From the
Interleukin-13 (IL-13) and interleukin-4 (IL-4) are related in
structure and function and are thought to share a common receptor
component. We have investigated the signal transduction pathways
activated by these two growth factors, as well as insulin, in
cell-lines and primary cells of lymphohemopoietic origin. All three
factors induced the tyrosine phosphorylation of a protein of 170 kDa
(p170), which coimmunoprecipitated with the p85 subunit of PI3`-kinase,
via high affinity interactions mediated by the SH2 domains of p85.
Antibodies raised against the entire insulin-receptor substrate-1
(IRS-1) protein immunoprecipitated p170 much less efficiently than they
did IRS-1 from 3T3 cells. However, antibodies directed against the
conserved pleckstrin homology domain of IRS-1 immunoprecipitated both
p170 and IRS-1 with similar efficiency, suggesting they share
structural similarities in this region. In lymphohemopoietic cells,
IL-13, IL-4, and insulin failed to induce increased tyrosine
phosphorylation of Shc, or its association with grb2, modification of
Sos1, or activation of erk-1 and erk-2
mitogen-activated protein kinases, suggesting that p170 mediates
downstream pathways distinct from those mediated by IRS-1. Both IL-13
and IL-4 induced low levels of tyrosine phosphorylation of Tyk-2 and
Jak-1. IL-4 also activated the Jak-3-kinase, but, despite other
similarities, IL-13 did not. Insulin failed to activate any of the
known members of the Janus family of kinases. In that Jak-3 is reported
to associate with the IL-2
The cytokines IL¹(¹)
-4 and IL-13 share
structural and functional features and define a newly defined subfamily
of cytokines
(1) . Both IL-4 and IL-13 are produced by activated
Th2 cells
(2) and have a similar range of target cells, with the
exception that only IL-4 affects T lymphocytes
(1) . Both share
important, specialized functions in promoting class switching in human
B cells to IgE production
(3, 4) and down-modulating
proinflammatory events in human monocytes and murine macrophages,
increasing production of IL-1 receptor antagonist, and decreasing
expression of IL-1
The IL-4
receptor is composed of at least two subunits. The first to be
characterized was a 140-kDa subunit, termed
IL-4R
(5, 6, 7) . A second subunit of the IL-4
high affinity receptor has recently been shown to be the IL-2
The nature of the
IL-13 receptor is unknown. Competitive binding studies on TF-1 cells,
which bind both IL-4 and IL-13, have demonstrated that IL-13 can
cross-compete with IL-4 for binding, leading to the suggestion that
their receptors share a common component (13). The
IL-4 is unusual in that it fails to activate many of
the pathways activated by other growth factors. IL-4 fails to activate
p21
Little is currently known about IL-13 signal
transduction. Two pieces of evidence point to similarities with IL-4
signal transduction, one being the cross-competition studies, pointing
to a shared receptor component
(13) , and the second being the
report that IL-13 activates NF-IL-4
(39) . We have characterized
some of the early events which occur following treatment of cells with
IL-13 and compared them to signals triggered by IL-4 and insulin in
cell lines and primary cultures of lymphohemopoietic cells.
We prepared antibodies against the conserved
pleckstrin homology domain of IRS-1 (
We also examined Jak kinase tyrosine
phosphorylation in FD-5-, B9-, and LPS-activated splenic B cells. The
results are summarized in . In B9 cells we detected low
levels of tyrosine-phosphorylated Jak-1 and Tyk-2 in response to IL-4
and IL-13, but not insulin. Jak-3 appeared to have a high constitutive
level of tyrosine phosphorylation, which did not alter after factor
deprivation or treatment with cytokines (see Fig. 8C).
In the LPS-activated splenic B cells, IL-4 induced Jak-1 and Jak-3
tyrosine phosphorylation, in contrast to insulin which failed to induce
tyrosine phosphorylation of either of these kinases. IL-13 treatment of
this population of murine B cells induced no detectable effects on Jak
kinases (Fig. 8D).
The similarities in
molecular mass, in tyrosine phosphorylation in response to insulin and
in association with the p85 subunit of PI3`-kinase via high affinity
interactions directed by the SH2 domains of p85, suggested that
p170/4PS was identical, or related to IRS-1. We found that antibodies
raised against the conserved pleckstrin homology domain of IRS-1 were
able to immunoprecipitate tyrosine-phosphorylated p170/4PS from myeloid
cells and a doublet in the 170 kDa range from insulin-treated 3T3 cells
with similar efficiencies (Fig. 5, A and B).
However, additional observations by ourselves and others
(27) suggest that p170 and IRS-1, while related, are distinct
molecules. A commercial anti-IRS-1 antibody was extremely inefficient
at immunoprecipitating tyrosine-phosphorylated p170 from FD-5 and TF-1
cells, but efficiently precipitated IRS-1 from insulin-treated Swiss
3T3 cells. Moreover, when immunoprecipitates from IL-4 or
insulin-treated FD-5 cells, made with the anti-IRS-1PH antibodies, were
subjected to a secondary immunoprecipitation with the commercial
anti-IRS-1 antibody, no tyrosine-phosphorylated p170 species could be
reprecipitated. In contrast, when the same procedure was performed on
extracts of insulin-treated 3T3 cells, clearly tyrosine-phosphorylated
IRS-1 (the lower species of the doublet) was reprecipitated. In
addition, in sensitive reverse transcription-polymerase chain reaction
analyses, it has proven extremely difficult to detect amplified
IRS-1-specific sequences from FD-5, B9, and TF-1 cells, whereas it was
consistently easy to detect amplified IRS-1 from Swiss 3T3 mRNA.² It has recently been reported that mice lacking IRS-1 exhibit a
second molecule of 170 kDa which is tyrosine phosphorylated in response
to insulin and which binds to PI3`-kinase p85
subunit
(56, 57) . The molecular mass of this protein was
In further characterization we found
that p170 was located in crude membrane preparations in a
salt-sensitive manner (Fig. 4). In the absence of NaCl, a greater
proportion of tyrosine-phosphorylated p170 was located at the membrane
following treatment of cells (TF-1 and FD-5), with either IL-4, IL-13,
or insulin than when fractionation of the same cells was performed in
the presence of NaCl, at 150 mM (isotonic conditions), when
the majority of tyrosine-phosphorylated p170 was located in the
cytosol. This was the case, irrespective of the mechanical disruption
procedure used. Similar, although less dramatic, alterations in p170
location were also observed in B9 cells, where treatment with insulin
had the most noticeable effects. The reason for these differences is
unclear and is under further investigation. These findings contrast
with a previous report in which 4PS was reported to be
membrane-associated
(20) . Other reports have demonstrated,
albeit at low stoichiometry, association of IRS-1 with the insulin
receptor
(58) and the IL-4 receptor
(29) . Our findings
suggest that these and other potential interactions of p170 with
membrane proteins are dependent on salt-sensitive interactions.
Molecular characterization of p170/4PS/IRS-2 and the development of
specific probes will enable these interactions to be characterized
further.
We propose that despite certain structural and functional
similarities, including association with p85 PI3`-kinase, p170/4PS and
IRS-1 mediate some distinct downstream signaling events. In myeloid
cells, which in our hands express barely detectable levels of IRS-1,
IL-13, IL-4, and insulin, all failed to induce tyrosine phosphorylation
of Shc or its association with grb2. Despite the fact that we observed
binding of grb2 to p170 in TF-1 cells treated with IL-13, IL-4, or
insulin none of these factors induced modification of mSos1 or
activation of erk-1 or erk-2 MAP kinases. This
indicates that association of p170 with grb2 was not sufficient for
activation of the ras/MAP kinase signaling pathway. We had
previously observed similar results with IL-4 in FD-5
cells
(16, 17) . Our findings with insulin were somewhat
surprising given that insulin has been shown to induce tyrosine
phosphorylation of Shc
(59, 60) , association of Shc and
IRS-1 with grb-2
(60, 61, 62) , and activation of
ras and MAP kinases
(63, 64) . Moreover, we were
able to detect activation of erk-1 and erk-2 MAP
kinases by insulin in Swiss 3T3 cells. In many of the previous reports,
cells over-expressing the insulin receptor (up to 1.4
Our observations on the activation of different Jak kinases
by IL-13 and IL-4 may be useful in clarifying which components of the
IL-4 receptor are shared by the IL-13 receptor. Both IL-4 and IL-13
were able to activate Jak-1 and Tyk-2 in TF-1 cells
(Fig. 8A) and B9 cells (). IL-4 has been
previously shown to activate Jak-1 in T
cells
(31, 32, 33) , but this is the first
demonstration that IL-4 and IL-13 can induce tyrosine phosphorylation
of Jak-1 in myeloid cells. It is also the first report that these
cytokines can activate Tyk-2. In other systems, there is some precedent
for what appears to be cell type-dependent activations of specific Jak
family kinases by particular growth factors
(65) .
IL-4 was
the only cytokine examined which was capable of activating Jak-3 in
TF-1, FD-5, and populations of normal murine splenic B cells. In TF-1
cells we demonstrated Jak-3 as being the major 115-kDa substrate
tyrosine phosphorylated in response to IL-4. Importantly, IL-13 failed
to induce tyrosine phosphorylation of Jak-3 and insulin failed to
induce significant tyrosine phosphorylation of any of the Jak kinases
examined.
These studies address two key issues. First, it appears
that Jak-3 alone is not responsible for tyrosine phosphorylation of
p170/4PS because it is not tyrosine phosphorylated in response to
IL-13, which induces p170 tyrosine phosphorylation in B9 and TF-1
cells. Moreover, while IL-2, IL-7, IL-9, and presumably IL-15 induce
tyrosine phosphorylation and activation of Jak-3 kinase, IL-2 fails to
induce consistently detectable tyrosine phosphorylation of p170/4PS
(Fig. 1, E and F).³ The levels of
tyrosine phosphorylation of Jak-1 and Tyk-2 were very low in the cells
examined, and we feel it unlikely that activation of either of these
kinases is related to tyrosine phosphorylation of p170. The case of
FD-5 is particularly instructive as in these cells IL-4 induces very
marked p170 tyrosine phosphorylation, but there is no detectable
tyrosine phosphorylation of Jak-1 or Tyk-2. Secondly, it has previously
been suggested that the cross-competition observed between IL-4 and
IL-13 for binding to TF-1 cells was due to both factors using the IL-2
The average of four independent experiments
are shown for the TF-1 and 3T3 samples, with the exception of the
erk-2 activity in TF-1 cells, which is the average of two
independent experiments. The average of at least three independent
experiments are shown for the FD-5 samples.
The symbols used are:
We thank Kevin Leslie for communication of unpublished
results and helpful discussions, Ian Clark-Lewis for synthetic cytokine
preparations, John O'Shea for anti-human Jak-3 antibodies, David
Bowtell for anti-Sos1 antibodies, and Immunex Corporation for anti-IL-4
receptor antibodies.
c chain, these data suggest that the
IL-13 receptor does not utilize this subunit. However, both IL-13 and
IL-4 induced tyrosine phosphorylation of the IL-4-140 kDa
receptor chain, suggesting that this is a component of both receptors
in these cells and accounts for the similarities in signaling pathways
shared by IL-13 and IL-4.
and
, tumor necrosis factor-
, and
IL-12 (reviewed in Ref. 1). Interestingly, the effects of IL-4 and
IL-13 are not synergistic, suggesting that many of these overlapping
functions are controlled by common signaling processes, potentially
emanating from common receptor components (see below).
chain, or
c
(8, 9) . Cells in which both IL-4R
140-kDa chain and
c are expressed bind IL-4 with affinities two to
three
greater than those measured in cells expressing only the
IL-4R 140-kDa chain
(8, 9) . At least two regions of the
cytoplasmic domain of the IL-4R 140-kDa molecule have been reported to
be essential for transmitting signals important for mediating cell
growth. These regions span residues 258-390 and
437-557
(10, 11, 12) .
c subunit has
been proposed as a likely candidate
(8, 9) . However, it
is also possible that the 140-kDa IL-4 receptor is the shared component
(see below).
(14, 15, 16) , induce
tyrosine phosphorylation of Shc
(17) , activate erk-1 and erk-2 kinases
(16, 18) , or activate
Raf-1
(19) . IL-4 also fails to induce tyrosine phosphorylation
of p120 GAP
(15, 20) , phospholipase C
1
(20) ,
or SH-PTP2
(21) . In addition, in some, but not all cells, IL-4
fails to activate protein kinase C
(22) , induce Ca
mobilization
(22, 23) , or activate phospholipase C
(22, 23, reviewed in Ref. 24). However, IL-4 does activate
PI3`-kinase
(20, 25, 26) . The p85 subunit of
PI3`-kinase has been shown to bind to the major substrate of
IL-4-induced tyrosine protein kinases that has been termed
4PS
(20, 25, 27) . 4PS is a 170-kDa protein,
which is reported to weakly react with antibodies directed against the
major insulin receptor substrate, IRS-1, suggesting some limited
homology of 4PS/p170 to IRS-1
(27) . In the factor-dependent
hemopoietic cell line 32D, IL-4 failed to induce tyrosine
phosphorylation of 4PS and also failed to promote proliferation.
However, after transfection of 32D with IRS-1, IL-4 stimulated
increased tyrosine phosphorylation of IRS-1 and cell
proliferation
(28) . Recently, a region of the IL-4R 140-kDa
subunit containing a motif shared with the insulin receptor has been
shown to be important for mediating IRS-1 phosphorylation and cell
growth of 32D cells
(29) . This region maps to that previously
shown to be important for IL-4 receptor mediated mitogenesis in Ba/F3
cells
(10, 11, 12) . The c-fes kinase
has been reported to associate with the IL-4 receptor
(30) ,
although this kinase was not activated by stimulation with IL-4. It has
also recently been shown that IL-4 activates Jak-1 and Jak-3 in T
cells
(31, 32) . Jak-1 may interact directly with the
140-kDa subunit of the IL-4 receptor
(33) , and Jak-3 has been
shown to associate with the IL-2
c subunit
(34, 35) .
Activation of the Jak family of kinases is likely to be related to the
ability of IL-4 to activate the Stat molecules termed NF-IL-4
(36) and Stat-IL-4
(37) or the recently cloned
IL-4-Stat
(38) .
Cell Culture
Cells were cultured in humidified
incubators at 37 °C, 5% CO (v/v) in either RPMI 1640
medium or Dulbecco's modified Eagle's medium (Life
Technologies, Inc.), supplemented with 10% (v/v) fetal bovine serum
(Life Technologies, Inc.), 20 µM 2-mercaptoethanol, 100
units of penicillin/streptomycin, and 2 mM glutamine.
FDMAC11/4.5 (FD-5) is a variant of FDMAC11 which will grow in response
to IL-4, as well as IL-3, granulocyte macrophage-colony stimulating
factor, and colony stimulating factor
(16) and was maintained in
3% (v/v) IL-4-conditioned medium derived from the AgX63/OMIL-4
cells
(40) , which were a gift of Dr. F. Melchers (Basel
Institute of Immunology, Basel, Switzerland). TF-1 is a human
erythroleukemia cell line
(41) and was maintained in RPMI
supplemented with 3% (v/v) gibbon IL-3-conditioned medium derived from
AgX63/gIL-3 cells. TF-1 cells respond to IL-3, IL-5, granulocyte
macrophage-colony stimulating factor, Epo, IL-4, IL-13, and
insulin
(13, 41) . B9 is a murine plasmacytoma cell line
and was grown in Dulbecco's modified Eagle's medium
supplemented with 2% (v/v) of a 10-fold concentrated stock of
conditioned medium from Swiss 3T3 cells as a source of
IL-6
(42) . These cells respond to IL-13 and IL-4
(1) .
(3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide) (MTT) assays were performed as described
previously
(43) . CT.4S, HDK-1, and D10 were grown as described
previously
(16, 44, 45) . Populations of primary
murine ConA-activated splenic T cells and murine
lipopolysaccharide-(LPS) activated splenic B cells were prepared as
described previously
(46, 47) .
Cell Stimulation and Growth Factors
Stimulation of
cells with different growth factors was carried out as described
previously (17). Synthetic forms of murine IL-3, IL-13, IL-4, and human
IL-4 were kindly provided by Dr. Ian Clark-Lewis (The Biomedical
Research Centre, Vancouver, British Columbia). mIL-13 has been
demonstrated to bind equally well to cells of both human and murine
origin
(13) . Porcine insulin was purchased from Sigma. mIL-2 was
purchased from Genzyme. Gibbon IL-3 expressed in AgX63 cells is fully
bioactive on human cells and was used as a source of IL-3 for studies
with TF-1 cells. Cells were stimulated for a length of time and with a
particular concentration of growth factor which had been previously
determined to induce maximal levels of tyrosine phosphorylation. These
were as follows, mIL-3 at 10 µg/ml
(17) , mIL-13 at 20
µg/ml, mIL-4 at 20 µg/ml
(16) , mIL-2 at 100
units/ml
(46) , hIL-4 at 20 µg/ml, and insulin at 5
µg/ml. All stimulations were carried out for 10 min, except for
those with insulin, which were carried out for 2 min. In some cases
time course analyses were performed to examine the kinetics of tyrosine
phosphorylation and are indicated in the appropriate figures. Cell
pellets were lysed in immunoprecipitation buffer (IP buffer: 50
mM Tris-HCl, pH 7.5, 10% (v/v) glycerol, 1% (v/v) Nonidet
P-40, 150 mM NaCl, 5 mM EDTA, 1 mM sodium
orthovanadate, 1 mM sodium molybdate, 10 mM sodium
fluoride, 40 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml
aprotinin, 10 µg/ml soyabean trypsin inhibitor, 10 µg/ml
leupeptin, 0.7 µg/ml pepstatin).
Subcellular Fractionations
Cytosol (C) and crude
membrane (M) fractions were prepared using sonication buffer (150
mM NaCl) or homogenization buffer (0 mM NaCl) as
described previously
(17) . Briefly, cells were disrupted on ice
by 4 2-s pulses using a Sonics and Materials ``Vibra
Cell'' (Danbury, CT), or by 100 strokes of a tight fitting Dounce
homogenizer. Similar results were obtained irrespective of the
mechanical procedure used. Nuclei and intact cells were removed by
centrifugation for 15 s, at 4 °C, in a microcentrifuge at full
speed. The supernatant was then subjected to centrifugation at 150,000
g at 4 °C for 20 min. The resulting supernatant
(S150) was designated the cytosol and the pellet (P150) the crude
membrane. The pellet was solubilized in the appropriate buffer
containing 1% Nonidet P-40 and 0.5% sodium deoxycholate and
recentrifuged to remove remaining insoluble material. The cytosolic
fraction was adjusted to contain 1% Nonidet P-40.
Immunoprecipitations
Extracts were precleared with
25 µl of a 50% (v/v) slurry of protein A-Sepharose beads
(Pharmacia) for 15 min, at 4 °C on a rotator prior to addition of
the appropriate antibody. The following antibodies were purchased from
UBI, Lake Placid, NY with the amounts used per IP stated in
parentheses: polyclonal rabbit antibodies to either the p85 subunit of
PI3`kinase (2 µl), Jak-1 (06-272, 3 µl), Jak-2 (2
µl), Jak-3 (5 µl), Shc (2 µl), or IRS-1 (2 µl). A
polyclonal antibody recognizing Tyk-2 was purchased from Santa Cruz
Biotechnology Inc (Santa Cruz, CA) and 2 µg/sample used for
immunoprecipitations. Anti-human Jak-3 antibodies (31) were a kind gift
of Dr. J. O'Shea (National Cancer Institute, Frederick, MD); 3
µl were used per sample. Monoclonal anti-IL-4 receptor antibody
(M2) was a kind gift of Immunex Corporation (Seattle, WA); 5 µg
were used per sample. Polyclonal rabbit antibodies to the pleckstrin
homology domain of IRS-1 were raised against a GST fusion protein
comprising amino acids 1-124 of murine IRS-1 fused to
GST²(²)
; 10 µl of antibody were used per
sample. After addition of antibody, the samples were vortexed and
incubated on ice for 30 min to 1 h. 40 µl of a 50% (v/v) slurry of
either protein A-Sepharose beads (for rabbit antibodies) or protein
G-Sepharose beads (for mouse monoclonal antibodies) were added to each
IP and samples incubated at 4 °C on a rotator for 1 h.
Immunoprecipitates were washed three times with IP buffer at 4 °C.
Bound proteins were eluted by boiling in SDS-PAGE sample buffer
containing 2-mercaptoethanol. Aliquots of cell extracts were removed
both prior to (``Pre IP'') and following (``Post
IP'') immunoprecipitation and were diluted with the same SDS-PAGE
sample buffer as the immunoprecipitates.
GST Fusion Proteins
The construction, expression,
and purification of full-length human grb2 and p85 PI3`-kinase SH2
domain GST fusion proteins have been previously
described
(17, 48) . 2 µg of either the PI3`-kinase
amino-terminal SH2 (N-SH2) or carboxyl-terminal SH2 (C-SH2) fused to
GST or 5 µg of the full-length human grb2-GST were used per
precipitation.
Kinase Assays
Assays were performed as described
previously (16). Anti erk-1 and anti erk-2 antibodies
conjugated to beads were purchased from Santa Cruz Biotechnology Inc.
(Santa Cruz, CA) and 30 µl of a 50% (v/v) slurry was used per
immunoprecipitation.
SDS-PAGE and Immunoblotting
SDS-PAGE and
immunoblotting were carried out as described
previously
(17, 49) . Primary antibodies were used at the
following concentrations: the antiphosphotyrosine monoclonal antibody
4G10 at 0.1 µg/ml, the polyclonal anti-Shc antibody at 0.25
µg/ml, polyclonal anti-Sos1 at 1:4000, anti-PI3`-kinase antibody at
1:4000, anti-Jak-1 and Jak-3 antibodies at 1:2000, and anti-Tyk-2
antibody at 0.2 µg/ml. Both goat anti-mouse and goat anti-rabbit
horseradish peroxidase-conjugated antibodies (Dako, Dimension
Laboratories, Missisauga, Ontario) were used at a concentration of 0.05
µg/ml. Immunoblots were developed using the ECL system (Amersham).
Kodak X-AR 5 film was used for detection of ECL signals. Blots were
stripped completely of antibodies by incubation at 55 °C for 60 min
with stripping solution (62.5 mM Tris-HCl, pH 6.8, 2% (w/v)
SDS, 100 mM 2-mercaptoethanol). After extensive washing, blots
were reblocked prior to reprobing. The results observed in different
experiments were independent of the sequence of antibodies used for
probing the immunoblots.
RESULTS
Induction of Tyrosine Phosphorylation in
Lymphohemopoietic Cells in Response to IL-13, IL-4, and
Insulin
We examined the responsiveness of a number of cell lines
of lymphohemopoietic origin to IL-13, IL-4, and insulin. In each case
the ability of the individual factors to induce tyrosine
phosphorylation of proteins was assessed by immunoblotting with
antiphosphotyrosine antibodies, and the results are shown in
Fig. 1
. In both TF-1 (Fig. 1A) and B9 cells
(Fig. 1C), IL-13, IL-4, and insulin all induced the
tyrosine phosphorylation of a major species of 170 kDa. This protein is
similar in molecular mass to the protein termed 4PS, which had been
shown by others to be the major substrate which becomes phosphorylated
on tyrosine residues following IL-4 and insulin treatment of murine
myeloid
cells
(20, 25, 27, 28, 29) . In
biological response MTT assays, both TF-1 and B9 responded to IL-13 and
IL-4 (data not shown). Interestingly, both IL-4 and IL-13 induced
tyrosine phosphorylation of a 112-kDa protein in TF-1 cells. In
addition, IL-4, but not IL-13, was also capable of inducing tyrosine
phosphorylation of a protein of 115 kDa in TF-1 (see below). Treatment
of the myeloid line FD-5 with either IL-4 or insulin, but not IL-13,
induced p170 tyrosine phosphorylation (Fig. 1B).
Treatment of two T cell lines, HDK-1 (of Th1 phenotype,
Fig. 1D) and D10 (of Th2 phenotype,
Fig. 1E) with either IL-4 or insulin (but not IL-2)
induced tyrosine phosphorylation of a 170-kDa protein. In both HDK-1
and D10, IL-4 also induced phosphorylation of a protein of
135-140 kDa, likely to be the IL-4 receptor. Importantly,
treatment with IL-4 of the T cell line CT.4S, which is absolutely
dependent on the presence of IL-4 for its continued proliferation, did
not induce detectable tyrosine phosphorylation of p170
(Fig. 1F), although both IL-4 and IL-2 induced tyrosine
phosphorylation of a 115-kDa protein.
Figure 1:
Induction of tyrosine phosphorylation
by IL-13, IL-4, and insulin in lymphohemopoietic cells. All cells were
factor deprived for 16 h prior to stimulations, with the exception of
B9 cells. All samples were separated by SDS-PAGE using 7.5% acrylamide
gels and immunoblotting performed with 4G10 antiphosphotyrosine
antibodies. Samples from control untreated cells are labeled C in each case. The position of molecular mass standards are shown
and expressed in kDa. The positions of p170, p140, p115, and p112 are
also indicated. A, TF-1 cells were treated for 10 min with
either IL-3 (3), hIL-4 (4), or mIL-13 (13)
or for 2 min with insulin (I). B, FDMAC11/4.5
(FD-5) cells were treated for 2 min with insulin (I)
or for 10 min with either mIL-4 (4) or mIL-13 (13).
C, B9 cells were treated for 2 min with insulin (I)
or for 10 min with either mIL-4 (4) or mIL-13 (13).
D-F, time course analyses were performed to examine kinetics
of tyrosine phosphorylation. Cells were treated with factor for the
indicated periods of time. D, HDK-1 cells were treated with
insulin (INS), IL-2, or IL-4. E, D10 cells were
treated with insulin (I), IL-2 (2), or IL-4.
F, CT.4S cells were treated with either IL-4 or
IL-2.
It was important to
investigate the effects of IL-13, IL-4, and insulin not only on cell
lines, but also on primary cells. Therefore, we generated populations
of murine ConA-activated splenic T cells or LPS-activated splenic B
cells, stimulated them with IL-2, IL-4, IL-13, or insulin, and
subjected cell extracts to SDS-PAGE and immunoblotting with 4G10. As
shown in Fig. 2, IL-4 and insulin both induced tyrosine
phosphorylation of a 170-kDa protein in primary populations of T cells
(Fig. 2A and data not shown) and B cells
(Fig. 2B). IL-13 failed to stimulate p170 tyrosine
phosphorylation in populations of T cells (data not shown) or B cells
(Fig. 2B), although it has been reported to be active on
human B cells
(1) .
Figure 2:
Induction of tyrosine phosphorylation in
primary T and B lymphocytes. A, primary murine ConA-activated
CD8-depleted splenic T cells were deprived of IL-2 for 16 h and then
either left untreated as a control (C) or treated with IL-4
for the indicated periods of time or with IL-2 for 10 min. B,
murine LPS-activated splenic B cells were either left untreated as a
control (C) or treated for 10 min with IL-4 (4), 10
min with IL-13 (13), or 2 min with insulin (I).
Samples were separated through 7.5% acrylamide gels by SDS-PAGE and
immunoblotting performed with 4G10. The molecular mass standards are
indicated in kDa. The position of p170 is also
indicated.
p170, Tyrosine Phosphorylated in Response to IL-13,
Co-immunoprecipitates with p85 PI3`-kinase.
We were interested
in determining if the p170 protein, tyrosine phosphorylated in response
to IL-13, had similar properties to the tyrosine-phosphorylated
170/4PS, which we³(³)
and others have shown to
coimmunoprecipitate with the p85 subunit of
PI3`-kinase
(20, 25, 27) . TF-1 cells were
stimulated with IL-13, IL-4, insulin, and IL-3 or left untreated as a
control. Immunoprecipitations were performed using antibodies directed
against the p85 subunit of PI3`-kinase, and resulting precipitates were
immunoblotted with antiphosphotyrosine antibodies. The results are
shown in Fig. 3A (upper panel, also see
Fig. 8B). In the IL-13-, IL-4-, and insulin-treated
samples, a tyrosine-phosphorylated 170-kDa protein coimmunoprecipitated
with the p85 subunit of PI3`-kinase. Similar results were observed in
B9 cells (see Fig. 3B, upper panel) and also in
FD-5, LPS-activated splenic B cells, and ConA-activated splenic T cells
in response to IL-4 or insulin (data not shown). In each case equal
amounts of p85 were immunoprecipitated (Fig. 3, A and
B, lower panels). Thus, the IL-13 p170 substrate
behaved in a similar manner to 4PS and IRS-1 in associating with
p85
(20, 50) .
Figure 3:
IL-13 induces coimmunoprecipitation of
p170 with PI3`-kinase. A, factor-deprived TF-1 cells were
either left untreated as a control (C) or treated for 10 min
with hIL-4 (4), 10 min with mIL-13 (13), 2 min with
insulin (I), or 10 min with gIL-3 (3). Cell extracts,
from the equivalent of 2 10
cells/sample, were
immunoprecipitated with 2 µl of anti-p85 PI3`-kinase antibody.
B, B9 cells were either left untreated as a control
(C) or stimulated for 10 min with mIL-4 (4), 10 min
with mIL-13 (13), or 2 min with insulin (I). The
equivalent cell extract from 2
10
cells were used
for immunoprecipitation with 2 µl of anti-PI3`-kinase antibody.
C, factor-deprived TF-1 cells were treated as follows:
control, untreated (C); 10 min IL-3 (3); 10 min hIL-4
(4); 10 min mIL-13 (13), or 2 min insulin
(I). Cell extracts were divided equally into two aliquots,
each containing the equivalent of 2.8
10
cells/sample and precipitates performed using 2 µg of either
purified N-SH2-GST or C-SH2-GST. Immunoblotting was performed first
with 4G10, antiphosphotyrosine antibody (A and B,
upper panels, and C). The same immunoblots as in
A and B were stripped and reprobed with antibodies
directed against p85 to monitor for efficiency of immunoprecipitation
in each case (A and B, lower panels). The
positions of molecular mass standards are shown and expressed in kDa.
The positions of immunoglobulin heavy chain (IgG
,
contributed by the immunoprecipitating antibody), p170, and p85 are
indicated.
Figure 8:
Activation of Jak family kinases by IL-13
and IL-4 in TF-1 cells. A, factor-deprived TF-1 cells were
either left untreated (C) or treated for 10 min with either
IL-3 (3), hIL-4 (4), or mIL-13 (13).
Extracts from the equivalent of 5 10
cells were
then immunoprecipitated with antibodies specifically recognizing either
Jak-1, Jak-3, or Tyk-2. After extensive washing, immunoprecipitates
were separated by SDS-PAGE through 7.5% acrylamide gels and
immunoblotting carried out first with 4G10 (upper panels in
each case). Blots were stripped and reprobed with the appropriate
immunoprecipitating antibody (indicated underneath the lower panel in
each case). Positions of Jak-1, Jak-3, and Tyk-2 are indicated, as are
the molecular mass standards (in kDa). The small arrowhead denotes the position of tyrosine phosphorylated Jak-1. B,
TF-1 cells were treated with IL-4 (4) or IL-13 (13)
for 10 min or left untreated as a control (C). Samples
containing the same number of cell equivalents were removed both prior
to (Pre IP) and following immunoprecipitation with either
Jak-3 or p85 PI3`-kinase antibodies (Post IP).
Immunoprecipitates are also shown. Immunoblotting was performed with
4G10. Blots were then stripped and sequentially reprobed with
anti-Jak-3 and anti-p85 antibodies. The positions of p170, Jak-3, and
p85 are indicated. The upper small arrowhead denotes p170 and
the lower small arrowhead p115/Jak-3. C, B9 cells,
and D, LPS-activated B cell blasts were treated for 10 min
with either mIL-4 (4) or mIL-13 (13), for 2 min with
insulin (I) or left untreated as a control (C).
Immunoprecipitations were performed with anti-Jak-3 antibodies (B9 and
LPS-activated B cells) or anti-Jak-1 antibodies (LPS-activated B cells)
and immunoblotted with 4G10. The same blots were stripped and reprobed
with anti-Jak-3 or anti Jak-1 antibodies as
appropriate.
The SH2 Domains of p85 PI3`-kinase Direct Interaction
with p170
We investigated the mechanism responsible for the
interaction between p170 and PI3`-kinase. The p85 subunit of
PI3`-kinase contains two SH2 domains, which mediate interaction with
phosphotyrosine residues having a consensus sequence of
Y*XXM
(51) . We investigated the ability of fusion
proteins, containing either the amino-terminal (N-SH2) or
carboxyl-terminal (C-SH2) SH2 domain of p85 fused to GST
(48) to
precipitate p170 from IL-13-, IL-4-, or insulin-treated TF-1 cells. The
results are shown in Fig. 3C. Both the amino-terminal
and the carboxyl-terminal SH2 domains of p85 immunoprecipitated
tyrosine phosphorylated p170 from IL-13, IL-4, and insulin-treated
cells. This complex was stable to 0.1% SDS and 0.5 M NaCl,
indicating that it was a stable, high affinity interaction (data not
shown). An additional protein of 115 kDa also coimmunoprecipitated with
the N-SH2 and C-SH2 constructs from IL-4-treated cells. Similar results
were obtained using the myeloid cell line FD-5 (data not shown).
Subcellular Localization of p170
Initial reports
suggested that 4PS was a membrane-associated molecule
(20) . We
investigated the localization of p170 in FD-5, TF-1, and B9. Cells were
fractionated into cytosol (C) and membrane components (M), either by
sonication in buffer containing 150 mM NaCl (isotonic) or in
homogenization buffer containing no NaCl (hypotonic). Cell fractions
were separated by SDS-PAGE and immunoblotting performed with
4G10-antiphosphotyrosine antibodies. The results are shown in
Fig. 4
. The subcellular location of tyrosine-phosphorylated p170
appeared to be dependent on the salt concentration of the buffer used
during the fractionation procedure. In the presence of 150 mM
NaCl (isotonic), the majority of tyrosine phosphorylated p170/4PS was
found in the cytosolic fractions of all the cells, with only small
amounts present at the membrane (Fig. 4, A-C, NaCl 150
mM). In the absence of NaCl (hypotonic conditions), a larger
percentage of tyrosine-phosphorylated p170 was located in the crude
membrane fraction (Fig. 4, A-C, NaCl 0 mM).
Comparable results were obtained for FD-5 (Fig. 4A) and
TF-1 (Fig. 4B) and suggest that tyrosine-phosphorylated
p170 interacts with membrane proteins in a salt-sensitive manner. In B9
cells the overall tyrosine phosphorylation of p170 appears to increase
in the homogenized samples, the reason for which is unclear, but may
simply reflect variability between stimulations. In addition in B9, the
association of tyrosine-phosphorylated p170 with the membrane following
IL-4 and IL-13 treatment in the absence of NaCl is less pronounced than
the results we observed with insulin in B9 cells. Further studies are
underway to investigate this in more detail. In previous studies we
have shown that fractionation in isotonic buffer does result in the
expected localization of other proteins in the crude membrane fraction.
Thus, integral membrane proteins, e.g. c-kit and IL-3
receptor c, were found only in crude membrane fractions, and we
could detect the characteristic PMA-induced translocation of PKC to the
plasma membrane in mast cells
(17) . Reprobing the immunoblots
shown here with anti-c-src antibodies demonstrated that
greater than 90% of c-src was associated with the crude
membrane fraction, irrespective of the buffer system used (data not
shown).
Figure 4:
Subcellular localization of p170. FD-5
(A), TF-1 (B), and B9 (C) cells were factor
deprived and then either left untreated (CON), or treated for
10 min with either IL-4, or IL-13, or for 2 min with insulin
(INS). Cells were fractionated using either sonication (150
mM NaCl) or homogenization buffer (0 mM NaCl) as
described under ``Materials and Methods.'' All samples were
disrupted by sonication except those in A (0 mM
NaCl), where the samples disrupted in a Dounce homogenizer. Similar
results were obtained in three independent experiments for each cell
line with a representative experiment shown in each case. Results were
independent of the mechanical procedure used. Total cytosol
(C) and crude membrane fractions (M), containing the
same cell equivalents were separated by SDS-PAGE and immunoblotted with
4G10. Note the order of the fractions in panel C. The
molecular mass standards are indicated (in kDa) and the position of
p170 is indicated.
p170 Is a Distinct Species from IRS-1
It has been
reported, based on weak antibody cross-reactivity, that 4PS/p170 is
distinct, but related, to the insulin receptor substrate-1 molecule,
IRS-1
(27) . However, these data can be interpreted in two ways;
either the myeloid cells being studied could have expressed extremely
low levels of authentic IRS-1 or the polyclonal anti-IRS-1 serum
contained a minor subset of antibodies which cross-reacted with 4PS.
One-dimensional phosphopeptide mapping of P-labeled
material demonstrated some differences between 4PS and
IRS-1
(27) , but these could simply reflect different patterns of
phosphorylation of IRS-1 in different cells. We used two approaches to
attempt to clarify this issue using antibodies with different
specificities.
-IRS-1PH) and compared these
antibodies with a commercially available polyclonal antibody raised
against the entire rat IRS-1 protein (
-IRS-1) for the ability to
immunoprecipitate tyrosine-phosphorylated p170/4PS from myeloid cells
and IRS-1 from 3T3 fibroblasts. The commercial antiserum
immunoprecipitated tyrosine-phosphorylated IRS-1 efficiently from
insulin-treated Swiss 3T3 cells, but precipitated only low amounts of
tyrosine-phosphorylated p170 from FD-5 cells or TF-1 cells
(Fig. 5A). Interestingly, tyrosine-phosphorylated p170
from FD-5 and TF-1 consistently migrated more slowly on SDS-PAGE than
did IRS-1 from Swiss 3T3 cells, suggesting that it may be a distinct
protein. The anti-IRS-1PH antibody was equally efficient at
immunoprecipitating a tyrosine-phosphorylated p170 species from both
FD-5 and Swiss 3T3 following insulin treatment (Fig. 5B)
and also precipitated tyrosine-phosphorylated p170 from IL-4-treated
FD-5 (Fig. 5B) and TF-1 cells (data not shown). The
immunoprecipitates from insulin-treated Swiss 3T3 cells appeared to
contain a doublet of tyrosine-phosphorylated proteins in the 170 kDa
range. In sequential immunoprecipitation analyses, extracts of 3T3 or
FD-5 were first immunoprecipitated with the anti-IRS-1PH antibody.
After extensive washing, samples were eluted, denatured by boiling in
SDS-PAGE sample buffer (without bromphenol blue), and half of the
sample subjected to a secondary immunoprecipitation using antibodies
raised against the entire IRS-1 protein. The primary immunoprecipitates
are those shown in Fig. 5B. After extensive washing,
reprecipitated proteins were eluted and immunoblotted with 4G10. The
results of the secondary immunoprecipitations are shown in
Fig. 5C. While the
IRS-1PH antibody precipitated
similar amounts of tyrosine-phosphorylated IRS-1 and p170 from 3T3 and
FD-5, respectively (Fig. 5B), the commercial anti-IRS-1
antibody reimmunoprecipitated only the lower of the
tyrosine-phosphorylated proteins, presumably IRS-1, from 3T3 cells
(Fig. 5C). This provides clear evidence that IRS-1 and
p170 are distinct species, and it is likely that p170/4PS plays a major
role in transducing IL-4, IL-13, and insulin signals in
lymphohemopoietic cells.
Figure 5:
p170 is distinct from IRS-1.
Factor-deprived FD-5 were either left untreated as a control
(C) or stimulated for 10 min with IL-4 (4) or 2 min
with insulin (I). Factor-deprived TF-1 cells were either left
untreated as a control (C), stimulated for 10 min with either
IL-3 (3), IL-4 (4), or IL-13 (13) or for 2
min with insulin (I). Swiss 3T3 cells (3T3) were
deprived of serum for 24 h prior to stimulation for 2 min with insulin
(I) or left untreated as a control (C). A,
cell extracts were immunoprecipitated using a commercially available
polyclonal antibody raised against the entire coding sequence of rat
IRS-1, termed -IRS-1. B, samples were removed prior to
immunoprecipitation (Pre IP) to demonstrate that the relative
levels of the tyrosine-phosphorylated 170-kDa protein (p170 or IRS-1)
in the different cells was similar. Cell extracts were then
immunoprecipitated with a polyclonal antibody raised against the
conserved pleckstrin homology domain of IRS-1, termed
-IRS-1PH.
C, aliquots of the same primary immunoprecipitations shown in
B were used for secondary immunoprecipitation with the aIRS-1
antibody. Primary
-IRS-1PH precipitates were eluted, denatured by
boiling in SDS sample buffer (without bromphenol blue) and
reimmunoprecipitated using the commercial
-IRS-1 antibody.
Immunoprecipitates were separated by SDS-PAGE and immunoblotting
performed with 4G10. The position of tyrosine phosphorylated p170 and
IRS-1 are indicated, as are the molecular mass standards (in
kDa).
Effects of IL-13 on Shc Tyrosine Phosphorylation and
Association of Shc and p170 with grb2
Given the similarities in
molecular weight, subcellular localization and the ability to
coimmunoprecipitate with p85 PI3`-kinase, it was important to examine
whether 4PS/p170 interacted with the same proteins as IRS-1 and was
involved in similar functions. We examined the ability of IL-13, IL-4,
and insulin to induce tyrosine phosphorylation of the adaptor molecule
Shc, which has been implicated as an upstream regulator of
ras(52, 53) . TF-1 cells were treated with
either IL-3 (which is known to induce Shc tyrosine phosphorylation,
17), IL-13, IL-4, or insulin or left untreated as a control.
Precipitations were performed with either anti-Shc polyclonal
antiserum, or with a grb2-GST fusion protein which only binds Shc that
has been tyrosine phosphorylated at Tyr (the grb2
recognition site, 54). Precipitates were subjected to SDS-PAGE, and
immunoblotting was performed with antiphosphotyrosine antibodies. The
results are shown in Fig. 6, A and B. As we had
observed in other cell types
(17) , IL-3 induced an increase in
tyrosine phosphorylation of p50
and p55
in
TF-1 cells (Fig. 6A), although there was a detectable
basal level of Shc tyrosine phosphorylation in these cells. This
tyrosine-phosphorylated Shc was also recognized by the grb2-GST fusion
protein, along with a number of other phosphotyrosine-containing
proteins (Fig. 6B). IL-13, IL-4, and insulin all failed
to induce increased tyrosine phosphorylation of Shc proteins as judged
by precipitation with anti-Shc antibodies, or with the grb2-GST fusion
protein. Reprobing the precipitates with anti-Shc antibodies
(Fig. 6, A and B, lower panels),
demonstrated equal amounts of Shc in the anti-Shc immunoprecipitates
(Fig. 6A) and an increase in Shc present in the grb2-GST
fusion protein precipitates from IL-3, but not IL-4-, IL-13-, or
insulin-treated TF-1 cells (Fig. 6B). However, the
grb2-GST fusion protein was able to coprecipitate p170 from cells
treated with IL-13, IL-4, or insulin, indicating that these factors
induce phosphorylation of p170/4PS on a tyrosine that is recognized by
grb2.
Figure 6:
IL-13,
IL-4, and insulin fail to induce tyrosine phosphorylation of Shc or its
association with grb2 in TF-1 cells. TF-1 cells were factor deprived
for 16 h, and cells were then either left untreated as a control
(C) or treated for 10 min with either hIL-4 (4),
mIL-13 (13), gIL-3 (3), or for 2 min with insulin
(I). A, cell extracts equivalent to 2
10
cells were subjected to immunoprecipitation with 2
µl/sample anti-human Shc antibodies. IPs were separated by SDS-PAGE
and immunoblotting performed first with 4G10 (upper panel).
The blot was stripped and reprobed with anti-Shc antibodies (lower
panel). B, cell extracts equivalent to 3
10
cells were used per sample, and following lysis 5 µg of
full-length human grb2-GST fusion protein was added per sample.
Coprecipitating proteins were analyzed by immunoblotting with 4G10
(upper panel). A duplicate immunoblot was probed with
antibodies to Shc (lower panel). The positions of the
molecular mass standards are shown (in kDa), as are the positions of
p170 and Shc. C, factor-deprived TF-1 cells were treated for
10 min with either gIL-3 (IL-3), hIL-4 (IL-4), or
mIL-13 (13) or treated with insulin for 2 min (INS).
Cells were fractionated into cytosol and membrane components using
sonication buffer and aliquots containing the equivalent of 2.6
10
cells/sample separated through 7.5% acrylamide gels by
SDS-PAGE. Immunoblotting was performed using anti Sos1 antibodies (17,
54).
Effects of IL-13 on mSos1
Shc and grb2 are
believed to be key adaptor molecules in mediating the relocation of the
ras nucleotide-exchange factor, Sos1, to the plasma membrane,
where ras is located
(55) . The observation that
tyrosine-phosphorylated p170 can be detected at the membrane (see
Fig. 4
) and that grb2 can bind to p170/4PS raised the possibility
that this interaction could mediate the relocation of Sos1 to the
plasma membrane. In addition, Sos1 is modified in its electrophoretic
mobility following stimulation of cells with epidermal growth
factor
(54) , IL-3, and SLF, but not IL-4
(17) . To
determine whether IL-13 induced either a translocation of Sos1 to the
membrane or a change in its electrophoretic mobility, TF-1 cells were
treated with either IL-3, IL-4, IL-13, or insulin, or left untreated as
controls and were fractionated into cytosol and crude membrane
components, in the presence of 150 mM NaCl. Aliquots,
containing equal cell equivalents, were subjected to SDS-PAGE and
immunoblotted with anti Sos1 antibodies
(54) . The results shown
in Fig. 6C indicate that IL-13, IL-4, and insulin all
failed to induce detectable translocation of Sos1 to the membrane
fraction. As expected from our previous experiments
(17) , IL-3,
but not IL-4, induced a retardation in electrophoretic mobility of
Sos1. Insulin and IL-13 both resembled IL-4 in failing to induce this
change in Sos1.
Effects of IL-13 on erk-1 and erk-2 MAP Kinase
Activity
The family of extracellular signal regulated
(erk) or mitogen-activated protein (MAP) kinases is a group of
serine/threonine kinases which are thought to play a central role in
mitogenesis. We had previously failed to detect any increases in either
erk-1 or erk-2 activity following treatment of
different lymphohemopoietic cells with IL-4
(16, 18) . To
examine the effects of IL-13 on regulation of activity of the erk-1 and erk-2 members of the MAP kinase family, TF-1 were
stimulated either with IL-3, PMA, IL-4, IL-13, or insulin or left
untreated as controls. MAP kinases were immunoprecipitated from cell
extracts and in vitro kinase assays performed on the
immunoprecipitates. Results of a typical experiment are shown in
Fig. 7
, and summarizes the results of multiple
analyses. In no case did we detect significant increases in erk-1 or erk-2 activity following treatment of TF-1 cells with
IL-13, IL-4, or insulin. erk-1 and erk-2 MAP kinases
were activated by IL-3 or PMA in TF-1 cells. Thus, IL-13 like
IL-4
(16, 18) , failed to activate erk-1 or
erk-2. In B9 cells, IL-13 and IL-4 also failed to induce
activation of either erk-1 or erk-2 (data not shown).
Interestingly, although insulin failed to activate erk-1 in
TF-1 cells, in Swiss 3T3 cells insulin induced activation of both
erk-1 and erk-2 (see ). In addition,
insulin did induce activation of erk-1 in another hemopoietic
cell line, FD-5 (see ), although insulin is not mitogenic
for these cells.(
)
Figure 7:
IL-13
does not induce erk-1 activity in TF-1 cells. 4.6
10
factor-deprived TF-1 cells were either left untreated as
controls (C) or treated for 10 min with either IL-3
(3), hIL-4 (4), IL-13 (13), or PMA
(P) or for 2 min with insulin (INS).
Immunoprecipitations were carried out with anti erk-1 beads,
and in vitro immune complex kinase assays were carried out in
the presence of MBP. A, the incorporation of
P
into MBP (indicated). B, immunoblotting with an anti-erk-1 antibody was performed to confirm immunoprecipitation of
erk-1. The positions of the heavy chain of IgG (from the
immunoprecipitating antibody) and p44
are indicated.
C, MBP bands were excised and counted in a scintillation
counter. The counts obtained were used to calculate the fold activation
in erk-1 activity following factor treatment. The levels in
the controls were taken as 1, and the results are depicted
graphically.
Effects of IL-13, IL-4, and Insulin on Jak
Family Kinases
It has recently been reported that IL-4 induces
activation of Jak-1 and Jak-3 kinases in human and murine T
cells
(31, 32) and that Jak-3 associates with the c
chain shared by the IL-2, 4, 7, and 9
receptors
(34, 35) . It has been suggested that the
c chain may also be part of the IL-13
receptor
(8, 9) . Therefore, we examined the repertoire
of Jak kinases activated by these individual growth factors in TF-1.
The results are shown in Fig. 8A and summarized in
. In all cases immunoblotting was carried out with 4G10
first (upper panels), and blots were then stripped and
reprobed with the appropriate anti-Jak antibody to control for
efficiency of immunoprecipitation (lower panels). Both IL-13
and IL-4 stimulated increased tyrosine phosphorylation of Tyk-2 and
Jak-1 kinases, although the levels of phosphorylated Jak-1 were
extremely low in TF-1 cells. Tyk-2 appeared to be constitutively
tyrosine phosphorylated, even in factor-deprived TF-1 cells, although
both IL-13 and IL-4 consistently induced increased tyrosine
phosphorylation of Tyk-2. As predicted from studies of IL-4 on T cells,
we observed increased tyrosine phosphorylation of Jak-3 in response to
IL-4 treatment in TF-1 cells. This tyrosine-phosphorylated Jak-3
appears to correspond to the prominent p115 protein substrate we had
observed in total cell extracts (Fig. 1A and
8B). In immunodepletion studies (Fig. 8B), the
intensity of the p115 band (denoted by the lower small arrowhead in IL-4 Post IP samples, Fig. 8B) was decreased in
extracts following Jak-3 immunoprecipitation, whereas p170 was not
depleted (denoted by the upper small arrowhead). In extracts
which had been immunoprecipitated with anti-p85 PI3`-kinase antibodies,
p170 was depleted but p115 was not (see Fig. 8B).
Reprobing the immunoblots with either anti-Jak-3
(Fig. 8B, centerpanel), or anti-p85
PI3`-kinase (Fig. 8B, lower panel) antibodies
demonstrated the expected partial immunodepletion of both Jak-3 and p85
in the Post IP samples. Therefore, in TF-1 cells Jak-3 appears to be
one of the major proteins phosphorylated on tyrosine in response to
IL-4. The p115 tyrosine-phosphorylated protein observed in CT.4S cells
following treatment with IL-4 or IL-2 (Fig. 1F) also
appears to correspond to Jak-3.
Interestingly, in repeated
experiments IL-13 failed to cause an increase in Jak-3 tyrosine
phosphorylation.
IL-13 Induces Tyrosine Phosphorylation of IL-4R 140-kDa
Subunit
The failure of IL-13 to induce tyrosine phosphorylation
of Jak-3 argued against the c chain having a role in the
functional IL-13 receptor complex. Jak-1 is reported to associate with
the IL-4 140-kDa receptor subunit
(33) and the activation of
Jak-1 by IL-13, albeit at low levels, prompted us to examine the
possible functional role of the 140-kDa IL-4 R chain in the IL-13
receptor complex. Therefore, we examined the ability of IL-13 to induce
tyrosine phosphorylation of the IL-4 140-kDa subunit in the murine cell
line B9. Cells were either left untreated as a control or stimulated
for 10 min with either IL-4 or IL-13 and immunoprecipitates prepared
using monoclonal antibodies specific for the 140-kDa subunit of the
IL-4 receptor. Immunoblotting was performed with 4G10. As is apparent
in Fig. 9A, both IL-4 and IL-13 induced tyrosine
phosphorylation of the 140-kDa IL-4 R in B9 cells. In LPS-activated
splenic B cells, only IL-4 was able to induce tyrosine phosphorylation
of the 140-kDa IL-4 receptor, consistent with the lack of effect of
IL-13 on these cells (Fig. 9B).
Figure 9:
IL-13 induces tyrosine phosphorylation of
the IL-4 receptor 140-kDa subunit. B9 cells (A) and
LPS-activated B cell blasts (B) were treated for 10 min with
either mIL-4 (4), or mIL-13 (13), or for 2 min with
insulin (I) or left untreated as controls (C).
Immunoprecipitations were performed with 5 µg of anti IL-4 receptor
antibody M2. Immunoblotting was performed with 4G10. The positions of
the molecular mass standards are indicated, as is the position of the
140-kDa IL-4 receptor.
DISCUSSION
In this report we have demonstrated that IL-13 shares major
signal transduction pathways with the structurally and functionally
related cytokine, IL-4. We have defined the major substrate of
IL-13-activated tyrosine kinases as the molecule termed p170, which is
likely to be equivalent to the IL-4 substrate termed 4PS
(20) .
IL-13, IL-4, and insulin induced tyrosine phosphorylation of a 170-kDa
protein in both human myeloid cells (TF-1) and murine lymphoid cells
(B9). IL-4 also induced the tyrosine phosphorylation of p170 in primary
cultures of murine splenic T cells and B cells and FD-5, whereas IL-13
was not active on these populations. The tyrosine-phosphorylated p170
molecule bound with high affinity to the p85 subunit of PI3`-kinase, an
interaction mediated by the SH2 domains of p85. Although p170 was also
bound by the adaptor protein grb2, and under certain conditions could
be found associated with the cell membrane, IL-13, like
IL-4
(16, 17, 18) failed to activate components
of the ras/MAP kinase pathway, including Shc, Sos1, and
erk-1 and erk-2 MAP kinases. IL-13 also resembled
IL-4 in that it activated the Janus family kinases Jak-1 and Tyk-2. The
one difference was that IL-13, in contrast with IL-4, failed to
activate the Jak-3 kinase. This is consistent with the notion that the
IL-13 receptor does not use the IL-2 receptor chain and
dissociates activation of Jak-3 alone from tyrosine phosphorylation of
p170. Importantly, we observed that IL-13-induced tyrosine
phosphorylation of the 140-kDa subunit of the IL-4 receptor in B9
cells, indicating that this receptor subunit may be responsible for the
shared effects mediated by IL-13 and IL-4.
10 kDa greater than IRS-1, a difference in size similar to that we
have observed when comparing p170 in FD-5 and TF-1 cells to IRS-1 in
Swiss 3T3 fibroblasts (see Fig. 5). This molecule has been termed
IRS-2 and may indeed be the same molecule as p170/4PS. Taken together
these results suggest that although p170 contains key structural
similarities, especially within the pleckstrin homology region, it is a
distinct molecule from IRS-1.
10
IR/cell; 59, 60) had been used, so the differences in our
findings could merely reflect differences in receptor numbers. However,
it is also formally possible that insulin may induce qualitatively
different signals in hemopoietic cells because its downstream effects
are mediated by p170/4PS and not IRS-1. With respect to this, it would
be interesting to determine if IL-4 treatment of the 32D cells
cotransfected with IRS-1 and the IL-4 receptor
(28, 29) transduce signals similar to those we have described here
and previously
(16, 17, 18, 21) , or
whether in such a cell IL-4 could activate the ras/MAP kinase
pathway.
c chain in their receptors
(8, 9) . It has recently
been shown that Jak-3 binds the IL-2
c
chain
(34, 35) , and all growth factors which use this
receptor subunit activate Jak-3
(34, 35) . The fact that
IL-13 failed to activate Jak-3 suggests that it does not use the
c
subunit in its receptor structure, and the similarities in signaling
observed between IL-13 and IL-4 must then be due to an alternate shared
receptor chain. Our evidence that in B9 cells both IL-4 and IL-13
induced tyrosine phosphorylation of the IL-4 receptor 140-kDa subunit
(Fig. 9) is more consistent with this being the IL-4/IL-13 shared
receptor subunit. We feel it is unlikely, but formally possible, that
IL-13 induces activation of a kinase, which cross-phosphorylates the
IL-4 receptor. Moreover, we propose that the shared features of IL-13
and IL-4 signaling are due to certain key signals being mediated by the
IL-4 140-kDa receptor subunit. Jak-1 is one candidate, since it is
found associated with the IL-4 receptor
(33) . Also, tyrosine 472
of the IL-4 receptor has been shown to be important for IL-4-induced
tyrosine phosphorylation of IRS-1 in 32D cell
transfectants
(29) . Differences in IL-4 and IL-13 signals,
e.g. involvement of Jak-3, may reflect the participation of
c in the IL-4 receptor and the probable involvement of a molecule
specific to the IL-13 receptor. Insights to how the early events in
signaling which we have characterized here relate to the specific
functions of IL-4 and IL-13, such as immunoglobulin class switching,
generation of Th2 helper cells, and anti-inflammatory effects, await
the molecular characterization of p170/4PS/IRS-2 and comparison of its
cell specific expression with that of IRS-1 and elucidation of the
structure of the IL-13 receptor.
Table:
Effects of IL-13, IL-4, and insulin on erk1
MAP kinase activation
Table:
Effects of IL-13, IL-4, and insulin on Jak
kinase tyrosine phosphorylation
, no
detectable tyrosine phosphorylation; +, detectable levels of
tyrosine phosphorylation; ++, good levels of tyrosine
phosphorylation; +++, high levels of tyrosine
phosphorylation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.