| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
: Y497 and Y713, But Not the STAT6-Docking Tyrosines, Signal Protection from Apoptosis1
Department of Immunology, Jerome Holland Laboratories, American Red Cross, Rockville, MD 20855
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
play in IL-4-mediated protection from
apoptosis. 32D cells expressing a wt huIL-4R or one truncated at aa
557 were protected by huIL-4 from apoptosis while cells expressing a
receptor truncated at aa 657 were not, suggesting that the
carboxyl-terminal domain signals protection from apoptosis. However,
changing Y713 within this region to phenylalanine had no effect. To
analyze the contribution of tyrosine-containing domains independently,
we transplanted regions of the huIL-4R to a truncated form of the
huIL-2Rß that could not signal protection from apoptosis.
Transplantation of the huIL-4R domains containing Y497 or Y713
partially prevented cell death and together signaled protection from
apoptosis in response to IL-2 as well as the wt IL-2Rß. Mutation of
Y497 and Y713 to phenylalanine inhibited protection. In contrast,
transplantation of the domain containing the potential STAT6-docking
tyrosines alone had no effect, yet it inhibited the protection mediated
by the other domains. Although IL-4R signals Shc and SH2-containing
inositol phosphatase (SHIP) phosphorylation, we could not establish an
association between their activation and protection from apoptosis.
Taken together, this study suggests that the domains of the huIL-4R
containing Y497 and Y713 positively regulate protection from apoptosis
while the domain containing the STAT6 docking sites suppresses this
protection, and that additional signaling molecules other than insulin
receptor substrate-1 (IRS1), Shc, or SHIP may be involved in
antiapoptotic signaling. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IL-4-mediates its effects by binding to a cell surface receptor
complex. This receptor complex consists of the IL-4-binding protein
(IL-4R) and the 
-chain of the IL-2 receptor complex (16) or
alternatively the low affinity IL-13 receptor (17). The IL-4R complex
does not contain any consensus sequences encoding enzymatic activity;
however, the binding of IL-4 to its receptor complex results in the
activation of the Janus family tyrosine kinases
Jak3-1 and Jak-3
(18, 19, 20). In addition, IL-4 induces the tyrosine phosphorylation of its
own receptor (21, 22), the insulin receptor substrates 1 and 2 (22, 23), the adapter protein shc (24), and STAT6 (25, 26).
The IL-4R cytoplasmic domain contains five tyrosine residues and a
surrounding amino acid sequence that are 100% conserved among the rat,
mouse, and human IL-4R (27, 28, 29, 30). These are Y497, Y575, Y603, Y631,
and Y713 in the huIL-4R. We and others have previously defined two
different functional tyrosine-containing domains in the huIL-4R, the
I4R, or growth-promoting, and the STAT6, or gene-induction, domains
(31, 32, 33, 34). The domain of the huIL-4R between aa 437 and 557 (I4R
domain) is important for IL-4-induced IRS phosphorylation, cell
proliferation, and protection from apoptosis (31, 35). This domain
contains Y497 surrounded by a sequence motif (I4R-motif) that is
homologous to sequences found in the insulin and insulin-like growth
factor (IGF)-1 receptors. Mutation of Y497 within the I4R-motif of the
IL-4R to phenylalanine (F) yields receptors that fail to signal IRS
phosphorylation, proliferation, and protection from apoptosis in
response to huIL-4 (31, 35). The STAT6 domain, between aa 558 and 657,
includes Y575, Y603, and Y631. These tyrosines are STAT6 docking sites
and have been implicated in gene regulation (32, 33, 34). In addition, the
most carboxyl-terminal domain of the huIL-4R contains the conserved
Y713 surrounded by a proline-rich sequence. However, the role that this
domain plays in IL-4R complex signaling has not yet been defined.
We have recently demonstrated that the I4R domain plays a major role in
IL-4-mediated protection from apoptosis in IL-3-dependent myeloid cells
(35). However, those studies do not preclude a role for other
tyrosine-containing domains of the huIL-4R in regulating this
process. Herein, we investigated the role that these domains play in
the signaling of protection from apoptosis in response to IL-4. We
analyzed the ability of several huIL-4R deletion mutants to prevent
cell death. To investigate the specific role of an individual domain,
we have also transplanted specific tyrosine-containing domains from the
huIL-4R to the cytoplasmic domain of a truncated form of the
huIL-2Rß that was not able to signal protection from apoptosis. We
found that two huIL-4R-specific domains containing either Y497 or
Y713 positively contribute to IL-4-mediated protection from apoptosis.
In contrast, the domain containing the three STAT6-recruiting tyrosine
residues would negatively regulate the protective signal mediated by
the I4R or the carboxyl-terminal domains. Although we have observed Shc
and SHIP phosphorylation in response to IL-4, we could not establish a
relationship between the activation of these proteins and the ability
of IL-4 to protect cells from apoptosis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The IL-3-dependent myeloid cell line 32D expressing IRS1 as a
result of transfection (32D/IRS1) was maintained in RPMI 1640 culture
medium supplemented with glutamine, penicillin, streptomycin, 5% FCS,
and 5% WEHI-3-conditioned medium. The 32D/IRS1 cells expressing the
huIL-4R wt, and d557, d437, and Y497F mutants have been previously
described (31). The 32D/IRS1 cells lacking or expressing the wt and the
truncated huIL-2Rß or the chimeric receptors chim1, chim2, and chim3
have been previously described (32). Recombinant muIL-4 expressed in
baculovirus was affinity purified as described (36). Recombinant huIL-4
was purchased from R&D Systems (Minneapolis, MN). Recombinant huIL-2
was a gift from Dr. Steven Rosenberg (National Institutes of Health,
Bethesda, MD).
Apoptosis assays
The percentage of apoptotic cells was determined by analyzing the nuclear DNA content by flow cytometry as indicated (10). After culture, 32D/IRS1 cells were resuspended in 0.25 ml of propidium iodide solution (50 µg/ml propidium iodide, 0.1% sodium citrate, 0.1% Nonidet P-40, and 50 µg/ml RNase (Sigma, St. Louis, MO) and incubated for 30 min at room temperature. DNA content was then analyzed by flow cytometer (FACScan, Becton Dickinson, Mountain View, CA). The apoptotic cells were defined as those with less than a 2 N DNA content.
Proliferation assays
The cells were incubated in 96-well plates at 20,000 cells in 0.2 ml of complete RPMI 1640 in the presence or absence of different concentrations of muIL-4 or huIL-4 for 28 h. Cells were pulsed with 1 µCi/well [3H]thymidine for the last 4 h of culture before harvesting using a Packard harvestor and the Matrix 9600 direct ß count system (Packard Instruments, Downers Grove, IL).
Construction of chimeric receptors and mutations
The truncated huIL-2Rß containing aa 1 to 378 of the
huIL-2Rß and the chimeric receptors chim1, chim2, and chim3 have been
previously described (32). The chimeric receptors chim4, chim5, chim6,
and chim7 were made using the same strategy. In addition to the aa 1 to
378 of the huIL-2Rß, the chim4 receptor contains the aa 666 to 768 of
the huIL-4R-chain. The chim5, chim6, and chim7 receptors were made
combining the different domains of the huIL-4R contained in chim1,
chim2, and chim4, as shown in Figure 4
A.
|
was accomplished using the Altered
Sites In Vitro Mutagenesis System from Promega (Madison, WI) following
the manufacturer’s protocol. To this end, the oligo
5'-AGTGGCATTGTCTTCTCAGCCCTTACC was used, in which the
original TAC encoding Y713 was changed to TTC (underline) to obtain a
phenylalanine. The correct introduction of the Y713F mutation in the
huIL-4R was verified by the Sanger dideoxy chain-termination method
analyzed on an automated sequencing machine. Transfections
Cells were washed and resuspended in PBS. For each transfection, 2 x 107 cells were mixed with 2 µg of vector carrying neomycin resistance and 20 µg of chimeric receptor cDNA and subjected to electroporation using a Bio-Rad (Hercules, CA)gene-pulsar set on 200 V and 960 µF. After transfection, cells were cultured overnight in appropriate media before selection with G418 (Life technologies, Grand Island, NY). Neomycin-resistant lines were tested for expression of huIL-2Rß by FACS analysis using biotin-anti-huIL-2Rß (Endogen, Boston, Ma) followed by streptavidin-phycoerythrin (Southern Biotechnology, Birmingham, Al). All clones used in this study demonstrated equivalent levels of huIL-2Rß expression (Ref. 30 and data not shown).
Immunoprecipitation and immunoblotting
Analysis of phosphotyrosine-containing proteins was performed as previously described (31). Briefly, cells were starved in RPMI 1640 for 2 h at 37°C. After washing, 20 x 106 cells were resuspended in media (RPMI 1640 plus 5% FCS). Where indicated, they were stimulated with muIL-4 (10 ng/ml), huIL-4 (10 ng/ml), or huIL-2 (200 U/ml) for 10 min. The reaction was terminated by 10-fold dilution in ice-cold PBS containing 100 µM Na3VO4. Cell pellets were lysed in HEPES lysis buffer (50 mM HEPES, 50 mM NaCl, 0.5% Nonidet P-40, 1 mM Na3VO4, 50 mM NaF, 10 mM pyrophosphate, 1 mM PMSF, and protease inhibitor mixture) and clarified. To detect Shc and SHIP phosphorylation, the soluble fraction was immunoprecipitated with a polyclonal anti-Shc (UBI, Lake Placid, NY) or anti-SHIP (Santa Cruz Biotechnology, Santa Cruz, CA). The precipitates were washed in lysis buffer and solubilized in SDS sample buffer. The samples were separated on a 7.5% SDS-polyacrylamide gels before transfer to a polyvinylidene difluoride (PVDF) membrane. The membranes were then probed with a monoclonal antiphosphotyrosine Ab, RC20 (Transduction Laboratories, Lexington, KY). To detect IRS1 phosphorylation, the cell lysates corresponding to 3 x 106 cells were separated by SDS-PAGE, and the transferred proteins were immunoblotted with the antiphosphotyrosine Ab. The bound Abs were detected using enhanced chemiluminescence (Amersham, Arlington, IL).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have previously used the murine IL-3-dependent myeloid cell
line 32D, which expresses IRS1 as a result of transfection (32D/IRS1),
to analyze functionally important domains of the huIL-4R (31, 32, 35). We found that the Y497 in the cytoplasmic tail of the huIL-4R
was important for signaling tyrosine phosphorylation of IRS1,
proliferation, and protection from IL-3 withdrawal-induced apoptosis in
response to huIL-4. In addition, we demonstrated that the activation of
the IRS1 pathway was partially responsible for IL-4-mediated protection
from apoptosis (35). These results established a role for the I4R
domain and Y497 in this process. To investigate the role that other
domains of the huIL-4R play in the protection from apoptosis
mediated by IL-4, we cultured 32D/IRS1 cells expressing the wt
huIL-4R or the deletion mutants d437, d557, or d657 (Fig. 1A) in the presence of media or
huIL-4. As a control, muIL-4 was used
since 32D cells express endogenous muIL-4 receptors that do not bind
huIL-4. The percentage of apoptotic cells was analyzed by measuring
nuclear DNA content with propidium iodide. Cells showing less than 2 N
DNA content were scored as apoptotic. As we have previously shown (35),
32D/IRS1 cells expressing wt huIL-4R or the deletion mutant d557,
which contains Y497, were protected by huIL-4 from the rapid onset of
apoptosis induced by IL-3 withdrawal (Fig. 1B).
Interestingly, those cells expressing the d657 form of the huIL-4R,
which contains not only Y497 but also Y575, Y603, and Y631, were not
protected from death by huIL-4. Cells expressing d657-huIL-4R
cultured in the presence of huIL-4 had a higher percentage of apoptotic
cells (47%) than those cells stimulated with muIL-4 (22%) and similar
to levels seen in unstimulated cells (52%). In contrast, engagement of
the wt, d557, and d657 receptors by huIL-4 induced comparable
proliferative responses in 32D/IRS1 cells (Ref. 31; Fig. 1C). These effects were observed with various
concentrations of huIL-4 (Refs. 31, 35; and data not shown). The
huIL-4R truncated form d437 lacking all tyrosine residues signaled
neither protection from apoptosis nor cell proliferation (Fig. 1, B and C). These data suggest that the
carboxyl-terminal domain of the huIL-4R can positively regulate
IL-4-induced protection from apoptosis and that the STAT6 domain
diminished the protection from apoptosis mediated by the I4R domain in
the absence of the carboxyl-terminal domain.
|
The carboxyl-terminal domain contains Y713, a conserved tyrosine
residue. To investigate the specific role of this tyrosine, we changed
Y713 to F and then analyzed the ability of huIL-4 to protect
transfected 32D/IRS1 cells from apoptosis. We found that this mutation
had no effect in huIL-4 signaling (Fig. 2). Treatment of Y713F-expressing cells
with huIL-4 resulted in protection from apoptosis similar to levels of
protection seen in muIL-4-treated cells (19% and 21%, respectively,
Fig. 2A). Similarly, Y713F-expressing cells
proliferated in response to both huIL-4 and muIL-4 (Fig. 2B). Moreover, the Y713F receptor was also able to
signal IRS1 phosphorylation (Fig. 2C). By contrast,
the Y497F receptor did not signal cell proliferation, protection from
apoptosis, or IRS1 phosphorylation (Refs. 31, 35; Fig. 2). These
results suggested that the regulation of apoptosis by the
carboxyl-terminal domain is independent of the Y713 and IRS1.
|
As mentioned above, IL-4 was able to signal protection from
apoptosis through IRS-dependent and -independent pathways, with both
dependent on Y497. Therefore, it is possible that the regulation of
apoptosis mediated by the carboxyl-terminal domain could be subordinate
to a signaling mechanism activated through Y497. Since the mutant d657
signals IRS1 phosphorylation (31), but not protection from apoptosis
(Fig. 1
), other proteins could be involved in the protective effect
mediated by the carboxyl-terminal domain. In addition to IRS1, Shc is
able to bind the NPXY motif of the I4R domain (37, 38). Furthermore,
the carboxyl-terminal domain contains an ITIM motif, IVYSAL, which
could theoretically act as a docking site for the SH2 domains of
phosphatases such as SHP1 and SHIP (39, 40, 41, 42, 43). Interestingly, SHIP has
been shown to interact with Shc after cytokine stimulation (43).
Therefore, we analyzed the ability of huIL-4R mutants to signal Shc
and SHIP phosphorylation to determine whether their activation might
account for the protection from apoptosis mediated by the
carboxyl-terminal domain (Fig. 3). We
observed that the wt and the mutants d657 and Y713F of the huIL-4R
were able to signal Shc phosphorylation (Fig. 3A). By
contrast, the mutant receptor Y497F did not promote Shc
phosphorylation, as expected, since Shc requires this tyrosine to bind
to IL-4R (Fig. 3A). These data correlate with the
ability of huIL-4R to signal proliferation but not protection from
apoptosis (see Figs. 1 and 2). On the other hand, all types of
huIL-4R used, including Y497F and Y713F, signaled SHIP
phosphorylation independently of their ability to signal protection
from apoptosis (Fig. 3B). These data also indicate
that additional sites instead of Y713 can recruit SHIP. Therefore, this
analysis did not establish a correlation between the ability of the
carboxyl-terminal domain to signal protection from apoptosis and Shc
and SHIP activation.
|
The inability of the Y713F mutation to affect IL-4 signaling may
be due to the strong influence that Y497 has on the 32D cell response
to IL-4. Moreover, the carboxyl-terminal domain of the huIL-4R
contains, in addition to Y713, a proline-rich sequence that could dock
potential cellular messengers (27, 28, 29, 30). Therefore, we utilized an
approach that allowed us to investigate the role that this
carboxyl-terminal sequence plays in the protection from apoptosis away
from the influence of the I4R domain. We have previously shown that
IL-4-specific biologic functions can be transplanted to a truncated
form of IL-2Rß (32). Herein, we transplanted domains of the
huIL-4R to the cytoplasmic domain of the truncated form of the
huIL-2Rß, and we then analyzed the ability of these chimeric
receptors to signal protection from apoptosis (Fig. 4). We transplanted the I4R domain, aa
439 to 555 containing the I4R motif; the STAT6 domain, aa 558 to 657
containing the potential STAT6 tyrosine docking sites; the
carboxyl-terminal domain, aa 666 to 768 containing Y713 and the
surrounding proline-rich domain; or various combinations of them (Fig. 4A). These huIL-2Rß/huIL-4R chimeric receptors
were transfected into 32D/IRS1 cells, and positive clones were selected
by FACS analysis using an anti-huIL2Rß Ab (32). All clones
expressed equivalent levels of huIL-2Rß (Ref. 32; data not
shown).
We took advantage of the fact that, although IL-2 was a survival factor
for those cells bearing the wt huIL-2Rß, it did not protect cells
expressing the truncated huIL-2Rß from apoptosis (Fig. 4B). Cells expressing the wt huIL-2Rß grew equally
well in IL-2 or IL-3. By comparison, cells expressing the truncated
huIL-2Rß could not be propagated in the presence of IL-2. They
started to die after 24 h of culture, and, by 72 to 96 h of
culture, most of the cells were dead (Fig. 4B; 47 . Therefore, the ability of IL-2 to protect 32D/IRS1 cells
expressing the chimeric receptors from apoptosis was examined after
86 h of culture (Fig. 4C). We observed that both
chimeras 1 and 4, containing the domain surrounding Y497 or Y713,
respectively, were partially protected from apoptosis in response to
IL-2. On average, only 21% of cells expressing chim1 and 25% of cells
expressing chim4 were dead after 86 h of culture in the presence
of IL-2 (Fig. 4C-I). Interestingly, cells expressing
chim2, containing the STAT6 domain, were not protected from apoptosis
at this time by IL-2 even though we have previously observed that this
chimera is competent to signal STAT6 activation and CD23 induction (32, 47). The results obtained with the chimeric receptors are consistent
with the observations made with the huIL-4R deletion mutants (see
Fig. 1). Therefore, not only the I4R domain, containing Y497, but also
the carboxyl-terminal domain, containing Y713, promoted antiapoptotic
signals.
Y497F and Y713F mutations inhibit protection from apoptosis
To analyze the specific role of Y713, we tested the ability of
chim1 and chim4 with Y497 and Y713 changed to F (chim1F and chim4F,
respectively) to signal protection from apoptosis in response to IL-2
(Fig. 4C-II). Both Y497F and Y713F mutations
inhibited the ability of IL-2 to protect transfected cells from
apoptosis. However, the effect in cells expressing chim1F was more
dramatic. The percentage of apoptotic cells increased from 21% in
chim1 to 78% in chim1F. In the case of chim4F, the percentage of
apoptotic cells increased from 25% in wt chim4 to 56% in chim4F.
These data suggest that, in addition to Y713, other sequences in the
carboxyl-terminal domain may be involved in signaling protection from
cell death.
The STAT6 domain inhibits the protection from apoptosis mediated by the I4R and the carboxyl-terminal domains
Since the results obtained with the d657 mutant of the huIL-4R
suggested that the STAT6 domain could diminish the protection from
apoptosis mediated by the I4R domain, we sought to determine whether
the STAT6 domain could also modulate the protection from apoptosis
mediated by the I4R and carboxyl-terminal domains in the chimeric
receptor system. We used a chimeric receptor containing the I4R domain
plus the STAT6 domain of the huIL-4R (chim3) or the STAT6 domain plus
the carboxyl-terminal domain (chim5). Similar to the huIL-4R
deletion mutant d657, the addition of the STAT6 domain inhibited the
ability of the I4R domain (chim3) to protect cells from apoptosis (Fig. 4C-III) even though this chimera is perfectly
competent to induce the tyrosine phosphorylation of IRS1 (32). On
average, 72% of cells expressing chim3 stimulated with IL-2 were dead
while only 21% of cells expressing chim1 were dead. The STAT6 domain
could also inhibit partially the protection from apoptosis mediated by
the carboxyl-terminal domain (Fig. 4C-III). The
percentage of apoptotic cells increased from 25% in cells expressing
the chimeric receptor containing only the carboxyl-terminal domain of
the huIL-4R (chim4) to 56% in cells expressing the STAT6 plus the
carboxyl-terminal domains (chim5) stimulated by IL-2. These data
indicate that the domain of the huIL-4R containing the STAT6 docking
site can play a negative role in the protection from apoptosis mediated
by IL-4.
The I4R and carboxyl-terminal domains cooperate in protecting from apoptosis
Since both domains containing Y497 and Y713 were able only
partially to protect cells from apoptosis, we next analyzed whether
they cooperated in this process. To this end, we made a chimera
containing both the I4R domain and the carboxyl-terminal domains of the
huIL-4R in the absence of STAT6 domain (chim6). This
chimera-mediated protection from apoptosis in response to IL-2 to the
same degree as wt huIL-2Rß or another chimera that contains all
tyrosine residues (chim7) (Fig. 4C-IV). For both chim6
and chim7, the percentage of apoptotic cells was similar to the
percentage observed in cultures stimulated with IL-3 with only 
5%
of apoptotic cells. Therefore, the cooperation between the I4R and the
carboxyl-terminal domains gives maximal protection from apoptosis.
Analysis of IRS1 phosphorylation in the chimeric system
Finally, we examined the correlation between the ability of the
different chimeric receptors to signal IRS1 phosphorylation, which was
associated with protection from apoptosis (35), and their ability to
protect cells from death. In the chimeric system, we did not analyze
Shc and SHIP since the truncated huL-2Rß was able to signal their
phosphorylation as well as the wt huIL-2Rß (J.Z., unpublished data).
We have previously shown that transplantation of the I4R domain
(chim1), but not the STAT6 domain (chim2), to the truncated huIL-2Rß
could signal IRS1 phosphorylation in response to IL-2 (32). Herein, we
found that chim4 containing only Y713 was not able to signal IRS1
phosphorylation in response to IL-2 (Fig. 5). By contrast, all chimeric
constructs containing Y497, chim1, chim6, chim7 (Fig. 5), and chim3
(32), could induce the tyrosine phosphorylation of IRS1. Therefore,
while the ability of the I4R domain to protect from apoptosis is linked
to its ability to activate IRS1, the protection from apoptosis mediated
by the carboxyl-terminal domain is not related to the induction of IRS1
phosphorylation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in this process. In the present study, we provide
evidence that the STAT6 and the carboxyl-terminal domains of the
cytoplasmic tail of IL-4R are also involved in the regulation of
protection from apoptosis mediated by IL-4.
The results presented in this study confirm the importance of Y497 in
IL-4 signaling. As we have previously shown (35), either the huIL-4R
deletion mutant lacking Y497 or the mutation Y497F completely eliminate
the ability of huIL-4 to protect cells from apoptosis. Furthermore, we
also show that the transplantation of the I4R-motif to the truncated
huIL-2Rß confers protection from apoptosis in response to IL-2 and
that this protection is also dependent on Y497. The ability of this
domain to prevent cell death is correlated with its ability to signal
IRS1 activation. In addition, these data indicate that the protection
from apoptosis mediated by the I4R domain is negatively regulated by
the STAT6 domain and positively by the carboxyl-terminal domain of the
huIL-4R.
These results suggest that the STAT6 domain of IL-4R can play a
negative role in protecting from apoptosis. This effect was first
manifested in the huIL-4R deletion mutants. The huIL-4R mutant
d657, which contains both the I4R and the carboxyl-terminal domains,
did not protect cells from apoptosis, while d557, which contains only
the I4R domain, was able to do so. Although structural conformational
changes in the huIL-4R constructs could be responsible for these
differences, it is unlikely, since the d657 receptor was able to signal
the phosphorylation of IRS1 (31), Shc, and SHIP (Fig. 3), induction of
STAT6-DNA binding activity (34), and cell proliferation (31). In
addition, the STAT6 domain blocked protection from apoptosis mediated
by the I4R in the IL-2/IL-4 receptors chimeric system, and to a lesser
extent, the protection mediated by the carboxyl-terminal domain. One
possible explanation is that the STAT6 domain activates a pathway that
negatively regulates protection from apoptosis (Fig. 6A). This pathway would
be counteracted by signals delivered simultaneously through the I4R and
carboxyl-terminal domains. The role that STAT6 plays in regulating
apoptosis has not yet been defined; however, cell viability has not
been reported to be affected in STAT6 knockout mice (44, 45), and we
have observed that B lymphocytes and stable cell lines derived from
these mice were well protected from apoptosis by IL-4 (J.Z.,
unpublished observation). In addition, chim2, which is able to induce
STAT6 activation (47), was not able to signal protection from
apoptosis. These data suggest that STAT6 does not mediate protection
from apoptosis. Nevertheless, it is possible that additional proteins
might interact with this domain of IL-4R and regulate apoptosis.
|
lacking this domain (d657) did not signal protection from apoptosis.
Second, transplantation of this domain to the truncated IL-2Rß
conferred protection from apoptosis. Although the Y713F mutation had no
effect on protection from apoptosis in the context of the full length
huIL-4R, such a mutation partially inhibited protection from
apoptosis in the chimeric receptor system, indicating that Y713 can be
involved in this process. In the full length huIL-4R, this mutation
could still allow partial protein binding, perhaps through its
proline-rich motifs, which could be enough to induce a positive
response to IL-4. The importance of Y713 is suggested by data
indicating that Y631, Y713, or Y821 may be the major phosphorylation
sites on the huIL-4R (46). In addition, this tyrosine is surrounded
by proline-rich residues, which suggests that they may be a docking
site for intracellular proteins (27, 28, 29, 30). Therefore, it would be of
interest to determine whether this domain docks a protein that could be
involved in apoptotic processes. Interesting candidates were SHIP and
SHP-1, which could theoretically bind the ITIM motif, including Y713
(27, 28, 29, 30, 37, 38, 39, 40, 41); however, we have detected SHIP phosphorylation in the
absence of this domain, and we have not been able to detect SHP-1
regulation by IL-4 in these cells (J.Z. and A.D.K., unpublished
observations).
The fact that Y497F mutation, but not Y713F, completely blocked
protection from apoptosis and that the STAT6 domain inhibited the
protection mediated by either the I4R or carboxyl-terminal domains
suggests that the pathways activated through the domain containing Y713
may be subordinated to Y497. We have recently shown that the protection
from apoptosis mediated by IL-4 is regulated by IRS1-dependent and
-independent mechanisms (35). Since the mutant d657 signals IRS1
phosphorylation but not protection from apoptosis, additional
protein(s) may be involved in the protection from death mediated by
this domain. Y713 is included in the ITIM sequence IVYSAL, which could
potentially dock SHIP, an inositol 5-phosphatase (39) containing two
NPXY sequences (43) that could potentially be docking sites for the
phosphotyrosine-binding (PTB) domains of IRS1. In addition, SHIP has
been shown to interact with Shc after cytokine stimulation (43).
Moreover, Shc can bind through its phosphotyrosine-binding domain to
the I4R motif of IL-4R (37, 38), and thus, it could, at least
theoretically, mediate the IRS1-independent pathway that IL-4 uses to
protect cells from apoptosis. Therefore, a network could be established
between IRS1, Shc, and SHIP after IL-4 stimulation which, in turn,
could regulate apoptosis (Fig. 6A). However, we have
not found a direct correlation between Shc or SHIP phosphorylation and
protection from apoptosis. Nevertheless, it appears that some
cooperation between the I4R and the carboxyl-terminal domains is
necessary to overcome the inhibition promoted by the STAT6 domain.
The data presented herein indicates that all tyrosine-containing
domains of the cytoplasmic tail of IL-4R participate in the
regulation of apoptosis by IL-4. In addition, they show that the
signals promoted by each domain are influenced by the others, and the
nature of the response depends on the balance between them. In Figure 6, we show two hypothetical models that illustrate this process.
Signals delivered from Y497 activate IRS and an additional pathway,
which could be through a molecule such as Shc, that mediate protection
from apoptosis. Simultaneously, an unknown pathway is activated through
the carboxyl-terminal domain, which also promotes cell survival.
Finally, an inhibitory pathway is activated through the STAT6 domain
that could block the protective signal mediated by the other domains.
When the three domains are activated, the receptor transmits a
protective signal; however, the STAT6 domain could block the protective
effect when one of the positive signals are off (Fig. 6A). Alternatively, a novel protein involved in
antiapoptotic signals could require both the I4R and the
carboxyl-terminal domains to dock to the receptor and perhaps bridge
Y497 and Y713 via a dual docking site (Fig. 6B).
Since we have not found a relationship between the activation of known
mediators other than IRS1 (35) and protection from apoptosis, it will
be interesting to determine whether additional proteins are involved in
IL-4R signaling protection from apoptosis.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Achsah D. Keegan, Immunology Department, Jerome Holland Laboratories, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. E-mail address:
3 Abbreviations used in this paper: JAK, Janus family tyrosine kinase; hu, human; SHIP, SH2-containing inositol phosphatase; IRS1, insulin receptor substrate-1; F, phenylalanine; mu, murine; wt, wild type; ITIM, immunoreceptor tyrosine-based inhibitory motif.
Received for publication December 31, 1997. Accepted for publication March 30, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chain: a functional component of the interleukin-4 receptor. Science 262:1880. chain. Immunity 4:113.[Medline]
This article has been cited by other articles:
|
|
 |
|
  M. S. Rahman, A. Yamasaki, J. Yang, L. Shan, A. J. Halayko, and A. S. Gounni IL-17A Induces Eotaxin-1/CC Chemokine Ligand 11 Expression in Human Airway Smooth Muscle Cells: Role of MAPK (Erk1/2, JNK, and p38) Pathways J. Immunol., September 15, 2006; 177(6): 4064 - 4071. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Stephenson, D.-S. Park, A. L. Mora, S. Goenka, and M. Boothby Sequence Motifs in IL-4R{alpha} Mediating Cell-Cycle Progression of Primary Lymphocytes J. Immunol., October 15, 2005; 175(8): 5178 - 5185. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zamorano, M. D. Rivas, A. Garcia-Trinidad, C.-K. Qu, and A. D. Keegan Phosphatidylcholine-Specific Phospholipase C Activity Is Necessary for the Activation of STAT6 J. Immunol., October 15, 2003; 171(8): 4203 - 4209. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Ravandi, M. Talpaz, and Z. Estrov Modulation of Cellular Signaling Pathways: Prospects for Targeted Therapy in Hematological Malignancies Clin. Cancer Res., February 1, 2003; 9(2): 535 - 550. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. M. Hanson, H. Dickensheets, C.-K. Qu, R. P. Donnelly, and A. D. Keegan Regulation of the Dephosphorylation of Stat6. PARTICIPATION OF TYR-713 IN THE INTERLEUKIN-4 RECEPTOR alpha , THE TYROSINE PHOSPHATASE SHP-1, AND THE PROTEASOME J. Biol. Chem., January 31, 2003; 278(6): 3903 - 3911. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Wurster, V. L. Rodgers, M. F. White, T. L. Rothstein, and M. J. Grusby Interleukin-4-mediated Protection of Primary B Cells from Apoptosis through Stat6-dependent Up-regulation of Bcl-xL J. Biol. Chem., July 19, 2002; 277(30): 27169 - 27175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Shen and M. H. Kaplan The Homeostasis But Not the Differentiation of T Cells Is Regulated by p27Kip1 J. Immunol., July 15, 2002; 169(2): 714 - 721. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Wurster, D. J. Withers, T. Uchida, M. F. White, and M. J. Grusby Stat6 and IRS-2 Cooperate in Interleukin 4 (IL-4)-Induced Proliferation and Differentiation but Are Dispensable for IL-4-Dependent Rescue from Apoptosis Mol. Cell. Biol., January 1, 2002; 22(1): 117 - 126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Zamorano, A. L. Mora, M. Boothby, and A. D. Keegan NF-{kappa}B activation plays an important role in the IL-4-induced protection from apoptosis Int. Immunol., December 1, 2001; 13(12): 1479 - 1487. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kashiwada, C. C. Giallourakis, P.-Y. Pan, and P. B. Rothman Immunoreceptor Tyrosine-Based Inhibitory Motif of the IL-4 Receptor Associates with SH2-Containing Phosphatases and Regulates IL-4-Induced Proliferation J. Immunol., December 1, 2001; 167(11): 6382 - 6387. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhu, L. Guo, C. J. Watson, J. Hu-Li, and W. E. Paul Stat6 Is Necessary and Sufficient for IL-4's Role in Th2 Differentiation and Cell Expansion J. Immunol., June 15, 2001; 166(12): 7276 - 7281. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. D. White, R. P. Andrews, and G. K. K. Hershey Sulfhydryl-2 domain-containing protein tyrosine phosphatase-1 is not a negative regulator of interleukin-4 signaling in murine mast cells J. Leukoc. Biol., May 1, 2001; 69(5): 825 - 830. [Abstract] [Full Text]
|
|
|
|
|
|
B. Relic, J. Guicheux, F. Mezin, E. Lubberts, D. Togninalli, I. Garcia, W. B. van den Berg, and P.-A. Guerne IL-4 and IL-13, But Not IL-10, Protect Human Synoviocytes from Apoptosis J. Immunol., February 15, 2001; 166(4): 2775 - 2782. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. F. Wagner, N. Hanna, L. D. Fast, N. Kouttab, P. R. Shank, A. Vazquez, and S. Sharma Novel Diversity in IL-4-Mediated Responses in Resting Human Naive B Cells Versus Germinal Center/Memory B Cells J. Immunol., November 15, 2000; 165(10): 5573 - 5579. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. D. Finkelman, S. C. Morris, T. Orekhova, M. Mori, D. Donaldson, S. L. Reiner, N. L. Reilly, L. Schopf, and J. F. Urban Jr. Stat6 Regulation of In Vivo IL-4 Responses J. Immunol., March 1, 2000; 164(5): 2303 - 2310. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Mason, B. K. Beattie, Q. Liu, D. J. Dumont, and D. L. Barber The SH2 Inositol 5-Phosphatase Ship1 Is Recruited in an SH2-dependent Manner to the Erythropoietin Receptor J. Biol. Chem., February 11, 2000; 275(6): 4398 - 4406. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Huang and W. E. Paul Protein Tyrosine Phosphatase Activity Is Required for IL-4 Induction of IL-4 Receptor {alpha}-Chain J. Immunol., February 1, 2000; 164(3): 1211 - 1215. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Pesu, K. Takaluoma, S. Aittomaki, A. Lagerstedt, K. Saksela, P. E. Kovanen, and O. Silvennoinen Interleukin-4-induced transcriptional activation by Stat6 involves multiple serine/threonine kinase pathways and serine phosphorylation of Stat6 Blood, January 15, 2000; 95(2): 494 - 502. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. J. Hinton and M. J. Welham Cytokine-Induced Protein Kinase B Activation and Bad Phosphorylation Do Not Correlate with Cell Survival of Hemopoietic Cells J. Immunol., June 15, 1999; 162(12): 7002 - 7009. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Y. Wang, C. P. Shelburne, J. Zamorano, A. E. Kelly, J. J. Ryan, and A. D. Keegan Cutting Edge: Effects of an Allergy-Associated Mutation in the Human IL-4R{alpha} (Q576R) on Human IL-4-Induced Signal Transduction J. Immunol., April 15, 1999; 162(8): 4385 - 4389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Minshall, S. Arkins, R. Dantzer, G. G. Freund, and K. W. Kelley Phosphatidylinositol 3'-Kinase, But Not S6-Kinase, Is Required for Insulin-Like Growth Factor-I and IL-4 To Maintain Expression of Bcl-2 and Promote Survival of Myeloid Progenitors J. Immunol., April 15, 1999; 162(8): 4542 - 4549. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Giallourakis, M. Kashiwada, P.-Y. Pan, N. Danial, H. Jiang, J. Cambier, K. M. Coggeshall, and P. Rothman Positive Regulation of Interleukin-4-mediated Proliferation by the SH2-containing Inositol-5'-phosphatase J. Biol. Chem., September 15, 2000; 275(38): 29275 - 29282. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
