From the Department of Immunology, Roswell Park
Cancer Institute, Buffalo, New York 14263 and the Departments
of § Oral Biology and ¶ Microbiology, State University
of New York at Buffalo, Buffalo, New York 14214
Received for publication, September 16, 2002, and in revised form, January 7, 2003
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ABSTRACT |
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The interleukin-2 receptor (IL-2R) is
composed of one affinity-modulating subunit (IL-2R Defining the molecular mechanisms by which cytokines and their
receptors trigger signal transduction has been the focus of intensive
research. Indeed, a detailed understanding of cytokine receptor
structure and dynamics at the molecular level has proven useful in
several therapeutic modalities to treat cancer and autoimmune diseases
(1, 2). This paper focuses on structure-function relationships in the
interleukin (IL)1-2 receptor
(IL-2R), which is a prototypical member of the type I cytokine receptor
superfamily (3). IL-2 is a crucial regulator of T cell proliferation,
survival, and programmed cell death (apoptosis) (for review, see
Ref. 4). The IL-2R is a highly complex receptor that employs multiple
subunits to activate a remarkable variety of cellular signaling
cascades (for review, see Ref. 5). Despite a wealth of information
about the IL-2R system gained in the last decade, it is still not fully
understood how the various subunits within the IL-2R act in a
coordinated fashion to trigger a coherent array of signaling pathways.
Most cytokine receptors are composed of multiple subunits, existing
either as homomers of identical subunits (such as the erythropoietin
(EPO) and tumor necrosis factor receptors) or as heteromers of distinct
proteins (such as the IL-2 and interferon receptors). In addition, many
cytokine receptor complexes share one or more subunits with other
receptors; this is especially true of the type I family of cytokine
receptors. The members of this family are characterized by several
conserved structural features, including spaced cysteines and
WSXWS motifs in their extracellular domains, and canonical
box 1 and box 2 domains and tyrosine residues in their cytoplasmic
tails. There are several subfamilies of the type I receptors, which are
defined by the subunits that they share. For instance, the IL-3, -5, and granulocyte-macrophage colony-stimulating factor receptors employ
an identical receptor subunit termed Upon interaction with their ligands, type I cytokine receptors activate
numerous downstream signaling molecules, including Janus kinases (JAK),
signal transducers and activators of transcription (STAT),
mitogen-activated protein kinases (MAPK), phosphatidylinositol 3'-kinase (PI3K), and/or suppressors of cytokine signaling.
Signaling pathways involving these molecules are initiated by
recruiting signaling intermediates to the receptor complex, usually to
phosphorylated tyrosines located on the receptor's cytoplasmic tail.
In some cases, signaling molecules bind directly to the receptor, via phosphotyrosine-binding motifs such as Src homology 2 or
phosphotyrosine binding domains. Alternatively, sometimes one or
more adaptor molecules are used to bridge the signaling intermediate to
the receptor. Therefore, tyrosines in cytokine receptor tails are crucial launching points for signaling pathways, and much effort has
been directed to mapping specific signaling pathways to individual tyrosine residues within cytokine receptors (e.g. Refs. 11
and 12).
At the level of the receptor, it is clear that signaling requires a
cooperative interaction between receptor subunits. Indeed, for nearly
all cytokines, dimerization or multimerization of receptor subunits is
essential for signaling (13). In the case of receptors belonging to the
type I family, mutational analyses have shown that both subunits in the
complex must have functional JAK binding domains, which are located in
the conserved box 1 and box 2 domains (14). Dimerization then permits
trans-phosphorylation and subsequent activation of the associated JAKs
(15, 16) followed by phosphorylation of the receptor tails on distal
tyrosine residues.
Apart from JAK activation, are all subunits within a multimeric
receptor complex necessary for signal transduction? In some circumstances, effective signaling is maintained even when large distal
regions of the receptors are deleted by mutagenesis. For example, in
the homodimeric erythropoietin receptor (EPOR), it has been shown that
both JAK2 binding sites within the receptor tail must be present for
STAT activation, but only one distal tail is required to be present
(16). Frequently, multiple tyrosines within the cytoplasmic tail of a
single receptor are able to activate identical pathways. For instance,
in the EPOR, four different tyrosine residues are capable of activating
the transcription factor STAT5 (17-19). Thus, homodimeric receptors
apparently contain duplicated signaling domains, any one of which can
activate downstream pathways such as STAT activation. Although these
duplicated domains may serve to amplify or fine tune signaling
responses (as has been demonstrated for tyrosine-containing motifs in
the T cell receptor (20)), they may serve as redundant or "backup"
mechanisms to ensure that essential pathways are activated.
Duplicated signaling domains also appear to exist in heteromeric
receptors such as the IL-2R, although the receptor subunits contribute
differentially to signaling (5, 12, 21-24). The IL-2R is composed of
three subunits, IL-2R Despite the central role of IL-2R Cell Culture, Cell Lines, and Cytokine Stimulations--
HT-2
cells (American Type Culture Collection, Rockville, MD) were maintained
in RPMI 1640, 10% fetal bovine serum (Gemini Bioproducts, Woodland,
CA), 2 mM glutamine, 0.05 mM 2-mercaptoethanol, penicillin/streptomycin, and 1 nM recombinant human IL-2
(generously provided by the Chiron Corporation, Emeryville, CA).
HT-2.EPO Plasmids--
All EPO-IL-2R chimeras were expressed in the pCMV4
vector series (33), kindly provided by Dr. M. A. Goldsmith.
Construction of EPO Proliferation Assays--
Conventional
[3H]thymidine incorporation assays were performed as
described previously (35). Briefly, 5 × 103
cells/well were washed twice in phosphate-buffered saline and incubated
with EPO or IL-2 for 24 or 48 h in triplicate. Four h prior to
harvesting, 1 µCi/well [3H]thymidine (PerkinElmer Life
Sciences) was added. Cells were harvested on a Skatron microwell
harvester (generously provided by Dr. W. C. Greene, Gladstone
Institute of Virology and Immunology, San Francisco) and analyzed on a
Wallac Microbeta counter (PerkinElmer Life Sciences).
Northern Blotting--
Cytoplasmic RNA was prepared from
1-2 × 107 cells using an RNeasy kit (Qiagen,
Valencia, CA) and quantified by spectrophotometry. Denaturing 1.4%
formaldehyde-agarose gels were prepared with 10 µg of RNA/lane,
blotted to Zeta probe membranes (Bio-Rad), and hybridized with
32P-labeled IL-2R Immunoprecipitations and Western Blotting--
For
immunoprecipitations, 2-3 × 107 cells were rested
for 3-4 h in RPMI 1640 and 1% bovine serum albumin and stimulated
with cytokines. Cells were lysed in buffer containing 1% Nonidet P-40, 20 mM Tris, pH 8.0, 150 mM NaCl, 50 mM NaF, 100 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and a 1% protease
inhibitor mixture (0.5 mg/ml antipain, 0.5 mg/ml aprotinin, 0.75 mg/ml
bestatin, 0.5 mg/ml leupeptin, 0.05 mg/ml pepstatin, 0.4 mg/ml
phosphoramidon, 0.5 mg/ml trypsin inhibitor). Immunoprecipitations were
performed with 1 µg of anti-JAK1 antibodies (Santa Cruz
Biotechnology, Santa Cruz, CA), 2.5 µg of anti-JAK3 antibodies
(Upstate Biotechnologies, Saranac NY), or 4 µl of anti-Akt1
antibodies (Cell Signaling Technology, Beverly, MA) and protein A- or
protein G-agarose (Roche Molecular Biochemicals). Western blots were
probed with 4G10 anti-phosphotyrosine (Upstate Biotechnologies),
anti-phospho-Akt1 (Ser-473), anti-Akt and Bcl-2 (Cell Signaling
Technology), anti-JAK3, or anti-JAK1 (Santa Cruz Biotechnology).
Flow Cytometry--
Apoptosis assays were performed as described
previously (36). Briefly, cells were incubated with a 1:100 dilution of
GFP-annexin V (a kind gift from Dr. Joel Ernst, University of
California, San Francisco (37)) for 20 min on ice, washed in staining
buffer (phosphate-buffered saline, 2% fetal bovine serum, 2.5 mM CaCl), and resuspended in 1 ml of staining buffer
containing 25 ng of propidium iodide (PI) (Molecular Probes, Eugene,
OR). Cells were analyzed on a FACScan using Cellquest software (BD
Biosciences). Intracellular staining for Bcl-2 was performed by
permeabilizing 106 cells/sample with a Cytoperm/Cytofix kit
(Pharmingen) for 30 min on ice. Cells were washed twice with 1× wash
buffer (from kit) and stained for 1-2 h with a 1:100 dilution of
anti-mouse Bcl-2-FITC (clone 3F11) or an isotype-matched control
antibody conjugated to FITC.
Cytoplasmic Tyrosine Residues within IL-2R To define regions within IL-2R subunits involved in engaging
anti-apoptotic signal transduction pathways, we used a chimeric receptor system previously established in a murine,
IL-2-dependent T cell line, HT-2 (22). To bypass signaling
by the endogenous IL-2R, HT-2 cells were stably transfected with
chimeric receptors composed of the extracellular domain of the EPOR
fused to the transmembrane and cytoplasmic tails of the IL-2R) and two
essential signaling subunits (IL-2R
and
c). Although most known
signaling events are mediated through tyrosine residues located within
IL-2R
, no functions have yet been ascribed to
c tyrosine
residues. In this study, we describe a role for
c tyrosines in
anti-apoptotic signal transduction. We have shown previously that a
tyrosine-deficient IL-2R
chain paired with wild type
c stimulated
enhancement of bcl-2 mRNA in IL-2-dependent
T cells, but it was not determined which region of the IL-2R or which
pathway was activated to direct this signaling response. Here we show
that up-regulation of Bcl-2 by an IL-2R lacking IL-2R
tyrosine
residues leads to increased cell survival after cytokine deprivation;
strikingly, this survival signal does not occur in the absence of
c
tyrosine residues. These
c-dependent signals are
revealed only in the absence of IL-2R
tyrosines, indicating that the
IL-2R engages at least two distinct signaling pathways to regulate
apoptosis and Bcl-2 expression. Mechanistically, the
c-dependent signal requires activation of Janus kinases
1 and 3 and is sensitive to wortmannin, implicating
phosphatidylinositol 3-kinase. Consistent with involvement of
phosphatidylinositol 3-kinase, Akt can be activated via tyrosine
residues on
c. Thus,
c mediates an anti-apoptotic signaling
pathway through Akt which cooperates with signals from its partner
chain, IL-2R
.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
c, and the IL-6 family of
receptors shares the gp130 subunit (for review, see Ref. 6). Similarly,
the IL-2 family of receptors is defined by their use of the common
(
c) subunit. Originally identified as the IL-2R
chain (7),
c is now recognized to be an essential component of the IL-2, -4, -7, -9, -15, and -21 receptors (8, 9). Humans with genetic deficiencies in
c
suffer X-linked severe combined immunodeficiency syndrome caused by a
phenotypic loss of these cytokines (for review, see Refs. 8 and
10).
(CD25 or Tac), IL-2R
, and
c. The
IL-2R
is an affinity modulator that is dispensable for signaling,
although it is essential in vivo for detection of
physiological levels of IL-2. In contrast, IL-2R
and
c are necessary and sufficient for signaling. Both IL-2R
and
c are phosphorylated after receptor ligation (25, 26), and both subunits are
highly conserved in mammalian evolution. The functional roles of
tyrosine residues within the IL-2R
chain have been carefully examined, and several important signaling pathways have been found to
be strictly dependent on tyrosines within this subunit. For example,
Tyr-338 recruits the adaptor molecule Shc and activates the p38-MAPK
pathway, leading to c-fos gene expression (27-29). Moreover, Tyr-338 is the only tyrosine within the IL-2R complex capable
of activating this pathway. In contrast, three different tyrosine
residues (Tyr-338, Tyr-392, and Tyr-510) all activate STAT5 and
proliferation independently (11, 12). Although multiple IL-2R
tyrosines can activate these pathways, tyrosines in
c cannot do so.
Both the MAPK and STAT5 activation proceed normally even in the absence
of tyrosine residues on
c (12, 30). Conversely, tyrosines within
c cannot compensate for an absence of tyrosine residues on IL-2R
(11, 22, 23). Furthermore, replacing
c with a heterologous cytokine
receptor (the EPOR) does not have a noticeably deleterious effect on
these signals, as long as IL-2R
tyrosines are retained (21). These
findings led to a model in which the only function of the
c
cytoplasmic tail is to recruit JAK3 to the receptor complex, whereupon
JAK3 is able to phosphorylate JAK1 and permit subsequent signaling via
phosphorylated tyrosine residues within IL-2R
.
tyrosine residues, a number of
signals proceed in the absence of tyrosine residues on the IL-2R
chain. In particular, the gene encoding Bcl-2, an important anti-apoptotic effector, is still enhanced by a mutant IL-2R
that
cannot be phosphorylated (12). Mutagenesis studies have indicated that
c appears to be crucial for regulating Bcl-2 in vivo even
in the absence of its JAK3 binding domain (31). Moreover, the immune
impairment observed in
c-deficient mice can be partially rescued by
a bcl-2 transgene (32). Therefore, given the widespread use
of
c as a signaling subunit and the evolutionary conservation of its
cytoplasmic tyrosine residues, we hypothesized that
c may engage
signaling pathways in addition to JAK3 activation, possibly through its
tyrosine residues. Accordingly, we show here that tyrosines within the
c subunit mediate a signaling cascade that leads to up-regulation of
Bcl-2 and inhibition of apoptosis, thus revealing a previously
unrecognized function for the
c subunit in signal transduction.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and HT-2.EPO
cells were maintained in this medium
supplemented with 0.5 mg/ml G418 (Invitrogen). HT-2.EPO
YF/
,
HT-2.EPO
ABC/
, HT-2.EPO
ABC/
YF, and
HT-2.EPO
D258A/
cells were maintained in this medium supplemented
with 0.5 mg/ml G418 and 0.4 mg/ml hygromycin B (Invitrogen).
HT-2.EPO
/
and HT-2.EPO
/
YF cells were maintained in this
medium with recombinant human EPO (5 units/ml, a kind gift from Amgen,
Thousand Oaks, CA) in place of IL-2. HT-2 transfectant cell lines were
prepared as described previously (22). For stimulations, cells were
washed twice in phosphate-buffered saline, stripped with 10 mM sodium citrate and 140 nM NaCl, and incubated in RPMI 1640 medium with 1% bovine serum albumin (Sigma) for
4 h. Stimulations were for indicated times with recombinant human
IL-2 (5-10 nM) or EPO (50 units/ml unless otherwise indicated).
, EPO
YF, EPO
, and EPO
YF has been
described elsewhere (22). EPO
ABC was made by subcloning a
BclI-XbaI fragment from IL-2R
ABC (34) into
corresponding sites within pCMV4.EPO
.
,
c, or glyceraldehyde-3-phosphate
dehydrogenase cDNA probes.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Are Not Required for
Induction of Bcl-2 or Anti-apoptotic Signaling
and
c subunits, termed EPO
and EPO
, respectively (Fig.
1A). Many prior studies have
confirmed that heterodimerization of the EPO
and EPO
cytoplasmic tails via EPO is both necessary and sufficient to direct signaling indistinguishable from the native IL-2R and that neither EPO
nor
EPO
co-opts endogenous IL-2R
or
c subunits for signaling (21,
22, 30). Notably, the EPO
/
complex represents both IL-2R- and
IL-15R-dependent signaling because these receptors both
contain the IL-2R
and
c subunits (38-40). Henceforth we shall
refer to our findings as IL-2-dependent, recognizing that these are presumably the same signals delivered by the IL-15R.
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Fig. 1.
A, schematic diagram of the EPO-IL-2R
chimeric receptor system. Chimeric receptors composed of the
extracellular domain of the murine EPOR were fused in-frame to the
transmembrane and cytoplasmic tails of the human IL-2R and
c
receptors (originally described in Ref. 22). Box 1 and box 2 regions
(which bind to JAKs) are shown. Tyrosine (Y) residues are
numbered according to Refs. 7 and 94 and are indicated by
circles; mutations to phenylalanine (F) are
indicated by straight lines. Aspartic acid
(D)-258 within IL-2R
is required for JAK1 activation, and
the IL-2R
ABC mutant is truncated after amino acid 312 (34).
YF indicates a tyrosine-deficient subunit of either IL-2R
or
c. Although these chimeric receptors do not incorporate IL-2R
,
it is well established that this subunit does not contribute to signal
transduction by the IL-2R (95). Note that similar IL-2R chimeric
receptor systems have been described by others (96-98). B,
expression of EPOR chimeras in HT-2 cells. Cytoplasmic mRNA was
made from the indicated cell lines and separated on a 1.4% denaturing
formaldehyde-agarose gel. The RNA was transferred to a nylon membrane
and probed with 32P-labeled cDNAs corresponding to
IL-2R
,
c, and glyceraldehyde-3-phosphate dehydrogenase
(GAPD). Arrows indicate endogenous
IL-2R
and
c bands. The EPO
YF construct migrates faster than
the EPO
construct because of differences in their 3'-untranslated
regions. C, activation of JAK1 by chimeric IL-2 receptors.
HT-2 cell lines stably transfected with the indicated pairs of chimeric
receptors were rested for 4 h in serum-free medium and incubated
without cytokines (U), 50 units/ml EPO (E), or 5 nM IL-2 (2) for 15 min. Lysates were
immunoprecipitated (IP) with anti-JAK1 antibodies, separated
by SDS-PAGE on an 8% gel, transferred to nitrocellulose, and probed
with anti-phosphotyrosine (P-Tyr) antibodies (4G10,
top panels). Blots were stripped and reprobed with anti-JAK1
antibodies to verify equivalent loading (bottom panels).
Experiments were performed multiple times with similar results and have
also been described elsewhere for several of these cell lines (12, 21,
22).
Plasmids encoding the EPO chimeras were stably transfected into HT-2
cells and monoclonal cell lines derived by limiting dilution. All
constructs were found to be highly expressed, especially compared with
endogenous IL-2R and
c levels, as assessed by Northern blotting
(Fig. 1B). To show that the chimeric receptors activated early responses equivalently, phosphorylation of JAK1 was monitored after treatment with EPO or IL-2 (Fig. 1C). Phosphorylation
of the JAKs requires dimerization of the IL-2R
with
c cytoplasmic tails (15, 22); therefore, EPO-inducible phosphorylation of JAK1
indicates that both chimeric receptors are expressed productively on
the cell surface. As shown, JAK1 was phosphorylated equivalently after
IL-2 and EPO treatment in all lines tested except HT-2.EPO
D258A/
cells (where the mutation at Asp-258 disrupts JAK1 activation (35, 41))
(Fig. 1C). These data indicate that the EPO chimeras expressed in HT-2 cells are competent to deliver EPO-inducible signals
essentially equivalent to those induced by the endogenous IL-2R.
IL-2 signaling is known to induce bcl-2 mRNA in
hematopoietic cells (12, 42, 43), and both the JAK-STAT and MAPK
pathways have been linked to control of bcl-2 expression
(44-47). However, we found previously that an IL-2R composed of a
tyrosine-deficient IL-2R chain paired with a wild type
c receptor
(i.e. the chimera EPO
YF/
) is able to stimulate
up-regulation of bcl-2 mRNA, thus implicating other
signaling pathways in Bcl-2 expression (12). To show that the
EPO
YF/
chimera could also up-regulate the Bcl-2 protein, we
performed intracellular staining and Western blotting experiments to
visualize Bcl-2 expression levels (Fig.
2). As expected, in HT-2 cells expressing
either the EPO
or the EPO
chimera alone, treatment with EPO did
not trigger significant increases in Bcl-2, whereas signaling through
the endogenous IL-2R enhanced Bcl-2 expression markedly (Fig. 2,
A and B). In contrast, in HT-2.EPO
/
cells,
almost equivalent levels of Bcl-2 were induced by both EPO and IL-2
(Fig. 2C). In HT-2.EPO
/
YF cells, EPO induced Bcl-2
equivalently to the wild type IL-2R, indicating that signaling could
proceed in the absence of
c tyrosines (Fig. 2D).
Strikingly, in HT-2.EPO
YF/
cells, EPO stimulation also caused
significant up-regulation of Bcl-2, albeit somewhat reduced compared
with the endogenous IL-2R (Fig. 2E). A similar pattern of
bcl-2 mRNA induction was observed in
HT-2.EPO
YF/
, HT-2.EPO
/
YF, and HT-2.EPO
/
cells (data
not shown), suggesting that Bcl-2 regulation occurs at the level of
mRNA.
|
To ascertain whether Bcl-2 levels were associated with productive
anti-apoptotic signals, cytokines were removed from the growth medium
of HT-2.EPO/
or HT-2.EPO
YF/
cells, and cell viability and
progression to apoptosis were monitored by flow cytometry. Cells were
treated with IL-2 or EPO and then stained with PI to test for viability
and annexin V coupled to GFP (GFP-annexin V (37)) to test for apoptosis
(Fig. 3). Note that stimulation of
parental HT-2 cells with EPO does not trigger a detectable anti-apoptotic signal because these cells do not express endogenous EPO
receptors (36). As expected, HT-2.EPO
/
cells exhibited strong
protection from apoptosis when incubated with EPO or IL-2. In addition,
only a small percentage of the viable cells underwent apoptosis in EPO
or IL-2, whereas most of the viable (PI-negative) cells were apoptotic
in the absence of cytokines (Fig. 3A). In HT-2.EPO
YF/
cells, EPO also induced significant protection from apoptosis (Fig.
3B). Similar to our observations with Bcl-2, the protection
afforded by EPO
YF/
was not as vigorous as that by the native
IL-2R or the wild type EPO
/
chimeras. Part of the basis for the
decreased survival is likely the reduced Bcl-2 in these cells (Fig. 2),
but additional events may also be required to maintain long term
survival (48). Together, these results agreed with previous findings
that not all signals by the IL-2R are regulated through IL-2R
tyrosine residues (12). These data also raised the question of whether
Bcl-2 induction and inhibition of apoptosis involve tyrosines on
c
or could occur through pathways completely independent of cytoplasmic
tyrosines.
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c Tyrosine Residues Are Required for Anti-apoptotic Signaling in
the Absence of IL-2R
Tyrosines
The mechanism by which the IL-2R controls Bcl-2 expression is
poorly defined. Most pathways implicated to date involve signals that
depend on tyrosines within IL-2R, such as p38-MAPK and STAT5 (44-47). To determine whether
c tyrosines can trigger additional pathways that regulate Bcl-2, we created cell lines expressing a
truncated form of the IL-2R
chain (EPO
ABC) paired with either a wild type
c tail (EPO
) or a tyrosine-deficient
c tail
(EPO
YF). The EPO
ABC mutant retains the ability to bind and
activate JAK1 (Fig. 1C) but does not contain cytoplasmic
tyrosine residues or other distal sequences (34). First, we found that
the HT-2.EPO
ABC/
cells behaved almost identically to the
HT-2.EPO
YF/
cells with respect to Bcl-2 induction and survival
signaling (Fig. 2 and Fig. 4,
A and B). Thus, there do not appear to be
"tyrosine-independent" signaling motifs in the IL-2R
cytoplasmic
tail required for promoting cell survival and Bcl-2 expression.
|
Strikingly, in the absence of IL-2R tyrosines,
c tyrosines appear
to be necessary to mediate up-regulation of Bcl-2. Specifically, treatment of HT-2.EPO
ABC/
YF cells with EPO failed to
up-regulate Bcl-2 detectably (Fig. 4A). This regulation
occurred at the level of mRNA because treatment with EPO resulted
in increased steady-state levels of bcl-2 message in
HT-2.EPO
ABC/
cells but not in HT-2.EPO
ABC/
YF cells
(data not shown). These results revealed that although maximal regulation of Bcl-2 probably requires signals from IL-2R
tyrosines, signals are also relayed from
c tyrosine residues. Moreover, signals
initiated directly from JAK1 and/or JAK3 are not sufficient for Bcl-2
up-regulation (as has been reported for PI3K activation (49)) because
HT-2.EPO
ABC/
YF cells activate these kinases efficiently.
Consistent with the involvement of c tyrosine residues in
up-regulating Bcl-2, we found that EPO stimulation failed to provide a
survival signal to HT-2.EPO
ABC/
YF cells, whereas EPO inhibited apoptosis in HT-2.EPO
ABC/
cells (Fig. 4B). As with
Bcl-2 expression, the anti-apoptotic signal was reduced compared with
that induced through the endogenous IL-2R or EPO
/
chimeras. The
degree of survival was very similar to that induced in
HT-2.EPO
YF/
cells (Fig. 3) and was consistent among independently
derived lines. Because HT-2.EPO
/
YF cells maintain the capacity to
up-regulate Bcl-2 and inhibit apoptosis even in the absence of
c
tyrosines (Figs. 2 and 4B), at least two distinct pathways
are initiated by the IL-2R to regulate Bcl-2. Although
IL-2R
-dependent anti-apoptotic pathways exist which
proceed in the absence of
c tyrosines, at least one anti-apoptotic
pathway is mediated through tyrosines within
c. These findings
highlight the complex interplay between the IL-2R
and
c chains in
controlling cell survival.
Mechanisms of Anti-apoptotic Signaling
JAK1 and JAK3 Activation--
Many signaling pathways have been
linked to the IL-2R, all of which depend on activation of JAK1 (for
review, see Ref. 50). To confirm that JAK1 was required for the
anti-apoptotic signal mediated by c, HT-2.EPO
cells were stably
transfected with a plasmid encoding EPO
D258A, which cannot activate
JAK1 (and consequently JAK3 (15, 34, 41)). EPO stimulation of
HT-2.EPO
D258A/
cells failed to induce Bcl-2 or protect cells from
apoptosis after cytokine withdrawal (Fig.
5, A and B). In the
c-mediated pathway described here, JAK1 is presumably required for
phosphorylation of JAK3 and subsequently EPO
, thus initiating the
anti-apoptotic cascade. In this regard, we have demonstrated that
EPO
is indeed phosphorylated on tyrosine(s) after stimulation of the
receptor (data not shown).
|
In contrast to JAK1, JAK3 has been suggested to be at least partly
dispensable for regulation of Bcl-2, both in cell lines and in
vivo (31, 51, 52). Therefore, to examine whether JAK3 activity is
required for survival signaling mediated by c tyrosines, we took
advantage of a pharmacological inhibitor of JAK3, WHI-P154 (53). First,
we confirmed that WHI-P154 inhibits EPO- and IL-2-induced proliferation
in HT-2.EPO
/
cells, a signal for which JAK3 is known to be
crucial (15, 54). Proliferation was blocked completely at a
concentration of 20 µg/ml WHI-P154, consistent with a published
report (53) (Fig. 5C). Next, we showed that WHI-P154 at this
concentration also blocked JAK3 phosphorylation (Fig. 5D).
Finally, we found that WHI-P154 blocked Bcl-2 induction and inhibition
of apoptosis through the EPO
ABC/
receptor (Fig. 5,
E and F). The slight decrease of Bcl-2 in
EPO-stimulated cells compared with unstimulated cells in the presence
of WHI-P154 was not consistently observed. Based on these results, the
anti-apoptotic signaling pathway mediated by
c tyrosines appears
to be JAK3-dependent and supports a model in which the JAKs
phosphorylate each other and then the
c tail, and
c
subsequently recruits downstream anti-apoptotic signaling pathways.
Interestingly, although WHI-P154 completely blocked IL-2-dependent proliferation (Fig. 5C), it only partially blocked anti-apoptotic signals mediated by the endogenous IL-2R (Fig. 5, E and F). It is possible that these signals simply show different sensitivities to this compound. Alternatively, this finding provides some support for previous (albeit controversial) suggestions that a poorly defined, JAK3-independent signaling pathway regulates Bcl-2 (see Fig. 7A and Ref. 55) (31, 51, 52).
Distal Signals--
Because tyrosines within IL-2R are not
involved in this anti-apoptotic signal, we inferred that neither the
STAT5A/B nor p38-MAPK pathway was responsible. This hypothesis was
confirmed in control experiments showing that HT-2.EPO
ABC/
cells did not activate STAT5 phosphorylation or nuclear import and that inhibitors of various MAPK pathways had no effect on the anti-apoptotic signal or Bcl-2 up-regulation (data not shown). In addition, we ruled
out involvement of the nuclear factor-
B pathway because IL-2
signaling does not activate this transcription factor in HT-2
cells.2
A major pathway implicated in anti-apoptotic signaling involves the
lipid kinase, PI3K (56, 57; for review, see Ref. 58), which activates
the serine-threonine kinase Akt (also called protein kinase B). Because
several studies have implicated Akt in anti-apoptotic signaling by IL-2
(59-62), we analyzed Akt activation in this system. HT-2.EPO/
,
HT-2.EPO
ABC/
, or HT-2.EPO
ABC/
YF cells were stimulated
with EPO or IL-2. Whole cell lysates were immunoprecipitated with
antibodies to Akt, separated on SDS-PAGE, and Western blots probed with
antibodies specific for the phosphorylated form of Akt (Ser-473) (Fig.
6A). In all cells there was
consistently a high background phosphorylation of Akt even after the
starving period, a feature common to many T cell lines. As expected,
stimulation of HT-2.EPO
/
cells with both EPO and IL-2 induced
phosphorylation of Akt over basal levels. EPO stimulation of
HT-2.EPO
ABC/
cells also triggered significant phosphorylation
of Akt, demonstrating that this pathway proceeds without distal
sequences and tyrosines within IL-2R
. In contrast,
HT-2.EPO
ABC/
YF cells did not trigger phosphorylation of Akt
(Fig. 6B), confirming that tyrosines within
c are
necessary for this signal. There was a more rapid decline in
phosphorylation of Akt in HT-2.EPO
ABC/
cells compared with HT-2.EPO
/
cells, suggesting that tyrosines within IL-2R
may be
needed to sustain optimal signaling. These data demonstrate that Akt
can be at least partly activated independently of tyrosines within
IL-2R
, and its activation correlates with the anti-apoptotic signaling cascade induced by
c.
|
Because several studies have shown that Akt contributes to
anti-apoptotic signaling in T cells (60, 63), we expected that blocking
this pathway would inhibit the anti-apoptotic pathway mediated by c
tyrosines. Indeed, treatment with the PI3K inhibitor wortmannin almost
completely prevented induction of Bcl-2 and survival signaling through
the EPO
ABC/
receptor (Fig. 6, C and D).
However, wortmannin exerted only a mild (though reproducible) inhibitory effect on survival signals induced through the endogenous IL-2R or wild type EPO
chimeras. Comparable results were obtained using another PI3K inhibitor, Ly294002 (not shown). These findings suggest a requirement for the PI3K/Akt family in anti-apoptotic signaling mediated by
c. Furthermore, because signaling through the
wild type receptor is only mildly impaired in the presence of PI3K
inhibitors, additional anti-apoptotic pathways independent of PI3K
apparently contribute to survival signaling in the context of the
intact IL-2R (Fig. 7).
|
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DISCUSSION |
---|
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---|
These data show for the first time that tyrosines within the
common subunit (
c) play a role in mediating signal transduction, as revealed in the IL-2 receptor system. We have addressed the role of
c tyrosines in survival signaling using chimeric EPOR-IL-2Rs expressed in IL-2-dependent HT-2 cells. The initial impetus
for this study was our previous report that a tyrosine-deficient
IL-2R
chain paired with a wild type
c subunit (EPO
YF/
)
stimulates significant enhancement of bcl-2 mRNA,
indicating that some intracellular signals can be transmitted
independently of IL-2R
tyrosines (12). However, that study did not
define the region within the IL-2R which mediated this signal, leaving
open the possibility that regions within IL-2R
or the JAKs might
serve to recruit signaling effectors that lead to bcl-2
up-regulation. In the present study, we found that that this signal
indeed requires the proximal, JAK1 binding region of
IL-2R
and JAK1 activity but does not require the IL-2R
distal
tail or cytoplasmic tyrosine residues. Instead, anti-apoptotic
signaling (in the absence of IL-2R
tyrosines) depends on tyrosines
within
c. Furthermore, the serine-threonine kinase Akt is induced by
an IL-2R complex that lacks IL-2R
tyrosine residues, and this also
requires
c tyrosines. Consistent with a role for the Akt pathway,
c-mediated up-regulation of Bcl-2 and inhibition of apoptosis are
sensitive to PI3K inhibitors. Thus, this work provides the first
evidence that
c activates specific signals through its tyrosine residues.
Functions of c--
Surprisingly, very few specific signals to
date have been linked directly to
c and none to its tyrosine
residues. The primary recognized role of
c is to bind JAK3, enabling
JAK3 to phosphorylate JAK1 and other substrates recruited to the
receptor complex (for review, see Ref. 14). The biological importance
of the
c-JAK3 association is underscored by the finding that
c
mutations in many X-severe combined immunodeficiency syndrome patients
lie in the JAK3 binding domain and also that JAK3
/
humans and mice exhibit an autosomal immunodeficiency syndrome very
similar to X-severe combined immunodeficiency syndrome (for review, see
Refs. 10 and 64). Although two tyrosine residues are present in the
JAK3 binding region of
c (65), none of the four
c tyrosines is
required for JAK3 activation (22, 23, 66, 67). Moreover, the
cytoplasmic domain of
c can be functionally replaced with that of a
heterologous receptor (the EPOR), which binds to a different Janus
kinase (JAK2) with no apparent detriment to signaling (21). Thus, it
has long been presumed that tyrosines within
c do not participate in
signaling at all, even though
c is phosphorylated after receptor
engagement, and its tyrosines and flanking regions are evolutionarily
conserved (25). However, the present report reveals a role for
c
tyrosines in anti-apoptotic signaling which was not identified
previously, apparently because of redundant signals transmitted through
IL-2R
tyrosines (Fig. 7).
Because c is shared by multiple receptors, there may be a role for
c tyrosine residues in the context of other cytokine receptors.
Similar to the IL-2/15R system,
c tyrosines are dispensable for
STAT5 activation by both the IL-7R and IL-9R (66, 67). Interestingly,
however,
c tyrosines may play a role in promoting cell growth by
IL-9 because proliferation is reduced substantially in HT-2 cells
expressing an EPO-IL-9R
chimera (EPO9) together with EPO
YF
compared with EPO9 paired with EPO
(66). It will be interesting to
determine whether close examination of other
c-containing receptors
identifies an anti-apoptotic role for
c which was not recognized in
previous analyses.
Convergent Survival Signals--
The emerging model of signal
transduction is that multiple, overlapping signals converge to create a
coherent and integrated cellular response. Because the survival signal
induced by c is neither as potent nor as prolonged as that induced
by the wild type IL-2R (Figs. 3 and 4), additional signals induced
through IL-2R
tyrosines are apparently required for a complete
signal. In support of this concept, it was recently shown that
cytokines control apoptosis by stimulating glucose metabolism, which
acts in concert with anti-apoptotic members of the Bcl-2 family to maintain cellular survival (48, 68, 69). Although ectopic Bcl-2
expression in vivo restores some aspects of T cell signaling when
c is absent (32), enforced Bcl-2 expression alone is not sufficient for all aspects of
c-mediated signaling (70, 71). Similarly, activation of the PI3K pathway by itself is not enough to
drive proliferation in a model T cell line (72).
It is probable that at least one complementary signal derives from the
p38-MAPK pathway. MAPK is activated through the most membrane-proximal
tyrosine within IL-2R (Tyr-338) (27, 29) and leads to transcription
of the c-fos gene (11, 12). In turn, c-fos
cooperates with STAT5, c-myc, and/or bcl-2 to
promote proliferation by IL-2 (43, 72, 73). Similar functional
cooperation among the STAT5, MAPK, and PI3K pathways is needed to
achieve full oncogenic activity by the BCR/ABL kinase (74).
Accordingly, although Bcl-2 is clearly an important target of
cytokine-derived signals, it must act in concert with other events to
regulate appropriate cellular outcomes.
Regulation of Bcl-2 expression by IL-2 is not well understood, and multiple signaling pathways have been implicated. For example, p38-MAPK regulates bcl-2 gene expression via the transcription factor Aiolos, which binds to specific sites in the bcl-2 promoter (75). STAT5 has also been shown to regulate Bcl-2 in response to IL-2 (47), IL-7, and IL-15 (46). However, because IL-2 can also enhance Bcl-2 expression without participation of the MAPK or STAT5 pathways (Figs. 2 and 4 and Refs. 12, 31, 51, 76), the IL-2R appears to activate a variety downstream signaling pathways that converge on regulation of Bcl-2 (Fig. 7A).
PI3K Signaling by the IL-2R--
It is still not entirely clear
how the IL-2R activates PI3K, which is presumably upstream of Akt. The
classic PI3K isoform is composed of a 110-kDa catalytic subunit (p110)
and an 85-kDa regulatory subunit (p85), although several other
isoforms of PI3K exist. Several studies have indicated that p85
is
recruited through Shc and Gab2, via Tyr-338 on IL-2R
(73, 77, 78). However, another report implicates IL-2R
-Tyr-392 in this process (56). Finally, a recent study suggests that p85
may bind directly to
JAK1 (49). However, if there is constitutive association of p85
with
JAK1 in HT-2 cells, it is not sufficient to mediate a detectable
anti-apoptotic signal in the absence of
c tyrosine residues because
the EPO
ABC/
YF receptors fail to activate Akt phosphorylation
and anti-apoptotic signaling (Figs. 4 and 6). Possibly
c serves as
the first recruitment point for some member of PI3K family and then
transfers it to JAK1 for subsequent activation, analogous to a model
proposed for JAK3 (41). Other isoforms of PI3K have been identified
which may perform equivalent functions to p85
/p110. Our attempts to
coimmunoprecipitate p85
with
c have been
unsuccessful,2 suggesting that the anti-apoptotic signal
induced by
c may be mediated by an alternative member of the PI3K family.
Physiological Significance of c-Mediated Signaling--
What is
the role of the
c-tyrosine-mediated response in vivo?
Although no studies have addressed this issue directly, several findings are consistent with this pathway being biologically
significant. It is clear that IL-2 and IL-15 play important roles in
maintaining immune homeostasis (79-81). IL-2R
/
mice
display severe dysregulation in immune homeostasis which results in
fatal autoimmunity (82), and they fail to develop natural killer cells
(83). Reconstitution studies in which deletion mutants of IL-2R
were
expressed as transgenes in IL-2R
/
mice demonstrated
that STAT5 was essential for natural killer cell development, but
immune homeostasis was maintained even when receptors that cannot
activate STAT5 or MAPK were expressed (84). Consistent with this
observation, long term survival and homeostasis of T cells do not
require STAT5 but instead correlate with Akt stimulation and
up-regulation of Bcl-2 (76). Collectively, these studies imply a role
for both
c and PI3K/Akt signaling in maintaining immune homeostasis.
IL-2R signaling has been proposed to regulate homeostasis by
controlling the persistence of activated peripheral T cells via Fas and
FasL expression, which is also regulated in part by STAT5 (80, 81, 85).
However, peripheral expression of IL-2R may be dispensable for
maintaining T cell homeostasis. When a wild type IL-2R
chain was
expressed as a transgene in the thymus of IL-2R
/
mice, they exhibited little or no autoimmunity even in the absence of
detectable peripheral IL-2R
(86), suggesting that IL-2 and/or IL-15
plays a role in developing thymocytes to maintain immune homeostasis.
In support of this model, IL-2 signaling may be directly involved in
negative selection of major histocompatibility complex class
II-restricted thymocytes (87). Putting these observations together, our
findings provide a potential mechanism for how
IL-2/15R-dependent signals contribute to immune balance.
Namely, anti-apoptotic signaling through
c (mediated by Akt and
Bcl-2) may confer prolonged survival on a subset of developing T cells
(e.g. regulatory CD4+/CD25+ T cells
(88)), which ultimately restricts autoreactive peripheral T cells.
Another reason for the IL-2R to activate seemingly redundant pathways
may be to allow for a continuum of Bcl-2 expression levels. It is clear
that alterations in Bcl-2 levels exert potent effects on cellular
survival; namely, Bcl-2 overexpression can be tumorigenic, and its
underexpression can cause apoptosis (for review, see Ref. 89). In
addition, several different signaling cascades influence Bcl-2
expression, such as MAPK (75), STAT5 (46, 47),
c-dependent pathways (Fig. 4), and JAK3-independent pathways (51, 52) (Fig. 7A). Perhaps each pathway
contributes partially to the levels of Bcl-2, and all are regulated
concurrently to ensure that appropriate expression is maintained. This
model has precedence in antigen receptors such as the T cell receptor, which encodes multiple, redundant immunoreceptor tyrosine-based activation motifs (ITAMs) that recruit similar signaling intermediates and thereby amplify signaling responses appropriately (20). Thus, the
contribution of
c pathways to regulation of Bcl-2 is likely to be
biologically meaningful.
Other Contributors to Anti-apoptotic Signaling--
There may be
other anti-apoptotic events triggered by c tyrosines which we have
not yet identified. First, PI3K can lead to activation of the
transcription factor c-myb, which mediates anti-apoptotic
effects including regulation of Bcl-2 (46, 90). Second, the PI3K/Akt
pathway prevents apoptosis through inhibitory phosphorylation of the
forkhead transcription factor, which controls expression of
pro-apoptotic factors such as Bim (91). Third, alterations in
intracellular pH have been linked to increases in both pro-apoptotic
and anti-apoptotic molecules such as Bcl-2 in the IL-7 system (92).
Finally, in some cells IL-2 regulates secretion of extracellular
apoptotic effector molecules, such as lipocalin (93). It will be
interesting to determine whether any of these events is regulated by
c tyrosine residues.
In summary, the IL-2R activates numerous signaling pathways, several of
which converge to maintain cell survival. We have shown that at least
one of these anti-apoptotic signals is triggered through tyrosine
residues within c and is mediated by the PI3K/Akt cascade.
Importantly, this is the first recognition of a specific signaling role
for
c tyrosines, although it appears to play a subordinate role to
the IL-2R
chain in IL-2- and IL-15-dependent signaling.
However, given the widespread use of
c among cytokine receptors,
this pathway is likely to have implications for anti-apoptotic signaling in a variety of contexts.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Mark Goldsmith and Dr. Warner Greene for plasmids, Dr. Joel Ernst for the kind gift of GFP-annexin V, and Dr. Warner Greene for generously donating the Skatron cell harvester. We thank Dr. Arvind Thakur and Dr. Philip Loverde for use of the Wallac beta counter. Technical assistance was provided by Stacey Printup and Grace Wong. EPO was a generous gift from Amgen, Inc., and IL-2 was kindly provided by Dr. Kirk Johnson of the Chiron Corporation. We thank Dr. Xin Lin, Dr. James Clements, and Dr. Thomas Melendy for helpful suggestions.
![]() |
FOOTNOTES |
---|
* This work was supported by the Departments of Oral Biology and Microbiology at the State University of New York at Buffalo, National Institutes of Health Training Grant DE07034 (to M. J. L.), and Immune Deficiency Foundation and National Institutes of Health Grant AI49329 (to S. L. G.).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: Dept. of Oral
Biology, 36 Foster Hall, 3435 Main St., Buffalo, NY 14214. E-mail: sgaffen@buffalo.edu.
Published, JBC Papers in Press, January 12, 2003, DOI 10.1074/jbc.M209471200
2 S. L. Gaffen, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: IL, interleukin; EPO, erythropoietin; EPOR, erythropoietin receptor; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; IL-2R, interleukin-2 receptor; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; STAT, signal transducer and activator of transcription.
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