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of June 18, 2017.
A Fas-Associated Death Domain
Protein/Caspase-8-Signaling Axis Promotes
S-Phase Entry and Maintains S6 Kinase
Activity in T Cells Responding to IL-2
Adrian F. Arechiga, Bryan D. Bell, Sabrina Leverrier, Brian
M. Weist, Melissa Porter, Zhengqi Wu, Yuka Kanno,
Stephanie J. Ramos, S. Tiong Ong, Richard Siegel and Craig
M. Walsh
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2007; 179:5291-5300; ;
doi: 10.4049/jimmunol.179.8.5291
http://www.jimmunol.org/content/179/8/5291
The Journal of Immunology
A Fas-Associated Death Domain Protein/Caspase-8-Signaling
Axis Promotes S-Phase Entry and Maintains S6 Kinase
Activity in T Cells Responding to IL-21
Adrian F. Arechiga,2* Bryan D. Bell,* Sabrina Leverrier,* Brian M. Weist,* Melissa Porter,†
Zhengqi Wu,† Yuka Kanno,‡ Stephanie J. Ramos,* S. Tiong Ong,§ Richard Siegel,†
and Craig M. Walsh3*
F
as-associated death domain protein (FADD)4 is an adaptor
protein for death receptors of the TNF family (including
Fas, TNFR1, and TRAIL) (1, 2). A death domain motif
(DD), present within the C terminus of FADD, binds to the cytoplasmic tail of TNFR family members, or to the DD-containing
adaptor TNFR-associated death domain protein (3–5). At the N
terminus, FADD contains a death effector domain which homotypically interacts with analogous motifs present within the prodomain of caspase-8, and in humans, its close relative caspase-10 (6).
When monomers of caspase-8 are brought into close proximity,
they become activated by autoproteolytic processing (7, 8). In this
*Department of Molecular Biology and Biochemistry, and Center for Immunology,
University of California, Irvine, CA 92697; †Immunoregulation Unit, Autoimmunity
Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases
(NIAMS), National Institutes of Health, Bethesda, MD 20892; ‡Molecular Immunology and Inflammation Branch, NIAMS, National Institutes of Health, Bethesda, MD
20892; and §Division of Hematology/Oncology, Department of Medicine, College of
Medicine, University of California, Irvine, CA 92697
Received for publication March 7, 2007. Accepted for publication August 7, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant AI050606 (to
C.M.W.) and Training Grants T32GM007311, T32CA09054, and T32AI060573 (to
A.F.A., B.D.B., and S.J.R., respectively).
2
Current address: Benaroya Research Institute at Virginia Mason, Seattle, WA
98101.
3
Address correspondence and reprint requests to Dr. Craig M. Walsh, Department of
Molecular Biology and Biochemistry, and Center for Immunology, University of
California, Irvine, CA 92697-3900. E-mail address: [email protected]
4
Abbreviations used in this paper: FADD, Fas-associated death domain protein; DD,
death domain protein; FADDdd, dominant-negative form of FADD; v-FLIP, viral
FLIP; S6K, S6 kinase; CDK, cyclin-dependent kinase; Rap, rapamycin; mTOR, mammalian target of Rap; Rb, retinoblastoma; Rosc, roscovitine; ChIP, chromatin immunoprecipitation; 7AAD, 7-aminoactinomycin D; SGK, serum and glucocorticoid-regulated kinase.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
way, FADD serves as an adaptor between the ligated death receptor and the caspases that directly convey apoptotic signals. Indeed,
FADD oligomerization plays a key role in the assembly of a
caspase-8-activating platform necessary for death receptor-dependent apoptosis (9 –11). In the past, the cellular function of this
adaptor has been described as exclusively apoptosis promoting.
However, parallel work has demonstrated that the function of
FADD, like other molecules recruited to ligated death receptors, is
far more complex (12, 13).
Although one would predict that mice with a deficiency in
FADD would lack death receptor-mediated apoptosis, thus leading
to an accumulation of nonapoptotic cells, transgenic mice expressing a dominant-negative form of FADD (denoted FADDdd) do not
manifest lymphoproliferative diseases (12). Paradoxically, previous results have shown that FADD mutant T cells fail to clonally
expand following mitogenic stimulation (14 –21). Patients possessing a mutant form of caspase-8 have an analogous defect in clonal
expansion (22). However, the defect in human caspase-8-deficient
T cells appears to be broader that that observed in FADDdd mice.
Whereas in the human T cells, caspase-8 activity was necessary for
IL-2 expression and NF-␬B activation following TCR cross-linking (22, 23), FADDdd T cells expressed normal levels of IL-2 and
induced the NF-␬B-signaling cascade to a similar extent as wildtype cells (24). Despite overtly normal TCR-proximal signaling,
FADDdd T cells are highly defective in clonal expansion. This
reduced clonal expansion is a consequence of diminished cell cycle entry and survival, particularly at low doses of TCR crosslinking Abs with exogenously supplied IL-2 (21). These findings
suggest that proliferative responses to IL-2, a cytokine known to
regulate both T cell proliferation as well as apoptosis (25), are
defective in FADDdd T cells. Mice with IL-2R deficiencies have
proliferative as well as apoptotic deficiencies, reminiscent of defects
observed in mice with a deficiency in Fas (25, 26). These findings
have lead to the hypothesis that there is an important connection
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Fas-associated death domain protein (FADD) constitutes an essential component of TNFR-induced apoptotic signaling. Paradoxically, FADD has also been shown to be crucial for lymphocyte development and activation. In this study, we report that FADD
is necessary for long-term maintenance of S6 kinase (S6K) activity. S6 phosphorylation at serines 240 and 244 was only observed
after long-term stimulation of wild-type cells, roughly corresponding to the time before S-phase entry, and was poorly induced in
T cells expressing a dominantly interfering form of FADD (FADDdd), viral FLIP, or possessing a deficiency in caspase-8. Defects
in S6K1 phosphorylation were also observed. However, defective S6K1 phosphorylation was not a consequence of a wholesale
defect in mammalian target of rapamycin function, because 4E-BP1 phosphorylation following T cell activation was unaffected by
FADDdd expression. Although cyclin D3 up-regulation and retinoblastoma hypophosphorylation occurred normally in FADDdd
T cells, cyclin E expression and cyclin-dependent kinase 2 activation were markedly impaired in FADDdd T cells. These results
demonstrate that a FADD/caspase-8-signaling axis promotes T cell cycle progression and sustained S6K activity. The Journal of
Immunology, 2007, 179: 5291–5300.
5292
Materials and Methods
Mice
1017-FADDdd-transgenic mice (Tg(Lck-FADD)1Hed) previously described
(16) were maintained on the C57BL/6 background. Mice bearing a floxed
allele of caspase-8 (32) and crossed onto a T cell-specific Cre-expressing transgenic line (CD4-Cre) were obtained from the laboratory of S. M. Hedrick
(University of California San Diego, La Jolla, CA). Mice were bred and maintained in accordance with the institutional animal use and care committee at the
University of California (Irvine, CA) vivarium. CD2-v-FLIP-transgenic mice
(Tg(CD2-v-FLIP)Siegl) were maintained at the National Institutes of Health,
and all animal procedures were done under approved animal protocols.
Cell culture
All cell culture was performed in RPMI 1640 (Irvine Scientific) supplemented with 100 U/ml penicillin, 100 ␮g/ml streptomycin, 1 mM glutamine, nonessential amino acids (Invitrogen Life Technologies), 0.11
mg/ml sodium pyruvate (Invitrogen Life Technologies), 5.5 ⫻ 10⫺5 M
2-ME (Invitrogen Life Technologies), and 10% FBS (HyClone, Logan,
UT). Cells were cultured at 37°C in a 5% CO2-buffered incubator. For
experiments with cells purified before culture, T cells were obtained by
negative selection with magnetic beads using a mixture of Abs to antimouse MHC class II (cat. no. 13-5321-85; Miltenyi Biotec), anti-mouse
CD45R (cat. no. 13-0452-85; Miltenyi Biotec), and anti-mouse CD11b
(cat. no. 13-0112-85; Miltenyi Biotec). For all assays, cells were activated
using recombinant human IL-2 at 50 U/ml, unless otherwise noted (a gift
of the Biological Resources Branch, Division of Cancer Treatment and
Diagnosis, National Cancer Institute, Frederick MD). In some cases,
anti-murine-IL-2 (10 ␮g/ml; eBioscience) was added during initial activation to prevent autocrine stimulation of the IL-2R. Anti-CD3 (1452C11; eBioscience) was used at 50, 200, or 1000 ng/ml, as indicated. In
all experiments, anti-CD3 was plate-bound by first binding anti-hamster
IgG in carbonate buffer, followed by 1 h incubation with the appropriate
amount of 2C11. zVAD-FMK was used at 150 ␮M (Alexis Biochemicals). For inhibition experiments, cells were pretreated before activation
for 20 min at 37°C with either LY294002 (LY; 10 ␮M) or rapamycin
(Rap; 10 ␮M); these inhibitors were left in the culture for the indicated
times. LY and Rap were obtained from Cell Signaling Technology. In
some experiments, Rap was added after 18 h stimulation to block mammalian target of Rap (mTOR) signaling after initial TCR stimulation,
but before the initial entry into S phase (18 h).
Western blot analysis
Following activation, splenocytes were harvested at times indicated. CD4
and CD8 T cells were isolated using anti-CD4 and/or anti-CD8-coated
Dynabeads (Dynal Biotech), followed by cell lysis (150 mM NaCl, 50 mM
NaF, 10 mM ␤-glycerophosphate, 20 mM HEPES (pH 7.4), 1% Triton
X-100, 1 mM PMSF, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, and 1 mM
NaVO4). Protein lysates were quantified using Bradford assays (Bio-Rad),
resolved by gradient SDS-PAGE, and transferred onto polyvinylidene difluoride membranes (Millipore). The following Abs were obtained from
Cell Signaling Technology and used for immunoblotting: phospho-(Ser/
Thr) AKT substrate (AKT-pSub) (cat. no. 9611), phospho-eIF4E (Ser209;
cat. no. 9741), phospho-S6 ribosomal protein (Ser235/236; cat. no. 2211),
phospho-S6 ribosomal protein (Ser240/244; cat. no. 2215), S6 ribosomal
protein (cat. no. 2212), phospho-p70 S6K (Thr389; cat. no. 9205), p70 S6K
(cat. no. 9202), 4E-BP1 (cat. no. 9452), phospho-retinoblastoma (Rb;
Ser807/Ser811; cat. no. 9308, Ser780; cat. no. 9307), Rb (cat. no. 9309),
cyclin D3 (cat. no. 2936). Polyclonal or mAbs to cyclin E (cat. no. 14 –
6714) were obtained from eBioscience. Immunoblotting was performed
according to instructions provided by the manufacturer. After incubating
with appropriate HRP-conjugated secondary Ab (Vector Laboratories), immunoreactive bands were visualized by chemiluminescence (Pierce Biochemicals). Where indicated, blots were stripped, according to the manufacturer’s instructions, using Restore-Western Blot Stripping Buffer
(Pierce), before reprobing with designated Abs. Monoclonal anti-mouse
␤-actin was used to verify equal protein loading (cat. no. ab6276; Abcam).
Chromatin immunoprecipitation (ChIP)
TCR/CD28 stimulated T cells were treated with or without IL-2 (100 U/ml)
and then incubated with 1% formaldehyde to cross-link DNA-bound
transcription factors to DNA. Cell lysates were sonicated and immunoprecipitated with anti-STAT5 Ab (R&D Systems) or control normal
rabbit serum (Upstate Biotechnology). After washing, precipitated
DNA was eluted, purified and the amount of DNA was quantitated by
quantitative PCR (ABI PRISM 7700 Sequence Detection System, Applied Biosystems) using Oncostatin M promoter site-specific primers as
previously described (33).
BrdU labeling
CD8⫹ cells were isolated by incubating pooled lymph nodes and splenocytes from wild-type, FADDdd, and Casp8 ⫻ LckCre mice with MACS
anti-murine CD8 beads (Miltenyi Biotec), followed by magnetic sorting.
Cells were activated as indicated and labeled with 10 mM BrdU for the
duration of the experiment. At given time points, cells were collected and
washed twice in PBS. Fluorescent Abs specific for cell surface markers
were added and incubated for 20 min, followed by washing in HBSS. Cells
were then fixed and permeabilized according to manufacturers recommendations using Cytofix/Cytoperm (BD Biosciences). Cells were treated with
DNase for 1 h at 37°C to expose incorporated BrdU. Cells were then
stained with FITC-conjugated anti-BrdU Ab for 20 min. After washing in
PBS, cells were stained for 20 min using 7-aminoactinomycin D (7AAD).
Cells were then washed and resuspended in HBSS and analyzed by flow
cytometry.
Flow cytometry analysis
A total of 1 ⫻ 107 spleen and lymph node cells were labeled with 2.5 ␮M
CFSE at room temperature for 8 min, washed three times with RPMI 1640
containing 10% FCS and then activated under the indicated conditions. At
various times after noted stimulation, cells were washed twice in 1⫻ PBS
before staining with allophycocyanin-CD4 (eBioscience), PerCP-CD8 (BD
Pharmingen), and PE-conjugated recombinant annexin-V (Caltag Laboratories). Cells were harvested using a FACSCalibur flow cytometer (BD
Biosciences) and this data were analyzed with FlowJo software (Tree Star).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
between IL-2 signaling and death receptor signaling for the regulation
of T cell homeostasis (26, 27).
As previously reported, FADDdd-expressing T cells had dramatic defects in clonal expansion to anti-CD3 and exogenously
supplied IL-2, despite normal STAT5 phosphorylation following
IL-2R stimulation (21). Similar defects as well as normal STAT5
phosphorylation have also been seen in transgenic mice expressing
the viral protein MC159-viral FLIP (v-FLIP), which blocks activation of caspase-8 in the Fas-signaling complex (28). A second
pathway activated by the IL-2R involves the recruitment of the Src
homology 2-containing adaptor SHC, and the consequent induction of PI3K (25). As assessed by anti-phospho-Akt immunoblotting, PI3K activity in response to IL-2 was overtly normal in
FADDdd T cells. However, using a proteomics approach with an Ab
generated to detect the phosphorylated substrates of Akt and other
members of the serum and glucocorticoid-regulated kinase (SGK)
family, we found that sustained activation of S6 kinase 1 (S6K1) and
phosphorylation of ribosomal S6 protein were defective in FADDddexpressing T cells following stimulation with IL-2. In accord with
this, FADDdd T cells completely failed to phosphorylate S6 on
serines 240 and 244, sites found to be phosphorylated late during T
cell activation. Induction of caspase activation has been reported to be
important for T cell expansion following antigenic stimulation (29 –
31). Strikingly, a similar defect in the phosphorylation of S6 was
observed in v-FLIP-transgenic T cells and in T cells bearing a conditional deletion of caspase-8 (casp8⫺/⫺). This failure to phosphorylate S6 in FADDdd T cells was observed even with acute stimulation of the IL-2R. Because blockade of cell cycle
progression in late G1 stage with the cyclin-dependent kinase
(CDK) inhibitor roscovitine (Rosc) also prevented S6 phosphorylation, our results demonstrate that the failure to phosphorylate S6 in FADDdd, v-FLIP, and casp8⫺/⫺ T cells is likely
a consequence of defective cell cycle progression during the
G1/S transition. Thus, in addition to accepted roles in promoting
apoptosis following death receptor binding, these results define
a novel signaling paradigm in which sustained S6K activation
depends upon FADD, cellular FLIP, and caspase-8.
DEFECTIVE S6 KINASE ACTIVITY IN FADDdd T CELLS
The Journal of Immunology
5293
Table I. Cell cycle progression analyzed by continuous BrdU labeling
of FADDdd vs wild-type (WT) cellsa
Genotype
WT
FADDdd
Time (h)
G0/G1
S
G2/M
Subdiploid
0
24
48
0
24
48
86
61
21
75
65
37
1
12
44
1
11
16
0
6
20
0
1
6
12
11
13
23
20
40
a
Percentages shown correspond to the gates labeled in Fig. 1A for T cells stimulated with anti-CD3 plus IL-2 for the indicated time.
CDK2 kinase assay
Wild-type, FADDdd, and casp8⫺/⫺ T cells were activated for 36 h using
anti-CD3 (200 ng/ml) and IL-2 (50 U/ml) in presence or absence of Rosc.
Following activation, CDK2 was immunoprecipitated overnight, then subjected to an in vitro kinase assay with [32P]ATP and histone H1 (Cell
Signaling Technology) as substrate. The reaction was resolved by SDSPAGE, and kinase activity was assessed by autoradiography. As a control
for loading, the gel was stained with Coomassie to detect total histone H1.
Results
Defective IL-2-induced S6K activity in FADDdd T cells
We have found that FADDdd T cells fail to undergo productive
proliferation in response to anti-CD3 plus anti-CD28 (21, 24), pos-
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FIGURE 1. Normal AKT phosphorylation in mitogenically stimulated
FADDdd T cells and phosphoprotein mapping of activated T cells using an
AKT phosphosubstrate-specific Ab. A, Defective cycle progression in
FADDdd T cells responding to IL-2. FADDdd or wild-type T cells were
stimulated with anti-CD3 plus IL-2 (50 U/ml) for the indicated times. BrdU
was present during the entire culture time. B, T cells were stimulated for
the indicated times with anti-CD3 (1 ␮g/ml) and IL-2 (50 U/ml), followed
by magnetic purification (using anti-CD4 and anti-CD8 Dynal beads), lysis, and Western blotting. Western blots were probed with Abs specific for
phosphorylated AKT (Ser473), then stripped and reprobed with anti-AKT to
control for loading. In some cultures, LY294002 (LY; 10 ␮M) or Rap (10
nM) was added to block PI3K or mTOR signaling, respectively. C, Defective phosphorylation of an ⬃32-kDa protein in FADDdd T cells. AntiAKT phospho-substrate (AKTpSub) Western blot of lysates from activated
wild-type and FADDdd-transgenic T cells. T cells were activated for 18 h
with anti-CD3 (“3low”; 50 ng/ml), human rIL-2 (50 U/ml), and/or LY (10
␮M), as indicated, followed by magnetic purification and Western blotting;
position of the ⬃32-kDa species is indicated with an arrow at the right.
Blots were also probed with anti-␤-actin to control for loading.
sibly due to a failure to respond to endogenously produced IL-2.
Addition of exogenous rIL-2 failed to restore proliferation (Fig.
1A). Whereas wild-type T cells effectively entered into S phase, a
diminished proportion of FADDdd T cells were observed to have
taken up BrdU at 24 and 48 h. Instead, many of these cells had an
apoptotic, subdiploid morphology, as assessed by 7AAD staining
(Table I). These results suggest a defect in signaling in response to
IL-2R ligation. Previously, we demonstrated that STAT5 tyrosine
phosphorylation occurs normally, indicating that the IL-2R complex is overtly functional in FADDdd T cells (21). A parallel and
essential signal transduction pathway activated by IL-2R stimulation involves the recruitment of SHC to the receptor, and subsequent activation of PI3K (25, 26, 34). PI3K promotes the membrane recruitment of protein kinase B/Akt, which itself plays a
central role in mediating critical cellular responses including cell
growth and survival (34). To assess the activation status of the
PI3K pathway in mitogenically stimulated FADDdd T cells, purified wild-type or FADDdd cells were cultured for the indicated
times with plate bound anti-CD3 and exogenous IL-2. Western
blots were prepared and serially probed with Abs for phospho-Akt
(Ser473) and compared with loading using an Ab specific for total
Akt. Despite difficulty observing induction of Akt phosphorylation
over basal levels, no consistent differences were observed in the
level of Akt phosphorylation in FADDdd T cells when compared
with wild-type cells (Fig. 1B) (the slightly elevated level of phospho-Akt observed at 90 m for FADDdd T cells was not consistent).
Furthermore, the PI3K inhibitor LY294002 (LY) eliminated both
induced and basal phospho-Akt to the same extent in wild-type and
FADDdd T cells, demonstrating that Akt dephosphorylation occurs
at a normal rate in FADDdd T cells. By comparison, treatment
with the mTOR inhibitor Rap, an agent not thought to interfere
with PI3K signaling, failed to inhibit Akt phosphorylation. These
results demonstrate that PI3K signaling induced via the TCR complex and/or IL-2R is not altered by FADDdd expression in T cells,
and are concordant with previously published results demonstrating normal Akt Ser473 phosphorylation following long-term stimulation of FADDdd-expressing T cells (17).
Although we observed normal Akt phosphorylation following
IL-2R stimulation, we considered the possibility that the signaling
defect imposed by FADDdd may exist further downstream of Akt
activation. Additionally, phosphorylation of Ser473 might not serve
as a direct readout of Akt activity. To address these considerations,
we used a proteomics-based strategy to characterize differential
phosphorylation of putative Akt substrates between FADDdd and
wild-type T cell lysates (35, 36). Western blots from FADDdd and
wild-type T cell cultures were probed with an Ab developed to
detect the phosphorylated substrates of Akt (35, 37), an Ab that has
been demonstrated to efficiently bind to phospho-proteins containing the Akt substrate recognition motif RxRxx(T*/S*) (38). With
this reagent, it is possible to classify, at least in theory, all of the
5294
potential substrates of Akt and related kinases under various stimulation conditions. The results of this experiment revealed a
roughly 32-kDa Akt-phospho-substrate-specific (AKTpSub) band
that was present in mitogenically stimulated wild-type cells (Fig.
1C). The induction of this species was dramatically reduced in
FADDdd T cells. Anti-CD3 or IL-2 alone were not sufficient to
induce this band, suggesting that activation of both TCR- and IL2R-linked signaling pathways is necessary to optimally elicit the
appearance of this putative phosphoprotein. Pretreatment of cells
with LY prevented the appearance of the 32-kDa AKTpSub species, demonstrating that PI3K activity is required for its induction.
Although many other bands were similarly detected with this Ab,
we did not observe a consistent dependence on FADD, PI3K or
mitogenic signaling for their appearance at this late time after activation (data not shown). Furthermore, because this band was not
enhanced in FADDdd lysates after treatment of the cells with okadaic acid or calyculin, its lack of appearance in these cells is not
likely to be due to hyperactive serine/threonine-directed phosphatase activity (data not shown).
Previously, we found that the AKTpSub Ab detected phosphorylated ribosomal S6 protein in BCR-Abl-transformed
chronic myelogenous leukemic tumors (39), yielding a band of
roughly 30 kDa. Additionally, S6Ks share motif specificity with
other SGK family kinases, including Akt (38). Thus, we hypothesized that the band detected by the AKTpSub Ab may be
S6 rather than a direct Akt substrate. To determine whether S6
phosphorylation was defective in FADDdd T cells, purified T
cells were activated with anti-CD3 plus IL-2 for various times,
lysed, and subjected to Western blotting with anti-phospho-S6.
Treatment of both wild-type and FADDdd T cells promoted
weak S6 phosphorylation at serines 235 and 236 as early as 30
min following stimulation (Fig. 2A). Pretreatment of cells with
LY or Rap reduced this level of phosphorylation to below basal
levels, as expected. With time, both total S6 and phospho-S6
(Ser235/236) accumulated, with highest levels observed after
FIGURE 3. Defective sustained S6K1 phosphorylation, but normal
mTOR function in FADDdd T cells. A, Lack of sustained S6K1 phosphorylation in FADDdd T cells. Cells were stimulated with anti-CD3 (50 ng/ml)
and IL-2 (50 U/ml) for the indicated times in the absence or presence of
LY, magnetically purified, and then used for Western blotting with antiphospho-S6K1 (Thr389) or total S6K1 Abs. B, Normal pattern of 4E-BP1
phosphorylation in FADDdd T cells. Cells were stimulated for the indicated times with and without LY. Immunoblots were probed with anti-4EBP1; lower mobility phosphorylated isoforms (␤ and ␥) detected by Western blotting are indicated. To control for loading, the blot was stripped and
reprobed with anti-␤-actin. C, Normal pattern of eIF4E phosphorylation in
mitogenically stimulated FADDdd T cells. Western blots of cells treated as
in A were probed with Abs to phospho-eIF4E (Ser209), or anti-␤-actin to
control for protein loading.
18 h of stimulation. Although phosphorylation of S6 at serines
235 and 236 was observed in FADDdd T cells at early times,
these cells possessed substantially reduced levels of phospho-S6 (Ser235/236) at 18 h.
S6 is sequentially phosphorylated, with initial phosphorylation
detected at serines 235 and 236, followed by subsequent phosphorylation at serines 240, 244, and 247 (40). Phosphorylation of S6 at
positions 240 and 244 was not observed at 5 h. After 18 h of
treatment with anti-CD3 plus IL-2, substantial phosphorylation of
S6 on these serines was observed. These data demonstrate that the
sustained activity of S6Ks is necessary to promote full phosphorylation of this riboprotein following mitogenic stimulation of primary, naive T cells (Fig. 2B). Consistent with the hypothesis that
phosphorylation at serines 240 and 244 requires sustained S6K
activity, FADDdd T cells failed to promote phosphorylation of S6
at these positions, even after 18 h mitogenic stimulation. Again,
incubation of activated FADDdd T cells in phosphatase inhibitors
did not impact S6 phosphorylation (data not shown). Thus, while
FADDdd T cells do not possess a general deficit in phosphorylation or hyperactive dephosphorylation of S6, they fail to sequentially phosphorylate serine residues necessary for maximal S6
function.
Defective S6K1 phosphorylation in FADDdd-transgenic T cells
Because a defect in S6 phosphorylation in FADDdd T cells was
observed, the phosphorylation status of S6K1 at position Thr389
was measured; phosphorylation at this site is essential for the activity of the kinase (41). Wild-type and FADDdd T cells were
activated for 2, 6, or 18 h in the absence or presence of LY and
lysates blotted and probed with an Ab to detect phosphorylated
S6K1 at Thr389 (Fig. 3A). Although early phosphorylation of S6K1
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 2. Defective phosphorylation of ribosomal protein S6 in mitogenically stimulated FADDdd T cells. A, Failure to maintain phosphorylation of ribosomal protein S6 at serines 235 and 236 in stimulated
FADDdd T cells. Immunoblot of phosphorylated (Ser235/236) in activated wild-type and FADDdd T cells following stimulation for the indicated times with anti-CD3 and human rIL-2 (50 U/ml). To block PI3K
or mTOR signaling, LY or Rap was added, respectively. Blots were
stripped and reprobed with anti-␤-actin to control for protein loading.
B, Defective S6 phosphorylation at serines 240 and 244 in activated
FADDdd T cells. Western blot of activated wild-type and FADDdd T
cells following 18 h of stimulation with anti-CD3 plus IL-2, in the
absence or presence of LY, probed with anti-phospho-S6 (240/244);
lysates were stripped and reprobed with anti-␤-actin to control for loading. LY was used at 10 ␮M, and Rap was used at 10 ␮M.
DEFECTIVE S6 KINASE ACTIVITY IN FADDdd T CELLS
The Journal of Immunology
FADDdd T cells have a diminished CDK2 activity during entry
into S phase
Due to the dramatic decrease in cell cycle progression and growth
observed in FADDdd T cells above, we wished to evaluate the
status of early cell cycle regulatory proteins. In particular, we focused our attention on those proteins known to be associated with
progression from G0 to S phase because FADDdd T cells exhibit
reduced progression through this stage of the cell cycle (24). Dtype cyclins promote activation of CDK4 and 6, kinases that induce exit from G0 (44). Consistent with previous results (45), activation of purified T cells with plate-bound anti-CD3 and IL-2
promoted increased expression of cyclin D3 after 18 h of stimulation (Fig. 4A). Increased expression of this early G1 cyclin was
not inhibited by FADDdd expression. No defect was observed in
p27Kip1 down-modulation, further suggesting that the early
G03 G1 transition occurred normally in FADDdd-expressing cells
(data not shown). Rap treatment substantially inhibited the expression of cyclin D3, consistent with a recent report demonstrating
that cyclin D3 translation requires mTOR activity (46). Similarly,
treatment of wild-type T cells with anti-CD3 and IL-2 promoted
up-regulation of cyclin E expression, although we could not detect
the presence of this G1/S cyclin until after 18 h of mitogenic stimulation. We did not detect the presence of cyclin E expression in
FADDdd T cells, even after 30 h of mitogenic stimulation. As was
the case for cyclin D3, cyclin E expression was also reduced by
pretreatment with Rap. The CDK2/cdc2 inhibitor Rosc inhibited
the up-regulation of cyclin E, while only having a moderate effect
on cyclin D3 induction, the latter result consistent with previous
reports in which the increased level of expression of cyclin E depends on CDK2 activity (47). Thus, based on these studies, we
hypothesize that FADDdd T cells progress normally through early
G1 phase, but have a defect in the CDK2-dependent transition to S
phase.
We also wished to determine the phosphorylation status of retinoblastoma (Rb), a key cell cycle inhibitory protein known to be
phosphorylated on multiple residues by G1 cyclin/CDK complexes
FIGURE 4. Defective G1/S cyclin expression and CDK2 activity in
FADDdd T cells. A, Expression of cyclins D3 and E in response to mitogenic stimulation of FADDdd and wild-type T cells. Wild-type or FADDdd
T cells were stimulated with anti-CD3 (200 ng/ml) and IL-2 (50 U/ml) for
the indicated times. In some cases, Rosc (10 ␮M) or Rap (10 ␮M) were
added. Blots were then serially probed with anti-cyclin D3, and anti-cyclin
E, followed by anti-␤-actin to control for loading. B, Normal hypophosphorylation but defective hyperphosphorylation of Rb in FADDdd T cells.
Cells were stimulated as above, some with Rosc added, then harvested at
the indicated times. Lysates were separated on a 7.5% SDS-PAGE gel,
blotted, and then serially probed for phospho-Rb (Ser807/Ser811), phospho-Rb (Ser780), total Rb, and ␤-actin as a loading control. C, Diminished
CDK2 activity in mitogenically stimulated FADDdd T cells. T cells were
stimulated with anti-CD3 plus IL-2 for 24 h, followed by lysis and immunoprecipitation with anti-CDK2. In vitro kinase reactions were conducted
with immunoprecipitates added to histone H1 plus radiolabeled ATP. In
some cases, Rosc was added to in vitro kinase reactions to demonstrate
blockade of histone H1 phosphorylation.
(47). Rb was phosphorylated at serines 807 and 811 as early as 6 h
following stimulation with anti-CD3 and IL-2, with maximal phosphorylation observed after 24 h (Fig. 4B, upper panel). Phosphorylation at these sites was similar in FADDdd T cells following
activation, a finding consistent with a recent report demonstrating
that CDK4/6:cyclin D complexes promote phosphorylation at
these sites (48). An additional site phosphorylated by CDK4/6:
cyclin D complexes is serine 780. However, this site was found to
be constitutively phosphorylated in untreated wild-type and
FADDdd T cells, regardless of activation status (Fig. 4B, middle
panel). During transition from early G1 to late G1, cyclin E levels
increase, promoting activation of CDK2. Elevated levels of active
cyclin E:CDK2 complexes promote hyperphosphorylation of Rb at
many additional sites (49). Although difficult to visualize, we observed a putative hyperphosphorylated Rb band in wild-type, but
not FADDdd T cells, following activation with anti-CD3 plus IL-2
for 24 h (Fig. 4B, lower panel). Treatment with the CDK2/cdc2
inhibitor Rosc at 10 ␮M failed to inhibit phosphorylation at serines
780, 807, and 811. However, like FADDdd expression, Rosc prevented the appearance of the hyperphosphorylated Rb band. These
results suggest that FADDdd T cells may have a defect in the
induction of CDK2 activity during the G1/S transition. To test this,
wild-type and FADDdd T cells were mitogenically stimulated for
24 h as above, in the absence and presence of Rosc. Lysates were
immunoprecipitated with anti-CDK2, followed by in vitro kinase
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was normal in FADDdd T cells, the level of S6K1 phosphorylation
was diminished at 18 h in FADDdd, but not wild-type cells. Together with results using anti-phospho-S6-specific Abs, this finding suggests that FADD function is required for the long-term
maintenance of S6K1 activity. To determine whether the failure to
maintain sustained S6K1 phosphorylation was due to a general
deficit in mTOR signaling, the phosphorylation status of 4E-BP1
was evaluated because this inhibitor of eIF4E is phosphorylated in
an mTOR-dependent manner. With stimulation, there was a
shift in the mobility of 4E-BP1 to slower migrating isoforms in
both wild-type and FADDdd T cells (Fig. 3B). Because treatment of FADDdd T cells with Rap blocked residual proliferation (data not shown), these results suggest that mTOR signaling was
unimpaired in FADDdd T cells after 18 h of stimulation. A parallel
signaling pathway crucial to growth-factor induced translation involves the phosphorylation of the eukaryotic cap-binding translation initiation factor eIF4E at serine 209 via a p38/Erk3 Mnk1
pathway (42, 43). Examination of this pathway revealed normal
phosphorylation of eIF4E in FADDdd T cells following stimulation with anti-CD3 and IL-2 (Fig. 3C). This phosphorylation was
not blocked by LY or Rap, consistent with previous results demonstrating that eIF4E phosphorylation at Ser209 depends on p38
and Mnk1, and occurs independently of PI3K or mTOR signaling
(42). Taken together, these results demonstrate that FADDdd T
cells fail to maintain S6K1 phosphorylation following mitogenic
stimulation, but that 4E-BP1- and eIF4E-signaling pathways are
intact.
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DEFECTIVE S6 KINASE ACTIVITY IN FADDdd T CELLS
Caspase-8 activity is necessary for efficient cell cycle
progression and sustained S6 phosphorylation in T cells
FIGURE 5. Sustained S6 phosphorylation depends upon IL-2-dependent cell cycle progression. A, Blockade of IL-2-driven S6 phosphorylation
by Rosc. Cells were preincubated with LY, Rosc, Rap, or left untreated,
followed by anti-CD3 plus IL-2 culture for 24 h. Lysates were probed with
anti-phospho-S6 (S240/S244). The blot was stripped and reprobed with
anti-S6 to control for loading. B, Blockade of S6K1 phosphorylation by
Rosc. Cells were stimulated for the indicated times in the absence or presence of Rosc, followed by immunoblotting as above. C, Acute IL-2 signaling leads to S6 phosphorylation. Cells were stimulated with anti-CD3
(50 ng/ml) in the absence or presence of anti-mouse IL-2 (5 ␮g/ml) to
block autocrine signaling. Human rIL-2 (100 U/ml) was added for the
indicated times, or provided continuously for the duration of the culture
(“cont”), followed by lysis and anti-phospho-S6 (S240/S244) staining. D,
Blockade of acute and long-term IL-2 induced S6 phosphorylation by
Rosc. Cells were incubated as in C, except that some cells were preincubated with Rosc before activation by plate-bound anti-CD3. Blot was
stripped and reprobed with anti-phospho-ERK1/2.
assays using histone H1 as substrate. Whereas wild-type cells possessed a high level of CDK2 activity, and this activity was blocked
by Rosc, FADDdd T cells lacked appreciable CDK2 activity (Fig.
4C). Taken together, these results demonstrate that while early G1
cell cycle progression occurs normally in FADDdd T cells, later
stages dependent upon cyclin E:CDK2 are defective in these mutant cells.
Sustained S6 phosphorylation depends on IL-2-driven CDK2
activity
To determine whether the defect in sustained S6 phosphorylation
might be a consequence of diminished CDK2 activity, we evaluated the potential that agents blocking cell cycle progression might
reduce S6 phosphorylation. Treatment of T cells with anti-CD3
plus IL-2 promoted S6 phosphorylation at S240/S244, as observed
previously. As expected, pretreatment with LY or Rap blocked
this. Pretreatment with Rosc prevented S-phase entry, as assessed
by BrdU analysis (data not shown), and also inhibited sustained S6
phosphorylation (Fig. 5A). Rosc pretreatment also blocked sus-
In addition to FADD, caspase activation has also been shown to be
essential for T cell proliferation (29, 30, 50, 51). To determine
whether a deficiency in caspase-8 activity might also impact S6
phosphorylation, we made use of two mutant lines of mice. First,
we tested mice expressing a T cell-specific transgene encoding
v-FLIP, a viral inhibitor of caspase-8. These mice have been previously shown to bear T cells with deficiencies in proliferation
similar to those observed in FADDdd mice (28). STAT5 activity
was assessed using a ChIP assay with the oncostatin M promoter,
a target of STAT5 activity (52). Because no defect in binding to
the oncostatin M promoter was observed (Fig. 6A), we conclude
that STAT5 activity downstream of the IL-2R is unimpeded by the
v-FLIP transgene. v-FLIP and wild-type T cells were preactivated
with anti-CD3 plus anti-CD28, followed by short-term stimulation
with IL-2 to assess STAT5 phosphorylation. As predicted by the
normal STAT5 binding to the oncostatin M promoter, no obvious
defects were observed in STAT5 phosphorylation in v-FLIP-transgenic T cells (Fig. 6B, upper panel). We also assessed the phosphorylation status of S6K1 and observed a modest but reproducible defect in S6K1 phosphorylation relative to total S6K1 (Fig.
6B, lower panel). When stimulated continuously with anti-CD3
plus IL-2 for 24 h, v-FLIP T cells failed to accumulate phosphorylation on serines 240 and 244, similar to results obtained with
FADDdd T cells (Fig. 6C). These results suggest that v-FLIP interferes with optimal S6K1 phosphorylation and activity following
IL-2R ligation.
To determine whether caspase-8 itself is necessary for sustained
S6 phosphorylation, we tested caspase-8-deficient T cells produced
by crossing mice bearing conditionally mutant caspase-8 (Casp8Flox) alleles (32) with mice expressing the Cre recombinase under
a T cell-specific promoter (CD4Cre). First, we assessed proliferation and cell cycle using CFSE labeling and BrdU/7AAD analysis.
As observed for FADDdd T cells, casp8⫺/⫺ T cells had a reduced
level of proliferation and survival, as assessed by CFSE dilution
analysis (Fig. 7A). This diminished proliferation was primarily a
consequence of decreased survival and was most obvious in the
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tained S6K1 phosphorylation (Fig. 5B). These results suggest that
sustained S6 phosphorylation induced by IL-2 requires CDK2 activity. To determine whether IL-2R signaling is responsible for
sustained S6 phosphorylation, T cells were preincubated with antimurine IL-2 to block autocrine signaling through the IL-2R via
endogenously produced cytokines, followed by 24 h stimulation
with plate-bound anti-CD3. This treatment greatly reduced the
number of cells entering into S phase at 24 h (data not shown). In
comparison to cells receiving continuous IL-2 during the 24 h
culture, cells pretreated with anti-IL-2 had diminished S6 phosphorylation (Fig. 5C). Treatment with human rIL-2 (which is not
blocked by the anti-mouse IL-2 Ab used in the assay) reversed the
S6 phosphorylation defect, indicating that S6 phosphorylation on
serines 240 and 244 is dependent on IL-2R signaling. Under all of
these conditions, S6 was only weakly phosphorylated in FADDdd
T cells (data not shown). Rosc blocked S6 phosphorylation in cells
receiving both short- and long-term IL-2 treatment after 18 h antiCD3 stimulation, suggesting that sustained S6 phosphorylation induced via IL-2R signaling depends upon cell cycle transit. Because
ERK phosphorylation induced by short-term IL-2 treatment of preactivated T cells was not inhibited by Rosc, it is unlikely that Rosc
blocked global IL-2R signaling. These results demonstrate that the
sustained S6 phosphorylation in T cells that is blocked in FADDdd
T cells depends upon IL-2R signaling, and that this process likely
requires active cell cycle entry.
The Journal of Immunology
CD8⫹ population, consistent with our previous results using
FADDdd expressing mice and those reported for another line of mice
bearing a conditional mutation in caspase-8 (21, 50). To determine
FIGURE 7. Cell cycle progression and S6K activity depends on
caspase-8 function in T cells. A, Defective proliferation of casp8⫺/⫺ T cells
following mitogenic stimulation with plate-bound anti-CD3 plus antiCD28. Wild-type and casp8⫺/⫺ T cells were labeled with CFSE before
culture for 3 days in the presence of anti-CD3 (50 ng/well) plus IL-2 (50
U/ml). B, Cell cycle analysis of magnetically purified casp8⫺/⫺ T cells
following stimulation with anti-CD3 plus IL-2. To assess DNA synthesis,
BrdU was added during the entire culture. After the indicated times, cells
were permeabilized, then stained with FITC-conjugated anti-BrdU Abs and
7AAD. C, Diminished S6 phosphorylation in casp8⫺/⫺ T cells following
mitogenic stimulation. Cells were stimulated for the indicated times using
plate-bound anti-CD3 plus IL-2. Where indicated, cells were preincubated
with LY. Lysates were then subjected to immunoblot analysis using antiphospho-S6 (S235/S236), anti-phospho-S6 (S240/S244) and anti-␤-actin
Abs. D, Diminished CDK2 activity in activated casp8⫺/⫺ T cells.
Casp8⫺/⫺ or wild-type T cells were activated with anti-CD3 plus IL-2 for
24 h, followed by lysis and immunoprecipitation with anti-CDK2. Immunoprecipitates were analyzed for histone H1 phosphorylation activity, without or with added Rosc, as in Fig. 4C. E, Potential model for FADD/
caspase-8-dependent S6K1 signaling. FADD and caspase-8 promote
inactivation of cell cycle inhibitor that interferes with CDK2/cyclin E (“E”)
function. This prevents CDK2/cyclin E-dependent phosphorylation of
S6K1, which along with mTOR-dependent phosphorylation, leads to full
S6K1 activity.
whether casp8⫺/⫺ T cells also possess a defect in cell cycle progression, we stimulated cells for 24 and 48 h with anti-CD3 plus
IL-2 in the presence of BrdU (Fig. 7B). Following mitogenic stimulation, cells were permeabilized and stained with anti-BrdU plus
7AAD. Casp8⫺/⫺ T cells had diminished entry into S phase and an
increased proportion of subdiploid cells when compared with wildtype cells (Table II), similar to results observed in FADDdd T cells
stimulated in this manner (24). We observed a slight decrease in S6
phosphorylation on serines 235 and 236, but a more substantial
decrease of phosphorylation on serines 240 and 244 following
stimulation of casp8⫺/⫺ T cells (Fig. 7C). As with FADDdd T
cells, activated casp8⫺/⫺ T cells possessed diminished CDK2 activity compared with wild-type T cells (Fig. 7D). Thus, deficiencies in either FADD or caspase-8 lead to decreased S-phase entry
and survival during T cell responses to mitogenic stimulation.
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FIGURE 6. IL-2 induces normal STAT5 activity but defective S6K1
and S6 phosphorylation in v-FLIP-transgenic T cells. A, Normal STAT5
DNA binding activity in v-FLIP-transgenic T cells, as assessed by ChIP
assays of the oncostatin M promoter. Purified T cells from wild-type and
v-FLIP-transgenic mice were stimulated with anti-CD3 plus anti-CD28 for
3d, followed by a 1 h stimulation with IL-2 (100 U/ml). After lysis and
cross-linking, chromatin was immunoprecipitated using anti-STAT5 or a
nonimmune rabbit serum (NRS) control. B, Slight defect in S6K1 phosphorylation in T cells expressing transgenic v-FLIP. Cells were prestimulated with anti-CD3 plus anti-CD28 for 48 h before addition of human
rIL-2 for the indicated times. Cells were lysed and serially immunoblotted
with anti-phospho-STAT5, anti-STAT5, anti-phospho-S6K1, and antiS6K1. C, Defective serine 240/244 phosphorylation of S6 ribosomal protein in v-FLIP-transgenic T cells. T cells from wild-type and v-FLIP-transgenic mice were stimulated for 18 h with plate-bound anti-CD3 (50 ng/ml)
plus recombinant human IL-2 (100 U/ml) in the absence and presence of
LY, followed by immunoblotting with anti-phospho-S6(240/244), anti-S6,
and anti-␤-actin. Numbers below lanes in B and C indicate the relative
levels of phosphorylated to total protein in each set of blots above as
determined by densitometry.
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DEFECTIVE S6 KINASE ACTIVITY IN FADDdd T CELLS
Table II. Cell cycle progression analyzed by continuous BrdU labeling
of Casp8⫺/⫺ vs wild-type (WT) cellsa
Genotype
WT
Casp8⫺/⫺
Time (h)
G0/G1
S
G2/M
Subdiploid
0
24
48
0
24
48
96
64
5
96
76
24
0
26
69
0
15
45
0
4
22
0
2
17
3
4
3
2
6
14
a
Percentages shown correspond to the gates labeled in Fig. 1A for T cells stimulated with anti-CD3 plus IL-2 for the indicated time. Percentages shown correspond
to the gates labeled in Fig. 1A for T cells stimulated with anti-CD3 plus IL-2 for the
indicated time. Percentages shown correspond to the gates labeled in Fig. 7B for T
cells stimulated with anti-CD3 plus IL-2 for the indicated time.
Discussion
We have established that, in addition to promoting death-receptor
dependent apoptotic pathways, FADD and caspase-8 play an essential role in maintaining S6K1 activity during T cell cycle progression. We have found that prolonged T cell activation is essential for complete serial phosphorylation of ribosomal protein S6,
and potentially other S6K1 substrates. In accord with this defective
S6K1 activity, we have found that naive FADDdd T cells failed to
efficiently enter S phase, and possessed defective CDK2 activity,
cyclin E induction, and Rb hyperphosphorylation (Fig. 4). Although FADDdd T cells failed to maintain S6K1 and S6 phosphorylation during late G1 phase, we found that mTOR function
was apparently normal because phosphorylation of 4E-BP1 was
unaltered in the mutant cells. Because FADDdd T cells had diminished CDK2 activity, and because a specific inhibitor of CDK2
reduced S6 phosphorylation on S240/S244, we propose that sustained S6 phosphorylation depends on active cell cycle progression
induced by IL-2. Our data also demonstrate similar defects in cell
cycle progression and S6K activity in T cells derived from v-FLIPtransgenic and caspase-8 conditionally null mice, suggesting that
FADD and caspase-8 are both essential for optimal cell cycle progression and survival following mitogenic stimulation. Taken together, these findings suggest that FADD, caspase-8 and cellular
FLIP operate together to promote efficient cell cycle progression in
mitogenically stimulated T cells.
The means by which FADD and caspase-8 participate in S6K1
function is currently unclear. Because S6K1 signaling was normal
immediately following TCR cross-linking (Fig. 3), our results
demonstrate that the effect of the FADDdd transgene is not likely
directed toward mTOR or other inputs into this signaling pathway
(53). Rather, we consider it likely that FADD and caspase-8 play
a distal role in maintaining S6K1 activity, likely during the late G1
phase of cell cycle. This defect is temporally distinct from the
reduced NF-␬B activation seen in human caspase-8-deficient T
cells, which occurs within minutes of TCR triggering (23, 54). Our
data demonstrate that in primary murine T cells, the long-term
activation of S6K1 is required for complete phosphorylation of
ribosomal protein S6 (Fig. 3). S6 possesses five distinct serine
residues that are known to be sequentially phosphorylated by
S6K1 and S6K2 (55). In response to mitogenic stimulation with
anti-CD3 and IL-2, serines 235 and 236 became phosphorylated
within 90 min, whereas phosphorylation at serines 240 and 244
was not observed until much later (Fig. 2). Recently, it was shown
Acknowledgments
We thank Stephen Hedrick, Daniel Beisner, and Irene Ch’en for providing
T cells from casp8⫺/⫺ ⫻ CD4Cre mice. We also thank David Fruman,
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Perhaps due to diminished cell cycle entry and decreased CDK2
activity, T cells with deficiencies in FADD and caspase-8 signaling also failed to maintain S6 phosphorylation at normal
levels, especially on serines 240 and 244.
that S6K1⫺/⫺/S6K2⫺/⫺ embryonic fibroblasts retained the capacity to phosphorylate S6 on serines 235 and 236, whereas phosphorylation at serines 240 and 244 was completely blocked (56).
Additionally, although phosphorylation of S6 on serines 235 and
236 is highly dependent upon p90/RSK and Ras/ERK signaling,
240 and 244 are exclusively phosphorylated by S6K1 and S6K2
(57). These results demonstrate that although phosphorylation of
S6 at serines 235 and 236 may occur via S6K1/S6K2-independent
means, complete phosphorylation of S6 depends on the function of
these kinases. Our results are consistent with the notion that defective phosphorylation of S6K1 at Thr389, a site essential for the
activity of this kinase, completely abrogates serial S6 phosphorylation at sites 240 and 244.
As described here, phosphorylation of S6 at S240/S244 was
blocked by the CDK2 inhibitor Rosc. This result suggests a potential feedback mechanism whereby induction of CDK2 before
S-phase entry leads to enhanced ribosome biogenesis and macromolecular synthesis required for later stages in cell cycle. Indeed,
recent evidence has demonstrated that both CDK1 and CDK5 have
the potential to phosphorylate S6K1 directly. In the case of CDK5,
phosphorylation of S6K1 on Thr411 has been demonstrated to be
required for its phosphorylation and activation by mTOR, suggesting that such a feedback loop between CDK2 and S6K1 may exist
in cycling T cells (58, 59). That S6 is itself important for T cell
proliferation has been established in mice expressing one defective
allele for S6 (60). In these mice, T cells bear a similar defect in
proliferation as we have observed in FADDdd and casp8⫺/⫺ T
cells. It is notable that S6⫹/⫺ T cells have diminished S-phase
entry and survival following mitogenic stimulation, and this is
most strikingly evident in CD8⫹ T cells. This selective defect may
be a consequence of the higher rate of cell cycle progression observed in CD8⫹ T cells.
One explanation may be that FADD and caspase-8 modulate the
activity of a key signaling molecule for late G1 progression, and
that this step maintains or enhances the active state of S6K1, likely
via active CDK2. S6K1 then facilitates cell growth during late G1
and promotes subsequent steps of cell cycle. For example, both
PI3K and S6K1 are essential for modulating E2F in response to
IL-2, an event necessary for promoting S-phase entry (61– 65). An
alternative explanation that may account for the results reported
here is that FADD and caspase-8 in some way govern the signaling
cascade that leads from the IL-2R to S6 phosphorylation. However, it is unlikely that FADDdd and v-FLIP-transgenic T cells
possess a global defect in IL-2R function, because we observed
normal STAT5 and Akt phosphorylation (Ref. 21 and Figs. 1, 3,
and 6). Rather, the defect appears to be selective, at a level between mTOR and S6K1 (Fig. 7E). Thus, an alternative scenario is
that the observed defects in CDK2 activity may be a consequence
rather than a cause for diminished S6K function. Recent studies
have uncovered an interaction between the anti-apoptotic factor survivin, mTOR, and aurora B kinase, an interaction essential for T cell
proliferation and survival (66). Death effector domain-containing protein (DEDD), a protein sharing a homologous death effector domain
with FADD, has also been shown to control cell cycle via the CDK1/
cyclin B1 complex (67). Our own recent work suggests that FADDdd
and casp8⫺/⫺ T cells possess a hyperautophagic phenotype (B. Bell
et al., manuscript in preparation). Because mTOR and S6Ks directly
impact autophagy (68), our studies here highlight an additional signaling paradigm in which the mTOR pathway is subject to regulation
by an apoptotic-signaling intermediate.
The Journal of Immunology
Thomas Lane, and Aimee Edinger for thoughtful suggestions and critical
evaluation of the manuscript.
Disclosures
The authors have no financial conflict of interest.
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