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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 46-53
c-Cbl Is Tyrosine-Phosphorylated by Interleukin-4 and Enhances
Mitogenic and Survival Signals of Interleukin-4 Receptor by
Linking With the Phosphatidylinositol 3 -Kinase Pathway
By
Hiroo Ueno,
Ko Sasaki,
Hiroaki Honda,
Tetsuya Nakamoto,
Tetsuya Yamagata,
Kiyoshi Miyagawa,
Kinuko Mitani,
Yoshio Yazaki, and
Hisamaru Hirai
From the Third Department of Internal Medicine, Faculty of Medicine,
University of Tokyo, Tokyo, Japan.
 |
ABSTRACT |
Interleukin-4 (IL-4) is a cytokine that induces both proliferation
and differentiation and suppresses apoptosis of B cells. Although IL-4
has been shown to activate the phosphatidylinositol 3
(PI3)-kinase pathway, the role of PI3 kinase in the IL-4 receptor
(IL-4R) signaling remains unclear. In this study, we demonstrated that
c-Cbl proto-oncogene product is inducibly phosphorylated on tyrosine
residues and is associated with the p85 subunit of PI3-kinase by IL-4
stimulation. Overexpression of c-Cbl enhances the PI3-kinase activity
and, at the same time, mitogenic activity and survival of cells in the
presence of IL-4. However, these effects of c-Cbl were abolished by
wortmannin, a specific inhibitor for the PI3 kinase pathway, or by a
point mutation at tyrosine 731 of c-Cbl, which is a major binding site
for p85. These results indicate that c-Cbl plays a role in linking
IL-4R with the PI3 kinase pathway and thus enhancing the mitogenic and
survival signals.
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INTRODUCTION |
INTERLEUKIN-4 (IL-4) is a cytokine that
possesses various biological activities. First of all, IL-4 induces
proliferation and differentiation of B cells.1 It induces
expression of class II major histocompatibility complex molecules on
resting B cells and enhances both secretion and cell surface expression
of IgE and IgG1. Furthermore, IL-4 upregulates the expression of CD23,
the low-affinity Fc receptor for IgE.2 IL-4 is also shown
to act not only on B cells, but also on T cells, mast cells,
macrophages, and even nonhematopoietic cells.3 The
activation of intracellular signaling by IL-4 depends on ligand binding
to the IL-4 receptor (IL-4R) complex, composed of IL-4R subunit and
 c subunit. IL-4R subunit is a 140-kD protein, which
is sufficient to permit high affinity binding of the IL-4
ligand.4,5 On the other hand, c subunit is a common
component of receptors for IL-2, IL-7, IL-9, and IL-15.6-9
Mutations in the c subunit have been shown to result in X-linked
severe combined immunodeficiency (X-SCID).10 The binding of
IL-4 to IL-4R induces activation of JAK1 and JAK3 tyrosine kinases,
leading to phosphorylation of STAT6.11-14 Another signaling
pathway activated by IL-4 is the phosphatidylinositol 3
(PI3)-kinase pathway. IL-4 induces tyrosine phosphorylation of IRS-1
and/or IRS-2/4PS and their association with the p85 subunit of
the PI3 kinase, resulting in activation of the PI3 kinase
pathway.15-17 However, the role of the PI3-kinase pathway
in the IL-4R signaling pathway remains unclear.
We report in this study that c-Cbl proto-oncogene product is a
substrate for the IL-4-induced intracellular signaling. The c-Cbl
proto-oncogene was originally identified as a cellular homologue of
v-Cbl oncogene, which was cloned from the Cas NS-1 murine leukemia
virus.18 The c-Cbl gene product is a 120-kD protein, which
contains an NH2-terminal domain with a nuclear localization signal,
followed by a RING finger motif.19 The
COOH-terminal half of the protein contains a proline-rich domain, which
has been shown to function as a ligand for the SH3 domains of several
signaling molecules.20-26 Although many growth factors
induce tyrosine phosphorylation of
c-Cbl,24,25,27-30 the role of this molecule is
not well-understood. As far as we know, phosphorylation of c-Cbl is
closely related to activation of the Ras signaling pathway. Recent
studies have demonstrated that Sli-1, Caenorhabditis elegans
(C elegans) homologue of c-Cbl, is a negative regulator
of the let-60-mediated signaling pathway, a C elegans
counterpart of the Ras pathway.31,32 However, it has been
demonstrated that IL-4 cannot activate the Ras pathway, at least in
hematopoietic tissues.33 Therefore, our data showing that
IL-4 phosphorylates c-Cbl is somewhat surprising. Thus, we were very
interested in the role of phosphorylation of c-Cbl in IL-4R signaling.
In the present study, we analyzed the role of c-Cbl in the IL-4R
signaling pathway by using various mutants of c-Cbl and obtained data
that it enhances the PI3-kinase activity, which is responsible for
transmitting mitogenic and antiapoptotic signals in the IL-4R signaling
pathway.
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MATERIALS AND METHODS |
Cells and antibodies.
Ba/F3 cells were cultured in RPMI 1640 containing 10% fetal calf serum
(FCS) and 0.25 ng/mL of murine interleukin-3 (mIL-3).
Anti-c-Cbl antibody, antiphosphotyrosine antibody 4G10, anti-
subunit of IL-4R and antiinfluenza hemagglutinin (HA) epitope tag
antibody 12CA5 were purchased from Santa Cruz Biotechnology Inc (Santa
Cruz, CA), Upstate Biotechnology Inc (Lake Placid, NY), Genzyme Inc
(Cambridge, MA), and Bohehringer Mannheim Inc (Mannheim,
Germany), respectively. Anti-p85 antibody was a gift from
Y. Fukui (University of Tokyo, Tokyo, Japan).
Construction of c-Cbl mutant cDNAs and transfection.
The human c-cbl cDNA epitope-tagged with a nine amino acid HA
peptide (YPYDVPDYA) from the human influenza virus was a kind gift from
W.Y. Langdon (University of Western Australia, Nedlands,
Australia). The construction of deletion mutants of c-Cbl,
 RING-Cbl, and PD-Cbl were described
previously.34 The tyrosine 731-phenylalanine mutation of
c-Cbl was generated by site-directed mutagenesis with the Chameleon
Site-Directed mutagenesis kit (Stratagene, La Jolla, CA)
according to the manufacturer's instruction. Retrovirus vector was
used to transfect cDNAs of c-Cbl and its mutants into Ba/F3 cells as
described previously.35
Immunoprecipitation and immunoblotting.
Before stimulation, cells were starved in RPMI 1640 containing 0.5%
FCS for 12 hours. Cells were then stimulated with 10 ng/mL of mIL-4
(PEPRO TECH EC LTD, London, UK) for 5 minutes at 37°C,
washed twice with ice-cold phosphate-buffered saline (PBS), and lysed
in Triton lysis buffer (0.5% (vol/vol) Triton X-100, 50 mmol/L
Tris-HCl pH 7.4, 2 mmol/L phenylmethylsulfonyl fluoride
(PMSF), 10 U/mL aprotinin, 1 mmol/L sodium orthovanadate,
1 mmol/L EDTA). Cell lysates were collected and subjected to
immunoprecipitation and immunoblotting as described
previously.36 The immunoblots were developed with the
ProtoBlot system (Promega, Madison, WI) or the ECL system
(Amersham, Arlington Heights, IL).
Measurement of PI3-kinase activity.
The kinase activity of PI3-kinase was measured as described
previously.37 Briefly, lysates from cells were
immunoprecipitated with indicated antibodies. The immunoprecipitates
were washed four times in PI3 kinase buffer (20 mmol/L, Tris-HCl pH
7.5, 100 mmol/L NaCl, 0.5 mmol/L EGTA). Then crude brain
phosphoinositides (Sigma, St Louis, MO, P-6023) were
added to the immunoprecipitates at a final concentration of 0.2 mg/mL.
Adenosine triphosphate (ATP), [- 32P]
ATP and MgCl2 were added and incubated for 20 minutes at
room temperature. The lipids were extracted into a chloroform/methanol
mixture (1:1, vol/vol). The lipids-containing organic phase was
resolved on thin layer chromatography (TLC) plates (Silica Gel 60,
Merck, Darmstadt, Germany), developed in
chloroform/methanol/ammonia solution (28%)/water (215:190:25:35
mixture) and visualized by autoradiography.
The [3H]thymidine incorporation assay.
Cells were deprived of mIL-3 for 24 hours. Then 1 ng/mL of mIL-4 was
added to the medium and incubated for 12 hours. The cells were then
labeled with 1 mCi of [3H]thymidine for 3 hours after
growth factor stimulation. The amount of nucleotide incorporated into
DNA was quantitated by scintillation counting. Each experiment was
repeated at least three times and a growth rate was expressed as a
ratio over the basal value at no mIL-4 stimulation.
Analysis of chromosomal DNA fragmentation.
To extract the fragmented DNA observed during apoptotic death, 5
× 106 cells were collected and lysed with 400 µL of
lysis buffer (10 mmol/L Tris-HCl, pH 7.5, 10 mmol/L EDTA, 0.2 % Triton
X-100). The lysate was kept on ice for 10 minutes and centrifuged at
15,000 rpm for 10 minutes. The supernatant was subjected to extraction
with phenol/chloroform/isoamylalcohol (25:24:1) followed
by ethanol precipitation. The precipitate was dissolved in 20 µL of
Tris-EDTA (TE) and incubated for 1 hour with RNase A (2
µg/mL ) at 37°C. DNA fragments were separated by 2% agarose gel
electrophoresis and visualized by ethidium bromide staining.
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RESULTS |
IL-4 induces tyrosine phosphorylation of c-Cbl proto-oncogene product
in Ba/F3 cells.
We examined whether c-Cbl is involved in the IL-4R signaling pathway.
First, we tested whether stimulation with IL-4 results in
phosphorylation of c-Cbl. We confirmed that Ba/F3 cells, a
mIL-3-dependent pro-B cell line, express endogenous IL-4R by
methionine labeling of cells followed by immunoprecipitation with
anti-IL-4R antibody. Because 32D cells are known to express endogenous
IL-4R, we used them as a positive control (Fig
1A). We then stimulated Ba/F3 cells with
mIL-4, lysed, and immunoprecipitated with anti-c-Cbl antibody. The
immunoprecipitates were subjected to the antiphosphotyrosine
immunoblotting. In this experiment, we found that c-Cbl was inducibly
phosphorylated on tyrosine residues by IL-4 stimulation (Fig 1B). This
result indicates that c-Cbl is one of the target molecules in the IL-4R
signaling pathway.
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| Fig 1.
IL-4 phosphorylates c-Cbl on tyrosine residues. (A) Ba/F3
cells express endogenous IL-4R. Methionine-labeled Ba/F3 and 32D cells
were lysed, immunoprecipitated with anti- subunit of IL-4R. The
immunoprecipitates were subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by
autoradiography. An arrow indicates the 140-kD subunit of IL-4R.
(B) Tyrosine phosphorylation of c-Cbl by IL-4. Ba/F3 cells were
serum-starved for 10 hours and stimulated with murine IL-4 (10 ng/mL)
for 5 minutes at 37°C, lysed, and immunoprecipitated with
anti-c-Cbl antibody. The immunoprecipitates were subjected to
immunoblotting with antiphosphotyrosine antibody, 4G10 (upper panel) or
anti-c-Cbl antibody (lower panel). An arrow indicates c-Cbl.
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Overexpression of c-Cbl enhances the survival effects and the
mitogenic signal of IL-4 in Ba/F3 cells, which are abolished by
wortmannin.
To examine the roles of c-Cbl in the signaling pathway through IL-4R,
we introduced cDNA of c-Cbl into Ba/F3 cells by the retroviral vector
and established a stable line, HCbl cells, which overexpress c-Cbl. We
confirmed by the anti-c-Cbl immunoblot and, as an internal control,
the anti-p85 immunoblot that HCbl cells express c-Cbl 10 times more
than parental Ba/F3 cells (Fig 2A). IL-4 is
known to generate antiapoptotic and growth signals in IL-4R expressing
cell lines38-40 and, as expected, the survival of Ba/F3
cells was prolonged in the presence of IL-4, even if IL-3 was depleted
(Fig 2B). However, when HCbl cells were cultured in the presence of
IL-4, transient proliferation and survival of cells was significantly
enhanced when compared with those of mock cells. Interestingly, both
HCbl and mock cells rapidly fell into apoptotic death in the presence
of wortmannin, a specific inhibitor of PI3 kinase (Fig 2B). The low
molecular weight DNAs were extracted from HCbl and parental Ba/F3 cells
cultured in the presence or absence of wortmannin and electrophoresed
in an agarose gel. Notably, the ladder pattern typical for apoptotic
death was observed for DNAs extracted from cells cultured in the
presence of wortmannin and IL-4 for 48 hours (Fig 2C). We performed the
thymidine incorporation assay to assess the mitogenic activities of
c-Cbl overexpressing cell lines treated with IL-4. Compared with mock
cells, the thymidine incorporation into HCbl cells in the presence of
IL-4 was significantly enhanced, but in the presence of wortmannin,
that into HCbl cells was reduced to the level without IL-4 (Fig 2D).
From these data, we can conclude that survival and mitogenic signals
were enhanced by overexpression of c-Cbl and that activation of the PI3
kinase pathway is required for generation of these signals.
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| Fig 2.
The effects of c-Cbl in IL-4R signaling. (A)
Establishment of HCbl cells that overexpress c-Cbl. Lysates from HCbl
and mock cells were subjected to the immunoblotting with anti-c-Cbl
antibody (upper panel) and anti-p85 antibody (lower panel). Arrows
indicate c-Cbl and p85. We think that bands located below p85 are
nonspecific bands. (B) Overexpression of c-Cbl enhances transient
growth and elongates survival of Ba/F3 cells in the presence of IL-4.
HCbl and mock cells were cultured in RPMI containing 5% FCS and IL-4
(1 ng/mL) in the presence (+WT, 50 nmol/L) or absence of wortmannin.
(C) Survival effects of IL-4 enhanced by c-Cbl is sensitive to
wortmannin. Low molecular weight DNA extracts from HCbl and mock cells
cultured with or without IL-4 (1 ng/mL) plus wortmannin (WT, 50 nmol/L)
for 48 hours were electrophoresed on an agarose gel (2%). (D) HCbl and
mock cells were deprived of IL-3 for 24 hours. IL-4 (1 ng/mL) and
wortmannin (WT, 50 nmol/L) were then added to the medium, incubated for
12 hours, and subjected to the [3H]thymidine
incorporation assay. The growth rate was expressed as a ratio over the
basal values at no IL-4 stimulation.
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The PD mutant of c-Cbl is not tyrosine-phosphorylated by IL-4,
whereas the RING mutant is tyrosine-phosphorylated.
To examine the mechanism of how c-Cbl enhances the survival and
mitogenic signals transmitted by IL-4R, we first tested which domain of
c-Cbl is phosphorylated on tyrosine residues by IL-4 treatment. We
constructed deletion mutants of c-Cbl, RING-Cbl, which lacks amino
acids (aa) 193-491 including the RING finger domain and PD-Cbl,
which lacks aa 492-877 including the proline-rich domain of c-Cbl (Fig
3A) and introduced them into Ba/F3 cells by
the retroviral vector and established stable transfectants, RING and
PD cells. The cDNAs for wild-type c-Cbl and both deletion constructs
were epitope-tagged with an HA peptide and therefore distinguishable
from endogenous c-Cbl protein (Fig 3B). We examined if these deletion
mutants of c-Cbl were phosphorylated by IL-4 treatment, finding that
the PD mutant of c-Cbl was not tyrosine-phosphorylated by IL-4,
whereas the RING mutant was tyrosine-phosphorylated (Fig 3B). This
result indicates that it is within aa 497-877 of c-Cbl, which is
phosphorylated by IL-4 stimulation. Next, HCbl, RING, PD, and
mock cells were cultured in the presence of IL-4 and subjected to the
thymidine incorporation assay. We found in this experiment that while
the levels of thymidine incorporated into HCbl and RING cells
treated with IL-4 were approximately the same, that of PD cells was
significantly decreased (Fig 4), suggesting
that the region within aa 497-877 of c-Cbl is required for generating
mitogenic signals.
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| Fig 3.
IL-4 phosphorylates the region within aa 492-877 of
c-Cbl. (A) Constructs of cDNAs for wild-type and deletion mutants of
c-Cbl epitope-tagged with an HA peptide.  RING-Cbl lacks aa 193-491
including the RING finger domain and PD-Cbl lacks aa 492-877
including the proline-rich domain of c-Cbl. HA, a nine amino acid HA
peptide (YPYDVPDYA) from the human influenza virus. RING, the RING
finger motif. LZ, the leucine zipper motif. (B) Expression and tyrosine
phosphorylation of deletion mutants of c-Cbl by IL-4 stimulation. The
cDNAs encoding deletion mutants, RING-Cbl and PD-Cbl, were
introduced into Ba/F3 cells by the retroviral vector, and stable
transfectants, RING and PD cells were established.
These cells were stimulated with IL-4 (10 ng/mL) for 5 minutes at
37°C, lysed, and immunoprecipitated with anti-c-Cbl antibody. The
immunoprecipitates were then subjected to the immunoblotting with
antiphosphotyrosine antibody (upper panel) or anti-HA monoclonal
antibody, 12CA5 (lower panel).
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| Fig 4.
The thymidine incorporation assay of deletion mutants of
c-Cbl in the presence of IL-4. HCbl, RING and PD cells were
subjected to the thymidine incorporation assay. The growth rates were
expressed as in Fig 2D. The data for HCbl cells are the same as
presented in Fig 2D.
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Tyrosine 731 of c-Cbl is required for transmission of mitogenic
signal in IL-4R signaling.
We hypothesized from the results presented above that the PI-3 kinase
pathway is required for transmission of survival mitogenic signals
enhanced by c-Cbl. The SH2 domain of p85 subunit of PI3 kinase is known
to bind to c-Cbl phosphorylated by T-cell receptor
activation,23 but the binding site on c-Cbl has not been
identified. There are two potential binding sequences for the SH2
domain of p85 on aa 371-374 and aa 731-734 of c-Cbl, which are known as
YXXM motifs.41,42 Because, as indicated above, the region
phosphorylated by IL-4R is located within aa 492-877 of c-Cbl, it is
most likely that the binding site for the SH2 domain of p85 is tyrosine
731. To confirm this hypothesis, we introduced a tyrosine
731-phenylalanine mutation into the c-Cbl cDNA and established a stable
Ba/F3 cell line, YF, which expresses the Y731F mutant of c-Cbl (Fig
5A). To confirm that tyrosine 731 of c-Cbl
is the major binding site for p85, HCbl and YF cells stimulated with
IL-4 were lysed and immunoprecipitated with anti-p85 antibody. The
immunoprecipitates were subjected to immunoblotting with anti-HA
antibody, which recognizes introduced c-Cbl and its mutants, but not
endogenous c-Cbl protein. In this experiment, while p85 could
coprecipitate wild-type c-Cbl, it could not coprecipitate the Y731F
mutant of c-Cbl (Fig 5B). These data support that the binding site for
p85 on c-Cbl is tyrosine 731. We also performed immunoprecipitation
with anti-HA followed by immunoblotting with anti-p85 antibody and
could observe that p85 was inducibly coimmunoprecipitated with the
wild-type c-Cbl, but not with the Y731F mutant of c-Cbl (data not
shown).
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| Fig 5.
The Y731F mutant of c-Cbl cannot associate with p85 by
IL-4 stimulation. (A) The establishment of YF cells, which stably
express Y731F mutant of c-Cbl. Lysates from mock, HCbl, and YF cells
were immunoprecipitated with anti-c-Cbl antibody and subjected to the
immunoblotting with anti-HA antibody, 12CA5. The construct of Y731F
mutant of c-Cbl is presented in Fig 3A. An arrow indicates c-Cbl. (B)
Y731F c-Cbl mutant cannot associate with p85. Lysates from HCbl and YF
stimulated with IL-4 (10 ng/mL) for 5 minutes at 37°C were
immunoprecipitated with anti-p85 antibody, followed by the
immunoblotting with antiphosphotyrosine antibody (upper panel) or
anti-p85 antibody (lower panel). An arrow indicates c-Cbl.
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The PI3 kinase activation by IL-4 treatment was enhanced in HCbl
cells, but not in YF-Ba/F3 cells.
First, we confirmed that IL-4 simulation enhances c-Cbl-associated PI3
kinase activity. Ba/F3 cells treated with or without IL-4 were lysed
and immunoprecipitated with anti-c-Cbl antibody. The
immunoprecipitates were subjected to the PI3 kinases assay. As
expected, enhancement of c-Cbl-associated PI3 kinase activity by IL-4
treatment was observed (Fig 6A). This
result indicates that c-Cbl can connect IL-4R with the PI3 kinase
pathway. If this is the case, overexpression of c-Cbl should enhance
the PI-3 kinase activity in Ba/F3 cells by IL-4 treatment and, to the
contrary, the Y731F mutant of c-Cbl, which cannot bind to p85, could
not. To examine this, HCbl, YF, and mock cells stimulated with IL-4
were lysed and immunoprecipitated with antiphosphotyrosine antibody.
The immunoprecipitates were then subjected to the PI3 kinase assay. As
expected, PI3 kinase activity in HCbl was significantly enhanced when
compared with that of mock cells, while that of YF cells was
approximately equal to the level of mock cells (Fig 6B). We next
performed the thymidine incorporation assay to assess the mitogenic
activities of HCbl and YF cells and found that the Y731F-Cbl mutant
could not enhance the DNA synthesis as did the wild-type c-Cbl (Fig
6C). If the c-Cbl-PI3 kinase pathway is responsible for enhancing
transient growth and survival effects of IL-4 observed in Fig 2, we can
expect that growth and survival of YF cells in the presence of IL-4
should not be enhanced as HCbl cells. As expected, growth of YF cells
in the presence of IL-4 was similar to that of parental Ba/F3 cells
(Fig 6D). All of these results indicate that tyrosine 731 of c-Cbl,
which is a binding site for p85, is critical for transmitting mitogenic
signal of IL-4R, suggesting that the PI3-kinase pathway plays a role in
enhancing DNA synthesis, transient growth, and survival of cells.
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| Fig 6.
Tyrosine 731 of c-Cbl is critical for enhancing PI3
kinase activity. (A) IL-4 activates c-Cbl-associated PI3 kinase in
Ba/F3 cells. Lysates from Ba/F3 cells stimulated with or without IL-4
were immunoprecipitated with anti-c-Cbl antibody. The
immunoprecipitates were subjected to the PI3 kinase assay. (B) The PI3
kinase assay of mock, HCbl, and YF cells stimulated with or without
IL-4. Lysates from mock, HCbl and YF cells stimulated with or without
IL-4 were immunoprecipitated with antiphosphotyrosine antibody. The
immunoprecipitates were then subjected to the PI-3 kinase assay. (C)
The thymidine incorporation assay of HCbl and YF cells in the presence
or absence of IL-4 (10 ng/mL). The growth rates were expressed as in
Fig 2D. The data for HCbl cells are the same as presented in Fig 2D.
(D) Growth of HCbl and YF cells in the presence of IL-4. HCbl and mock
cells were cultured in RPMI containing 5% FCS and IL-4 (1 ng/mL), but
without IL-3. The data for HCbl cells are the same as presented in
Fig 2B.
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The signaling pathway of IL-4R is summarized in Fig
7. In this study, we demonstrated that,
like IRS-1 and IRS-2, c-Cbl plays a role in linking IL-4R with the PI3
kinase pathway, which enhances mitogenic and antiapoptotic signals of
IL-4R.
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| Fig 7.
Schematic representation of the signaling pathway of
IL-4R. In this study, we showed that c-Cbl serves as a second pathway
linking IL-4R with the PI3 kinase pathway. , the subunit of
IL-4R; c, the subunit of IL-4R; c-Akt, c-Akt serine/threonine
kinase.49
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DISCUSSION |
In this study, we presented data showing that c-Cbl is a substrate for
IL-4R signaling. IL-4 phosphorylates c-Cbl on tyrosine residues and
then c-Cbl enhances mitogenic and survival signals evoked by IL-4R.
These effects of c-Cbl are considered to be generated by linking IL-4R
with the PI3 kinase pathway, because wortmannin, a specific inhibitor
for the PI3 kinase pathway, abolished the effects of c-Cbl. It is
well-known that IL-4 stimulation of IL-4R-expressing cells
phosphorylates IRS-1 and IRS-2 on tyrosine residues, resulting in their
association with the p85 subunit of PI3 kinase. Thus, one of the roles
of IRS protein in the IL-4R signaling pathway is to link IL-4R with the
PI3 kinase pathway. Our data demonstrated in the present study indicate
that c-Cbl functions as a second molecule that links IL-4R with the PI3
kinase pathway.
A recent study on c-Cbl demonstrated that the SH3 domain of p85
constitutively binds to the proline rich domain of c-Cbl, but this
interaction is weak. On tyrosine-phosphorylation of c-Cbl, the SH2
domain of p85 also binds to c-Cbl, achieving a more tight
association.43 There are two YXXM motifs, which are
potential binding motifs for the SH2 domain of p85 on human c-Cbl
protein, at aa 371-374 and aa 731-734. From the data that IL-4 did not
phosphorylate the PD-Cbl mutant, which lacks aa 492-877, we can
speculate that the binding site of c-Cbl for the SH2 domain of p85
should be tyrosine 731. Therefore, we constructed tyrosine-731
phenylalanine mutant of c-Cbl. As expected, IL-4-induced association
between p85 and c-Cbl was considerably reduced by introducing the Y731F
mutation. Moreover, the Y731F mutant of c-Cbl could not enhance PI3
kinase activity.
The next question is what is the role of the PI3 kinase pathway in the
IL-4R signaling pathway. Interestingly, Yao et al44
reported that the PI3 kinase pathway is responsible for generating
survival signals activated by nerve growth factor (NGF)
and platelet-derived growth factor (PDGF) in PC12 cells.
Moreover, recent studies indicate that c-Akt serine/threonine kinase,
which is a direct target of PI-3 kinase, regulates neuronal
survival.45-47 Because IL-4 is known to suppress apoptosis
of B cells,38-40 it is likely that the PI-3 kinase-c-Akt
pathway plays a critical role in enhancing the survival signal through
IL-4R. Combined with our data that c-Cbl-overexpressing cells showed
enhanced PI-3 kinase activity in the presence of IL-4, it is likely
that c-Cbl contributes to the survival effects of IL-4 by connecting
with the PI3 kinase-c-Akt pathway (Fig 7). Moreover, transient
elevation of DNA synthesis was observed in c-Cbl-overexpressing cells
when treated with IL-4, which was abolished in the presence of
wortmannin or by generating the Y731F mutation into c-Cbl. These data
indicate that the PI3 kinase pathway contributes to the mitogenic
signal of IL-4.
We do not know yet the tyrosine kinase, which phosphorylates c-Cbl or
which region of IL-4R is required for phosphorylation of c-Cbl. Because
IL-4 is known to activate JAK1 and JAK3 tyrosine
kinases,11-13 it is possible that c-Cbl is phosphorylated
by JAK kinases, but we have not obtained any supporting data. By using
various IL-4R mutants, Deutsch et al48 reported that
distinct regions of IL-4R differentially regulate apoptosis inhibition
and cell growth. They demonstrated that the proline- rich motif (PRM)
(P242-K264) and the acidic region (S330-S365) are required for both
growth and apoptosis inhibition effects.48 These regions
are distinct from the NPXY motif, which is required for IRS protein
phosphorylation. Because our data in the present study indicate that
c-Cbl enhances both growth and survival signals of IL-4, the
PRM or the acidic region on IL-4R might be involved in
c-Cbl phosphorylation. Currently, the mechanism of how IL-4R
phosphorylates c-Cbl and what differential roles c-Cbl and IRS protein
play are under investigation.
| |
FOOTNOTES |
Submitted February 13, 1997;
accepted August 22, 1997.
Supported in part by grants-in-aid from the Ministry of Education,
Science, and Culture of Japan and from the Ministry of Health and
Welfare of Japan, Tokyo, Japan.
Address reprint requests to Hisamaru Hirai, MD, Third Department of
Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The human c-Cbl cDNA was a kind gift of W.Y. Langdon. We thank Y. Fukui
for the anti-p85 antibody. We also thank O.N. Witte for the expression
vector.
| |
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Abstract
Introduction
Abstract