(Received for publication, August 30, 1996, and in revised form, January 6, 1997)
From the Laboratory of Molecular Immunology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1674
The interleukin-2 receptor chain (IL-2R
)
is potently induced by antigens, mitogens, and certain cytokines that
include IL-2 itself. This induction leads to the formation of high
affinity IL-2 receptors when IL-2R
is co-expressed with the
(IL-2R
) and
(
c) chains of this receptor. We
investigated the signaling pathways mediating IL-2-induced IL-2R
mRNA expression using 32D myeloid progenitor cells stably
transfected with either wild type IL-2R
or mutants of IL-2R
containing tyrosine to phenylalanine substitutions. Of the six
cytoplasmic tyrosines in IL-2R
, we have found that only the two
tyrosines that mediate Stat5 activation (Tyr-392 and Tyr-510)
contribute to IL-2-induced IL-2R
gene expression and that either
tyrosine alone is sufficient for this process. Interestingly, the IL-7
receptor contains a tyrosine (Tyr-429)-based sequence resembling the
motifs encompassing Tyr-392 and Tyr-510 of IL-2R
. Further
paralleling the IL-2 system, IL-7 could activate Stat5 and drive
expression of IL-2R
mRNA in 32D cells transfected with the human
IL-7R. However, IL-3 could not induce IL-2R
mRNA in 32D cells,
despite its ability to activate Stat5 via the endogenous IL-3 receptor.
Moreover, the combination of IL-3 and IL-2 could not "rescue"
IL-2R
mRNA expression in cells containing an IL-2R
mutant
with phenylalanine substitutions at Tyr-392 and Tyr-510. These data
suggest that Tyr-392 and Tyr-510 couple to an additional signaling
pathway beyond STAT protein activation in IL-2-mediated induction of
the IL-2R
gene.
Interleukin-2 (IL-2)1 is a pivotal
cytokine that influences several arms of the immune system, including T
cells, B cells, natural killer cells, and monocytes (1-4). Three
classes of IL-2 receptors are known to exist: low affinity receptors
contain the chain, intermediate affinity receptors contain the
and
chains, and high affinity receptors contain all three chains
(2-4). Interestingly, the
chain is also a component of the IL-15
receptor, whereas the
chain is a common chain (
c)
shared by the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 (2-5).
Mutations in
c form the genetic basis of X-linked severe
combined immunodeficiency, a disease state affecting both cellular and
humoral immunity (5, 6).
In lymphocytes, IL-2R and
c are constitutively
expressed, while IL-2R
is only expressed following activation by a
variety of stimuli that include IL-2 itself (1, 7, 8). When induced, IL-2R
complexes with
and
chains to convert intermediate
affinity to high affinity receptors (2-4). Although the intermediate
and high affinity receptors are both functional (8-11), the induction of IL-2R
is essential for normal immune function as evidenced by the
autoimmunity, inflammatory bowel disease, and premature death occurring
in IL-2R
knockout mice (12). The regulation of IL-2R
expression
is tightly controlled at the level of transcription, relying on the
interaction of positive regulatory elements with multiple transcription
factors that include Stat5, Elf-1, HMG-I(Y), and NF-
B (Refs. 13 and
14 and references therein).
While many of the regulatory proteins and binding sequences influencing
expression of the IL-2R gene have been defined, the more proximal
events leading from IL-2 binding to IL-2R
gene transcription are
less clear. Previous work with the tyrosine kinase inhibitor herbimycin
A has demonstrated the importance of tyrosine phosphorylation in
IL-2-signaling (15). Among the proteins tyrosine-phosphorylated in
response to IL-2 is IL-2R
itself (16-19). This receptor chain
contains six cytoplasmic tyrosines (Fig. 1), at least
some of which can serve as phosphotyrosine docking sites for signaling
proteins containing SH2 and PTB domains (20, 21).
Examination of signaling via mutant IL-2R chains containing
different tyrosine to phenylalanine substitutions therefore represents a method for mapping proximal events induced by IL-2 and has previously been used to investigate key functions such as proliferation (22-24). This analysis has demonstrated that tyrosine 338 (Tyr-338) mediates phosphorylation of the adaptor protein Shc (22, 25) (involved in
ras activation), while Tyr-392 and Tyr-510 can independently direct activation of Stat5 (22-23, 26-28). To elucidate the role of
these or other pathways in IL-2-induced expression of the IL-2R
gene, we have examined multiple combinations of these tyrosine to
phenylalanine substitutions in IL-2R
. These experiments show that
IL-2R
mutants possessing Tyr-392 or Tyr-510 alone can augment expression of the IL-2R
gene, whereas no IL-2R
induction occurs when both Tyr-392 and Tyr-510 are mutated. Although previous studies link these tyrosines to Stat5 activation, our data suggest that Stat5
activation is not sufficient for IL-2R
induction and that the same
tyrosines may couple to additional signaling pathway(s).
Expression of IL-2R was assessed
using a polyclonal antiserum to IL-2R
(ErdA antiserum, Ref. 9) or
4G10 monoclonal antibody to phosphotyrosine (Upstate Biotechnology,
Inc.).
Human IL-2R was mutated
using the altered sites in vitro mutagenesis system
(Promega) and oligonucleotides designed to change tyrosine (TAC) to
phenylalanine (TTC), as described previously (22). Following sequence
confirmation (Sequenase, U. S. Biochemical Corp.), mutant constructs
were subcloned into the vector pME18S in which expression is driven by
the SR
promoter (29). The human IL-7R cDNA has been described
(30).
32D cells were grown in RPMI
1640 medium supplemented with 10% fetal bovine serum,
105 M 2-mercaptoethanol, 5% WEHI-3B
conditioned medium (WEHI-CM) as a source of IL-3, 2 mM
glutamine, and 100 units/ml each of penicillin and streptomycin. The
transfectants expressing wild type and most of the mutant IL-2R
constructs were described previously (22). Additional transfectants
were generated by electroporating cells (5 × 106
cells/400 µl) with linearized plasmids consisting of IL-2R
constructs and pCDNA3neo (InVitrogen) using a gene pulser (Bio-Rad;
300 V, 960 microfarads; time constants were approximately 30 ms). After 24 h, cells were aliquoted into 24-well plates and selected in 1 mg/ml G418 (Life Technologies, Inc.) for 2-3 weeks. Resistant clones
were analyzed for IL-2R
expression by flow cytometry with fluorescein isothiocyanate-conjugated anti-IL-2R
monoclonal antibody (Endogen) or a control IgG2a (Becton Dickinson) on a FACSort FST (Becton Dickinson). In some cases, Western blotting with ErdA antiserum
to IL-2R
was used to further evaluate receptor expression. IL-7R
transfectants were established in a similar fashion.
32D transfectants grown to a density of 2-6 × 105 cells/ml were washed three times in acidified RPMI to
strip any growth factors bound to the cell surface. Cells were then
incubated for 16-18 h in medium containing either IL-3 (5% WEHI-CM),
IL-2 (2 nM), IL-2 plus IL-3, or IL-7 (5 nM)
plus IL-3, and RNA was prepared using TRIzol reagent (Life
Technologies, Inc.) according to the established protocol. 30 µg of
total RNA from each sample were run overnight in a 1% agarose
formaldehyde gel and transferred to nylon membranes (Amersham Hybond).
Equivalent loading and transfer of RNA was confirmed by ethidium
bromide staining of the membrane. A 1.2-kilobase pair murine IL-2R
cDNA fragment (31) was labeled with Life Technologies, Inc.'s
random primer labeling kit and used to probe the blots. As an
additional control for loading and expression of RNA, blots were also
hybridized with a 1-kilobase pair murine glyceraldehyde-3-phosphate
dehydrogenase cDNA fragment. All hybridizations were carried out
for 20 h at 43 °C. Following multiple washes in SDS/SSPE-based
solutions, blots were autoradiographed for 48-72 h at
70 °C.
EMSAs were performed as described previously (22).
Wild type or transfected 32D cells (~5 × 105/ml)
were washed and then depleted of growth factor for 4 h in phosphate-buffered saline or RPMI 1640 medium lacking serum and cytokines. Cells were then incubated at 37 °C for 20-30 min in RPMI, 10% fetal bovine serum medium with or without cytokines (2 nM IL-2, 5 nM IL-7, or 5% WEHI-CM). Following
one wash in ice-cold phosphate-buffered saline, cells were lysed with
three freeze thaw cycles to generate total cell extracts.
Alternatively, nuclear extracts were prepared by standard methods. In
EMSAs, 10 µg of total cell extract or 7 µg of nuclear extract were
preincubated with 1 µg of poly(dI-dC) for 25 min and then combined
with 20,000 cpm of a double-stranded 32P-labeled
oligonucleotide probe corresponding to the -interferon-activated site (GAS) of the Fc
RI gene (22) or the upstream (
1375 to
1334)
GAS motif region of the murine IL-2R
IL-2-response element (5
-GTGCAGTTTCGTACCAGACATGAG-3
;
canonical and noncanonical GAS sites are underlined) (32).
After an additional 20-min incubation on ice, samples were
electrophoresed on a 5% polyacrylamide gel (0.5 × TBE (0.045 M Tris borate, 0.001 M EDTA)) and
autoradiographed. Where indicated, polyclonal anti-Stat5 (26) or
anti-Stat3 (gift of S. Chen-King) antiserum was added for antibody supershifting experiments. In the case of anti-Stat3, the binding reaction was performed prior to addition of antibody, and all reactions
were carried out at room temperature. DNA affinity purification of
Stat5a and Stat5b was performed using 400 µg of total cell extract
and a biotinylated oligonucleotide containing a trimer of the
-casein GAS motif, as described previously (33).
32D cells lack IL-2R and are
IL-3-dependent, but they can proliferate in response to
IL-2 after IL-2R
is transfected and expressed (22, 34). To assess
the potential contribution of different IL-2R
tyrosines in the
signaling pathway(s) for IL-2-mediated IL-2R
mRNA induction, we
therefore examined 32D cells transfected with wild type or mutated
forms of IL-2R
. As shown previously (34), IL-3 could not mediate
IL-2R
gene expression in 32D cells transfected with wild type
IL-2R
, while IL-2 could induce IL-2R
mRNA in the same cell
line (Fig. 2A, lanes 3-4). The Northern blot
analysis presented in Fig. 2A further demonstrated that IL-2 could potently induce IL-2R
mRNA in all transfectants expressing IL-2R
chains retaining the terminal two tyrosines (Tyr-392, Tyr-510) (lanes 4, 8, and 10). Simultaneous mutation of
Tyr-392 and Tyr-510 completely blocked the induction of IL-2R
mRNA (compare
YYYYYY and
YYYYFF, lanes 3-4 versus
11-12), indicating that at least one of these tyrosines is
required for IL-2-mediated IL-2R
gene expression. Further analysis
revealed that either Tyr-392 or Tyr-510 alone is sufficient for this
process (Fig. 2B), analogous to the functional overlap of
Tyr-392 and Tyr-510 in mediating IL-2-induced proliferation (22). Of
note, the fact that either of these tyrosines can mediate IL-2R
gene
expression, but Tyr-338 cannot, indicates that IL-2R
induction is
not absolutely required for proliferation (which can occur at
suboptimal levels with Tyr-338 alone (22)) and further supports
observations that pathways linked to Tyr-338 are not functionally
equivalent to signals directed from Tyr-392 and Tyr-510 (22).
Consistent with the functional redundancy of Tyr-392 and Tyr-510,
previous work has shown that both Tyr-392 and Tyr-510 can independently
mediate IL-2-induced Stat5 activation in 32D cells transfected with
appropriate IL-2R constructs (22). Since each of these tyrosines
shares a motif similar to that surrounding Tyr-429 of the IL-7 receptor
(26) (Fig. 3A), we speculated that IL-7 could
trigger IL-2R
gene expression in 32D cells transfected with the IL-7
receptor. This hypothesis seemed plausible given the expression of
IL-2R
in double negative thymocytes normally exposed to IL-7
in vivo. Indeed, IL-7 induced both Stat5 activation (Fig.
3B) and IL-2R
mRNA expression (Fig. 3C) in
32D-IL-7R transfectants.
Stat5 Induction Alone Is Not Sufficient to Induce IL-2R
Collectively, these results suggested an important
role for Stat5 activation in the induction of IL-2R mRNA. Hence,
the inability of IL-3 (which also can activate Stat5 (35, 36)) to
augment expression of IL-2R
mRNA (Fig. 2A) was
surprising, particularly since IL-2 and IL-3 induced indistinguishable
STAT complexes in IL-2R
-transfected 32D cells when performing EMSAs
with a probe comprised of the murine IL-2R
GAS motifs. As shown in
Fig. 4, the complexes resulting from IL-2 or IL-3
treatment (lanes 2 and 3) supershifted with
anti-Stat5 antiserum (lanes 5 and 6), but not
with anti-Stat3 (lanes 8 and 9). Similar results
were obtained using the Fc
RI GAS site as a probe (data not shown).
Because the anti-Stat5 antiserum used in these experiments recognizes two closely related proteins termed Stat5a and Stat5b, we performed additional supershift experiments with Stat5a- and Stat5b-specific antisera to exclude the possibility that IL-2 and IL-3 differed in
their induction of these proteins. In fact, this analysis demonstrated that both cytokines could independently activate Stat5a and Stat5b in
32D cells transfected with wild type IL-2R
(Fig.
5A). DNA affinity purification of STAT
proteins with an oligonucleotide probe containing a trimer of the
-casein GAS motif confirmed these results (Fig. 5B).
Overall, these data indicate that Stat3 is not required for
IL-2-induced IL-2R
expression in 32D cells and that Stat5a/Stat5b
activation alone is not sufficient to mediate IL-2R
mRNA
induction, supporting a role for additional cooperating signal(s). Of
note, the fact that IL-2 and IL-3 (which normally exert effects on
different cell types) differ in their capacity to induce IL-2R
gene
expression within the same cell line indicates that such
cooperating signals depend on the receptor system rather than cell
lineage alone.
IL-3 Is Unable to Rescue IL-2R
Given the possibility that IL-2R gene expression
requires the coupling of Stat5 activation with other IL-2-derived
signals, we examined the ability of IL-3 to complement IL-2 and rescue expression of the IL-2R
gene in 32D cells transfected with
YYYYFF. Surprisingly, however, the combination of IL-2 and IL-3 did
not induce IL-2R
mRNA in cells transfected with
YYYYFF (Fig.
6A, lanes 3-5), despite the activation of
STAT complexes indistinguishable from those induced by IL-2 in cells
containing
YYYYYY (Fig. 6B, lanes 4-6 versus 9-11).
This experiment demonstrates that the four proximal tyrosines as well
as the nonphosphorylated regions of IL-2R
cannot provide signals
sufficient to cooperate with IL-3-mediated Stat5 activation in
directing expression of IL-2R
mRNA. These data therefore suggest
that the additional factor(s) involved in IL-2-mediated IL-2R
gene
induction may also be linked functionally to Tyr-392 and Tyr-510.
Using IL-2R constructs containing various combinations of
tyrosine to phenylalanine substitutions, we have demonstrated that in
the context of full-length IL-2R
either Tyr-392 or Tyr-510 is
sufficient to direct IL-2-mediated induction of the IL-2R
gene.
Based on these results, at least four different models potentially explain the proximal events in IL-2R
gene induction following binding of IL-2 to its receptor. In the first model, STATs are activated after docking at phosphorylated Tyr-392 or Tyr-510 and by
themselves are sufficient for IL-2R
gene induction (Fig.
7A). However, the failure of IL-3 to promote
IL-2R
gene expression in 32D transfectants minimizes this
possibility, as the same STAT proteins are activated by IL-2 and IL-3
(Figs. 4 and 5).
The remaining models therefore assume that STAT protein activation is
not the sole pathway triggered by IL-2 in the induction of IL-2R
mRNA. For example, the second model (Fig. 7B) portrays a
direct interaction between an undefined "X" factor and Tyr-392 or
Tyr-510, without the need for concomitant STAT activation. However,
this paradigm is unlikely given the existence of consensus STAT binding
sequences in the upstream region of the human and murine IL-2R
promoters and the inability of IL-2 to induce IL-2R
promoter
activity when these binding sequences are mutated (14, 32).
Incorporating these facts, the third model (Fig. 7C) depicts the interaction of STAT proteins activated at Tyr-392 or Tyr-510 with
an unknown factor X bound to a different portion of IL-2R
. In this
scheme, the requirement for these signaling molecules to functionally
collaborate in cis accounts for the inability of IL-3 to
rescue IL-2R
gene induction when providing activated STATs in
trans. Finally, the fourth model (Fig. 7D) links
Tyr-392 and Tyr-510 to the activation of both STAT proteins
and an additional signaling molecule (factor X). This scheme
effectively couples both Tyr-392 and Tyr-510 to two separate pathways
that cooperate in IL-2R
gene induction. In turn, the "non-STAT"
pathway may influence activation of other transcription factors
required for IL-2R
mRNA expression, consistent with the complex
regulation of this gene (13, 14, 32).
Although we favor the final model, the identity of the putative
additional signaling molecule for IL-2-induced IL-2R gene expression
is unknown. Examination of the IL-7 receptor system may provide some
insights in view of its functional overlap with IL-2R
. For example,
in addition to Stat5 activation (26), Tyr-429 of the IL-7R has been
implicated in the activation of PI 3-kinase (37). Along these lines,
one published report proposed a direct interaction between Tyr-392 of
IL-2R
and PI 3-kinase based on phosphopeptide competition
experiments (38). Yet, because the same analysis failed to demonstrate
any interaction between PI 3-kinase and Tyr-510 of IL-2R
(38), PI
3-kinase is unlikely to represent the missing molecule depicted in Fig.
7D that must be capable of interacting with Tyr-510 as well as
Tyr-392. Further comparison of the IL-7 and IL-2 receptor systems
reveals that both receptors employ Jak1 and Jak3. In contrast, IL-3
signaling involves Jak2. However, the use of Jak2 instead of Jak1 and
Jak3 does not fully explain the inability of IL-3 to induce the
IL-2R
gene, as erythropoietin also activates Jak2 but can
induce IL-2R
mRNA in 32D cells transfected with the murine
erythropoietin receptor (data not shown).
In conclusion, we have demonstrated that Tyr-392 and Tyr-510 play vital
but redundant roles in IL-2R gene induction. Although more work is
required to clarify the proximal events that culminate in IL-2-mediated
expression of this gene, the evidence supports the existence of
additional signaling molecule(s)/pathway(s) linked to Tyr-392 and
Tyr-510. Defining the missing element(s) may reveal yet another
IL-2-mediated signaling pathway and provide insight to critical
processes regulating the immune response.
We thank Cetus Corp. as well as G. Ju and J. Hakimi, Hoffmann La Roche for recombinant IL-2; S. Chen-King for anti-Stat3 antiserum; A. Miyajima for pME18S; S. Ziegler and Immunex Corp. for the human IL-7R cDNA; J.-X. Lin for Stat5, Stat5a, and Stat5b antisera, as well as the DNA affinity purification experiment; J. Pierce for 32D-EpoR transfectants; and S. John for critical comments.