(Received for publication, March 18, 1995; and in revised form, June 1, 1995)
From the
To evaluate the possible role for receptor-based tyrosine
phosphorylation in growth signaling induced by interleukin-2 (IL-2), a
series of substitution tyrosine mutants of the IL-2 receptor and
chains was prepared and analyzed. Concurrent mutation
of all six of the cytoplasmic tyrosines present in the
chain
markedly inhibited IL-2-induced growth signaling in both pro-B and T
cell lines. Growth signaling in a pro-B cell line was substantially
reconstituted when either of the two distal tyrosines (Tyr-392,
Tyr-510) was selectively restored in the tyrosine-negative
mutant, whereas reconstitution of the proximal tyrosines (Tyr-338,
Tyr-355, Tyr-358, Tyr-361) did not restore this signaling function.
Furthermore, at least one of the two cytoplasmic tyrosines that is
required for
chain function was found to serve as a phosphate
acceptor site upon induction with IL-2. Studies employing a chimeric
receptor system revealed that tyrosine residues of the
chain
likewise were important for growth signaling in T cells. In contrast,
although the
subunit is a target for tyrosine
phosphorylation in vivo, concurrent substitution of all four
cytoplasmic tyrosines of this chain produced no significant effect on
growth signaling by chimeric IL-2 receptors. However, deletion of
either the Box 1, Box 2, or intervening (V-Box) regions of
abrogated receptor function. Therefore, tyrosine residues of
but not of
appear to play a pivotal role in
regulating growth signal transduction through the IL-2 receptor, either
by influencing cytoplasmic domain folding or by serving as sites for
phosphorylation and subsequent association with signaling
intermediates. These findings thus highlight a fundamental difference
in the structural requirements for IL-2R
and
in
receptor-mediated signal transduction.
Interleukin-2 (IL-2) ()is a helical cytokine that
induces the proliferation of T and B lymphocytes as well as the
expression of a number of immune effector functions by binding to the
heterotrimeric IL-2 receptor complex (IL-2R). The 70-75-kDa
(IL-2R
) and 64-kDa
(
) subunits of the IL-2R
share structural homology with other members of a cytokine receptor
superfamily (1) and together form a receptor complex that is
competent to bind IL-2 with intermediate affinity and to transduce
growth and differentiation signals (reviewed in (2) ). As in
other receptor systems, evidence has accumulated indicating that signal
transduction is initiated upon ligand-induced heterodimerization of the
and
cytoplasmic tails(3, 4) .
Interestingly, IL-2R
is also employed in the receptor for
IL-15(5, 6) , whereas
participates
in the formation of the receptors for IL-4(7) ,
IL-7(8, 9) , IL-9(10) , and IL-15(6) .
Among the earliest biochemical changes induced by ligation of the IL-2 receptor is activation of cytoplasmic tyrosine kinases resulting in the phosphorylation of certain recognized and unrecognized cellular substrates. The biologic relevance of IL-2-induced tyrosine kinase activity is supported by the finding that selective tyrosine kinase inhibitors (herbimycin A and genistein) concomitantly block these intracellular phosphorylation events as well as growth signal transduction(11, 12) . Although none of the known IL-2R subunits contain recognizable kinase catalytic domains, tyrosine kinase activity has been coimmunoprecipitated with the IL-2R(13, 14, 15, 16, 17, 18) . Recent evidence indicates that the Janus kinases JAK1 and JAK3 (19, 20, 21) as well as various src family kinases (13, 15, 18, 22) are among the signaling molecules that are physically and functionally linked to the IL-2R. However, the specific role of each of these kinases and their substrates in IL-2R signal transduction remains to be defined.
Like
many growth factor receptors containing intrinsic tyrosine kinase
activity (for review, see (23) ), the cytoplasmic domains of
the and
subunits of the interleukin-2 receptor itself
undergo inducible tyrosine phosphorylation upon engagement by
IL-2(24, 25, 26) . The biological
significance of such receptor phosphorylation is poorly defined for
cytokine receptors lacking intrinsic tyrosine kinase activity. Since
the IL-2 receptor itself is a major substrate of tyrosine
phosphorylation following the binding of IL-2, the present
investigation was undertaken to determine the potential regulatory role
played by the cytoplasmic tyrosine residues of the IL-2R
and
subunits. Our results demonstrate that tyrosines
within the cytoplasmic tail of IL-2R
are critical for full growth
signaling in pro-B and T cells. In contrast, the tyrosine residues of
the
chain are dispensable for this function,
revealing an important distinction between the IL-2R
and
subunits. These findings, along with a delineation of
essential membrane-proximal domains of
, may have
general implications for the functional design of cytokine receptors,
particularly those employing the common
subunit.
The tyrosine substitution
mutants of IL-2R and
(tyrosine (TAC) to
phenylalanine (TTC)) were prepared by a combination of
oligonucleotide-directed mutagenesis in M13 bacteriophage and PCR-based
methods. For constructs involving the
cytoplasmic
tail, a full-length cDNA was obtained by reverse transcription PCR
based on the IL-2R
sequence reported by Takeshita et
al.(32) . Deletion and substitution mutants described
under ``Results'' (see figure legends) were prepared by PCR
using IL-2R
or
cDNAs as templates.
pEPOneo, constructed by PCR using an NheI site at the
fusion junction, encodes a chimeric receptor (see Fig. 6A) containing the extracellular domain of the
EPOR fused just above the transmembrane segment to the human IL-2R
transmembrane and cytoplasmic segments (resulting sequence: . . .
(EPOR-T-A-S)-(G-K-D-IL-2R
)
. . . ). pEPO
neo, also constructed by PCR using the NheI
site, encodes a receptor (see Fig. 6A) containing the
extracellular domain of the EPOR fused to the human
transmembrane and cytoplasmic segments (resulting sequence: . .
.
(EPOR-T-A-S)-(S-K-E-
)
. . . ). Expression plasmids encoding the mutants described in the text
were prepared by subcloning appropriate DNA fragments spanning the
indicated mutations into the parental pEPO
neo and pEPO
neo
plasmids.
Figure 6:
Chimeric EPOR/IL-2R receptor subunits. A, schematic representation of native (IL-2R,
, and EPOR) and chimeric (EPO
and
EPO
) subunits fused at a unique NheI site located
immediately N-terminal to the transmembrane segment. Some conserved
features of the cytokine receptor superfamily shown are indicated (the
extracellular WSXWS sequence and the intracellular signaling
domain, including Box 1, Box 2, and the intervening V-(variable) Box). B, immunoblot analysis of native and chimeric receptor
subunits. The EPOR and EPO
variants are shown on the left, and the EPO
variants are shown on the right. The
A,
AB, and
D258A mutations
have been described(28) ; in the
YF and
YF
cytoplasmic tails all tyrosines (TAC) have been replaced with
phenylalanines (TTC). See Fig. 9legend for description of
additional mutant
cytoplasmic tails. C,
parental HT-2 cells(-) or stable transfectants expressing EPOR,
EPO
, or EPO
were stimulated with either EPO (closed
bars, 50 units/ml) or IL-2 (open bars, 10 nM)
for 24 h, and [
H]thymidine incorporation was
assessed as in Fig. 2. Results are expressed relative to the
level of incorporation occurring with IL-2 stimulation (100%); error bars represent standard errors of the mean (n = 3).
Figure 9:
Functional analyses of EPOR/ chimeras in transfection assays of proliferation. Transfection
growth assays using the HT-2EPO
line as a host to assess the
responses of the the indicated EPO
mutants. The
336,
294, and
TM mutants are truncated immediately after amino
acids 336, 294, and 286, respectively, in the mature
protein.
Box 1 is deleted of residues 281-294,
V-Box is deleted of residues 295-320, and
Box
2 is deleted of residues 321-334. Results are expressed as the
incorporation of [
H]thymidine for each line
relative to that of the wild type (
wt) cytoplasmic tail,
with standard errors of the mean (n
3).
Figure 2:
Growth signaling and expression
characteristics of stable transfectants expressing tyrosine-negative
IL-2R chains. BA/F3 cells expressing wild type IL-2R
(Baf
WT) or tyrosine-negative mutant IL-2R
chains (Baf
YF) were analyzed. Cells stimulated for 24 h with
IL-2 at the indicated concentration (in the absence of IL-3) were
pulsed with [
H]thymidine for the final 4 h of the
culture and harvested. Results are expressed relative to the level of
incorporation occurring with IL-3 stimulation (100%); error bars represent standard errors of the mean (n =
3).
Figure 1:
Growth signal transduction properties
and expression of tyrosine-negative mutant IL-2 chains in
transient assays. A, proliferation of BA/F3 cells transfected
with expression vector encoding wild type IL-2R
(
WT,
closed circles) or empty vector (Vector, open circles),
as measured by incorporation of
[
H]thymidine on the indicated days
following initiation of IL-2 selection (10 nM) in the absence
of IL-3. Each data point is the mean of triplicates, and each
experiment shown is representative of several independent experiments.
The relative positions of the six cytoplasmic tyrosine residues of
IL-2R
are indicated by closed symbols in the schematic: 1, Tyr-338; 2, Tyr-355; 3, Tyr-358; 4, Tyr-361; 5, Tyr-392; 6, Tyr-510. B, proliferation of cells transfected with vector encoding
tyrosine-negative mutant of IL-2R
(
YF, closed
squares). C, surface expression of IL-2R
and
YF
mutant receptors. Autoradiograph of immunoprecipitates of COS cells
cotransfected with vectors encoding wild type human IL-2R
and
either IL-2R
(
WT) or the tyrosine-negative mutant (
YF) prepared following disuccinimidyl suberate-mediated
cross-linking with
I-IL-2. Molecular mass markers are
shown on left (kDa). D and E, equilibrium
I-IL-2
binding analysis of COS cells cotransfected with vectors encoding wild
type human IL-2R
and either IL-2R
(D) or
YF (E).
Two independent types
of experiments were performed to ensure that the impaired function of
YF was not simply the result of ineffective surface expression or
faulty binding of ligand. First, to monitor surface expression COS
cells were transiently transfected with expression vectors encoding the
IL-2R
chain and either native IL-2R
or
YF, followed by
incubation with
I-IL-2, chemical cross-linking with
disuccinimidyl suberate, and immunoprecipitation with the anti-
monoclonal antibody DU-2(14) . Following SDS-polyacrylamide gel
electrophoresis, bands of comparable intensity and migration were
observed for cells transfected with the wild type
and
YF,
indicating the unimpaired surface expression of the mutant
YF
receptor (Fig. 1C). To investigate potential changes in
receptor affinity, radioligand binding analyses were performed with
I-IL-2 in COS cells transfected with IL-2R
and
WT or
YF. These studies revealed the expected single class of
intermediate affinity IL-2 binding sites for both
WT and
YF (K
300-400 pM) (Fig. 1, D and E). Thus, surface expression
and ligand binding by
YF appeared indistinguishable from wild type
and therefore do not account for its impaired signaling function
in the transfection assay system.
Analysis of
[H]thymidine incorporation in response to IL-2
revealed marked unresponsiveness of the stable Baf
YF cell line to
IL-2 compared with Baf
WT (Fig. 2). As expected, the
Baf
WT cell line demonstrated detectable proliferation even at very
low doses of IL-2 (10 pM) well below the K
of IL-2 binding to IL-2R
complexes, whereas the
Baf
YF line demonstrated no response even at very high doses of
IL-2 (100 nM) vastly exceeding the measured K
. These findings confirmed the impaired
proliferation signaling exhibited by the
YF mutant initially
detected in the transient system.
Figure 3:
Peak
proliferative responses of various tyrosine and deletion mutants of
IL-2R. Transient proliferation assays were performed as in Fig. 1. Peak [
H]thymidine incorporation is
shown as a percentage of the peak response by
WT transfectants in
each assay. Each value is the mean of triplicate determinations with
standard errors of the mean, and results shown are representative of
multiple independent experiments.
WT, wild type
IL-2R
;
YF, tyrosine-negative IL-2R
;
Y1F,
with Tyr-338 (1) mutated to Phe;
Y234F,
with Tyr-355(2) ,
Tyr-358(3) , and Tyr-361 (4) mutated to Phe;
Y5F,
with Tyr-392 (5) mutated to Phe;
Y6F,
with Tyr-510 (6) mutated to Phe;
YF:56Y,
with Tyr-338(1) ,
Tyr-355(2) , Tyr-358(3) , and Tyr-361 (4) mutated to Phe;
Y56F,
with Tyr-392 (5) and Tyr-510 (6) mutated to Phe;
AB, IL-2R
deleted from amino acid
313-431;
ABC, IL-2R
truncated after amino
acid 312.
We further observed that internal deletion of a
119-amino acid cytoplasmic region of IL-2R spanning the A region
as well as the contiguous ``B'' segment exhibited fully
preserved growth signaling (Fig. 3,
AB),
suggesting that the first five tyrosines are dispensable. In contrast,
extension of this deletion to include the C-terminal region containing
the sixth tyrosine (
ABC) abrogated receptor function (Fig. 3). These results suggested that the sixth tyrosine
(Tyr-510) is sufficient to permit growth signal transduction. Indeed,
an IL-2R
mutant in which only this single tyrosine was restored in
the
YF background (
YF:6Y) exhibited substantial IL-2 growth
signaling (Fig. 4A).
Figure 4:
Proliferative responses of IL-2R
mutants with selective reconstitution of cytoplasmic tyrosines.
Transient proliferation assays were performed as in Fig. 1. A,
YF:6Y,
YF with Phe-510 (6) back-mutated to Tyr; B,
YF:5Y,
YF with Phe-392 (5) back-mutated to Tyr; C,
YF:234Y,
YF with Phe-355(2) ,
Tyr-358(3) , and Tyr-361 (4) back-mutated to Tyr; D,
YF:1Y,
YF with Phe-338 (1) back-mutated to Tyr. Constructs were prepared by
recombination of cytoplasmic restriction fragments derived from mutants
shown in Fig. 3and were verified by DNA sequence
analysis.
Although Tyr-510 alone is
sufficient for receptor competence, selective substitution of
phenylalanine at this position had little effect on the signaling
function (Fig. 3). These results strongly implied that at least
one other tyrosine site also could support growth signal transduction,
a hypothesis that was tested by evaluating additional tyrosine add-back
mutants. Interestingly, reconstitution of Tyr-392 (YF:5Y)
substantially restored the IL-2R
signaling function (Fig. 4B). In contrast, restoration of tyrosines in the
first four positions in two additional add-back mutants (
YF:234Y
and
YF:1Y) failed to reconstitute receptor function (Fig. 4, C and D, respectively). Importantly,
the
YF:56Y,
Y56F,
YF:1Y,
YF234Y,
YF:5Y, and
YF:6Y proteins were all expressed abundantly as detected by
immunoblotting analysis (data not shown). Thus, either the fifth
tyrosine (Tyr-392) or sixth tyrosine (Tyr-510) is necessary and
sufficient for IL-2 growth signaling in BA/F3 cells.
Figure 5:
Signaling function and receptor
phosphorylation in stable transfectants expressing IL-2R tyrosine
add-back mutants. A, BA/F3 cells expressing tyrosine-negative
IL-2R
(Baf
YF) or IL-2R
tyrosine add-back
mutants with restoration of Tyr-392 (Baf
YF:5Y) or Tyr-510 (Baf
YF:6Y) IL-2R
chains (Baf
YF) were
analyzed. A, [
H]Thymidine incorporation
assay with the indicated doses of IL-2, as described in the legend to Fig. 2. B, tyrosine phosphorylation analysis of
Baf
WT (
wt), Baf
YF (
YF), and
Baf
YF:5Y (
YF:5Y). Anti-IL-2R
immunoprecipitates
from cells stimulated with IL-2 for 10 min were subjected to
immunoblotting with anti-phosphotyrosine
antibody.
Phosphorylation studies were next performed using these stable
transfectants. In these experiments, cell lines were rested without
growth factors and then exposed to IL-2. Stimulated cells were lysed,
immunoprecipitated with anti-IL-2R monoclonal antibody, and then
subjected to immunoblot analysis with anti-phosphotyrosine antibody.
Upon induction with IL-2 the Baf
WT line yielded a strong
phosphotyrosine signal at the appropriate molecular weight for
IL-2R
chains, whereas the Baf
YF line yielded no discernible
signal (Fig. 5). Like Baf
WT, Baf
YF:5Y cells also
yielded a phosphotyrosine-containing protein band (Fig. 5B). Since this add-back cell line expresses
IL-2R
chains containing only a single cytoplasmic tyrosine residue
(Tyr-392) with all others replaced by phenylalanine, a phosphotyrosine
signal generated in the immunoblot experiment is clearly attributable
to this tyrosine. These results thus indicated that Tyr-392 of
IL-2R
serves as a phosphate acceptor site during receptor
activation.
Similar experiments were performed with the
BafYF:6Y line to assess the role of Tyr-510 in receptor
phosphorylation. Surprisingly, no IL-2R
chain tyrosine
phosphorylation was detectable in experiments with cells expressing the
Tyr-510 add-back mutant (data not shown). Such experiments were
performed with multiple, independently derived lines, and stimulations
were performed for various lengths of time ranging from 3 to 30 min. It
remains possible that this functional tyrosine residue of IL-2R
does indeed undergo phosphorylation and that this site is perhaps
particularly sensitive to phosphatase attack after detergent
solubilization of the cells. Nonetheless, phosphorylation of this
tyrosine has not yet been detected (see ``Discussion'').
The
IL-2-dependent murine helper T cell line, HT-2, was employed for
analysis of EPO and EPO
signaling. Initially, stable HT-2
transfectants expressing the EPOR, EPO
, or EPO
subunits were
established. In 24-h [
H]thymidine incorporation
assays, the EPOR was found to mediate a modest response to EPO, whereas
neither of the chimeric receptor subunits alone produced a detectable
response in multiple transfected clones (Fig. 6C). The
failure of EPO
and EPO
to mediate a response was not due to
lack of expression, since Northern blotting, Western blotting, and
radioligand binding analyses with
I-EPO confirmed the
expression and ligand binding competence of these chimeras in the
HT-2EPO
and HT-2EPO
cell lines (data not shown).
Since
neither chimera alone (EPO or EPO
) demonstrated detectable
growth signal transduction, combinations of these chimeras in HT-2
cells were tested for growth signaling in response to EPO as a means of
promoting heterodimerization of the IL-2R
and
cytoplasmic tails. For these studies the transfection assay
originally described for BA/F3 cells (28) was adapted to HT-2
cells. When the EPO
expression plasmid was introduced by
electroporation into multiple HT-2 clones stably expressing EPO
(HT-2EPO
), addition of EPO without IL-2 produced marked
proliferation and vigorous incorporation of
[
H]thymidine during the 12-day assay (Fig. 7A). Similarly, multiple HT-2 clones stably
expressing EPO
(HT-2EPO
) displayed marked proliferative
responses to EPO following introduction of the EPO
expression
plasmid (Fig. 7B). Additionally, double transfectants
arising from such experiments were easily maintained in long term
culture by addition of EPO alone, allowing the isolation of a stable
transfected cell line (HT-2EPO
) for further studies of early
signal transduction events. Thus, concurrent engagement of both the
and
chimeras is required for effective growth
signaling, as has been reported in studies with other chimeric
receptors(3, 4) .
Figure 7:
Functional analyses of EPOR/IL-2R
chimeras. A, parental HT-2 cells or HT-2EPO cells
described in the legend to Fig. 6were transfected with the
EPO
expression plasmid, selected in EPO (50 units/ml) without
other cytokines, and assayed for growth by measuring
[
H]thymidine incorporation on the indicated days. B, parental HT-2 cells or HT-2EPO
cells described in Fig. 6were transfected with the EPO
expression plasmid,
selected in EPO, and assayed for growth. C, HT-2EPO
cells
were transfected with the EPO
YF expression plasmid, selected in
EPO, and assayed for growth. Each experiment shown was performed
multiple times with similar results. D, to assess
phosphorylation of JAK1 during receptor activation, the stable
transfectants HT-2EPO
and HT-2EPO
YF/
were
stimulated with no cytokine(-), EPO (E), or IL-2 (2) followed by immunoprecipitation with the anti-JAK1
antiserum and immunoblotting with the anti-phosphotyrosine
antibody.
To analyze further the
disruption in signal transduction by the YF mutant, Janus kinase
induction in response to receptor engagement was assessed. Lysates
prepared from HT-2 cells stimulated with no cytokine, IL-2, or EPO were
subjected to immunoprecipitation with an anti-JAK1 antiserum followed
by immunoblot analysis with an anti-phosphotyrosine antibody. Cells
expressing chimeric
chains and either wild type
chimeric
chains (HT-2EPO
) or tyrosine-negative
chains (HT-2EPO
YF/
) both exhibited strong induction of JAK1
phosphorylation in response to either ligand (Fig. 7D).
Likewise, preserved induction of JAK3 phosphorylation by receptor
complexes containing EPO
YF was observed in parallel experiments
employing an anti-JAK3 antiserum (data not shown). Therefore, at least
one early phase of receptor-mediated signaling by the
YF mutant is
intact despite the failure to achieve full growth signaling.
Since tyrosine
phosphorylation of the subunit upon ligand binding
has been well described(26) , we investigated the putative role
of the tyrosine residues present in the
subunit by
phenylalanine substitution of all four tyrosine residues (EPO
YF).
Surprisingly, growth signal transduction by EPO
YF was nearly
indistinguishable from that by EPO
both in transfection assays (Fig. 8, A and B) and in 24-h
[
H]thymidine incorporation assays of stable
transfectants arising from transfection of HT-2EPO
cells with the
EPO
YF expression plasmid (Fig. 8, C and D). Thus, the cytoplasmic tyrosine residues of
appeared to be dispensable for growth signaling, which stands in
sharp contrast to their importance in the IL-2R
subunit.
Figure 8:
Preserved growth signal transduction
function of tyrosine-negative chains. A and B, transfection growth assays were performed by transfecting
HT-2EPO
cells with expression vectors encoding either EPO
(A) or EPO
YF (B) and selecting in EPO, as in Fig. 7. C and D, stable HT-2 transfectants
expressing EPO
and either EPO
(C) or EPO
YF (D) were analyzed by 24-h [
H]thymidine
incorporation assays, as in Fig. 6C. Shown is the dose
response to EPO for each cell line relative to the response to IL-2,
with closed bars representing the means and error bars
indicating the standard errors of the mean (n =
3).
Although the tyrosine residues are non-essential, other regions of
the cytoplasmic tail proved important for growth
signaling. EPO
mutants truncated at the cell membrane (EPO
TM)
or at the end of the Box 1 (39) homology region (EPO
294)
mediated no detectable proliferation signaling (Fig. 9).
Similarly, internal deletion of Box 1 (EPO
Box1), of a segment
with distant relationship to the Box 2 motif (EPO
Box2), or of
the segment connecting Box 1 to Box 2 (EPO
V-Box), also
abolished proliferation signaling. However, truncation of the
subunit at the C-terminal end of the Box 2 region
(EPO
336) resulted in levels of growth signaling similar to that
obtained with the wild type subunit. Thus, unlike the IL-2R
subunit, the distal portion of the
subunit is
dispensable for proliferation signal transduction, and full
growth-signaling function resides in the proximal 53 amino acids
containing the Box 1, Box 2, and intervening (V-Box) segments.
Like many other cytokine receptor systems, the binding of
IL-2 to the IL-2R induces the tyrosine phosphorylation of a variety of
intracellular substrates, including the IL-2R and
chains(24, 25, 26) . Although no
tyrosine kinase domain is identifiable within the recognized
ligand-binding subunits of the IL-2R, the Janus kinases JAK1 and JAK3
as well as the src family kinase p56
and p59
are now recognized to
associate noncovalently with the cytoplasmic tails of IL-2R
subunits(10, 15, 19, 40) . The
activation of such receptor-associated kinases may represent a
mechanism for signal transduction that is fundamentally the same as
that for receptors containing intrinsic kinase activity. Indeed, as in
such kinase-containing receptors, some evidence has accumulated from
mutagenesis and in vitro analyses that certain tyrosine
residues of the IL-4 and interferon receptors are crucial for signal
transduction
competence(41, 42, 43, 44) .
The
present studies were undertaken to evaluate the potential regulatory
role of cytoplasmic tyrosines of the IL-2R and
chains. In these studies employing both native and chimeric
receptors, substitution of phenylalanine for all six cytoplasmic
tyrosine residues of IL-2R
substantially impaired growth signaling
in both a pro-B and a mature T cell line ( Fig. 1and Fig. 7). A panel of add-back mutants revealed that both Tyr-392
and Tyr-510 individually exhibit signaling potential in the BA/F3 pro-B
cell line while the four more proximal tyrosines demonstrate no
functional capacity in this specific cellular environment (Fig. 4). We conclude from these experiments that, in BA/F3
cells, the two C-terminal cytoplasmic tyrosines serve important but
redundant functions in determining the signal transduction competence
of the IL-2R
chain.
The finding that C-terminal tyrosines of
IL-2R influence growth signaling in this system appears to
contrast with an earlier report that the IL-2R
segment
encompassing these tyrosines is dispensable for proliferative
signaling(38) . However, point substitutions and deletions of
identical regions may have different phenotypic consequences,
particularly if the protein region in question exerts regulatory
effects via conformational changes. For example, the C terminus of
IL-2R
may negatively regulate proximal domains through steric
hindrance, which might be relieved by receptor activation. Such a model
would also explain the negative regulatory domain identified within the
EPOR C terminus(45) . A deletion mutant thus may obscure a role
of tyrosine residues within this region. Therefore, we conclude that
tyrosines within the IL-2R
cytoplasmic tail are indeed important
for the growth signaling competence of IL-2R
.
The mechanism(s)
underlying the importance of Tyr-392 and Tyr-510 to IL-2R function
remain uncertain. In the platelet-derived growth factor receptor
system, several distinct signaling pathways are activated selectively
by individual phosphotyrosine residues through interactions with
proteins via SH2 domains(46, 47) . Recent reports have
described the inducible binding of p52 to the
IL-2R
chain upon the binding of IL-2(48, 49) ,
although the molecular basis of this interaction is unknown. Similarly,
phosphatidylinositol 3-kinase has also been found to associate with the
IL-2R
chain in the presence of IL-2(50, 51) , an
event which may be facilitated by phosphorylation of IL-2R
Tyr-392
as revealed in studies with phosphopeptides(51) . Finally,
following completion of the present work, we (52) and others (53) have demonstrated that phosphopeptides encompassing either
Tyr-392 or Tyr-510 are potent and specific inhibitors of the in
vitro DNA binding activity of STAT-5, a STAT factor that is
regulated by the IL-2R(52, 53, 54) .
Interestingly, tyrosine residues of IL-2R
are dispensable for
Janus kinase activation by the IL-2R (Fig. 7D) but are
essential for the effective induction of STAT-5 (52) .
Together, these findings are consistent with the popular model of
cytokine receptor function (55) in which ligand-induced
phosphorylation of certain tyrosine residues of the receptor is a
critical step in the generation of downstream intracellular signals.
Convincing demonstration of the significance of this model for IL-2R
function requires identification of the sites of IL-2-induced tyrosine
phosphorylation of IL-2R in vivo. The present studies
demonstrated that Tyr-392 serves as a phosphate acceptor site upon
exposure of BA/F3 transfectants to IL-2 (Fig. 5). Unexpectedly
we failed to detect phosphorylation of Tyr-510 in parallel experiments.
It is possible that this lack of detection results from technical
problems, such as insensitivity of the assay method or contaminating
phosphatase activity released during cell lysis. Alternatively, this
observation may indicate that Tyr-510 function is entirely independent
of its phosphorylation status. Indeed, the published evidence
supporting a critical role for receptor phosphotyrosines in the
JAK-STAT pathway is largely circumstantial. For example, experimental
demonstration of direct interactions between STAT factors and
phosphotyrosine-containing receptor segments has proven difficult in
most circumstances, and heavy emphasis has been placed instead on in vitro peptide approaches(44) . Therefore, the lack
of detectable phosphorylation of Tyr-510 in the present studies raises
the possibility that this and perhaps other tyrosine residues of
IL-2R
exert crucial influences on the tertiary conformation of
IL-2R
independently of their phosphorylation status. Although we
tend to favor the tyrosine phosphorylation model, rigorous
consideration of the published data demands further studies to
distinguish effectively between these interpretations.
Other
cytokine receptor superfamily members (1) may similarly be
influenced by tyrosines. Functionally important tyrosine residues
within the cytoplasmic domains of the IL-4 and interferon-
receptors have been described
recently(41, 42, 43) , although the
significance of IL-4R phosphorylation has been disputed(56) .
The functional redundancy described here for the distal IL-2R
tyrosines may also be a feature of the human IL-4 receptor that could
explain the incomplete impairment of function reported upon
substitution of phenylalanine for Tyr-497 in the IL-4
receptor(41) . Further investigation is needed to clarify these
events within the IL-2R.
The EPOR/IL-2R chimeric system also
permitted an assessment of the role of tyrosine and other residues
within the cytoplasmic tail for growth signaling in T
cells. In contrast to the IL-2R
chain, the
subunit functioned fully in the absence of all four of its
cytoplasmic tyrosine residues (Fig. 8). This finding indicates
that growth signaling intermediates interacting with the
tail do so independently of phosphotyrosine docking sites, even
though one or more of these tyrosine sites is phosphorylated after IL-2
stimulation in vivo. In view of the fact that both the IL-4
and IL-2 receptors employ the
subunit, these
observations raise the intriguing possibility that the longer, unique
chain in each receptor provides the docking sites for the specific
signaling intermediates engaged by each receptor complex. In this
arrangement, the shared
subunit would participate in
general initiation of the signaling process, whereas the specialized
subunits would contain unique sites for the inducible binding of
specific components, such as STAT factors. Other cytokine receptors
might employ a similar functional configuration. Of course, it remains
possible that components involved in other pathways not measured here
(such as differentiation) do indeed depend upon these
tyrosine sites.
Although the tyrosines of proved to be dispensable for growth signaling by the IL-2R, a
panel of truncation and internal deletion mutants revealed other
elements within
that are critical for growth
signaling in the T cell line. Remarkably, the C-terminal 33 amino acids
of
are fully dispensable for growth signaling (Fig. 9), indicating that the proximal 53 amino acids are
sufficient for full growth signal transduction. Mutations within this
membrane-proximal region abrogated signaling function. For example,
extension of the truncation N-terminal to a vestigial ``Box
2'' motif (39) abolished the signaling function, as did
internal deletion of the 14 amino acids constituting a ``Box
1'' motif, the 14 amino acids constituting this vestigial Box 2
motif, or the 26 amino acids connecting Box 1 to Box 2 (V-Box) (Fig. 5). These observations in T cells extend the studies by
others which employed certain truncated
subunits
expressed in heterologous cell types(57, 58, 59) and demonstrate clearly that the
tail is
needed for growth signal transduction by IL-2R heterodimers in T cells.
Importantly, the impairment of these
domains
undoubtedly contributes to the pathologic effects manifested in the
X-linked severe combined immunodeficiency syndrome(60) .
The
recognition that the growth signaling function of resides in a relatively small portion of the cytoplasmic tail and
that this segment functions independently of tyrosine residues is
consistent with the receptor model described above. The essential,
membrane-proximal region of
has been shown to be
crucial for the assembly of the Janus kinase JAK3 with
(10, 40) . Perhaps the primary function of
in the IL-2, IL-4, and other receptors is to convey
JAK3 into the receptor complex upon engagement of the appropriate
ligand, which would thus allow trans-activation of JAK1 and
JAK3 bound to their respective receptor subunits. Subsequent signaling
activities may focus primarily upon the extended cytoplasmic tail of
the unique IL-2R
chain, including the inducible binding and
activation of specific factors. Further studies are needed to determine
whether or not the
chain has additional functions in
addition to its conveyance role. One or both of the Janus kinases may
be involved in phosphorylation substrates within the receptor complex.
The present findings provide a rationale for further investigation of
these intracellular events.