(Received for publication, June 6, 1995; and in revised form, September 5, 1995)
From the
The interleukin (IL)-2 receptor system has previously been shown
to signal through the association and tyrosine phosphorylation of Shc.
This study demonstrates that the IL-2 receptor (IL-2R
) chain
is the critical receptor component required to mediate this effect. The
use of IL-2R
chain deletion mutants transfected into a Ba/F3
murine cell model describes a requirement for the IL-2R
``acid-rich'' domain between amino acids 315 and 384 for Shc
tyrosine phosphorylation and receptor association. COS cell
co-transfection studies of IL-2R
chain constructs containing point
mutations of tyrosine to phenylalanine along with the tyrosine kinase
Jak-1 and a hemagglutinin-tagged Shc revealed that the motif
surrounding phosphorylated tyrosine 338 within the acid-rich domain of
the IL-2R
is a binding site for Shc. Deletion of this domain has
previously been shown to abrogate the ability of IL-2 to activate Ras
but does not affect IL-2-dependent mitogenesis in the presence of
serum. Proliferation assays of Ba/F3 cells containing IL-2R
chain
deletion mutants in serum-free medium with or without insulin shows
that deletion of the acid-rich domain does not affect IL-2-driven
mitogenesis regardless of the culture conditions. This study thus
defines the critical domain within the IL-2R
chain required to
mediate Shc binding and Shc tyrosine phosphorylation and further shows
that Shc binding and phosphorylation are not required for
IL-2-dependent mitogenesis. Neither serum nor insulin is required to
supplement the loss of induction of the Shc adapter or Ras pathways,
which therefore suggests a novel mechanism for mitogenic signal
transduction mediated by this hematopoietin receptor.
Interleukin-2 (IL-2) ()is a multifunctional cytokine
that has been shown to affect the physiology of cells of immune and
nonimmune tissue (reviewed in (1) ) by direct interaction with
a high or intermediate affinity IL-2 receptor (IL-2R). The high
affinity IL-2R (K
=
10
) is composed of three receptor subunits
(reviewed in (2) );
(55 kDa),
(75 kDa), and the
common
chain (
, 64 kDa), which is shared by
several hematopoietic cytokine receptors(3) . The intermediate
affinity receptor (K
=
10
) is a
-
complex
only(2) . Signal transduction by IL-2 minimally requires the
intermediate affinity
-
receptor complex and does
not require the
chain(2) . Thus the apparent function of
the
chain is to affect IL-2 affinity only and not the mechanism
of IL-2 signal transduction.
Signal transduction by IL-2 is
initiated by the activation of several tyrosine kinases associated with
specific receptor molecules of the IL-2R. Among these are members of
the Src family of tyrosine kinases, which have been shown to
functionally couple to the IL-2R chain (4, 5, 6) ; the Janus family tyrosine kinase
Jak-1, which associates with the IL-2R
chain(7, 8) ; and
Jak-3(9, 10, 11) , which associates with the
chain(7, 8) . The first detectable
event following treatment of T cells with IL-2 is the formation of an
activated
-
-
IL-2R complex containing
tyrosine-phosphorylated and activated Jak-1 and
Jak-3(7, 8, 10) . This is soon followed by
the activation of Src family tyrosine kinases (4, 5, 6) and the tyrosine phosphorylation of
multiple substrates.
Among the proteins that are tyrosine phosphorylated in response to IL-2 is the recently identified protein Shc(12) . Shc is an SH2 domain containing protein found as two dominant, widely expressed, and tyrosine-phosphorylated forms of 52 and 46 kDa as well as a 66-kDa protein with more restricted expression(13) . Shc functions to link receptor tyrosine kinase activation and tyrosine phosphorylation to the downstream activation of Ras and Ras-like pathways (reviewed in (14) ) and accomplishes this by recognizing and binding to a phosphotyrosine-containing motif within the receptor or a receptor-associated protein. Binding to the receptor complex brings Shc into proximity for phosphorylation by an activated tyrosine kinase and establishes a secondary ``docking'' site for the protein Grb2(15) . The subsequent binding of Grb2 to the receptor complex brings guanine nucleotide releasing factor activity, such as Sos (16) or Vav(17) , to the membrane where it can catalyze the release of GDP from inactive Ras-GDP, allowing the formation of an activated Ras-GTP complex. Thus it is clear that the activation of Ras via the Shc/Grb2 adapter pathway is dependent on the ability of Shc to recognize and bind to a tyrosine-phosphorylated receptor complex.
Tyrosine phosphorylation of p52Shc and activation of the Shc/Grb2 adapter pathway can be induced by a number of mitogenic cytokines and growth factors(13, 18, 19, 20, 21, 22) and involves the ligand-dependent association of Shc with the receptor complex(23, 24) . As in other systems, Shc has been found associated with the IL-2R complex (25) and the induction of this pathway by IL-2 has been reported to correlate with the ability of IL-2 to elicit a mitogenic response(26) . Because of this and the multi-protein nature of the IL-2R complex, we were interested in determining the precise mechanism by which IL-2 stimulates Shc/receptor binding and tyrosine phosphorylation.
Ba/F3 cells transfected
with various IL-2R chain deletion mutants (FL, AD, BD, SD, and BS)
were maintained at 37 °C in a humidified CO
incubator
in RPMI 1640 medium supplemented with 10% fetal calf serum, 5% WEHI-3B
culture supernatant(30) , glutamine, antibiotics, and 250
µg/ml hygromycin-B (Calbiochem). IL-2 treatments were performed on
these cells using 100 nM recombinant IL-2 at a cell density of
50
10
cells/ml in RPMI 1640 medium supplemented
with 5% fetal calf serum. Cells were incubated at 37 °C for 15 min,
recovered by centrifugation, and prepared for lysis and immune
precipitation.
Anti-human Jak-3 and anti-Shc immune precipitation of
human T cell lysates were performed on a 100 10
cell lysate equivalent using 2 µl of anti-Jak-3 antibody (9) or 1 µl of anti-Shc antibody (Upstate Biotechnology
Inc.). Anti-Shc immune precipitation of Ba/F3 cells was performed using
a 300
10
cell lysate equivalent and 4 µl of
anti-Shc antibody/sample. All immune precipitations were performed for
4 h to overnight at 4 °C in the presence of 20 µl of protein
A-conjugated Sepharose (Sigma). Immune precipitations were washed 6
times in lysis buffer, and proteins were eluted by boiling in 50 µl
of 2
SDS-polyacrylamide gel electrophoresis sample buffer.
Proteins were resolved by SDS-polyacrylamide gel electrophoresis,
transferred to Immobilon polyvinylidene difluoride membranes
(Millipore), and probed using anti-phosphotyrosine immunoblotting.
IL-2R immune precipitation was performed using anti-IL-2R
monoclonal antibody 561 (provided by R. Robb) directly conjugated to
protein A-Sepharose (PAS). Preparation of 561-PAS was initiated by
incubating 1 mg of 561 with 1 ml of packed PAS beads in 3 ml of lysis
buffer overnight at 4 °C. The beads were washed extensively with
lysis buffer, 2 times with 0.1 M borate, pH 9.0 (BB) and
resuspended in 10 mls of BB. Dimethyl pimelimidate (Sigma) was added to
20 mM, and beads were incubated at room temperature for 1 h.
561-PAS was washed once with BB, resuspended in 40 mM ethanolamine pH 8.0, and incubated for 1 h at room temperature.
Beads were washed several times with alternating cycles of BB and 0.1 M glycine, pH 3.0. 561-PAS was resuspended at a final
concentration of 1 mg/ml 561/packed beads in phosphate-buffered saline,
supplemented with 0.1% sodium azide, and stored at 4 °C.
For
IL-2R chain immune precipitations, treated Ba/F3 cells containing
mutant IL-2R
chains or transfected COS-7 cells were lysed,
clarified as described, and immune precipitated using 30 µl of
561-PAS (30 µg of antibody equivalent). Immune precipitations were
incubated for 4 h at 4 °C and washed six times with lysis buffer.
Proteins were eluted by boiling in 50 µl of nonreducing 2
SDS-polyacrylamide gel electrophoresis sample buffer (no
-mercaptoethanol). Samples were centrifuged, and the supernatant
was removed and adjusted to 5%
-mercaptoethanol. Proteins were
resolved by SDS-polyacrylamide gel electrophoresis, transferred to
Immobilon polyvinylidene difluoride membranes (Millipore), and probed
for Shc or Shc-HA by anti-Shc or anti-HA immunoblotting.
The IL-2 receptor complex
contains four dominant tyrosine-phosphorylated proteins: the IL-2R
chain, the common
chain, and the receptor-associated tyrosine
kinases Jak-1 and Jak-3. To determine which of these phosphotyrosine
containing proteins was most important in mediating Shc phosphorylation
we used the IL-4 receptor system, which also contains the common
chain and activates Jak-1 and Jak-3(7) , and asked whether IL-4
was capable of inducing Shc phosphorylation in a manner similar to that
observed with IL-2.
Activated human T cells were treated with IL-2
or IL-4 and assayed for receptor tyrosine kinase activation, as
represented by increased tyrosine phosphorylation of Jak-3, and
factor-dependent tyrosine phosphorylation of Shc. This analysis clearly
demonstrates that IL-4 does not induce Shc tyrosine phosphorylation in
spite of inducing receptor tyrosine kinase activity and phosphorylation
of Jak-3 in human T cells (Fig. 1). This observation is
supported by additional results that describe an inability of IL-4 to
induce Shc tyrosine phosphorylation in other cell types(37) .
This therefore implies a direct role for the IL-2R chain in
controlling IL-2-dependent Shc phosphorylation.
Figure 1:
Differential induction
of Shc tyrosine phosphorylation by IL-2 and IL-4 establishes a
requirement for the IL-2R chain. G
-enriched activated
human T cells were treated with or without IL-2 or IL-4 for the
indicated times. Lysates were prepared and immune precipitated using
antibody specific for Jak-3 (upper panel) or Shc (lower
panel). Samples were then analyzed for relative changes in
tyrosine phosphorylation by anti-phosphotyrosine
immunoblotting.
Figure 2:
Structure of the IL-2R chain deletion
mutants. Schematic representation of IL-2R
chain deletion mutants.
Each construct contains an extracellular domain (214 amino acids), a
transmembrane domain (25 amino acids), and a cytoplasmic domain
(26-286 amino acids). The acid-rich domain is composed of amino
acids 315-384. Box 1 and Box 2 domains
represent regions of homology shared among many hematopoietin
receptors. The name of each mutant receptor is depicted above its representation.
Figure 3:
IL-2-dependent tyrosine phosphorylation of
Shc requires the IL-2R acid-rich domain. Ba/F3 cells containing
FL, AD, or BD receptors were treated with (+) or without(-)
IL-2 for 15 min, lysed, and immune precipitated with anti-Shc antibody.
Relative tyrosine phosphorylation of Shc was determined by
anti-phosphotyrosine immunoblot of anti-Shc immune precipitates. The
position of p52
is indicated on the left.
FL-, AD-, and
BD-containing cells were treated with IL-2, the IL-2R chain was
immune precipitated, and an immunoblot was performed using an anti-Shc
antibody. Results from this experiment show an IL-2-dependent
association of Shc with FL and BD receptors but not in cells containing
the AD mutant (Fig. 4). This clearly shows that IL-2-dependent
tyrosine phosphorylation of Shc involves direct binding of Shc to a
motif within the acid-rich domain of the receptor and implies a
requirement for IL-2-dependent tyrosine phosphorylation of this region
as a prerequisite for Shc association.
Figure 4:
IL-2-dependent association of Shc with the
IL-2R chain requires the IL-2R
acid-rich domain. Ba/F3 cells
containing FL, AD, or BD receptors were treated with (+) or
without(-) IL-2 for 15 min, lysed, and immune precipitated with
anti-IL-2R
chain antibody 561 conjugated to protein-A Sepharose.
Immune precipitates were probed for the presence of Shc by anti-Shc
immunoblot. The position of p52
is indicated on the left.
Figure 5:
Detailed map of tyrosines contained within
the IL-2R cytoplasmic domain. Schematic representation of
tyrosines within the cytoplasmic domain of the IL-2R
chain. Four
tyrosines at amino acid positions 338, 355, 358, and 361 are within the
acid-rich domain. Tyrosines 392 and 510 are in the distal portion of
the cytoplasmic domain.
Figure 6:
IL-2R point mutations of tyrosine to
phenylalanine establish the motif surrounding phosphorylated tyrosine
338 as the IL-2R
Shc binding site. COS-7 cells were transfected
with constructs containing Shc-HA, Jak-1, and/or wild type IL-2R
chain (
WT) or tyrosine to phenylalanine mutants of the
IL-2R
chain containing only tyrosine 338 (
YF:1Y),
tyrosines 355, 358, and 361 as a group (
YF:234Y),
tyrosine 392 (
YF:5Y), or tyrosine 510 (
YF:6Y). Cells were cultured, lysed, and immune
precipitated with antibody 561 and probed for Shc-HA association using
anti-HA immunoblot (upper panel). To verify equivalent
expression in transfected cells, lysates were subjected to anti-HA
immunoblot (middle panel) or anti-
chain immunoblot using
antibody 561 (lower panel).
This observation is in sharp contrast to several reports that have clearly shown that the tyrosine phosphorylation of Shc and downstream activation of Ras are closely coupled to ligand-induced mitogenesis (14, 39) . For example, mutant granulocyte macrophage colony-stimulating factor receptors that do not signal through Ras pathways have been shown to require serum for granulocyte macrophage colony-stimulating factor-induced proliferation(39) . The addition of serum in this system results in supplemental Ras activation such that these cells can now respond mitogenically to granulocyte macrophage colony-stimulating factor(39) . To determine whether serum supplementation is required for IL-2-induced mitogenesis of Ba/F3 cells containing the AD receptor mutant, the proliferative response to IL-2 in wild type (FL) and AD-containing cells under serum-free conditions in the presence or the absence of insulin was analyzed. Although we have previously described minor differences in the IL-2-induced proliferative response of FL and AD receptor-containing cells(31) , these results show that FL and AD mutants support increased DNA synthesis (Fig. 7) as well as increases in cell number (data not shown) in response to IL-2 in both serum-free and insulin-free conditions. Although mitogenesis is enhanced in the presence of insulin in these cells, there is no significant difference between FL and AD receptors. The AD mutants fail to activate Ras (38) and fail to activate the Shc adapter pathway, which may result in the activation of other Ras-like pathways, but are capable of supporting IL-2-induced mitogenesis in the presence of serum or in serum-free and insulin-free conditions in a manner similar to that observed with the wild type FL receptors. These results thus suggest that IL-2 signal transduction leading toward mitogenesis does not require Shc phosphorylation or the activation of Shc/Grb2 or Ras pathways(38) .
Figure 7:
IL-2-induced mitogenesis in FL and AD
receptor-containing Ba/F3 cells does not require serum or insulin.
Ba/F3 cells containing either the full-length wild type IL-2R (FL)
or the acid-rich domain deletion mutant (AD) were treated with 50 ng/ml
IL-2 in serum-free medium with or without insulin. Proliferation was
measured by [
H]thymidine incorporation. Note that
there is no significant difference between FL or AD receptors regarding
their ability to transduce IL-2-dependent mitogenic signal in Ba/F3
cells.
Using mutational analysis of the IL-2R system, we have shown
that IL-2 induction of Shc tyrosine phosphorylation is dependent on Shc
association with the IL-2R. We further show that this association
is dependent on the presence of phosphorylated tyrosine 1 at amino acid
338 within the acid-rich region of the IL-2R
cytoplasmic domain.
This tyrosine is found within a motif containing the sequence
TNQGpYFFF. Structural studies of motifs required for Shc binding to
various receptor complexes (40, 41, 42) have
revealed a consensus binding site for the Shc phosphotyrosine binding
domain(40) , also referred to as the SAIN (Shc and IRS-1
NPXY binding) domain (42) of NPXpY.
Considering the structural similarities between proline and glutamine,
the motif within the IL-2R
chain of NQGY appears quite similar to
the predicted SAIN domain recognition consensus of NPXY. Using
the predicted IL-2 receptor binding domain for Shc, NQXY, a
search was initiated for proteins containing this motif(43) .
Generally, this search identified proteins that have been shown to have
regulatory functions in various signal transduction pathways including
Ras-Gap (motif from amino acid
PTNQWYH-), which is
involved in the regulation of Ras and Ras-like pathways, and several
receptor molecules such as the fibroblast growth factor receptor (motif
from amino acid
TSNQEYL-). Experiments aimed at
investigating the potential role that Shc association with these and
other proteins may play in regulating signal transduction are presently
underway.
It has been shown (31, 38) that deletion
of the acid-rich region of the IL-2R chain abrogates the ability
of IL-2 to induce Ras activation as well as increased transcription of fos and jun but did not affect IL-2-dependent
mitogenesis in the murine IL-3-dependent cell Ba/F3. This analysis did
not preclude the possibility of IL-2-dependent activation of Ras-like
but Ras-distinct pathways, which would support IL-2-driven cell growth.
By focussing on the analysis of IL-2 activation of upstream Ras pathway
regulators (Shc adapter pathway), it becomes much easier to define the
potential role that these pathways play in IL-2 signal transduction.
Results from this study clearly show that Shc phosphorylation that acts
to initiate this pathway is not required for mitogenesis. This analysis
does not preclude the possibility that Grb2 may interact directly with
this receptor complex and function to activate other guanine nucleotide
releasing factor activities in the AD mutant, which may lead to
mitogenesis. If this were the case, however, one would expect AD
receptors to also activate Ras. This clearly does not occur in this
mutant(38) . Additionally, in the insulin receptor system,
which has been shown to associate independently with both Shc and Grb2,
Shc binding and tyrosine phosphorylation is the primary mechanism
utilized in vivo to bring Grb2 and guanine nucleotide
releasing factor activity to the membrane resulting in Ras
activation(44) . These data thus suggest that Shc binding to
the IL-2R
chain, which results in its tyrosine phosphorylation and
initiation of the Shc/Grb2 adapter pathway, is the mechanism used by
IL-2 to activate Ras and Ras effector pathways.
These data also strongly suggest that IL-2 receptor signal transduction functions to activate specific mitogenic signals without a strict requirement for Ras in IL-2 receptor-containing Ba/F3 cells. This does not preclude the possibility, however, that in the context of a different cellular background IL-2 may have different requirements for Ras and Ras signaling pathways.
It is well described that Ras activation and regulation of downstream pathways is critical in controlling proliferation(14) . It seems plausible therefore that mitogenic signal transduction by IL-2 in Ba/F3 cells may activate one or more of these downstream pathways by functionally bypassing Ras activation. A key mitogenic pathway activated by upstream activation of Ras is the Raf/Map kinase cascade (45, 46) . This pathway has been described as a ``mitogenic bottleneck'' due to the wide variety of mitogenic signals that funnel into Raf activation (46) and because antisense elimination of Raf blocks cytokine-triggered mitogenesis(47) . IL-2 has been shown to activate Raf, and Raf has been found to be associated with the IL-2 receptor complex by ourselves (data not shown) and others(48, 49) . Furthermore, IL-2-dependent activation of Raf involves a tyrosine kinase-dependent mechanism(48) . As has been shown in other systems, this is presumed to involve the tyrosine phosphorylation of Shc, the subsequent activation of Ras, and the downstream activation of Raf and Map kinases(14, 46) . However, Raf can also be activated by direct tyrosine phosphorylation(50) . The possibility exists, therefore, that IL-2 signaling may involve the direct tyrosine phosphorylation of Raf as an activating mechanism. This would theoretically abrogate the requirement for Ras activation in an IL-2-driven mitogenic response. To further elucidate this, the role that Raf activation plays in IL-2 signal transduction in IL-2 receptor mutants is being investigated.
With the data presented here, a
potentially novel mechanism by which IL-2, and perhaps other IL-2R
chain containing receptors, controls cell growth is being established.
In contrast to the results presented from studies within the
granulocyte macrophage colony-stimulating factor receptor
system(39) , which clearly establishes an absolute requirement
for some level of Ras activation in order for this receptor system to
support cytokine-induced mitogenesis, the model of IL-2 signal
transduction that has been generated in Ba/F3 cells establishes that
mitogenesis can be fully supported by IL-2 receptors that lack the
ability to activate this or similar pathways through utilization of the
Shc adapter pathway. IL-2 signal transduction thus forms a novel
paradigm for mitogenic signaling by hematopoietic cytokines.