From the
The tyrosine kinases JAK1 and JAK3 have been shown to undergo
tyrosine phosphorylation in response to interleukin-2 (IL), IL4, IL7,
and IL9, cytokines which share the common IL2 receptor
Interleukin-4 (IL4)
Interestingly, the tyrosine
kinase JAK3 has been shown to undergo marked tyrosine phosphorylation
in response to all cytokines tested that use the common IL2R
Indirect support for this model has been
provided by evidence for a selective preference of JAK3 for IL2R
A key to understanding the mechanism of JAK
activation by cytokine receptors will be to define the nature of
receptor-JAK interactions. Moderately conserved membrane-proximal
regions of cytoplasmic receptor domains have emerged as probable
binding sites that are critical for JAK activation and signal
transduction
(13, 17, 24, 25) . More
specifically, a membrane-proximal region of the shared IL2R
The
current investigation was undertaken to characterize the involvement of
JAK tyrosine kinases in IL4 receptor signal transduction, addressing
not only the relative IL4-induced tyrosine phosphorylation of various
JAK enzymes, but also evaluating their catalytic activation and
receptor association. For these initial studies of IL4 receptor
function we primarily used the human IL4-responsive premyeloid cell
line TF-1
(32) . Moreover, we sought to determine the importance
of cytoplasmic regions of IL4R
Immunoblotting of anti-phosphotyrosine immunoprecipitated
proteins revealed that JAK3 was the predominant protein inducibly
tyrosine phosphorylated in TF-1 cells after 5 min of IL4 treatment
(Fig. 1). Specifically, IL4 stimulated the tyrosine phosphorylation of
a protein migrating with an apparent molecular mass of 116 kDa by
SDS-polyacrylamide gel electrophoresis (Fig. 1, lane b),
and this protein was recognized by anti-JAK3 serum (Fig. 1,
lane j). Moreover, the 116-kDa protein comigrated with
inducibly phosphorylated JAK3 which had been immunoprecipitated with
anti-JAK3 serum (Fig. 1, lane d), as well as with
positive controls of immunoprecipitated and immunoblotted JAK3
(Fig. 1, lanes k and l). These results
corresponded well with our previous demonstration of IL2-induced
tyrosine phosphorylation of JAK3
(11) . Phosphorylated JAK3 was
not readily detectable in total cell lysates (Fig. 1, lanes g and h), demonstrating the necessity of amplifying the
signal by prior immunoprecipitation. The IL4-induced tyrosine
phosphorylation of JAK3 was time- and dose-dependent as judged from
parallel anti-phosphotyrosine immunoblots of either anti-JAK3 or
anti-phosphotyrosine immunoprecipitates from lysates of stimulated TF-1
cells (Fig. 2). Peak phosphorylation levels were reached after
5-10 min of IL4 stimulation (Fig. 2 A) and with an
EC
A comparison
of the ability of the engineered IL4R
The present study verified that IL4 may induce parallel
tyrosine phosphorylation and activation of JAK1 and JAK3, but found
that the recruitment of individual JAK enzymes by IL4 in human TF-1
cells was highly skewed in favor of JAK3. These results therefore did
not favor the concept of equimolar transphosphorylation and activation
of JAK1 and JAK3 following IL4-induced heterodimerization of IL4R
The
overall quality and efficiency of the anti-JAK1 and anti-JAK3 sera used
for these experiments were comparable, justifying semiquantitative
assessments to be made. In addition, this evaluation was in part based
on methods that did not rely directly on anti-JAK sera, including the
dominant degree of JAK3 tyrosine phosphorylation observed in
anti-phosphotyrosine immunoprecipitated material from IL4-stimulated
cells (Figs. 1 and 2), and the detectable catalytic activation of JAK3,
but not JAK1, in biotin-IL4 receptor complexes
(Fig. 6 B). Neither of these two approaches is biased
toward the detection of one specific JAK enzyme. The possibility that
the antiserum to the cytoplasmatic domain of IL2R
Another question of significance for understanding the mechanism of
JAK activation by IL4 is to determine the basis for the interdependence
of IL4R
Mutational analyses of IL4R
One principal mechanism of signal transduction
subsequent to JAK kinase activation and phosphorylation of tyrosine
residues of cytoplasmic receptor domains and JAK molecules, is the
secondary recruitment of a variety of phosphotyrosyl-binding effector
proteins with SRC-homology 2 (SH2) domains
(36) . However, it
has been argued that tyrosine residues of the IL4R
It also interesting that tyrosine
kinase activity from IL4-stimulated cells spontaneously associates with
I4R-containing fusion proteins
(30) , indicating that tyrosine
kinases other than JAKs may also be activated by IL4. Since an
involvement of SRC family tyrosine kinases have been implicated in
signaling downstream of JAK enzymes by several hematopoietin receptors,
it is possible that SRC kinases or other SH2 domain kinases such as FES
(14) associate with IRS-1 or 4PS, and may indeed phosphorylate
these docking proteins.
The present paper has demonstrated that a
membrane-proximal region of human IL4R
We thank Dr. Richard Robb for providing generous
amounts of monoclonal anti-human IL2R
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-chain
(IL2R
), and evidence has been found for a preferential coupling of
JAK3 to IL2R
and JAK1 to IL2R
. Here we show, using human
premyeloid TF-1 cells, that IL4 stimulates JAK3 to a larger extent than
JAK1, based upon three different evaluation criteria. These include a
more vigorous tyrosine phosphorylation of JAK3 as measured by
anti-phosphotyrosine immunoblotting, a more marked activation of JAK3
as determined by in vitro tyrosine kinase assays and a more
manifest presence of JAK3 in activated IL4- receptor complexes. These
observations suggest that IL4 receptor signal transduction does not
depend on equimolar heterodimerization of JAK1 and JAK3 following
IL4-induced heterodimerization of IL4R
and IL2R
. Indeed, when
human IL4R
was stably expressed in mouse BA/F3 cells, robust
IL4-induced proliferation and JAK3 activation occurred without
detectable involvement of JAK1, JAK2, or TYK2. The present study
suggests that JAK1 plays a subordinate role in IL4 receptor signaling,
and that in certain cells exclusive JAK3 activation may mediate
IL4-induced cell growth. Moreover, mutational analysis of human
IL4R
showed that a membrane-proximal cytoplasmic region was
critical for JAK3 activation, while the I4R motif was not, which is
compatible with a role of JAK3 upstream of the recruitment of the
insulin receptor substrate-1/4PS signaling proteins by IL4 receptors.
(
)
is a T-cell derived
growth factor and differentiation agent that acts on a variety of
cells, including T cells, B cells, thymocytes, mast cells, and
granulocytes
(1) . IL4 signals across the cell membrane via two
receptor proteins which belong to the hematopoietin receptor
superfamily, the unique IL4R
which is exclusive to IL4, and the
shared IL2R
, which is also used by IL2, IL7, IL9, and IL15
(2, 3, 4, 5, 6) . All of these
cytokines are lymphoid growth factors, but each factor may also induce
distinct and even opposing effects in certain cells. For example, IL4
has been shown to inhibit IL2-induced proliferation in peripheral blood
mononuclear cells, monoclonal B-cells, natural killer cells, and
TALL-103/2 cells
(7, 8, 9, 10) .
Dissimilar cellular impact of these individual cytokines can most
likely be attributed to recruitment of separate intracellular effector
proteins by their unique receptor chains, while shared functions may be
mediated by the common receptor subunit.
,
including IL2, IL4, IL7, and IL9
(4, 11, 12, 13) .
(
)
In addition, JAK1 but not JAK2 or TYK2 has also been found
to undergo tyrosine phosphorylation in response to these cytokines in T
cells and NK cells
(4) .
Similar paired activation
of different JAK enzymes has been shown for interferons and IL6-related
ligands
(15, 16) . A series of initial studies have
indicated that cytokines which cause homodimerization of their
respective receptors are able to signal via a single form of JAK,
i.e. JAK2 by erythropoietin, growth hormone, and prolactin
(17, 18, 19) , while cytokines which induce
heterooligomerization of at least two distinct receptor subunits may
rely on the activation of more than one JAK kinase
(20) .
Genetic complementation studies of interferon receptor signaling have
shown that simultaneous involvement of two separate JAKs is indeed
required for signal to occur
(21, 22) . It has been
proposed that ligand-induced hetero- or homooligomerization of receptor
subunits cause preassociated JAK molecules to hetero- or
homooligomerize, resulting in intermolecular transphosphorylation and
activation in a fashion similar to that reported for receptor tyrosine
kinases
(20) .
(5)
and JAK1 for IL2R
(5, 23) .
On the other hand, comparison of the relative involvement of JAK1 and
JAK3 in IL2 receptor signaling in human T cells and NK cells showed a
disproportionately higher recruitment of JAK3 than JAK1, and did not
favor the concept of equimolar stoichiometry of the two enzymes in the
IL2 receptor complex.
Indeed, semiquantitative analysis
indicated at least a 10-fold higher recruitment of JAK3 over JAK1 by
IL2 receptors. It is therefore currently unclear whether both JAK
enzymes are needed for signal transduction via IL2R
-containing
receptor complexes.
has
been shown to be essential for IL2-induced JAK3 activation and growth
signal
(26) . Similarly, mutational mapping of the IL4R
has
indicated that a cytoplasmic region of 130-140 membrane-proximal
amino acids is indispensible for IL4-induced growth signaling
(27, 28, 29) . This part of IL4R
contains
the proline-rich homology box 1 and two conserved acid-rich elements.
Intriguingly, a second separate motif has been shown to be important
for the growth-stimulatory effect of IL4
(27, 30) . This
motif contains a positionally conserved tyrosine residue that is also
found in the receptors for insulin and insulin-like growth factor-I and
has been designated I4R, because it couples IL4R
to the insulin
receptor substrate-1 (IRS-1) or its homologue, 4PS
(30) .
However, the relative contribution of the I4R-motif to growth induction
by IL4 in different cell lines may largely depend on the cellular
expression levels of IRS-1 or 4PS
(30, 31) .
for IL4-induced JAK tyrosine
phosphorylation and activation, and to what extent JAK activation
correlated with mitogenesis. Functional testing of a series of human
IL4R
variants with internal deletions was carried out by stable
expression in murine lymphoblastoid BA/F3 cells, which consequently
proliferate in response to human IL4
(27) .
Materials
Polyclonal rabbit antisera to
synthetic peptides derived from human/mouse JAK1, mouse JAK2, mouse
JAK3, and human TYK2 sequences were purchased from UBI (catalog no.
06-272, 06-255, 06-342, and 06-275, respectively). These antibodies
recognize both human and mouse forms with the exception of the
anti-mouse JAK3 serum, and each could be used for immunoprecipitation
and immunoblotting independent of the phosphorylation state of the JAK
enzymes. Antiserum to human JAK3, previously named L-JAK, was raised
against a peptide corresponding to the 20 COOH-terminal amino acids
(amino acids 1104-1124) which are unique to human JAK3
(33) . We also generated polyclonal rabbit antibodies against an
eight-residue peptide corresponding to the COOH terminus of human
IL2R (NH
-CYTLKPET-COOH) and affinity purified the
antibodies as described by others
(4) . Monoclonal mouse
anti-phosphotyrosine antibodies were purchased from UBI (4G10; catalog
no. 05-321) and anti-human IL2R
(561) was a generous gift
from Dr. Richard Robb
(34) . Biotinylation of IL4 was carried
out by incubating 1 mg of human IL4 (PeproTech, Rock Hill, NJ) in 1 ml
of carbonate buffer (pH 8.5) with 1 m
M NHS-LC-biotin (Pierce,
catalog no. 21335) for 4 h at room temperature. The reaction was
stopped by addition of 20 µl of 1
M NH
Cl and
uncoupled biotin was removed by dialysis against phosphate-buffered
saline.
Cell Culture and Treatment
Human T lymphocytes
from normal donors were grown in RPMI 1640 medium containing 10% fetal
calf serum (Sigma, catalog no. F2442), 2 m
M
L-glutamine, 5 m
M HEPES buffer (pH 7.3), and
penicillin-streptomycin (50 IU/ml and 50 mg/ml, respectively), while
the human erythroleukemic cell line, TF-1, was grown in the same medium
supplemented with 5 ng/ml granulocyte macrophage-colony stimulating
factor. T lymphocytes were activated for 72 h with phytohemagglutinin
(1 µg/ml) and were subsequently made quiescent by washing and
incubating for 24 h in RPMI 1640 medium containing only 1% fetal calf
serum before exposure to cytokines, while TF-1 cells were made
quiescent in 5% gelded horse serum (Sigma, catalog no. H1885). The
IL3-dependent murine BA/F3 cell line containing the IL4R mutants
were grown in RPMI 1640 medium with 10% fetal calf serum supplemented
with 0.8 mg/ml geneticin sulfate (G-418 sulfate, Life Technologies,
Inc.), 10 m
M HEPES and 2% WEHI-3B supernatant as a source of
IL3. Cells were normally stimulated with 100 n
M recombinant
human IL4 (PeproTech, Rock Hill, NJ) at 37 °C as indicated in the
corresponding figure legends. Cell pellets were frozen at -70
°C.
Solubilization of Membrane Proteins and
Immunoprecipitation
Frozen cells were thawed on ice and
solubilized in lysis buffer (10cells/ml) containing 10
m
M Tris-HCl (pH 7.6), 5 m
M EDTA, 50 m
M NaCl,
30 m
M sodium pyrophosphate, 50 m
M sodium fluoride,
200 µ
M sodium orthovanadate, 1% Triton X-100, 1
m
M phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 1
µg/ml pepstatin A, and 2 µg/ml leupeptin. Cell lysates were
rotated end over end at 4 °C for 60 min, and insoluble material was
pelleted at 12,000
g for 20 min. Depending on the
experiment, supernatants were incubated rotating end over end for 2 h
at 4 °C with anti-phosphotyrosine monoclonal antibody (4G10; 4
µg/ml), anti-IL2R
monoclonal antibody (561; 5 µg/ml),
normal rabbit serum or polyclonal sera to individual JAK enzymes (5
µl/ml). Antibodies were captured by incubation for 30 min with
protein A-Sepharose beads. Cells incubated with biotinylated IL4 were
lysed and treated as described above and biotin-IL4 was subsequently
captured with streptavidin-agarose beads (Life Technologies, Inc.,
BRL). For immunoblotting, anti-phosphotyrosine and anti-IL2R
antibodies were used at 1 µg/ml, while all anti-JAK sera were used
at a dilution of 1:1000. Precipitated material was eluted by boiling in
SDS sample buffer for 4 min, subjected to 7.5% SDS-polyacrylamide gel
electrophoresis under reducing conditions, and transferred to
polyvinylidene difluoride membrane (Immobilion, Millipore, catalog no.
1PVH 00010). Immunoblot analysis was performed as described previously
(35) .
Tyrosine Kinase Assays
JAK1 and JAK3
immune-complex tyrosine kinase assays were carried out by incubating
the individually immunoprecipitated tyrosine kinases from lysates of
unstimulated and IL4-stimulated cells in the presence and absence of
ATP, and visualizing incorporated phosphate on tyrosines by
immunoblotting. Receptor complex tyrosine kinase assays were carried
out in a similar manner, but were based on capture of activated
receptor complexes from cell lysates by means of either anti-IL2R
antibodies or biotinylated IL4. Immobilized proteins were washed three
times with lysis buffer followed by a single wash with kinase buffer
containing 25 m
M HEPES (pH 7.3), 0.1% Triton X-100, 100
m
M NaCl, 10 m
M MgCl
, 3 m
M
MnCl
, and 200 µ
M sodium orthovanadate.
Isotope-free tyrosine kinase reactions were initiated by the addition
of 15 µ
M unlabeled ATP and allowed to incubate at 37
°C for 15 min. The reactions were quenched by washing the
streptavidin-agarose or protein A-Sepharose beads with lysis buffer and
eluting bound material by boiling in SDS sample buffer for 4 min. The
material in each lane represents immunoprecipitates from approximately
2
10
cells unless specified otherwise.
Proliferation Assay
Quiescent BA/F3 cells (2.5
10
/well) were plated in flat-bottom 96-well
microtiter plates in starvation medium (100 µl final) in the
presence of various concentrations of human IL4 or IL3. Cells were
pulsed for 4 h with [
H]thymidine (0.5 µCi/100
µl) after 24 h of stimulation, harvested onto glass fiber filters,
and analyzed for [
H]thymidine uptake by liquid
scintillation counting.
value of 1-10 n
M (Fig. 2 B).
Figure 1:
IL4-induced tyrosine phosphorylation of
JAK3 in human TF-1 cells. Anti-phosphotyrosine ( PY;
lanes a-h) or anti-JAK3 (
JAK3; lanes i-p)
immunoblots of two parallel sets of protein samples from lysates of
human TF-1 cells which had been stimulated without (-) or with
(+) 100 n
M IL4 for 10 min at 37 °C. Individual lanes
represent immunoprecipitates ( IP) with anti-phosphotyrosine
(
PY; lanes a, b, i, and j), anti-JAK3
(
JAK3; lanes c, d, k, and l), control antibodies
( CTRL; lanes e, f, m, and n), or total cellular
lysates ( TCL; lanes g, h, o, and p). Arrow denotes JAK3 and bracket indicates immunoglobulin heavy
chains ( IgG
). Molecular size markers are
indicated on the left (kDa).
Figure 2:
Time kinetics and concentration dependence
of JAK3 tyrosine phosphorylation induced by IL4 in human TF-1 cells.
Anti-phosphotyrosine immunoblots of proteins that had been
immunoprecipitated with either anti-phosphotyrosine ( PY)
or anti-JAK3 antibodies (
JAK3) from lysates of TF-1 cells
(1
10
cells/lane) which had been stimulated with
100 n
M hIL4 from 0 to 20 min ( Panel A) or with
varying concentrations of hIL4 (0-100 n
M) for 10 min
( Panel B). Arrows denote JAK3 and brackets indicate immunoglobulin heavy chains ( IgG
).
Molecular size markers are indicated on the left (kDa).
Anti-phosphotyrosine immunoprecipitates in these first two
experiments did not indicate the presence of significant quantities of
a second IL4-modulated JAK of higher molecular weight than JAK3.
However, specific testing with antibodies to other known JAK kinases
showed that JAK1 was also phosphorylated to a certain extent in TF-1
cells in response to IL4 (Fig. 3, lane d). In contrast, the
phosphorylation state of JAK2 and TYK2 was not modulated by IL4. Since
the anti-JAK2 serum shows partial cross-reactivity with the faster
migrating JAK3
(5) , phosphorylated JAK3 was detected to some
extent in the JAK2 immunoprecipitates from IL4-stimulated cells (Fig.
3, lane f). Moreover JAK2, but not TYK2, for unknown reasons
showed high basal tyrosine phosphorylation levels in quiescent TF-1
cells (Fig. 3, lanes e and f).
Figure 3:
IL4-induced tyrosine phosphorylation of
JAK1 and JAK3 but not JAK2 or TYK2 in human TF-1 cells.
Anti-phosphotyrosine ( PY) immunoblot of TF-1 cells (1
10
cells/lane) which had been incubated in the
absence (-) or presence (+) of 100 n
M hIL4 for 10
min at 37 °C. Lysates were immunoprecipitated with anti-JAK3
(
JAK3; lanes a and b), anti-JAK1 (
JAK1;
lanes c and d), anti-JAK2 (
JAK2; lanes e and f), or anti-TYK2 (
TYK2; lanes g and
h) antibodies. Arrows denote JAK1, JAK2, or JAK3, and
bracket indicates immunoglobulin heavy chains
( IgG
). Molecular size markers are indicated on
the left (kDa).
We next
sought to establish the relative presence of JAK kinases in activated
IL4-receptor complexes. Since no antibody was available that recognized
IL4-complexed human IL4R, we used biotinylated IL4 (biotin-IL4) to
assess indirectly the binding of JAK3 and JAK1 to IL4 receptors.
Initial testing of the biotin-IL4 showed that it retained its
bioactivity and induced tyrosine phosphorylation of JAK3 and JAK1 to
the same extent as unconjugated IL4 (data not shown). While JAK3
coprecipitated with biotin-IL4 from lysates of cells after
immobilization on streptavidin-agarose beads and subsequent
visualization by anti-JAK3 immunoblotting (Fig. 4 A, lanes b and d), biotin-IL4 receptor complexes did not contain
detectable amounts of JAK1 (data not shown). This observation combined
with the observed lower degree of IL4-induced tyrosine phosphorylation
of JAK1 indicated a quantitatively lower recruitment of JAK1 than JAK3
by IL4. Biotin-IL4 complexes also contained IL2R
as demonstrated
by immunoblotting of parallel samples with anti-IL2R
antibodies
(Fig. 4 B, lane b) in agreement with previous reports
(2, 4) .
Figure 4:
Association of JAK3 with the IL4-receptor
complex. Panel A, anti-JAK3 immunoblot of proteins captured
with streptavidin-agarose beads from lysates of TF-1 cells (2
10
cells/lane) which had been incubated with (+) or
without (-) 100 n
M biotinylated hIL4 (bIL4) for 10 min
( lanes a and b) or 30 min ( lanes c and
d) at 37 °C. Panel B, anti-IL2R
immunoblot
of proteins captured with streptavidin-agarose beads from lysates of
TF-1 cells treated with bIL4 for 10 min ( lane b).
Anti-IL2R
immunoprecipitated material from human YT cells treated
with IL2 provided a positive control ( lane d). Lanes a and c represent untreated control cells ( C).
IL2R
chain is denoted by arrow, bracket indicates immunoglobulin heavy chains ( IgG
).
Molecular size markers are indicated on the left (kDa).
The effect of IL4 on the catalytic
activities of JAK1 and JAK3 in immune-complex autokinase assays was
also evaluated. Several studies have revealed a close correlation
between cytokine-induced tyrosine phosphorylation and catalytic
activation of JAK kinases
(12, 13, 16, 17, 18, 19) .We here extend this correlation to include IL4-modulated JAK3 and
JAK1 in TF-1 cells by demonstrating that both JAK kinases were
activated by IL4 (Fig. 5, lanes a-d). Although a certain
degree of basal JAK3 activity was present in unstimulated cells
(Fig. 5, lane b), a significant increase in phosphate
incorporation on tyrosine residues was seen when JAK3 from
IL4-stimulated cells was incubated with ATP in vitro (Fig. 5, lanes c and d). Control
immunoprecipitates were negative (Fig. 5, lanes e-h),
and reprobing of the blots with JAK3 antibody verified equal loading
(data not shown). In order to detect IL4-induced JAK1 autokinase
activity in vitro, three times as many cells had to be used,
so that immunoprecipitates from as many as 6
10
cells per lane had to be loaded (Fig. 5 A, lanes
i-l). A similar need for higher cell numbers had also been
required for the detection of IL2-induced JAK1 activity in YT
cells.
These results provided a third line of evidence for
a quantitatively lower involvement of JAK1 in IL4 receptor signaling.
Moreover, we observed comparably disproportionate activity levels of
JAK3 and JAK1 in IL4-stimulated human T-lymphocytes and several
lymphoid cell lines, including HuT-101, YT, A-301, rat Nb2-11C,
and mouse CTLL-2,
(
)
suggesting that a skewed
recruitment of JAK3 over JAK1 is a widespread phenomenon in IL4 target
cells.
Figure 5:
IL4-induced activation of JAK3 analyzed by
autophosphorylation assay using unlabeled ATP and anti-phosphotyrosine
immunoblotting. Anti-phosphotyrosine immunoblot of immunoprecipitated
lysates of TF-1 cells, which had been incubated with (+) or
without (-) 100 n
M IL4 for 5 min at 37 °C. Anti-JAK3
( JAK3; lanes a-d) or control antibody ( CTRL; lanes
e-h) immunoprecipitates were washed and subsequently incubated for
20 min at 37 °C in the absence (-) or presence (+) of 15
µ
M unlabeled ATP. For detection of JAK1 activity,
immunoprecipitates from 6
10
cells were loaded per
lane, in contrast to 2
10
cells per lane for JAK3
and CTRL. Arrows denote JAK1 and JAK3. Molecular size markers
are indicated on the left (kDa).
The presence of catalytically active JAK enzymes in activated
IL4 receptor complexes was also assessed, using either anti-IL2R
antibodies or biotin-IL4 to purify receptor complexes for in vitro tyrosine kinase assays. IL4-stimulated the in vitro activity of JAK3 which could be coprecipitated with anti-IL2R
antibodies, whereas no detectable JAK1 activity was observed (Fig.
6 A, lane d). Parallel analysis of biotin-IL4 receptor
complexes revealed similar selective detection of JAK3 activity
(Fig. 6 B, lane d). The identity of the 116-kDa protein
as JAK3 was verified by reprobing of the stripped blot with anti-JAK3
serum (data not shown). The absence of detectable amounts of
catalytically active JAK1 from IL4-receptor complexes represents
another indication of a subordinate role for JAK1 in IL4 signaling.
Figure 6:
Detection of JAK3 autokinase activity in
IL4 receptor complexes. Panel A, anti-phosphotyrosine
immunoblot of proteins coprecipitating with IL2R captured with
protein A-Sepharose beads from lysates of TF-1 cells which had been
treated treated with (+) or without (-) 100 n
M IL4
and for 5 min at 37 °C, subsequently incubated in the absence
(-) or presence (+) of 15 µ
M unlabeled ATP.
Arrow denotes JAK3 and molecular size markers are indicated on
the left (kDa). Panel B, anti-phosphotyrosine
immunoblot of proteins captured with streptavidin-agarose beads from
lysates of TF-1 cells which had been treated treated with (+) or
without (-) 100 n
M biotinylated IL4 ( bIL4) for
5 min at 37 °C, and subsequently incubated for 20 min at 37 °C
in the absence (-) or presence (+) of 15 µ
M
unlabeled ATP. Arrow denotes JAK3 and molecular size markers
are indicated on the left (kDa).
To analyze the role of the cytoplasmic domain of human IL4R in
the IL4-induced activation of JAK enzymes, we tested a set of
previously described IL4R
variants with systematic internal
deletions which had been individually introduced in the murine
IL3-dependent lymphoblastoma line BA/F3
(27) . The human
IL4R
can form functional complexes with the murine IL2R
and
is able to mediate proliferative signals in these cells
(27, 28, 29) . The structures of wild type
IL4R
and the mutant variants
Cyt,
R1,
R2,
R3,
and
R4, are reviewed in Fig. 7 A. Previous Scatchard
analysis of the various cloned cell populations revealed between 2,000
and 14,000 binding sites for IL4 per cell
(27) .
variants to mediate
IL4-stimulated thymidine incorporation in BA/F3 cells is shown in
Fig. 7B. In parental BA/F3 cells and each of the
sublines expressing IL4R
, IL3 induced comparable increases in
thymidine incorporation after 24 h of stimulation. However, only cells
expressing wild type,
R4,
R3, and
R2 forms of IL4R
responded with IL4-induced thymidine incorporation. The data shown in
Fig. 7 B represent maximal incorporation levels induced by 1
n
M IL4, and repeated dose-response studies ( n = 3) established that the four active receptor variants had
similar EC
values of 5-50 p
M IL4 (data not
shown). Of the four functional forms of IL4R
, wild type,
R4,
and
R2 mediated average IL4 responses that were 52, 67, and 39% of
the corresponding IL3-induced effects, respectively. In contrast,
IL4-induced proliferation mediated by
R3, which lacks the I4R
motif, was as low as 13% of the corresponding IL3-induced response.
These results are compatible with the suboptimal IL4-induced thymidine
incorporation observed in several BA/F3 clones which were stably
expressing another I4R-deficient form of human IL4R
, the P-mutant
reported by Harada and colleagues
(29) . This P-mutant only
included the first 176 cytoplasmic amino acids of the IL4R
.
However, BA/F3 cells expressing
R3 did not survive prolonged
culture in IL4 in the absence of IL3 as reported previously
(27) .
Figure 7:
Ability of IL4R mutants to mediate
IL4-induced thymidine incorporation. Panel A, structural
review of the various human IL4R
mutants that have been stably
introduced into the murine IL3-dependent BA/F3 cell line. The wild type
( wt) receptor was not modified, while
Cyt lacks the
entire cytoplasmic domain. The internal deletion mutants
R1,
R2,
R3, and
R4 are defined by the following excluded
segments which are indicated in the sketch:
Ile
-Ser
(
R1),
Ser
-Thr
(
R2),
Thr
-Aal
(
R3), and
Ala
-Thr
(
R4), respectively. The
diagram also depicts the relative position of homology box 1 ( Box
1), acidic domains 1 ( Acid1) and 2 ( Acid2), as
well as conserved tyrosine residues. Panel B, thymidine
incorporation assay comparing the ability of the various IL4R
variants to mediate IL4-induced proliferation. Quiescent cells (2
10
cells/well) were incubated with or without 1
n
M human IL4 or murine IL3 for 24 h, followed by addition of
[
H]thymidine (0.5 µCi/well) for 4 h, before
harvesting and quantification of the incorporated thymidine. Results
are expressed as counts/min, reflecting incorporation into 2
10
cells. BA/F3 indicates the parental cell line,
whereas the denotation of the IL4R
constructs are used to indicate
the various stably transfected BA/F3
subclones.
Subsequent analyses ( n = 4) of the
ability of the IL4R variants to mediate IL4-induced tyrosine
phosphorylation of JAK3 demonstrated that wild type,
R4,
R3,
and
R2 were equally efficient, while the growth-defective mutants
R1 and
Cyt mediated no JAK3 phosphorylation. The results from
one representative experiment is shown in Fig. 8. Intriguingly,
repeated analyses failed to detect any IL4-induced tyrosine
phosphorylation of a second JAK enzyme, including JAK1, JAK2, or TYK2,
in these BA/F3 cells (data not shown), raising the possibility that IL4
receptors under certain circumstances may signal via only one JAK
kinase. Moreover, the ability of wild type and mutant forms of
IL4R
to mediate IL4-induced JAK3 tyrosine phosphorylation
coincided with their ability to mediate catalytic activation of JAK3,
as assessed by in vitro JAK3 immune-complex tyrosine kinase
assay (Fig. 9).
and IL2R
, which has been proposed as a generic activation model
for cytokine receptor complexes
(20) . Our conclusion of a
predominant involvement of JAK3 in IL4 receptor signaling was based
upon several independent evaluation criteria. These included a
preferential tyrosine phosphorylation of JAK3 as measured by
anti-phosphotyrosine immunoblotting, a more marked activation of JAK3
as determined by in vitro tyrosine kinase assays, and a more
manifest presence of JAK3 in activated IL4 receptor complexes.
might
selectively displace JAK1 and not JAK3 from the activated IL4 receptor
complex (Fig. 6 A) is unlikely in light of the recent
demonstration that in the IL2 receptor complexes JAK3 associates with
IL2R
and JAK1 with IL2R
(5) . We have also observed
similar predominant JAK3 recruitment by IL4 in activated human T cells
and several lymphoid cell lines (data not shown), indicating that this
phenomenon is not specific to TF-1 cells. Moreover, a more extreme
imbalance of JAK activation was observed in murine BA/F3 cells
expressing human IL4R
. In these cells IL4 induced detectable
tyrosine phosphorylation and activation exclusively of JAK3, and not of
JAK1, JAK2, or TYK2. Nonetheless, IL4 induced robust proliferation in
IL4R
-expressing BA/F3 cells, suggesting that activation of JAK3
alone is sufficient to mediate growth signal by IL4 receptors. However,
it is possible that the assay is not sensitive enough to detect minor,
but essential phosphorylation of JAK1 in these cells. Specific
immunoblotting did indeed reveal the presence of low amounts JAK1 in
BA/F3 cells (data not shown), but it cannot at present be excluded that
the human IL4R
does not recognize mouse JAK1. Parallel
anti-phosphotyrosine immunoblotting of anti-phosphotyrosine
immunoprecipitated proteins from IL4-stimulated BA/F3 cells did not
indicate any other modulated protein than JAK3 in the size range of
known JAK kinases (110-150 kDa; data not shown), arguing against
the possibility that a novel JAK kinase might be activated in these
cells. Reconstitution of IL4 receptors in JAK1-deficient cells will
help to determine the importance of JAK1 in IL4 receptor signaling.
and IL2R
. As mentioned previously, a preferential
coupling of JAK3 to IL2R
has been proposed
(5, 11)
and a membrane-proximal region of
IL2R
is critical for JAK3 activation by IL2
(26) . In the
present study we demonstrate that a membrane-proximal region of 132
amino acids of IL4R
is also critical for the mediation IL4-induced
JAK3 activation. In agreement with previous reports, this region is
essential for IL4-induced growth signal. Interestingly, a corresponding
membrane-proximal region of the IL2R
has recently been shown to be
of similar importance for JAK3 activation by IL2
(13) . These
observations therefore suggest that although the common IL2R
may
serve as the principal interaction partner for JAK3, the cytoplasmic
domains of the unique components of IL4 and IL2 receptor complexes,
i.e. IL4R
and IL2R
, are also indispensible for JAK3
activation. Studies directed toward identifying the exact localization
and nature of the interaction sites between receptors and JAKs will be
needed to understand how JAK kinases become activated by cytokines.
have previously shown that a second
more distally located motif of the cytoplasmic domain of IL4R
is
important for IL4-induced growth signal transduction
(27, 30) . This motif has been designated I4R, and
couples IL4R
to the IRS-1 or 4PS signaling proteins
(30) .
The present study found that selective deletion from IL4R
of a
region containing the I4R motif did not affect JAK3 activation, but
caused a profound reduction in IL4-induced proliferation as measured by
thymidine incorporation assay. However, a residual growth promotional
activity remained that was approximately 22% of the response mediated
by wild type IL4R
, suggesting that a JAK-activated
IRS-1/4PS-independent proliferative pathway exists. In support of this
view, two previous studies have shown moderate IL4-induced thymidine
incorporation in BA/F3 cells mediated by human IL4R
mutants that
had been truncated above the I4R-motif
(28, 29) . On the
other hand, since
R3 expressing cells do not survive long-term
culture (>1 week) in medium containing only IL4
(27) , it
appears that this residual IL4-stimulated growth signal mediated by the
R3 mutant is incapable of overcoming the apoptotic drive induced
by IL3 depletion.
are not
essential to IL4-induced signal transduction, based upon the ability of
a tyrosine-deficient truncated form of IL4R
to mediate IL4-induced
thymidine incorporation
(28) . The present paper supports the
view that at least a portion of the IL4-induced growth signal may not
depend on phosphorylated tyrosine residues of IL4R
or the I4R
motif, suggesting that recruitment of some growth-inducing SH2 domain
proteins may occur directly via phosphorylated tyrosine residues of
IL2R
or JAK3. However, it has been shown that IL4R
becomes
tyrosine phosphorylated after IL4 stimulation
(37) , and it is
evident that the I4R motif contributes significantly to an enhanced
IL4-induced growth signal, both in BA/F3 cells as shown in the present
work (Fig. 7 B) and in 32D cells
(31) . Indeed,
overexpression of IRS-1 in 32D cells dramatically increased the growth
induction by IL4 in these cells
(30) . It is possible that
Tyr-472 of the I4R motif is phosphorylated by JAK3 and serves as a
docking site for IRS-1 via the SH2 domain of an unidentified adaptor
protein, since IRS-1 does not itself contain an SH2 domain. It will
therefore be interesting to determine whether a Y472F substitution will
abolish the recruitment of IRS1 and 4PS by IL4R
. Other conserved
tyrosine residues in the cytoplasmic domain of IL4R
may interact
selectively with different SH2 domain proteins upon phosphorylation,
and contribute to cell-specific effects of IL4 that do not overlap with
those induced by IL2 and IL7.
is essential for
IL4-induced activation of the tyrosine kinase JAK3 in murine myeloid
BA/F3 cells. In these cells, no detectable tyrosine phosphorylation of
other JAK kinases than JAK3 was induced by IL4, although robust
IL4-stimulated cell proliferation was observed. In human T lymphocytes
and human TF-1 cells minor, but consistent, IL4-induced tyrosine
phosphorylation of JAK1 was found in addition to the more dominant JAK3
phosphorylation. We have recently observed similar disproportionate
tyrosine phosphorylation and activation of JAK1 and JAK3 by IL2 in
human T cells and YT cells.
Whereas interferon receptor
signal transduction may depend on the simultaneous presence of two
distinct JAK kinases
(21, 22) , the present work
suggests that IL4 receptors can mediate growth signal solely through
JAK3 activation. This study therefore does not support a symmetric
model of interdependent and equimolar transactivation of JAK3 and JAK1
associated with each of the heterodimerizing IL4R
and IL2R
.
Ongoing studies are addressing the qualitative and quantitative roles
of individual JAK enzymes in IL4 receptor signal transduction.
/
, IL2 receptor
/
; IL4R
, IL4 receptor
; JAK, Janus kinase.
and Terry Williams for
expert technical help with the preparation of the figures. We also
acknowledge the support and critical review of the manuscript by Dr.
Joost Oppenheim and Dr. Dan Longo.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.