From the
The IL-2, IL-4, and IL-7 signaling pathways have been shown to
utilize shared components. The receptors for these cytokines are
composed of ligand-specific binding chains that associate with a shared
signaling subunit, the common
Cytokines modulate cellular proliferation and differentiation
through their interaction with specific cell-surface receptors. A
comparison of the genes that encode the cytokine receptors indicates
that most are members of a related family, termed the hematopoietin
family of receptors(1) . Functional cytokine receptors consist
of multicomponent complexes composed of two or more chains. Frequently,
one of these chains is responsible for high affinity ligand binding,
while the other(s) plays a role in signal transduction(2) .
Within the hematopoietin family of receptors, different subfamilies
have been identified based on conserved features including the
employment of common signaling subunits(3, 4) . For
instance, each component of the IL-6 family of receptors (the receptors
for IL-6, IL-11, LIF, CNTF, and oncostatin M) associates with
gp130(5, 6, 7, 8) . Recently, a new
subfamily of cytokine receptors, which includes the IL-2, IL-4, and
IL-7 receptors, has been recognized. The ligand binding units of the
IL-4 and IL-7 receptors have been found to heterodimerize with the
The ability of cytokines to rapidly activate genes appears to
involve the activation, via tyrosine phosphorylation, of a set of
latent cytoplasmic factors which belong to the STAT
Interestingly, cytokine receptors which share
a common signaling subunit (e.g. IL-6 family) induce
activation of a common set of JAK kinases and of identical STFs (e.g. STF-IL-6) upon binding ligand(22) . By analogy
with these receptors, employment of a common signaling chain and
activation of the same set of JAK kinases (Jak-1 and Jak-3) (23, 24, 25, 26) by the receptors for
IL-2, IL-4, and IL-7 would be expected to lead to the activation of the
same STF. In this report, we demonstrate that, unlike the IL-6 family,
activation of STFs by the members of the
Cytokine treatments were as
follows. M12.4.1 and RAMOS 266 were stimulated, respectively, with
recombinant murine IL-4 (400 units/ml; DNAX) or human IL-4 (10
units/ml; Schering-Plough). D1.1 and CTLL cells were stimulated with
recombinant human IL-2 (10 units/ml; Eurocetus) or murine IL-4. Clone K
cells were stimulated with recombinant murine IL-7 (10 ng/ml; Genzyme).
All cytokine treatments were performed for 15 min at 37 °C. Prior
to treatment with cytokines, growth factor-dependent cells were starved
of these factors for 4 h.
Peptides from the IL-4 and IFN
STAT antisera were added (final dilution 1:20) for 30-60 min
(at 4 °C), after a standard 20-min (25 °C) incubation of
extracts with shift probe. Antiphosphotyrosine antibodies were added to
the extracts for 30-60 min (at 4 °C), prior to the addition
of the shift probe.
Whole cell extracts (WCE) were prepared as
described previously(31, 33, 34) . Whole cell
extracts from Clone K, splenocytes, and thymocytes were prepared using
a detergent-free buffer (Buffer C) as described previously(35) .
Oligonucleotide precipitation assays were carried out
with a 3-unit tandem multimer of the
If
these factors are activated by tyrosine phosphorylation, then the IL-2-
and IL-7-induced GAS binding activities should contain phosphotyrosyl
residues. To examine this possibility, extracts from stimulated cells
were incubated with an antiphosphotyrosine antibody or with a control
antibody prior to analysis by EMSA (Fig. 2B). In a
pattern similar to STF-IL-4 and other STFs(17, 45) ,
addition of an antiphosphotyrosine antibody, but not of equivalent
amounts of an isotype-matched control antibody, prevented the
appearance of these STFs. These results provide further evidence that
STF-IL-2 and STF-IL-7 contain phosphotyrosyl residues which are
essential for their binding to specific DNA elements. These results are
consistent with the paradigm of activation of these latent cytoplasmic
factors that has been delineated for the IFNs.
In order to
better define whether STF-IL-2 and STF-IL-7 are distinct from STF-IL-4,
we took advantage of the recent finding that the ability of purified
Stat6 to bind GAS elements is inhibited by phosphopeptides derived from
the IL-4 receptor, but not by an IFN-
To determine whether p94 has
GAS binding activity, as has been observed with other STAT
proteins(17, 38, 46, 48) , we
precipitated extracts from IL-2- and IL-7-stimulated cells using an
agarose-linked GAS element. These precipitants were immunoblotted with
an antiphosphotyrosine antibody. The precipitants from both
IL-2-stimulated CTLL and IL-7-stimulated Clone K contained the same
94-kDa tyrosine-phosphorylated protein detected by immunoprecipitations
using p91-ab4 (Fig. 5). In addition, both murine splenocytes and
thymocytes also activate a protein of identical size in response to
IL-7 (Fig. 5). These results indicate that the 94-kDa protein
(p94) recognized by p91-ab4 is part of the complex that binds to the
GAS element that we have used to characterize STF-IL-2 and STF-IL-7.
Our studies demonstrate that, although the IL-2, IL-4, and
IL-7 signaling pathways share common components, only IL-2 and IL-7
activate closely related STFs. By several criteria including mobility
on EMSA, competition profile by GAS-related sequences as well as by
IL-4 receptor phosphopeptides, and reactivity to Stat1 and Stat6
antisera, the Stat6 homodimer induced by IL-4 behaves clearly
differently from STF-IL-2 and STF-IL-7. In addition, only IL-2 and
IL-7, but not IL-4, induce the rapid tyrosine phosphorylation of a
94-kDa protein (p94). The fact that this p94 protein possesses GAS
binding activity (which has also been shown for Stat1, Stat3, Stat4,
and Stat6(22, 38, 46, 49) ) along with
its cross-reactivity with some, but not all, Stat1 antisera suggests
that it is a member of the STAT family of proteins.
The receptors
for the cytokines we have studied are composed of multicomponent
complexes consisting of the common
The data presented above indicate that
the STFs activated by IL-2 and IL-7, but not IL-4, contain a 94-kDa
protein (p94) that is related to Stat1. Interestingly, on EMSA, the
mobility displayed by STF-IL-2 and STF-IL-7 is identical with that
exhibited by the IL-3-inducible complex (data not shown), which appears
to be composed of a Stat5 homodimer (50). In addition, preliminary
results reveal that p94 reacts with a monoclonal antibody directed
against Stat5. These findings raise the possibility that the
STAT-related protein activated by IL-2 and IL-7 is indeed Stat5 or a
Stat5-related protein. Activation of p94 is detected in a variety of
IL-7 responsive cell types. We have shown that p94 is activated in
response to IL-7 in thymocytes, splenocytes, and an IL-7-dependent
pre-B cell line (Clone K). Interestingly, the mobility of STF-IL-7 in
Clone K is slightly slower than the one detected in murine splenocytes
and thymocytes (data not shown), raising the possibility that
additional components of STF-IL-7 exist that are specific for different
stages of differentiation and/or cell type. In addition, it is possible
that STF-IL-2 and STF-IL-7 may contain proteins that complex with p94
and are unique for each STF. Further characterization and cloning of
these factors will allow a more careful analysis of the component
proteins of these complexes.
Cytokine-induced STFs have been shown
to bind to unique enhancer elements in the promoter of target genes and
are responsible for the activation of early response
genes(14, 15) . The fact that IL-2 and IL-7 induce
similar STFs may indicate that the early transcriptional events induced
by these two cytokines are identical. Our data are consistent with
prior work which shows that the pattern of tyrosine-phosphorylated
proteins induced by IL-2 overlaps with that stimulated by
IL-7(51) , but differs from that induced by IL-4(52) .
Distinctions between the IL-2 and the IL-4 signaling pathways have also
been noted in the activation of p21
We thank Robert Coffman and Jan de Vries of DNAX
Research Institute for recombinant IL-4 and IL-13 and Doug Fisher of
Pfizer Corp. for cytokine receptor peptides. We also thank Kathryn
Calame and Ned Braunstein for their critical reading of this
manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
) chain. In
addition, IL-2, IL-4, and IL-7 induce activation of a common set of
nonreceptor tyrosine kinases, Jak-1 and Jak-3. We have further
investigated the signaling events induced by these cytokines and find
that the
-associated receptors activate distinct
signal transducing factors (STFs). In addition, we show that a 94-kDa
STAT-related protein (p94) is activated in response to IL-2 and IL-7,
but not IL-4. These data indicate that IL-2, IL-4, and IL-7 activate
distinct signaling molecules which might be differentially recruited to
the receptor complex by the ligand-specific units of the IL-2, IL-4,
and IL-7 receptors.
chain of the IL-2
receptor(9, 10, 11, 12) . This common
(
) chain appears to be essential for mitogenic
signaling mediated by the IL-2, IL-4, and IL-7
receptors(9, 10, 11, 12, 13) .
(
)(Signal Transducers and Activators of Transcription)
family of proteins(14, 15) . Since the hematopoietin
receptors lack kinase domains, tyrosine phosphorylation of these
factors is hypothesized to be mediated by specific members of the Janus
kinase family(16) . These kinases can associate with the
hematopoietin receptors and, upon ligand binding, become activated.
Phosphorylation of the STATs renders them competent for complex
association. These complexes, termed signal transducing factors (STFs),
can then undergo nuclear translocation. Once in the nucleus, many of
these factors, like the recently characterized IL-4-inducible factor
(17-19) (which is composed of a Stat6 homodimer (20),
(
)bind to DNA sequences related to the interferon (IFN)
gamma-activation site (GAS). These elements are conserved DNA sequences
which mediate transcriptional activation by several STFs, including
STF-IFN
(a p91
Stat1 homodimer) (15) and
STF-IL-3(21) .
family
(IL-2, IL-4, and IL-7) is regulated in a more complex manner. IL-2 and
IL-7, in fact, induce GAS binding complexes (STF-IL-2 and STF-IL-7,
respectively) which are similar by several criteria including GAS
binding specificity and antibody reactivity. STF-IL-2 and STF-IL-7,
however, are clearly distinct from the STF activated by IL-4
(STF-IL-4). We further demonstrate that IL-2- and IL-7-stimulated
extracts contain a 94-kDa tyrosine-phosphorylated protein that is not
detected in IL-4-stimulated extracts. This protein, which binds to GAS
sequences, appears to be a member of the STAT family of STF component
proteins.
Cell Lines
The M12.4.1 (M12) cells and their
culture conditions have been described previously(17) . Ramos
266 cells were grown in Iscove's modified Dulbecco's medium
supplemented with 10% fetal calf serum (Atlanta Biologicals, Inc.).
CTLL (ATCC) cells were grown in RPMI 1640 supplemented with 10% fetal
calf serum, 50 µM -mercaptoethanol, and recombinant
human IL-2 (10 units/ml; Eurocetus). Clone K cells (27) were
grown in RPMI 1640 supplemented with 10% fetal calf serum, 50
µM
-mercaptoethanol, 1% L-glutamine, and 1%
sodium pyruvate on a feeder layer of AC6.21 cells. The derivation of
the murine TH1 cell clone, D1.1, and its culture conditions have been
described previously(28) .
Cell Isolation and Culture
Peripheral blood
mononuclear cells (PBM or PBL) were prepared from freshly drawn blood
of healthy volunteers by centrifugation on Ficoll-Hypaque (Sigma). The
isolation of murine splenocytes has been described
previously(29) . Thymocytes were harvested from BALB/c mice. PBM
were stimulated for 15 min at 37 °C with the following cytokines:
human IL-2 (10 units/ml; Eurocetus), murine IL-7 (10 ng/ml; Genzyme),
human IL-4 (10 units/ml; DNAX), murine IL-13 (500 units/ml; DNAX), or
human IFN (5 ng/ml; Hoffman-LaRoche). In cycloheximide
experiments, PBM were cultured with 10 µg/ml cycloheximide (Sigma)
for 1 h prior to the addition of the cytokines. In staurosporine
experiments, PBM were cultured in medium containing 500 µM staurosporine (Biomol) for 20 min prior to the addition of the
cytokines. Murine splenocytes and thymocytes were stimulated with
murine IL-7 (10 ng/ml).
DNA Binding Assays and Cell Extracts
The
preparation and employment of DNA oligonucleotide probes for mobility
shift assays have been described previously.(30, 31) .
The IRF-1 GAS probe employed in these studies was as follows:
5`gatcGATTTCCCGAAAT3`. Double-stranded oligonucleotides used as cold
competitors were prepared from single-stranded oligonucleotides (Oligos
Etc.) with the following sequences: CAS-GAS
(5`gatcGACTTCTTGGAATT3`), Ly6E-GAS (5`gatcATATTCCTGTAAGT3`), and I
(-119 to -104) (17) (5`gatctAACTTCCCAAGAACAG3`)(17, 18) .
receptors were obtained from Dr.
Douglas Fisher from Pfizer Inc. The peptide sequences were as follows:
1) unphosphorylated IL-4 receptor: CASSGEEGYKPFQDLI(20) , 2)
phosphorylated IL-4 receptor: CASSGEEGY
KPFQDLI(20) ,
3) unphosphorylated IFN
receptor: CTSFGYDKPH(32) , 4)
phosphorylated IFN
receptor: CTSFGY
DKPH(32) .
Peptides were added in increasing concentrations (30 µM,
100 µM, 300 µM) to whole cell extracts for 30
min at room temperature prior to the addition of the shift probe.
Immunoprecipitations and
Oligoprecipitations
Extracts were immunoprecipitated by p91-ab4
as described previously(31, 33) . The precipitates were
fractionated in a 7% SDS-PAGE prior to immunoblotting with an
antiphosphotyrosine antibody (4G10, UBI). Bands were detected by ECL
(Amersham).
CAS-GAS element. The 11-base
pair GAS elements were separated by 10-base pair spacers(21) .
This oligomer was biotinylated (Sigma) by a fill-in reaction and linked
to streptavidin agarose beads (Sigma) as per recommendations by the
manufacturer, at 1-2 µg of DNA per µl of beads. 300 to
1000 µl of extract were incubated with the immobilized
oligonucleotides for 2 to 12 h at 4 °C and then eluted with 1 or 2 M NaCl.
Antibodies
Polyclonal rabbit antiserum were
generated against various domains of p91 with GST fusion proteins as
described previously(33, 36) . p91-ab1 recognizes the
carboxyl terminus of p91 (aa-aa
), p91-ab2
recognizes the amino-terminal (aa
-aa
), and
p91-ab4 recognizes the SH2 domain of denatured p91
(aa
-aa
). Stat2 antiserum recognizes p113
(aa
-aa
)(33, 36) . Polyclonal
rabbit antisera against the carboxyl terminus of Stat3 or Stat6 were
purchased from Santa Cruz Biotechnology, Inc. The antiphosphotyrosine
antibody was purchased from UBI (4G10), and the isotype-matched control
OKT4 (ATCC) was a generous gift from M. Yellin.
IL-2 and IL-7 Rapidly Stimulate Signal Transducing
Factors
In an effort to determine whether IL-2 and IL-7 can
activate specific signal transducing factors (STFs), we stimulated
human peripheral blood mononuclear cells (PBMs) with either IL-2, IL-4,
IL-7, IL-13, or IFN- for 15 min. We prepared whole cell extracts
and assayed them for the induction of DNA binding activity by
electrophoretic mobility shift assay (EMSA) with the GAS element from
the IRF-1 gene (Fig. 1A). We have previously shown that
a wide variety of STFs bind this element(21) . Extracts from
untreated PBMs do not contain any DNA binding activities. However,
extracts from IL-2- or IL-7-stimulated cells reveal novel complexes,
which migrate with similar mobilities. The pattern and mobility of
these complexes are clearly different from those of either STF-IL-4 or
STF-IFN
. As already reported, the DNA binding complex induced by
IL-13 migrates with a mobility identical with
STF-IL-4(17, 37) . Assaying these extracts by EMSA
utilizing a different GAS probe (from the
-casein promoter)
revealed identical results (data not shown). These data suggest that
IL-2 and IL-7 activate unique factors (STF-IL-2 and STF-IL-7,
respectively) that are distinct from the Stat6 homodimer induced by
IL-4.
Figure 1:
A,
induction of IRF-1 GAS binding activities in PBM by IL-2, IL-4, IL-7,
and IL-13. WCE were prepared from PBM with or without a 15-min
stimulation at 37 °C with human IL-2 (10 units/ml), murine IL-7 (10
ng/ml), human IL-4 (10 units/ml), murine IL-13 (500 units/ml), or human
IFN (5 ng/ml). B, comparison of IL-2-, IL-4-, and
IL-7-inducible GAS binding activities in different cell types. WCE were
prepared and stimulated as described under ``Materials and
Methods.'' Lanes are as follows: 1, unstimulated PBM; 2, PBM stimulated with human IL-2 (10 units/ml); 3,
PBM stimulated with murine IL-7 (10 ng/ml); 4, PBM stimulated
with human IL-4 (10 units/ml); 5, unstimulated D1.1; 6, D1.1 stimulated with human IL-2 (10 units/ml); 7,
D1.1 stimulated with murine IL-7 (10 ng/ml); 8, D1.1
stimulated with murine IL-4 (400 units/ml); 9, unstimulated
CTLL; 10, CTLL stimulated with human IL-2 (10 units/ml); 11, unstimulated Clone K; 12, Clone K stimulated with
murine IL-7 (10 ng/ml). The WCE were examined by EMSA using an IRF-1
GAS probe.
Since PBM represent a complex population of cells and the
activation of STF-IL-2, STF-IL-4, and STF-IL-7 may be occurring in
different cell types, we were concerned that some of the differences in
GAS binding activity might be cell-type-specific. Therefore, we assayed
clonal cell populations for their ability to induce GAS binding
activities after stimulation with IL-2, IL-4, or IL-7 (Fig. 1B). Extracts from cells of a murine T cell clone
(D1.1) stimulated with IL-2, or with IL-7, exhibited complexes which
migrated with a mobility identical with that of the IL-2- and
IL-7-inducible factors detected in human PBM. In contrast, as already
observed in PBM, extracts of D1.1 cells cultured with IL-4 displayed a
complex whose mobility was clearly distinct from that of STF-IL-2 or
STF-IL-7. Interestingly, the murine STF-IL-4 displayed a different
ratio of individual bands as well as a slightly different mobility from
its human counterpart. Since different tissues have been shown to
contain different Stat6 mRNA species(20) , it is possible that
different forms of Stat6 protein are present in different cell types.
Thus, the multiple bands induced by IL-4 in PBM may be related to
different forms of Stat6 found in this heterogeneous cell population.
Extracts from another IL-2-stimulated murine T cell line, CTLL, also
revealed the appearance of a factor that on EMSA migrates with a
mobility identical with that of the IL-2-inducible activities in PBMs
and in D1.1. The IL-2-inducible complex in D1.1, in addition, contains
a faster migrating band which is not consistently detected. The nature
of this band is unclear. Interestingly, extracts of an IL-7-stimulated
pre-B cell line (Clone K) exhibit a GAS binding activity with slightly
slower mobility than the other IL-2- or IL-7-treated extracts. By
several criteria, including competition by unlabeled GAS-related
oligonucleotides and antibody supershifting, the IL-7-inducible complex
detected in Clone K behaves exactly like the STF-IL7 present in PBMs
and D1.1 (data not shown). Taken together, these results illustrate
that, both in murine and human cells, IL-2 and IL-7 induce similar GAS
binding activities which are distinct from STF-IL-4. However, STF-IL-2
and STF-IL-7 from different cell types can exhibit slight variations in
both mobility and pattern of bands. This might be secondary either to
the presence of additional cell-type-specific factors or to different
forms of the same factor(s).
STF-IL-2 and STF-IL-7 Are Activated by Tyrosine
Phosphorylation
In order to better characterize the GAS binding
activities induced by IL-2 and IL-7, we studied whether the activation
of STF-IL-2 and STF-IL-7 follows the pattern delineated for other STFs.
Previous studies have demonstrated that the component proteins of STFs
reside in the cytoplasm prior to activation. After receptor-cytokine
interaction, and in the absence of new protein synthesis, these factors
are quickly activated. Once activated, they rapidly translocate to the
nucleus(17, 38) . In order to assess the requirement for
new protein synthesis in the induction of STF-IL-2, and STF-IL-7, we
pretreated cells with cycloheximide for 1 h prior to cytokine
stimulation. As in the case of STF-IL-4 and other STFs characterized to
date, the appearance of STF-IL-2 and STF-IL-7 is resistant to
pretreatment with cycloheximide (Fig. 2A), demonstrating
that the protein components of these complexes are present prior to
activation by cytokines.
Figure 2:
Mode of activation of STF-IL-2 and
STF-IL-7. A, effects of cycloheximide or staurosporine on
STF-IL-2 and STF-IL-7 induction. Extracts from PBM stimulated with IL-2
or IL-7 alone or after pretreatment with cycloheximide (C) (10
µg/ml) for 1 h or with staurosporine (S) (500
µM) for 20 min were assayed as described in Fig. 1. B, STF-IL-2 and STF-IL-7 contain phosphotyrosyl residues.
Extracts were incubated with either an antiphosphotyrosine antibody
(4G10) or a control antibody (OKT4) for 60 min at 4 °C prior to
analysis by EMSA. 3 µg of antibodies were added for each reaction
except for the IL-7-stimulated extracts to which 4 µg of antibody
were added.
The activation of STFs has been shown to be
dependent on the Janus family of tyrosine kinases for a number of
cytokines(39, 40, 41, 42) . For example,
the activation of STF-IFN is dependent on the presence of Jak-1
and Jak-2(43, 44) . The induction of STF-IL-4 also
requires tyrosine phosphorylation(17, 18) . The
observation that STF-IL-2 and STF-IL-7 contain STAT-related proteins
(see below) prompted us to investigate whether these factors are also
dependent on the activity of tyrosine kinases. When cells are
pretreated with the kinase inhibitor staurosporine, which is known to
block the Janus kinases, the induction of STF-IL-2 and STF-IL-7 is
inhibited (Fig. 2A). This finding suggests that
phosphorylation is critical for the activation of these factors.
IL-2 and IL-7 Stimulate Signal Transducing Factors That
Are Distinct from STF-IL-4
Although STF-IFN and STF-IL-4
bind common DNA elements (e.g. GAS from the IRF-1 and
-casein (
CAS) genes), these unique factors can be
distinguished by their differential binding affinity for unique
GAS-like elements (e.g. GAS from the Ly6E gene and the I
element from the Ig germ line
promoter)(17) . In order to
further define the relationship between the factors induced by IL-2,
IL-4, and IL-7, we assessed the ability of the
CAS-GAS, Ly6E-GAS,
and I
(high affinity STF-IL-4 binding site) elements to compete
for binding of these STFs (Fig. 3A). The binding of
STF-IL-2 and STF-IL-7 to IRF-1 GAS is competed by a similar pattern of
GAS elements, i.e. with high affinity by the
CAS-GAS, but
only weakly by the Ly6E-GAS or by I
. This pattern differs from the
one exhibited by STF-IL-4, which is strongly competed with the
CAS-GAS and the I
element, but not the Ly6E-GAS. The
competition pattern exhibited by STF-IL-2 and STF-IL-7 is also distinct
from that shown by STF-IFN
, which is strongly competed by the
Ly6E-GAS(17) . These findings provide additional evidence that
IL-2 and IL-7 induce similar factors, which differ from the factor
induced by IL-4.
Figure 3:
Comparison of STF-IL-2, STF-IL-4, and
STF-IL-7 by oligonucleotide or IL-4 receptor phosphopeptide
competition, as well as antisera reactivity. Cells were stimulated, and
extracts were prepared and examined as in Fig. 1. A,
oligonucleotide competition assays were performed on WCE from IL-2-,
IL-4-, or IL-7-stimulated D1.1 either in the absence (lanes 1, 5, and 9) or in the presence of a 100-fold molar
excess of cold GAS oligonucleotides added to the shift reaction as
indicated (lanes 2-4, 6-8, and 10-12). Competitors included CAS-GAS (lanes
2, 6, and 10), I
(lanes 3, 7, and 11), and Ly6E-GAS (lanes 4, 8, and 12). B, antibody interference
mobility shift assays were carried out by the addition of either an
antiserum raised against a conserved domain of Stat1 (p91-ab4) (lanes 2, 5, and 8) (17) or preimmune serum (lanes 3, 6, and 9) to WCE derived from
IL-2-, IL-4-, or IL-7-stimulated PBM. Antisera were added at a final
dilution of 1:20 for 30 min at 4 °C to extracts which had been
incubated with the IRF-1 GAS probe for 20 min. at 25 °C. C, antibody interference mobility shift assays were carried
out, as described above, by the addition of either an antiserum raised
against the carboxyl terminus of Stat6 (lanes 2, 5, 8, and 11) (17) or preimmune serum (lanes 3, 6, 9, and 12) to WCE derived from untreated
or from IL-2-, IL-4-, or IL-7-stimulated PBM. D, IL-4 receptor
peptide competition assay were performed by adding either a
phosphorylated (lanes 2-4) or an unphosphorylated (lanes 5-7) form of this peptide to WCE from human PBL
which had been stimulated with either IL-2 (panel a), IL-7 (panel b), or IL-4 (panel c). The final
concentrations of peptide added were 30 µM (lanes 2 and 5), 100 µM (lanes 3 and 6), and 300 µM (lanes 4 and 7).
It has recently been shown that several STFs
contain p91 (Stat1) or other p91-related (STAT family)
proteins(30, 33, 38, 41, 45, 46, 47) .
In order to determine whether STAT family members participate in the
formation of STF-IL-2 and STF-IL-7, we assayed these GAS binding
activities for reactivity to a panel of antibodies raised against
various conserved domains of the human Stat1 protein. We found that an
antiserum (p91-ab4) which recognizes the denatured, but not the native,
Stat1 strongly supershifts both STF-IL-2 and STF-IL-7 as assayed by
EMSA using an IRF-1 GAS probe (Fig. 3B). Although this
antiserum can inhibit STF-IL-4 binding in certain human cell lines
(21), it did not interfere with PBM-derived STF-IL-4 binding (Fig. 3B) or with D1.1-derived STF-IL-4 binding (data
not shown). In contrast, an antiserum directed against the carboxyl
terminus of Stat6 was able to supershift the STF-IL-4 complex but did
not affect either STF-IL-2 or STF-IL-7 GAS binding activity (Fig. 3C). Other antisera directed against Stat1
(p91-ab1 and p91-ab2) or against Stat3 had no effects on either
STF-IL-2 or STF-IL-7, while Stat2 (p113) antiserum only very weakly
supershifted these GAS binding activities (data not shown). The ability
of a Stat1 antiserum to supershift STF-IL-2 and STF-IL-7 suggests that
these complexes contain a STAT-related protein. In addition, the
finding that a Stat6 antiserum reacts with STF-IL-4 but not with
STF-IL-2 or STF-IL-7 implies that STF-IL-2 and STF-IL-7 are not
composed of Stat6 homodimers. Taken together, these data point to the
fact that the component proteins of STF-IL-2/STF-IL-7 appear to be
distinct from those of the IL-4-inducible complex.
receptor-derived
phosphopeptide known to inhibit Stat1 activation(20) . This
inhibition is believed to be due to the fact that SH2-phosphotyrosine
interactions are essential for the recruitment of STATs to specific
cytokine receptors as well as for the dimerization of STAT
proteins(20, 32) . We, therefore, ran a series of
experiments to determine whether one of the IL-4 receptor-derived
phosphopeptides would affect GAS binding by STF-IL-2 and STF-IL-7.
Consistent with previous reports, the addition of the
tyrosine-phosphorylated, but not the unphosphorylated, peptide blocked
DNA binding by STF-IL-4 in a dose-dependent manner (Fig. 3D). In contrast, GAS binding by either STF-IL-2
or STF-IL-7 was inhibited only weakly by the highest concentration of
the IL-4 receptor-derived phosphopeptide (300 µM) (Fig. 3D). A slight inhibitory effect on STF-IL-2 and
STF-IL-7 DNA binding was, however, also seen by the addition of a
similar concentration of an IFN-
receptor phosphopeptide, which is
known to inhibit Stat1 GAS binding (data not shown). Since binding of
STATs to cytokine receptors is believed to be mediated by specific
SH2-phosphotyrosine interactions, these results suggest that the
STF-IL-2
STF-IL-7 complexes will be recruited to sites on the
receptor chains distinct from those involved in STF-IL-4 binding.
STF-IL-2 and STF-IL-7 Contain a Novel 94-kDa STAT-related
Protein
The ability of p91-ab4 to supershift STF-IL-2 and
STF-IL-7 GAS binding activities permitted us to investigate the nature
of the component(s) this antiserum recognizes. Extracts prepared from
cells stimulated with IL-2, IL-4, and IL-7 were immunoprecipitated with
p91-ab4. The precipitants were separated on SDS-PAGE and immunoblotted
with an antiphosphotyrosine antibody. We found that both IL-2 and IL-7
induce the tyrosine phosphorylation of a 94-kDa protein (Fig. 4A). Since this protein (p94) is not activated by
IL-4, these results provide additional evidence that the similarities
between STF-IL-2 and STF-IL-7 demonstrated by EMSA (see above) are
likely to be due to the ability of IL-2 and IL-7 to activate common
components. Furthermore, these studies show that activation of STF-IL-2
and STF-IL-7 involves tyrosine phosphorylation.
Figure 4:
A 94-kDa tyrosine-phosphorylated protein
can be immunoprecipitated using an anti-Stat1 antibody from IL-2- and
IL-7-, but not from IL-4-, stimulated cells. Extracts were prepared
from cells as described in Fig. 1. Extracts were then examined by
immunoprecipitation using p91-ab4. A, IL-2-stimulated CTLL,
IL-7-stimulated Clone K, PBM, IL-2-stimulated PBM, IL-7-stimulated PBM,
IL-4-stimulated PBM. B, CTLL, IL-2-stimulated CTLL,
IL-4-stimulated CTLL, M12 (murine B cell line), IL-4-stimulated M12,
RAMOS 266 (human B cell line), IL-4-stimulated RAMOS 266, Clone K, and
IL-7-stimulated Clone K.
In order to
ascertain whether p94 can be activated by IL-4, we immunoprecipitated
extracts from various cell lines stimulated with IL-4 and assayed for
tyrosine phosphorylation of p94 (Fig. 4B). p94 is not
tyrosine-phosphorylated in response to IL-4 in either a human (RAMOS
266) or a murine (M12) B cell line which contain STF-IL-4 binding
activity(17, 21) . In addition, CTLL cells, which
activate p94 in response to IL-2 (Fig. 4, A and B), do not tyrosine-phosphorylate p94 in response to IL-4 (Fig. 4B). These data confirm our previous results that
STF-IL-2 and STF-IL-7 contain a common, if not identical, component
which is not a constituent of STF-IL-4.
Figure 5:
A 94-kDa tyrosine-phosphorylated protein
can be identified in IL-2- and IL-7-stimulated extracts by GAS
oligonucleotide affinity precipitations. Extracts were precipitated
using agarose-immobilized CAS-GAS multimers except for the last
two lanes which represents CTLL cells immunoprecipitated with p91-ab4.
The protein components were then separated on 7% SDS-PAGE and analyzed
by Western blotting using an antiphosphotyrosine antibody. Lanes are as
follows: splenocytes, IL-7-stimulated splenocytes, thymocytes,
IL-7-stimulated thymocytes, Clone K, IL-7-stimulated Clone K, CTLL,
IL-2-stimulated CTLL, CTLL immunoprecipitated using p91-ab4,
IL-2-stimulated CTLL immunoprecipitated using
p91-ab4.
chain in addition to
ligand-specific chain(s). Our data, which demonstrate that there are
distinct complexes induced by different
chain-associated receptors, in spite of their association with
the same set of JAK kinases, suggest that factors other than the
kinases themselves, perhaps the ligand-binding chain, play a critical
role in defining the specificity of the transduced signal. This notion
is supported by the observation that a phosphorylated peptide derived
from the IL-4 receptor ligand-specific chain is able to block GAS
binding by STF-IL-4 more effectively than that by STF-IL-2 or STF-IL-7.
Hence, complex interactions between the individual components of the
IL-2, IL-4, and IL-7 receptors appear to dictate the recruitment and
activation of specific signal transducing factors by
receptor-associated kinases.
which is
activated in response to IL-2, but not IL-4(53) . The early
divergence of the signaling pathways among these cytokines might
explain their ability to perform their distinctive biologic functions.
activation
site; IFN, interferon; PBM, peripheral blood mononuclear cells; WCE,
whole cell extracts; PAGE, polyacrylamide gel electrophoresis; EMSA,
electrophoretic mobility shift assay.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.