(Received for publication, July 8, 1994; and in revised form, November 8, 1994)
From the
We have investigated tyrosine phosphorylation of cellular
proteins induced by interleukin (IL) 4 and compared it with the effects
of three related cytokines, IL-2, IL-7, and IL-13. We show here that
both IL-4 and IL-13 stimulate tyrosine phosphorylation of the 140-kDa
IL-4 receptor subunit, which suggests that this receptor protein is
used by both cytokines. Receptor phosphorylation induced by IL-13 was
both weaker and slower than with IL-4. Stimulation of cells with IL-2
and IL-7 induced identical phosphorylation patterns to each other but
not phosphorylation of the 140-kDa IL-4 receptor subunit. The only
signal appearing upon stimulation with any of the four cytokines was
the weak phosphorylation of an unidentified protein of 160 kDa. SH2
domains of p56 and p59
precipitated the same proteins as anti-phosphotyrosine
antibodies after IL-4 stimulation, which suggests that a src-type kinase may be involved in signal transduction through
the IL-4 receptor.
Sharing of receptor subunits is an increasingly recognized
property of cytokines(1, 2) . One family of growth
factors, comprising leukemia inhibiting factor, oncostatin M, ciliary
neurotrophic growth factor, IL()-6, and IL-11, uses the
gp130 molecule as receptor subunit, while IL-3, IL-5, and GM-CSF share
the KH97 receptor protein, also designated
c. A third family of
cytokines has emerged with the discovery that
IL-4(3, 4) , IL-7(5, 6) , and IL-15 (7) all bind to receptor complexes that contain the
subunit of the IL-2 receptor(8) . The IL-2R
, a member of
the cytokine-type receptor superfamily(8) , has been named
c(4, 5) , in analogy to the
c protein of the
IL-3/IL-5/GM-CSF group. The same component may also be a part of the
receptor for IL-13. Two findings suggest that the receptor for IL-13
shares at least one subunit with the IL-4 receptor: the IL-4 mutant
protein Y124D, which inhibits IL-4-dependent reactions(9) ,
also inhibits effects of IL-13(10, 11) , and IL-13
competes with radiolabeled IL-4 for binding to intact TF-1
cells(10) . It is not clear whether these cytokines share the
subunit or the
subunit or both.
IL-4 is a pleiotropic
immunoregulatory cytokine(12) . Its receptor has at least two
subunits, an IL-4 binding receptor protein of 140 kDa (IL-4R) and
the
c chain(13) . Specific functions of IL-4 include
induction of a T
2 phenotype in peripheral T-helper cells (14, 15) and stimulation of IgE synthesis by activated
B-cells(16) . Based on size, gene organization, and sequence
homologies, IL-13 has been classified as member of a cytokine subgroup
designated the IL-4 family(17) . IL-4 and IL-13 have very
similar effects on B-cells and monocytes, but in contrast to IL-4,
IL-13 cannot stimulate T-cells (18, 19, 20) .
It is particularly interesting that IL-13, like IL-4, can induce IgE
synthesis by B-cells (18, 21) because this suggests a
role for IL-13 in the allergic response.
Transfection of cells with
truncated versions of c has shown that its cytoplasmic domain is
involved in two different IL-2-induced signaling pathways, one leading
to expression of c-fos and c-jun and the other
leading to activation of a tyrosine kinase and expression of
c-myc(22) . We have compared tyrosine phosphorylation
induced by cytokines that have been shown or suggested to use the
c subunit. Our results demonstrate that IL-2 and IL-7 have
identical effects, while IL-4 and IL-13 induce a second type of
phosphorylation pattern. In the course of these studies, we have
identified IL-4R
as a substrate of IL-13-dependent tyrosine
phosphorylation.
Peripheral blood lymphocytes were obtained from lymphocyte
concentrates of healthy blood donors by Ficoll centrifugation. Cells
(10/ml) were prestimulated with 9 µg/ml PHA (Wellcome
Diagnostics, Dartford, UK) for 5 days. The cells were washed twice and
incubated two days without PHA before use.
The human pre-myeloid erythroleukemic cell line TF-1 was cultured in RFP containing 100 ng/ml GM-CSF.
The human monocyte-like histiocytic lymphoma U937 was grown in RFP without additions.
The IL-3-dependent murine hematopoietic cell line FDCP-2 (a gift of J. Pierce, Bethesda, MD) was cultured in RFP with 5% of X63 Ag 8-653 BPV-mIL-3-conditioned medium(23) .
Anti-phosphotyrosine antibodies used were 4G10 (UBI, Lake Placid, NY) for immunoprecipitation and horseradish peroxidase-coupled RC20 (Affiniti, Nottingham, UK) for detection on Western blots. RC20 consists of the bacterially expressed variable regions of the anti-phosphotyrosine antibody PY20, which were mutated for higher affinity(26) . The monoclonal anti-IL-4 receptor antibody A3/10 (27) was a gift from P. Reusch.
The recombinant
extracellular domain of the IL-4R was expressed in Chinese hamster
ovary cells (9) and was a gift from S. Arnold.
The proteins were expressed
in E. coli. Cultures of 800 ml with an absorbance of
0.8-1.0 (550 nm) were induced for 3 h with 1 mM isopropyl-1-thio--D-galactopyranoside. Bacteria were
lysed, and the fusion proteins were purified as described by Lavan et al.(28) .
IL-4, like most other cytokines, induces changes of protein tyrosine phosphorylation in responsive cells(29, 30, 31) . We have compared tyrosine phosphorylation patterns in three human cell types: PHA-stimulated peripheral blood lymphocytes, the erythroleukemic cell line TF-1, and the histiocytic lymphoma U937. The murine myeloid precursor cell line FDCP-2 was used for comparison. When any of the human cell types was stimulated with IL-4, changes in tyrosine phosphorylation were observed (Fig. 1). There was consistently strong phosphorylation of a protein at 140 kDa and, at least in peripheral blood lymphocytes and TF-1 cells, also a weak signal from a protein at 160 kDa. Other changes observed were less reproducible. Stimulation of mouse cells with murine IL-4 induced phosphorylation of several bands, most prominently at 160, 130, 100, and 65 kDa (Fig. 1).
Figure 1: Tyrosine phosphorylation in different cell types after stimulation with IL-4. Human peripheral blood lymphocytes and the human cell lines TF-1 and U937 were stimulated with 100 ng/ml IL-4 for 10 min or left untreated as indicated. The murine cell line FDCP-2 was stimulated with 5% of mIL-4 containing cell supernatant. Cells were lysed, the cleared cell lysates were immunoprecipitated with anti-phosphotyrosine antibody 4G10, and the immune complexes were analyzed by immunoblotting with anti-phosphotyrosine antibody RC20. Lines to the left in this and all other figures indicate positions of marker proteins in kDa, while arrows indicate positions of bands discussed in the text.
When PHA-prestimulated peripheral blood lymphocytes were treated with IL-2 or IL-7, changes in tyrosine phosphorylation were similar but clearly different to the response upon IL-4 (Fig. 2). Both cytokines induced strong phosphorylation of bands at 160 and 85 kDa along with some weaker signals. The kinetics of IL-2- and IL-7-induced phosphorylation were also identical with maximal signals 10 min after stimulation (not shown). The only band appearing with IL-2, IL-4, and IL-7 was the one at 160 kDa, which was weakly phosphorylated in response to all three cytokines.
Figure 2: Tyrosine phosphoproteins after stimulation with IL-2, IL-4, and IL-7 in peripheral blood lymphocytes. Cells were stimulated with 100 ng/ml IL-2, IL-4, or IL-7 for 10 min and lysed; phosphoproteins were analyzed as in Fig. 1.
The cell line TF-1 is dependent on GM-CSF but can also respond to IL-4 and IL-13 with transient proliferation(10, 32) . We used this line to compare the effects of IL-4 and IL-13. Tyrosine phosphorylation after different stimulation times is shown in Fig. 3. IL-4 and IL-13 induced tyrosine phosphorylation of proteins at 160 and 140 kDa. With IL-4, both bands were seen after only 30 s of stimulation. Phosphorylation was maximal after 5-10 min and declined afterwards. Signals obtained with IL-13 were consistently weaker and slower than with IL-4. Both the 160- and the 140-kDa band were detectable after 1-5 min and were maximal after 15 min. The band at 180-190 kDa was sometimes already present in unstimulated cells.
Figure 3: Time course of tyrosine phosphorylation during treatment with IL-4 (A) or IL-13 (B). TF-1 cells were stimulated with 100 ng/ml of IL-4 (A) or IL-13 (B) for the indicated time. Cells were lysed, and phosphoproteins were analyzed as in Fig. 1. In the IL-13 experiment (B), the blot had to be exposed for a longer time during ECL development to obtain acceptable signal strength.
A protein of
approximately 140 kDa that has been previously identified as
IL-4-dependent phosphorylation substrate in murine cells is IL-4R (29, 30) . We used the monoclonal antibody A3/10,
which was raised against the recombinant extracellular domain of the
human IL-4R
(27) to precipitate IL-4R
from stimulated
cell lysate. The precipitate was probed with an anti-phosphotyrosine
antibody. We found that both in IL-4- and IL-13-stimulated lysates from
TF-1 cells the IL-4R
was tyrosine phosphorylated, while no band
was obtained from unstimulated cell lysate (Fig. 4A).
Competition experiments with the recombinant extracellular domain
confirmed that the precipitated protein was the
subunit of the
IL-4R. These results proved that human IL-4R
is phosphorylated on
tyrosine residues in response to both IL-4 and IL-13. The IL-4R
was identified as phosphorylation substrate in peripheral blood
lymphocytes as well (Fig. 4B). Tyrosine phosphorylation
induced by IL-13 was weaker than with IL-4, regardless whether
precipitation was with anti-phosphotyrosine (Fig. 3) or with
anti-IL-4R
(Fig. 4A).
Figure 4:
Both IL-4 and IL-13 induce tyrosine
phosphorylation of the IL-4R. A, TF-1 cells were
stimulated with 100 ng/ml IL-4 or IL-13 for 10 min or left
unstimulated. B, peripheral blood lymphocytes (PBL)
were stimulated in the same way with IL-4. Both cell types were lysed,
and the lysates were immunoprecipitated with antibodies against the
IL-4R
in the absence (A, lanes1-3; B, lanes1-2)
or presence (A, lanes4-6, B, lanes3 and 4) of the recombinant
extracellular domain of the IL-4R
. The precipitates were blotted
and probed with the anti-phosphotyrosine antibody
RC20.
The identity of the
tyrosine kinase phosphorylating IL-4R is not known. A src-type kinase would be a likely candidate because the IL-2R
-subunit and the IL-7 receptor complex are associated with
p56
(33, 34) and
p59
(35) , respectively. We expressed the SH2
domains of these two kinases as fusion proteins with glutathione S-transferase and used them to precipitate phosphorylated
proteins from lysates of IL-2-, IL-4-, and IL-7-stimulated peripheral
blood lymphocytes (Fig. 5). The protein patterns precipitated
were identical to the ones observed when using the anti-phosphotyrosine
antibody 4G10 (Fig. 2). Both lck-SH2 and fyn-SH2 domains precipitated the same proteins.
Figure 5: Binding of cellular phosphoproteins to fusion proteins of glutathione S-transferase (GST) and fyn-SH2 (A) or lck-SH2 (B). Lysates from unstimulated peripheral blood lymphocytes or cells stimulated for 10 min with 100 ng/ml of IL-2, IL-4, or IL-7 were incubated with fyn-SH2 (A) or lck-SH2 fusion proteins (B) bound to glutathione-Sepharose beads. The immune complexes were analyzed by immunoblotting with anti-phosphotyrosine antibody RC20.
The pleiotropic and redundant effects displayed by an ever increasing number of identified cytokines make the assignment of specific functions to individual members of the cytokine network a difficult enterprise. Promiscuous receptor subunits may account for some of this complexity. Both cross-competition between cytokines for a receptor and initiation of common signal transduction pathways have to be considered.
IL-4 and IL-13 have very similar functions and share
at least one receptor subunit(10, 20) . Data presented
in this paper show that IL-4R is phosphorylated on tyrosine
residues following stimulation with either IL-4 or IL-13, which argues
for direct participitation of IL-4R
in the IL-13 receptor complex.
It has indeed been shown that a monoclonal antibody raised against the
extracellular domain of the IL-4R
inhibits IL-4- and
IL-13-dependent responses of TF-1 and B-cells(27) . The slower
and weaker response observed with IL-13 compared with IL-4 could be due
to the unknown specific subunit of the IL-13 receptor, which may result
in a lower and limiting number of functional receptors. Another
possible explanation could be the use of different src-type
kinases by the receptors for IL-4 and IL-13.
Available data
therefore prove sharing of IL-4R between IL-4 and IL-13, but it is
not clear whether
c is shared as well. Receptors for IL-4 and
IL-13 cannot be identical because some IL-4 responsive cells
(peripheral T-cells and the SP-B21 cell line) fail to respond to IL-13,
presumably because they lack an IL-13-specific receptor subunit (10) . IL-13 does not bind to COS cells transfected with the
human IL-4R
, apparently for lack of another receptor
component(10) . The molecule lacking is not
c because
SP-B21 cells fail to bind IL-13, despite the fact that they are IL-4
responsive and must therefore have
c(10) . So far, it is
not clear whether the IL-13 receptor uses both IL-4R
and
c.
IL-4 belongs to the family of cytokines sharing the c chain,
and IL-13 is a candidate to this club. To compare signaling pathways
within this family, we stimulated peripheral blood lymphocytes with two
other interleukins known to use
c, IL-2 (8) and
IL-7(5, 6) . IL-2 and IL-7 induced tyrosine
phosphorylation of the same proteins with identical kinetics. These
cytokines seem to be equivalent stimuli for T-cells because extent and
kinetics of cell proliferation induced by IL-2 and IL-7 are also very
similar to each other but clearly distinct to effects of
IL-4(37, 38) .
Only one band, at 160 kDa, appeared
to be common to all four cytokines studied here. In mouse cells, a
protein of 170 kDa, named 4PS, is strongly phosphorylated in response
to IL-4(30, 39) . This protein is related to IRS-1,
the major phosphorylation substrate of the insulin receptor, because it
cross-reacts weakly with some anti-IRS-1 antibodies(40) , and a
cell line deficient in responses to insulin and IL-4 could be
reconstituted in both aspects by transfection with IRS-1 (41) .
A binding motif for IRS-1 was identified in receptors for insulin,
insulin-like growth factor, and the IL-4R(42) . A highly
tyrosine-phosphorylated IRS-1-like protein can therefore be involved in
IL-4-induced signal transduction, but it seems to be absent in some
IL-4-responsive cell types(13) . The 4PS protein has not yet
been further characterized, and neither sequence nor antibodies are
available.
In human cells (29) and in murine cells
transfected with the human IL-4R(44) , no phosphorylation
in the range of 150-170 kDa has been found. In our hands, murine
FDCP-2 cells showed IL-4 induced tyrosine phosphorylation patterns
similar to those reported in the literature (30) , including a
prominent band at 160 kDa, but the pattern was quite different to those
found in human cells (see Fig. 1). The 160-kDa band from human
cells appeared at the same molecular weight as the putative 4PS band
from FDCP-2 cells, but we hesitate to identify this band as the human
homologue of 4PS because the tyrosine phosphorylation observed was much
weaker than found in mouse cells and, more importantly, because it also
appeared after stimulation with IL-2 and IL-7, which have so far not
been suspected to use IRS-1 or 4PS for signal transduction. The 160-kDa
band may well represent a signaling element common to the members of
the
c family, but its identity remains unclear.
It is unclear
which tyrosine kinases participate in IL-4-dependent signal
transduction. Jak3 is activated by IL-4(45) . A Jak-type kinase
probably mediates IL-4-stimulated tyrosine phosphorylation of a
transcription factor that binds to sequences resembling the
interferon- activation site and increases transcriptional activity
in reporter gene assays(46, 47, 48) . IL-13
activates the same transcription factor as IL-4(49) , which may
be a consequence of the shared IL-4R
subunit.
Another candidate kinase is a 92-kDa protein found associated with the IL-4 receptor that was recognized by an anti-Fes antibody, which shows that either c-Fes or a closely related protein may be involved in the IL-4 signal transduction pathway(49) . The protein is tyrosine phosphorylated after IL-4 stimulation(50) .
Kinases of the src family could well be involved in IL-4 signaling because
p56 binds to IL-2R
and is activated by IL-2
stimulation(33, 34) . Similarly, IL-7 stimulation
induces activation of p59
and its binding to the IL-7
receptor complex(35) . We have precipitated phosphorylated
proteins from IL-4-stimulated cells with SH2 domains from p56
and p59
. Both SH2 domains precipitated similar
bands from stimulated cell lysate. A possible explanation is that the
precipitated proteins are substrates for src-type kinases,
which after phosphorylation can bind to the SH2 domain of the kinase.
SH2 domains from different proteins have pronounced specificities, but
the binding affinities of SH2 domains from src-type kinases to
phosphotyrosine-containing peptides are very
similar(51, 52) , so the equivalence of fyn-SH2 and lck-SH2 in our assay is not surprising.
Furthermore, it is known that different src-type kinases can
be recruited for IL-2 signaling, like p53/56
and
p59
in pro-B-cells lacking p56
(53, 54) and p59
in cells
transfected with an IL-2R
mutant lacking the p56
binding site(55) . Three src-type kinases are
expressed in peripheral T-cells, p56
, p59
,
and to a low level p62
(43) . We have
recently found that yes-SH2 does not bind proteins from PHA
blasts after stimulation with IL-2, IL-4, or IL-7. (
)This
suggests that p56
or p59
(or both) may be
activated by stimulation of peripheral blood lymphocytes with IL-4,
just as in the case of IL-2 and IL-7. Because all three cytokines share
the
c subunit, activation of src-type kinases could be
linked to this common chain.