ser473 (data not shown).
PKB Ser473 phosphorylation accurately reflects the activation state of PKB
To confirm that phosphorylation of Ser473 of PKB
accurately reflects the level of activation of the enzyme, in vitro
kinase assays were performed using H2B as a substrate with the results
shown in Fig. 4. For particular cytokines
and cell combinations, we observed the same relative levels of
activation in the in vitro kinase assays as seen in the immunoblots
with the phosphospecific PKB Ab (see Fig. 3). In addition, pretreatment
with LY294002 reduced the ability of all of the cytokines examined to
activate PKB, indicating that the cytokine-induced activation of PKB is
PI3K-dependent. Hence, in all cases, activation of PKB is consistently
found to be dependent on functional PI3K activity.
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Bad, a member of the Bcl-2 family, is Ser-phosphorylated at two sites, Ser112 and Ser136 (31). The latter site is phosphorylated by PKB in response to IL-3 stimulation, and it has been proposed that this accounts for the survival signal generated by IL-3 and PKB (19, 31). Phosphorylation of both sites causes a shift in the migration of Bad on SDS-PAGE (31), and this feature can be used to examine the phosphorylation status of Bad. In MC/9 cells, it has previously been reported that IL-3, GM-CSF, and SCF all induce the conversion of Bad to its slower migrating phosphorylated form, while IL-4 does not (11). Given the established link between PKB activation and Bad phosphorylation, we were interested to determine whether 1) activation of PKB was sufficient to induce dual phosphorylation of Bad in the cells in our study and 2) to determine whether a particular cytokine had the same effect on different cell lines. Therefore, we examined the ability of different cytokines to induce a mobility shift of Bad in MC/9, BaF/3, and FD-6.
In FD-6, as shown in Fig. 5A,
IL-3 stimulation resulted in the complete conversion of Bad to its
slower migrating form. IL-4 treatment resulted in a low level, but
consistently observed partial phosphorylation of Bad. Interestingly,
although insulin was the most potent inducer of PKB activity in FD-6
cells (see Figs. 3 and 4), it only induced a shift in mobility of a
very low level of Bad. Pretreatment of FD-6 with the PI3K inhibitor
LY294002, partially inhibited the IL-3-induced mobility shift of Bad
and almost completely inhibited the IL-4 and insulin-induced
phosphorylation of Bad (Fig. 5A, middle panel).
We noted in repeated experiments that the combination of Abs, which we
were using, preferentially detected Bad in cytokine-treated samples
(particularly apparent after IL-3 treatment). The cells were only
treated for between 2 and 10 min with cytokine, and it would be
unlikely that the levels of Bad protein would be affected over such a
short period of time. Therefore, we feel the most likely explanation is
that the Abs have increased affinity for phosphorylated forms of Bad.
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A, middle
panel, were stripped and reprobed with an Ab that specifically
recognizes phosphorylated Ser112 of Bad. The results are
shown in the lower panel of Fig. 5A. IL-3 induced
significant phosphorylation of Ser112 of Bad, and both IL-4
and insulin induced low levels of Ser112 phosphorylation.
These results complement, and are consistent with, the mobility shift
data, which together demonstrate that in FD-6 cells, IL-4 and insulin
induce only low levels of dual phosphorylation of Bad. In all cases,
pretreatment with LY294002 resulted in decreased Ser112
phosphorylation, showing its dependence on PI3K activity. Attempts to
investigate phosphorylation of Ser136, with the Abs
currently available, have proved unsuccessful.
We also investigated the phosphorylation of Bad in BaF/3 cells. Similar
results were observed for IL-3 (see Fig. 5B). IL-4
consistently did not induce a detectable shift in Bad mobility or
increase Ser112 phosphorylation above levels in control
samples. Insulin induced a small shift in Bad mobility and a low level
of Ser112 phosphorylation, both of which were inhibited by
LY294002 (Fig. 5B). The results we observed in MC/9 cells
(data not shown) were similar to those previously reported (11), with
IL-4 inducing no detectable Bad phosphorylation. The results from the
BaF/3 and FD-6 cells demonstrate that there is a lack of correlation
between cytokine-induced PKB activation and phosphorylation of Bad and
show that different cytokines, e.g., IL-4, can induce Bad
phosphorylation in one cell type, but not another.
Activation of PKB does not correlate with cytokine-induced survival and growth
The results described above indicate that the ability of cytokines
to induce PKB activity does not always correlate with the ability to
induce Bad phosphorylation. The factor-dependent cell lines we have
used in this study provide a good model to examine whether there is a
correlation between cytokine-induced PKB activation and cell
survival/proliferation, because they will die by apoptosis in the
absence of a survival signal. XTT dye-reduction assays were used for
these analyses, as they are good indicators of cell survival and growth
(38, 39). First, we examined the effects of different cytokines on
FD-6. We observed a dose-dependent response of FD-6 to IL-3 and IL-4,
but, interestingly, no response to insulin was observed (see Fig. 6A). Given that insulin had
induced such a potent activation of PKB in FD-6, we then examined its
ability to sustain survival of FD-6 cells using cellular viability
assays. Cells were plated in the absence of any exogenous growth factor
or in the presence of insulin or IL-4. The results in Fig. 6B, right panel, demonstrate that IL-4 maintains
cell viability at levels of >90%. At 24 and 48 h, cells cultured
in the presence of insulin showed 
10% greater viability than cells
in media alone, but this effect was limited, and, after 72 h,
there was no difference in viability among these two populations. These
results suggested that PKB activation by insulin is not sufficient to
transduce a significant survival signal to FD-6 cells. We also examined
the ability of IL-4 and insulin to protect BaF/3 cells from apoptosis.
Insulin was unable to provide any survival signal, whereas IL-4
afforded a partial protection at 24 and 40 h, see Fig. 6B, left panel. These results again show that
insulin cannot act as a survival factor, despite the fact it can
activate PKB. Additionally, IL-4 can transmit a partial survival
signal, despite only inducing very low levels of PKB activity in BaF/3
and no detectable dual phosphorylation of Bad.
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C, with IL-4-induced proliferation being noticeably more
sensitive to inhibition than IL-3-induced proliferation. In the case of
the IL-3 dose-response analyses, at concentrations of LY294002 10 µM
or less, no significant effects on cell viability were apparent (data
not shown), suggesting that the primary effect of inhibiting PI3K
activity is on proliferation. Similar results were observed in BaF/3
cells with IL-3 (data not shown) and correlate well with our findings
from other experiments, where we have inducibly expressed a dominant
negative version of the p85 subunit of PI3K, termed p85. We showed
that expression of p85 resulted in a dramatic reduction in
IL-3-induced proliferation of BaF/3 cells, without significantly
affecting levels of apoptosis (32), consistent with the data we report
here using the chemical PI3K inhibitor, LY294002. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytokines have long been documented to act as growth and survival factors for hemopoietic cells, many acting in both capacities. To test the link between PKB activation and proliferation/survival, we have examined the response of three different cell lines to a number of different cytokines and measured PKB phosphorylation and activation, Bad phosphorylation, the effect of the different factors on cell survival/proliferation, and the dependence of these events on PI3K activity. All the cytokines we examined induced activation of PKB in a PI3K-dependent manner, since PKB activation was sensitive to LY294002 pretreatment. However, not all of these cytokines induced a detectable shift in the mobility of Bad, which is indicative of its phosphorylation on serine residues 112 and 136 (31), or displayed the ability to promote cell survival. In addition, there was no apparent link between the kinetics or levels of PKB activation induced by different cytokines in the different cells and the ability to induce Bad phosphorylation or cell survival. Therefore, our results demonstrate a distinct lack of correlation between PKB activity, Bad phosphorylation, and survival signals in hemopoietic cells, suggesting other pathways must also be involved, functioning either in concert with or independently of PI3K/PKB activities. Interestingly, PKB-independent survival signaling pathways have recently been suggested to be utlized by insulin-like growth factor 1, indicating there are multiple pathways involved in survival signaling (42).
In all the three cell types examined, MC/9, which are mast cells, FD-6, which are myeloid progenitor cells and BaF/3, which are pro-B cells, IL-4 was able to activate PKB in a PI3K dependent manner. In MC/9 cells, it has previously been reported that IL-4 fails to induce dual phosphorylation of Bad (11), and we observed similar results. However, we now show that this may be a cell-type-dependent event, because dual phosphorylation of Bad could be consistently detected in FD-6 cells, albeit at low levels, and Ser112 of Bad was also shown to be directly phosphorylated in response to IL-4. FD-6 are grown long term in IL-4, so one interpretation of these results is that Bad phosphorylation is required for long-term IL-4-driven proliferation to proceed. We show that the PI3K inhibitor, LY294002, can reduce IL-4-dependent proliferation of FD-6 cells in a dose-dependent manner, and this correlates with inhibition of PKB and Bad phosphorylation. In BaF/3 cells, IL-4 does not support proliferation, but does show some ability to protect the cells from apoptosis. The levels of PKB activation in these cells was very low, and no dual Bad, or Ser112, phosphorylation could be detected. Taken together, the results described above suggest that PI3K, PKB, and Bad may be involved in IL-4-driven proliferation, are possibly less important for survival of these cells, and that additional pathways are activated by IL-4 to prevent apoptosis. Recent studies by others have implicated both PI3K and insulin receptor substrate-dependent and independent pathways in IL-4-induced cell survival and growth (43, 44).
The results we have obtained with insulin also support the view that significant levels of PKB activation alone do not necessarily result in efficient phosphorylation of Bad or confer a survival signal to the treated cells. In FD-6, insulin was the most potent inducer of PKB phosphorylation and activity, with >10-fold increases in in vitro PKB kinase activity measured. However, the ability of insulin to induce a shift in the mobility of Bad was very low, although it correlated with low levels of phosphorylation of Ser112. It is possible that phosphorylation of Ser112 is limiting, and, with respect to this, it has been suggested that mitogen-activated protein/extracellular signal-related kinase kinase (MEK) may also be required for Bad phosphorylation (11). However, we have previously shown in FD cells that insulin induces activation of both erk1 and erk2 (45), hence all the kinases so far implicated in Bad phosphorylation are activated by insulin in FD-6 cells, and we still only detect very limited Bad phosphorylation following insulin treatment. When we examined the ability of insulin to promote survival of FD-6 cells over a 72-h time course using XTT assays, no survival was observed and insulin was very limited in its efficacy at promoting cell survival when measured in viability assays. These results were somewhat surprising given the demonstration that insulin-like growth factor-I has been shown to act as a survival factor for cell lines related to FD-6 (9, 46), the reason for the difference in the observations are unclear. It is also puzzling that IL-4 and insulin should have such different outcomes on cell metabolism given that both IL-4 and insulin signal through the insulin receptor substrate 2 molecule in FD-6 (47) and both activate PKB in a PI3K-dependent manner (these studies). Also, insulin can induce activation of erk1 and erk2 in FD cells (45), but fails to provide either a proliferative or survival signal, whereas IL-4 fails to induce activation of erk1 or erk2 in either MC/9 (48) or FD cells (35) and yet can promote survival and proliferation in both. Clearly, additional pathways must be triggered by IL-4 in these cells, which promotes survival and proliferation.
It is interesting to note that, in the cases where cytokine treatment induced phosphorylation of Ser112 of Bad, inhibition of PI3Ks by LY294002 resulted in greatly decreased levels of Ser112 phosphorylation. It has previously been shown that Ser136 is phosphorylated by PKB in a PI3K-dependent manner (19, 20), and, in platelet-derived growth factor signaling, it has been reported that only phosphorylation of Ser136 is PI3K/PKB-dependent (20). Our results suggest that in the hemopoietic cells under investigation, phosphorylation of both Ser112 and Ser136 are dependent on PI3K activity. Therefore, it will be interesting to determine the nature of the kinase responsible for phosphorylating Bad at Ser112 in hemopoietic cells.
In other recent studies, we have shown that expression of dominant negative PI3K mutants in BaF/3 cells reduced IL-3-induced PKB phosphorylation and activation and Bad phosphorylation, without leading to increased apoptosis. Instead, the major physiological consequence of inhibiting PI3K activity on IL-3 signaling was a dramatic reduction in IL-3-induced proliferation (32). These findings correlate with those presented here showing that LY294002 treatment reduces IL-3- and IL-4-induced proliferation. In studies using FDC-P1/Mac1 cells, Minshall et al. (9) have shown that treatment with the PI3K inhibitor wortmannin does not induce apoptosis in the presence of IL-3 during a 24-h incubation period, but this group did not report on whether wortmannin had any effect on IL-3-induced cell proliferation. In addition, insulin-like growth factor-I utilizes both PKB-dependent and -independent survival signaling pathways (42). The summation of these findings, including the data presented here, suggest that activation of PI3K/PKB by cytokines may not be absolutely required to prevent hemopoietic cell apoptosis. In addition, our data demonstrate that the simple activation of PKB alone is not sufficient to confer a survival signal, implying the involvement of alternative pathways. More detailed investigations into the roles played by PKB in cytokine-dependent hemopoietic cells should help to clarify its functional importance.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Melanie J. Welham, Department of Pharmacology, University of Bath, Bath, BA2 7AY, U.K. E-mail address:
3 Abbreviations used in this paper: PI3K, phosphoinositide-3 kinase; PKB, protein kinase B; SCF, stem cell factor; FD, factor-dependent.
Received for publication December 14, 1998. Accepted for publication March 22, 1999.
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C. C. Davies, J. Mason, M. J. O. Wakelam, L. S. Young, and A. G. Eliopoulos Inhibition of Phosphatidylinositol 3-Kinase- and ERK MAPK-regulated Protein Synthesis Reveals the Pro-apoptotic Properties of CD40 Ligation in Carcinoma Cells J. Biol. Chem., January 9, 2004; 279(2): 1010 - 1019. [Abstract] [Full Text] [PDF]
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B. Yan, M. Zemskova, S. Holder, V. Chin, A. Kraft, P. J. Koskinen, and M. Lilly The PIM-2 Kinase Phosphorylates BAD on Serine 112 and Reverses BAD-induced Cell Death J. Biol. Chem., November 14, 2003; 278(46): 45358 - 45367. [Abstract] [Full Text] [PDF]
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 R. Karlsson, M. Engstrom, M. Jonsson, P. Karlberg, C. J.H. Pronk, J. Richter, and J.-I. Jonsson Phosphatidylinositol 3-kinase is essential for kit ligand-mediated survival, whereas interleukin-3 and flt3 ligand induce expression of antiapoptotic Bcl-2 family genes J. Leukoc. Biol., November 1, 2003; 74(5): 923 - 931. [Abstract] [Full Text] [PDF]
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 K. Wang, D. Scheel-Toellner, S. H. Wong, R. Craddock, J. Caamano, A. N. Akbar, M. Salmon, and J. M. Lord Inhibition of Neutrophil Apoptosis by Type 1 IFN Depends on Cross-Talk Between Phosphoinositol 3-Kinase, Protein Kinase C-{delta}, and NF-{kappa}B Signaling Pathways J. Immunol., July 15, 2003; 171(2): 1035 - 1041. [Abstract] [Full Text] [PDF]
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S. Dramsi, M. P. Scheid, A. Maiti, P. Hojabrpour, X. Chen, K. Schubert, D. R. Goodlett, R. Aebersold, and V. Duronio Identification of a Novel Phosphorylation Site, Ser-170, as a Regulator of Bad Pro-apoptotic Activity J. Biol. Chem., February 15, 2002; 277(8): 6399 - 6405. [Abstract] [Full Text] [PDF]
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J.-H. Zhou, S. R. Broussard, K. Strle, G. G. Freund, R. W. Johnson, R. Dantzer, and K. W. Kelley IL-10 Inhibits Apoptosis of Promyeloid Cells by Activating Insulin Receptor Substrate-2 and Phosphatidylinositol 3'-Kinase J. Immunol., October 15, 2001; 167(8): 4436 - 4442. [Abstract] [Full Text] [PDF]
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R. Chian, S. Young, A. Danilkovitch-Miagkova, L. Ronnstrand, E. Leonard, P. Ferrao, L. Ashman, and D. Linnekin Phosphatidylinositol 3 kinase contributes to the transformation of hematopoietic cells by the D816V c-Kit mutant Blood, September 1, 2001; 98(5): 1365 - 1373. [Abstract] [Full Text] [PDF]
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T. Shinjyo, R. Kuribara, T. Inukai, H. Hosoi, T. Kinoshita, A. Miyajima, P. J. Houghton, A. T. Look, K. Ozawa, and T. Inaba Downregulation of Bim, a Proapoptotic Relative of Bcl-2, Is a Pivotal Step in Cytokine-Initiated Survival Signaling in Murine Hematopoietic Progenitors Mol. Cell. Biol., February 1, 2001; 21(3): 854 - 864. [Abstract] [Full Text]
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P. F. Dijkers, R. H. Medema, C. Pals, L. Banerji, N. S. B. Thomas, E. W.-F. Lam, B. M. T. Burgering, J. A. M. Raaijmakers, J.-W. J. Lammers, L. Koenderman, et al. Forkhead Transcription Factor FKHR-L1 Modulates Cytokine-Dependent Transcriptional Regulation of p27KIP1 Mol. Cell. Biol., December 15, 2000; 20(24): 9138 - 9148. [Abstract] [Full Text]
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M. Majka, A. Janowska-Wieczorek, J. Ratajczak, M. A. Kowalska, G. Vilaire, Z. K. Pan, M. Honczarenko, L. A. Marquez, M. Poncz, and M. Z. Ratajczak Stromal-derived factor 1 and thrombopoietin regulate distinct aspects of human megakaryopoiesis Blood, December 15, 2000; 96(13): 4142 - 4151. [Abstract] [Full Text] [PDF]
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 J. Kitaura, K. Asai, M. Maeda-Yamamoto, Y. Kawakami, U. Kikkawa, and T. Kawakami Akt-Dependent Cytokine Production in Mast Cells J. Exp. Med., September 5, 2000; 192(5): 729 - 740. [Abstract] [Full Text] [PDF]
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K. M. Schubert, M. P. Scheid, and V. Duronio Ceramide Inhibits Protein Kinase B/Akt by Promoting Dephosphorylation of Serine 473 J. Biol. Chem., April 28, 2000; 275(18): 13330 - 13335. [Abstract] [Full Text] [PDF]
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J. Li, S. Yang, and T. R. Billiar Cyclic Nucleotides Suppress Tumor Necrosis Factor alpha -Mediated Apoptosis by Inhibiting Caspase Activation and Cytochrome c Release in Primary Hepatocytes via a Mechanism Independent of Akt Activation J. Biol. Chem., April 21, 2000; 275(17): 13026 - 13034. [Abstract] [Full Text] [PDF]
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X. Tang, C. P. Downes, A. D. Whetton, and P. J. Owen-Lynch Role of Phosphatidylinositol 3-Kinase and Specific Protein Kinase B Isoforms in the Suppression of Apoptosis Mediated by the Abelson Protein-tyrosine Kinase J. Biol. Chem., April 21, 2000; 275(17): 13142 - 13148. [Abstract] [Full Text] [PDF]
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S. Pugazhenthi, A. Nesterova, C. Sable, K. A. Heidenreich, L. M. Boxer, L. E. Heasley, and J. E.-B. Reusch Akt/Protein Kinase B Up-regulates Bcl-2 Expression through cAMP-response Element-binding Protein J. Biol. Chem., April 6, 2000; 275(15): 10761 - 10766. [Abstract] [Full Text] [PDF]
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T. Biwa, M. Sakai, T. Matsumura, S. Kobori, K. Kaneko, A. Miyazaki, H. Hakamata, S. Horiuchi, and M. Shichiri Sites of Action of Protein Kinase C and Phosphatidylinositol 3-Kinase Are Distinct in Oxidized Low Density Lipoprotein-induced Macrophage Proliferation J. Biol. Chem., February 25, 2000; 275(8): 5810 - 5816. [Abstract] [Full Text] [PDF]
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A. Schürmann, A. F. Mooney, L. C. Sanders, M. A. Sells, H. G. Wang, J. C. Reed, and G. M. Bokoch p21-Activated Kinase 1 Phosphorylates the Death Agonist Bad and Protects Cells from Apoptosis Mol. Cell. Biol., January 15, 2000; 20(2): 453 - 461. [Abstract] [Full Text]
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M. P. Scheid, K. M. Schubert, and V. Duronio Regulation of Bad Phosphorylation and Association with Bcl-xL by the MAPK/Erk Kinase J. Biol. Chem., October 22, 1999; 274(43): 31108 - 31113. [Abstract] [Full Text] [PDF]
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C. Giallourakis, M. Kashiwada, P.-Y. Pan, N. Danial, H. Jiang, J. Cambier, K. M. Coggeshall, and P. Rothman Positive Regulation of Interleukin-4-mediated Proliferation by the SH2-containing Inositol-5'-phosphatase J. Biol. Chem., September 15, 2000; 275(38): 29275 - 29282. [Abstract] [Full Text] [PDF]
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A. Masuda, T. Matsuguchi, K. Yamaki, T. Hayakawa, and Y. Yoshikai Interleukin-15 Prevents Mouse Mast Cell Apoptosis through STAT6-mediated Bcl-xL Expression J. Biol. Chem., July 6, 2001; 276(28): 26107 - 26113. [Abstract] [Full Text] [PDF]
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