From the Laboratory of Molecular Immunology, NHLBI, National
Institutes of Health, Bethesda, Maryland 20892
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INTRODUCTION |
Interleukin-2 (IL-2)1 is
the principal growth factor for T lymphocytes and is responsible for
regulating the magnitude and duration of the T cell immune response
following antigen encounter (1-4). Three classes of IL-2 receptors
exist, binding IL-2 with low (Kd = 10
8
M), intermediate (Kd = 10
9
M), and high (Kd = 10
11
M) affinity. The low affinity receptors contain only the
IL-2 receptor
chain (IL-2R
); intermediate affinity receptors
contain IL-2R
and the common cytokine receptor
chain,
c; and high affinity receptors contain all three chains
(3, 4). The intermediate and high affinity receptors are the functional
forms, and heterodimerization of the IL-2R
and
c
cytoplasmic domains is necessary and sufficient for IL-2 signaling
(5-7). The highly inducible
chain has a very short cytoplasmic
domain (8, 9) and presumably mainly functions to increase the affinity
for IL-2, allowing cellular responsiveness to the low levels of IL-2
that are physiologically present in vivo. In contrast,
IL-2R
and
c have longer cytoplasmic domains that can
associate with a number of signaling molecules, allowing the activation
of signaling pathways (2-4). Stimulation of lymphocytes with IL-2
results in the rapid activation of the Janus family tyrosine kinases,
Jak1 and Jak3 (10-14). Activated Jaks are critical for inducing rapid
tyrosine phosphorylation of downstream substrates, including STATs
(signal transducers and activators of transcription), which then
dimerize, translocate into the nucleus, and regulate the transcription
of target genes (4, 13-15).
It has been reported that IL-2R
and
c constitutively
associate with two of the four Jak family kinases in a selective
manner, IL-2R
with Jak1 and
c with Jak3 (10, 16, 17).
The S region (amino acids 267-322) of IL-2R
has been shown to be
important for Jak1 association (17). In addition to its ability to
constitutively interact with Jak1, although it is not well appreciated,
IL-2R
can also associate with Jak3 following IL-2 stimulation of
lymphoid cells (10), but the regions of interaction between IL-2R
and Jak3 have not previously been investigated.
A number of membrane proximal cytoplasmic point mutants of IL-2R
that diminish IL-2-induced proliferation have been identified (18-20).
We found that these mutants also diminish IL-2-induced STAT protein
activation and the association of both Jak1 and Jak3 with IL-2R
.
This led us to further characterize the regions of IL-2R
required
for the binding of Jak1 and Jak3, and we demonstrate that membrane
distal as well as membrane proximal regions of IL-2R
are vital for
Jak kinase interaction. Moreover, the association between Jak3 and
IL-2R
is Jak1-independent and both Jak3 and Jak1 can be
coprecipitated only in the presence of IL-2R
. Finally, we provide
evidence indicating that the association between IL-2R
and Jak3 is
important for potent Stat5 activation in response to IL-2 and, thus,
that more than one IL-2R
-Jak kinase interaction is involved in IL-2
signaling.
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MATERIALS AND METHODS |
Constructs and in Vitro Mutagenesis--
The IL-2R
constructs
with point mutations in the cytoplasmic chain were prepared using an
in vitro mutagenesis kit (5 Prime-3 Prime, Inc.) and
wild-type IL-2R
as the template. The oligonucleotides used for
mutagenesis were as follows (mutant nucleotides are underlined): 5'-AAGTGTAACACCTCAGACCCCTCG-3' (for P257S);
5'-GTAACACCCCAGCGCCCTCGAAGTTC-3' (for D258A);
5'-GACGTCCAGAAGGGGCTCTCTTCG-3' (for W277G); and
5'-CTGGCACCTGAGATATCGCCAGCAGAAGTGCTGGAG-3' (for L299A).
Successful mutagenesis was confirmed by DNA sequencing.
Wild-type IL-2R
or IL-2R
constructs containing internal deletion
or truncation mutations in its cytoplasmic domain (see Fig.
3A), were cloned in the expression vector, pME18S, in which transcription is directed by the SR
promoter. pME18S also contains the SV40 origin of replication and is expressed at high copy number in
either 293T+ or COS-7 cells, both of which express the SV40
large T antigen. Internal deletion mutants of IL-2R
(
A,

S) were generated by loop-out mutagenesis using single-stranded
M13 bacteriophage as a template. IL-2R
truncation mutants (
379,
371,
362,
350,
330,
313,
300,
290, and
267)
were prepared using the polymerase chain reaction to amplify
BclI to XbaI fragments of IL-2R
with premature
termination codons, followed by subcloning into pME18S-IL-2R
in
which the BclI to XbaI fragment was excised. The
IL-2R
construct with four tyrosines changed to phenylalanines
(
FFFFYY) was described previously (21).
The murine Jak1 cDNA in pMLCMV was provided by Dr. J. Ihle; the
human Jak3 cDNA was provided by Dr. J. O'Shea. Jak3 was subcloned in pME18S. Wild-type
c (
c-wt) and
truncated mutant of
c (
c-
CT) were
previously described (22, 23). Human Stat5a and Stat5b cDNAs (24) were
cloned into pCi (Promega).
Cell Lines and Transfections--
COS-7 cells (ATCC),
293T+ cells (provided by Dr. N. Rice, National Cancer
Institute), and E1C3 cells (Jak1-deficient HeLa cells, provided by Dr.
R. Flavell, Yale University) were cultured in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum, 2 mM
glutamine, 100 units/ml each of penicillin and streptomycin. Transient
transfections were performed using either DEAE-dextran (for COS-7
cells) or calcium phosphate (for 293T+ and E1C3 cells)
methods. For immunoprecipitation experiments, cells were transfected in
150-mm dishes using 2-3 µg of each plasmid. Transfectants were
harvested 36-48 h later. For experiments in which IL-2-induced STAT
DNA binding activity was reconstituted, cells were transfected in
100-mm dishes using 1-2 µg of each plasmid, and nuclear extracts
were made 36-48 h later.
32D cells were maintained in RPMI 1640 medium supplemented with 10%
fetal bovine serum, 5% from WEHI-3B cells conditioned medium (WEHI-CM)
as a source of IL-3, 2 mM glutamine, and 100 units/ml each
of penicillin and streptomycin. Stable transfectants expressing
wild-type or mutant forms of IL-2R
were created by electroporation
using a Gene Pulser (Bio-Rad) at 300 V and 960 microfarads. Cells
(5 × 106/condition) were cotransfected with
linearized IL-2R
constructs and pcDNA3neo. 24 h after
electroporation, cells were aliquoted into 24-well plates using medium
containing 1 mg/ml G418 (Life Technologies, Inc.). Resistant clones
were stained for IL-2R
expression with fluorescein
isothiocyanate-conjugated anti-p75 (IL-2R
) monoclonal antibody
(Endogen) and analyzed on a FACSort (Becton-Dickinson).
Reagents and Antibodies--
Anti-IL-2R
hMik
1 (humanized
Mik
1; Ref. 25) or anti-Jak3 antibody (provided by J. O'Shea) and
protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) were used for
immunoprecipitation. Immunoblots were performed using antibodies to
Jak1 (Transduction Labs and Santa Cruz Biotechnology), Jak3, or
IL-2R
(goat anti-human IL-2R
, R&D Systems).
Immunoprecipitation and Western Blotting--
COS-7,
293T+, or E1C3 transient transfectants (one 150-mm culture
dish) or 32D stable transfectants (5-10 × 106 cells)
were harvested by washing with phosphate-buffered saline and lysed with
lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 0.4 mM Na3VO4,
1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride
hydrochloride, 1 mM leupeptin, 1 mM aprotinin).
IL-2R
-Jak3 or Jak3-IL-2R
-Jak1 associations were assessed by
immunoprecipitation with hMik
1 or anti-Jak3 at 4 °C for 1-2 h.
Samples were washed four times with lysis buffer, analyzed on 8 or 10%
SDS-polyacrylamide gel electrophoresis (NOVEX), transferred to
Immobilon-P membranes (Millipore Corp.), immunoblotted with different
antibodies, and developed with ECL (Amersham Pharmacia biotech or
Pierce).
Preparation of Nuclear Extracts and Electrophoresis Mobility
Shift Assays (EMSAs)--
Extracts were prepared from
293T+ or COS-7 transfectants (cells from one 100-mm culture
dish) or from 32D cells (1 × 107 cells) that were
starved of growth factor for 4 h in RPMI 1640 medium and treated
with 2 nM IL-2 for 30 min at 37 °C. Cells were washed
with ice-cold phosphate-buffered saline, nuclear extracts were prepared
as described previously (26), and 1 µg of protein from
293T+ or COS-7 transfectants or 5-10 µg of protein from
32D cells were used in EMSAs. For EMSAs, 1 µg of poly(dI-dC) was used
as a nonspecific competitor and 15,000 cpm of 32P-labeled
double-stranded oligonucleotide containing a trimer of the GAS sequence
(5'-AGATTTCTAGGAATTC-3') from the
-casein promoter (a motif capable
of binding IL-2-activated STAT proteins) was used as the probe. The
reactions were separated on 6% polyacrylamide gels in 0.5 × Tris
borate-EDTA and autoradiographed.
Thymidine Incorporation Assays--
32D cells were washed and
starved of growth factor for 4 h in RPMI medium. Cells were
aliquoted at 2-4 × 104 cells/well in a 96-well plate
in triplicate in 200 µl of medium or medium containing 2 nM IL-2 or 5% WEHI-CM. After 20 h of incubation at
37 °C, 1 µCi of 3H-labeled thymidine (NEN Life Science
Products) was added, and the cells were incubated at 37 °C for
4 h. Cells were harvested using a cell harvester (Tom Tec), and
thymidine incorporation was assayed using a Betaplate 1205 counter
(Wallac). For each transfectant, at least three clones with similar
IL-2R
expression were assayed.
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RESULTS |
IL-2R
Point Mutants That Affect Proliferation Diminish
IL-2-induced Stat5 DNA Binding Activity--
Four IL-2R
mutants,
including P257S (proline 257 replaced by serine), D258A, W277G, and
L299A, have been reported to impair IL-2-induced proliferation in Ba/F3
or MOLT4 cells (18-20) even though they exhibit similar surface
expression and IL-2 binding affinities (19). We sought to investigate
the basis for the decreased proliferation of these mutants. We first
made stable transfectants of each of these mutants in 32D cells and
confirmed similar cell surface expression by flow cytometry (Fig.
1A). As expected, we confirmed
that these mutants mediated greatly diminished proliferation, as
compared with wild-type IL-2R
, in 32D cells, which lack IL-2R
but
can proliferate in response to IL-2 after IL-2R
is transfected and
expressed (Refs. 21 and 27; Fig. 1B). Moreover, each of
these mutations also diminished IL-2-induced STAT binding activity in
transfected 32D cells (Fig. 1C) as well as in transiently
transfected COS-7 cells (Fig. 1D). In 32D cells, previous
studies indicate that the IL-2-induced STAT binding activity is due to
Stat5 rather than Stat3 (28). For the COS-7 cell experiments, cells
were transfected with
c, Jak3, Stat5a, Stat5b, and the different IL-2R
constructs using a system previously shown to reconstitute IL-2-induced Stat5 DNA binding activity with
wild-type IL-2R
(24).

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Fig. 1.
IL-2R mutants containing individual
substitutions at Pro-257, Asp-258, Trp-277, or Leu-299 each exhibit
diminished IL-2-induced proliferation and STAT activation.
A, flow cytometric analyses indicating IL-2R expression
of representative 32D cell clones transfected with wild-type or mutant
IL-2R constructs. The mean fluorescent intensity for each construct
is indicated. B, diminished proliferation by IL-2R
mutants. Untransfected 32D cells (32D), 32D cells
transfected with mutant IL-2R constructs (P257S, D258A, W277G, and
L299A), or wild-type IL-2R construct ( wt) were treated
with medium alone (open bars), 2 nM IL-2
(stippled bars), or 5% WEHI-CM as a source of IL-3
(hatched bars). 3H-labeled thymidine
incorporation was determined. Three independent stable transfectants
with similar expression levels of IL-2R (selected by flow cytometry
with fluorescein isothiocyanate-conjugated anti-IL-2R monoclonal
anitbody and similar IL-2R expression confirmed by Western blotting
with a polyclonal anti-IL-2R antiserum) were assayed for each
construct. C and D, defective STAT binding
activity in the cells transfected with IL-2R mutants P257S, D258A,
W277G, and L299A. C, untransfected 32D cells or stable 32D
transfectants expressing either wild-type or mutant IL-2R constructs
were washed, starved in medium without any growth factors, and then
left untreated (lanes 1, 3, 5, 7, 9, and 11) or
treated with 2 nM IL-2 for 15 min (lanes 2, 4, 6, 8, 10, and 12). D, COS-7 cells were transfected
with Stat5a, Stat5b, Jak3, c, and either wild-type or
mutant forms (P257S, D258A, W277G, and L299A) of IL-2R . Two days
after transfection, nuclear extracts were prepared from cells not
treated (lanes 1, 3, 5, 7, 9, and 11) or treated
with 2 nM IL-2 for 15 min (lane 2, 4, 6, 8, 10,
and 12). C and D, EMSAs were performed
using the -casein probe.
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IL-2R
Point Mutants Also Exhibit Diminished Association with
Both Jak1 and Jak3--
Given the diminished STAT activation and that
each of these mutations are contained in a region of IL-2R
where Jak
kinase interactions might be affected (Box B1/Box B2 region, see Refs. 29-32), we tested if these mutations diminished the association of
Jak1 or Jak3 as a possible explanation for the decreased IL-2 signaling. Because the IL-2R
-Jak3 interaction is only well seen in T
cells following IL-2 stimulation, we used an overexpression system to
map the regions of IL-2R
that mediate association with Jak1 and
Jak3. COS-7 cells were transfected with Jak1 or Jak3 and IL-2R
mutants, cells were lysed, and lysates were immunoprecipitated with
hMik
1 antibody to IL-2R
and then blotted with antibodies to
IL-2R
(Fig. 2A), Jak1 (Fig.
2B), or Jak3 (Fig. 2C). Jak1 and Jak3 each
exhibited less binding to each of the IL-2R
mutants than to
wild-type IL-2R
(Fig. 2, B and C).

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Fig. 2.
IL-2R mutants containing individual
substitutions at Pro-257, Asp-258, Trp-277, or Leu-299 decreased
interaction with Jak1 and Jak3. A, similar expression levels
for the mutant and wild-type IL-2R . Cell lysates were
immunoprecipitated with hMik 1 and then Western blotted with
anti-IL-2R . Cell surface expression of each IL-2R construct was
confirmed by flow cytometry (data not shown). B, the P257S,
D258A, W277G, and L299A IL-2R point mutants exhibit decreased
association with Jak1. COS-7 cells were transfected with Jak1, Jak3,
and either mutant or wild-type IL-2R , were immunoprecipitated with
hMik 1, and then were Western blotted with anti-Jak1 (top
panel). Lysates were Western blotted with anti-Jak1 to confirm the
expression of Jak1 in different transfectants (bottom
panel). C, the P257S, D258A, W277G, and L299A point
mutations in IL-2R also resulted in decreased association of Jak3.
The blots in panel C were stripped and reblotted with
anti-Jak3.
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The Regions of IL-2R
Required for Jak1 and Jak3 Binding
Partially Overlap--
Because each of the IL-2R
point mutations
interfered with the association of both Jak1 and Jak3, we hypothesized
that the regions of IL-2R
that were important for Jak kinase
interaction might be similar. To investigate this possibility and to
map the regions of IL-2R
involved in the binding of both Jak
kinases, COS-7 cells were transiently transfected with Jak1, Jak3, and wild-type IL-2R
or a series of IL-2R
truncation mutants (Fig. 3A). Cells were lysed, and
lysates were immunoprecipitated with anti-IL-2R
antibody, followed
by Western blotting with antibodies to IL-2R
(to control for
expression and the efficiency of immunoprecipitation, Fig.
3B), Jak1 (Fig. 3C), or Jak3 (Fig.
3D). IL-2R
truncation mutants retaining 350 (
350
construct) or more residues (
362,
371, and
379 constructs)
could bind efficiently to Jak1 (Fig. 3C);
330 and
313
bound to Jak1 weakly; whereas
300,
290, and
267 could not bind
to Jak1. Therefore, the region between residues 300 and 350 of IL-2R
is important for its interaction with Jak1. In contrast, the region
between residues 330 and 362 was important for the Jak3-IL-2R
interaction, given that there was efficient coprecipitation of Jak3
with
362 but no detectable coprecipitation of Jak3 with
330 even
at longer exposure times (Fig. 3D and data not shown).
Wild-type IL-2R
and IL-2R
mutants retaining the first 362, 371, or 379 amino acids could associate with both Jak1 and Jak3. These
results in COS-7 cells were confirmed using 293T+ cells
(data not shown). Thus, the 300-350 and 330-362 regions of IL-2R
are important for Jak1 and Jak3 binding, respectively (summarized below
in Fig. 8).

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Fig. 3.
Interaction of Jak1 and Jak3 with
non-identical regions of IL-2R . A, schematic of IL-2R
and its truncated mutants showing the extracellular domain,
transmembrane (TM) domain, and cytoplasmic domains. In the
cytoplasmic domain, the S, A, Box B1, and Box B2 regions are shown.
B, expression of wild-type and truncated forms of IL-2R .
COS-7 cells were transfected with Jak1, Jak3, and either wild-type
IL-2R ( wt) or truncated mutants of IL-2R , were
immunoprecipitated with hMik 1, and then were Western blotted with
anti-IL-2R . Cell surface expression of each IL-2R construct was
confirmed by flow cytometry (data not shown). C, importance
of the amino acids 300 to 350 region of IL-2R for Jak1 binding.
Top panel, lysates from COS-7 cells transfected with Jak1,
Jak3, and either wild-type or mutant forms of IL-2R were
immunoprecipitated with hMik 1 and then Western blotted with
anti-Jak1. Bottom panel, the lysates were Western blotted
with anti-Jak1 to confirm the expression of Jak1 in different
transfectants. D, importance of the amino acids 330 to 362 region of IL-2R for Jak3 binding. The blots described in panel
C were stripped and reblotted with anti-Jak3.
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We next tested the effect of internal deletions of the S region
(residues 267 to 322) or the A region (residues 313 to 382) on the
binding of Jak1 (Fig. 4A) and
Jak3 (Fig. 4B). Deletion of the S region (
S) resulted
in a dramatic decrease in IL-2R
association with Jak1, consistent
with previously reported results (17), whereas deletion of the A region
only modestly decreased Jak1 association (Fig. 4A). In
contrast to the findings for Jak1, deletion of the A region had a much
greater effect on the association of Jak3 than did deletion of the S
region (Fig. 4B). Thus, the A region of IL-2R
is more
important for Jak3 association, whereas the S region is more important
for Jak1 association. Consistent with the data in Fig. 3, these data
indicate that Jak3 binding extends to a more distal region of the
IL-2R
cytoplasmic domain than does Jak1. Therefore, the data
contained in Figs. 3 and 4 demonstrate that Jak1 and Jak3 interact with
different, albeit overlapping regions of IL-2R
.

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Fig. 4.
The A and S regions of IL-2R are
differentially important for binding Jak3 and Jak1, respectively.
A, deletion of either the A or S regions of IL-2R
diminished the association of Jak1. COS-7 cells were transfected with
Jak1, Jak3, and either mutant or wild-type IL-2R , were
immunoprecipitated with hMik 1, and then were Western blotted with
anti-Jak1. Lysates were Western blotted with anti-Jak1 to confirm the
expression of Jak1 in different transfectants. B, deletion
of the A region of IL-2R more greatly diminished the association
with Jak3 than did the S region. The blots described in panel
A were stripped and reblotted with anti-Jak3. C,
similar expression levels for wt,  A, and  S. The cell
lysates were immunoprecipitated with hMik 1 and then Western blotted
with anti-IL-2R . Cell surface expression of each IL-2R construct
was confirmed by flow cytometry (data not shown). D,
association of IL-2R with Jak1 and Jak3 does not depend on the
phosphorylation of the tyrosine residues located on the A region. Cells
were transfected with Jak1 + Jak3 + either wt, FFFFYY, or pME18S.
Cell lysates were immunoprecipitated with hMik 1 and then blotted
with either anti-Jak3 (top panel), anti-Jak1 (middle
panel), or anti-IL-2R (bottom panel). The bands
corresponding to Jak3, Jak1, and IL-2R are indicated.
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As the A region contains four tyrosines (Tyr-338, Tyr-355, Tyr-358, and
Tyr-361), we evaluated the ability of Jak3 to associate with IL-2R
containing mutations in these tyrosines (IL-2R
FFFFYY). As shown in
Fig. 4D, Jak3 efficiently associated with this mutant, indicating that the interaction does not depend on phosphorylated tyrosine residues.
Jak3 Can Bind to IL-2R
in Jak1-deficient HeLa Cells--
Given
that Jak1 is ubiquitously expressed, it was possible that the
interaction of Jak3 with IL-2R
required Jak1. To investigate this
possibility, we transfected Jak1-deficient HeLa cells (E1C3 cells) with
Jak3 + wild-type IL-2R
± Jak1. Transfected cells were lysed and
immunoprecipitated with hMik
1, followed by blotting with an
antiserum to Jak3. We found that IL-2R
and Jak3 could interact even
in the absence of Jak1, and the presence of Jak1 did not enhance this
interaction (Fig. 5A,
first two lanes). The uniformity of expression of Jak3,
Jak1, and IL-2R
was verified by immunoblotting with appropriate
antibodies (Fig. 5B). We also used E1C3 cells to map the
region of IL-2R
required for its interaction with Jak3, and
confirmed the findings reported above in Figs. 3 and 4 (data not
shown).

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Fig. 5.
Jak3 can interact with IL-2R in the
absence of Jak1. A, HeLa cells lacking Jak1 (E1C3 cells)
were transfected with wt or pME18S and Jak3, Jak1, or Jak3 + Jak1.
Cell lysates were immunoprecipitated with hMik 1 and then blotted
with either anti-Jak3. B, lysates of the E1C3 transfectants
were Western blotted with anti-Jak3, anti-Jak1, or anti-IL-2R to
confirm the expression levels of transfected cDNAs.
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Jak1 and Jak3 Can Only Be Coprecipitated in the Presence of
IL-2R
--
Because the association between Jak3 and IL-2R
was
Jak1-independent, and Jak1 could be coprecipitated with IL-2R
in the absence of Jak3, we next investigated whether Jak1 and Jak3 could be
coprecipitated through IL-2R
. COS-7 cells were transfected with
Jak1, Jak3, and either pME18S, wild-type IL-2R
, or IL-2R
deletion
constructs (
A, 
S, and
350) that were missing regions important for the interaction of either Jak1 and/or Jak3 (see Figs. 3
and 4). Coprecipitation of Jak3 and Jak1 required IL-2R
(Fig.
6A, lane 2 versus lane 1);
this association was markedly decreased when the 
A, 
S, or
350 mutants were used instead of wild type IL-2R
(lanes
3-5), further confirming that the association between Jak1 and
Jak3 is dependent on the presence of IL-2R
.

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Fig. 6.
Jak3 can associate with Jak1 in the presence
of wild-type IL-2R . COS-7 cells were transfected with Jak1,
Jak3, and either wild-type IL-2R , mutant IL-2R , or pME18S. Cell
lysates were immunoprecipitated with anti-Jak3 and then blotted with
anti-Jak1 (A). The blot was stripped and reblotted with
anti-Jak3 to control the immunoprecipitation of Jak3 (B).
The expression of Jak1 is shown in (C).
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Association between IL-2R
and Jak3 Is Required for IL-2-induced
Stat5 DNA Binding Activity--
It has previously been shown that
disruption of the Jak1-IL-2R
interaction diminished IL-2 signaling
(2). To investigate the functional significance of the association
between IL-2R
and Jak3, we used Jak3-deficient 293T+
cells in which IL-2-induced Stat5 DNA binding activity could be
reconstituted following transfection with IL-2R
,
c,
Jak3, Stat5a, and Stat5b (Fig. 7A,
lanes 5 and 6; Fig. 7B, lanes 1 and 2). Previous studies indicate the vital role of Jak3 for
IL-2-induced STAT activation (24). Both IL-2R
and
c
were required since little, if any, IL-2-induced Stat5 DNA binding
activity was seen in the absence of either
c (Fig.
7A, lanes 1 and 2) or IL-2R
(Fig. 7A,
lanes 3 and 4). However, a truncated form of
c (
c-
CT) that is missing 80 of 86 amino acids of the
c cytoplasmic domain and contributes to IL-2
binding (22, 23) but does not interact with Jak3 (10) still allowed
partial IL-2-induced DNA binding activity (Fig. 7B, lanes 3 and 4). This activity was diminished when Jak3 (Fig.
7B, lanes 5 and 6) was deleted, implicating the IL-2R
-Jak3 interaction as being important for STAT activation.

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Fig. 7.
Reconstitution of Stat5 DNA binding
activation in 293T+ cells. Jak3 association with
IL-2R is required for IL-2-induced Stat5 activation. A,
293T+ cells were transfected with IL-2R , Jak3, Stat5a,
and Stat5b (lanes 1 and 2); c,
Jak3, Stat5a, and Stat5b (lanes 3 and 4); or
IL-2R , c, Stat5a and Stat5b (lanes 5 and
6). 36-48 h after transfection, cells were either not
treated (lanes 1, 3, and 5) or treated with 2 nM IL-2 for 30 min (lanes 2, 4, and
6), and nuclear extracts were made. EMSAs were performed
using the -casein probe. B, 293T+ cells were
transfected with IL-2R , c, Jak3, Stat5a and Stat5b
(lanes 1 and 2); IL-2R ,
c- CT, Jak3, Stat5a, and Stat5b (lanes 3 and 4); or IL-2R , c- CT, Stat5a, and
Stat5b (lanes 5 and 6). IL-2 treatment, nuclear extracts,
and EMSAs were performed as described in panel A.
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DISCUSSION |
IL-2 signaling requires the dimerization of both IL-2R
and
c. As Jak1 has been shown to associate with IL-2R
and
Jak3 with
c, an attractive model has been that each
receptor chain associates with a different Jak family kinase in a
selective manner and that IL-2-mediated activation of Jak1 and Jak3
initiates a signaling cascade(s). It is well established that the
c-Jak3 interaction (10) and Jak3 activation (33, 34) are
vital for signaling. We now provide evidence that Jak3 and IL-2R
can
associate with each other in a Jak1-independent fashion. The fact that
IL-2R
provides interaction sites for Jak3 as well as Jak1 (see Figs. 8 and 9)
suggests that a function of
c might be not only to
recruit Jak3 but also to facilitate the "delivery" of Jak3 to
IL-2R
. Moreover, the ability of Jak3 to associate with both IL-2R
and
c suggests that Jak3 might stabilize the receptor
complex and promote downstream signaling. Our studies on the
reconstitution of IL-2-induced Stat5 activation in 293T+
cells provide evidence that the full activation of Stat5 requires IL-2R
association with both Jak1 and Jak3, and that the heretofore poorly appreciated IL-2R
-Jak3 association has physiological
significance.

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Fig. 8.
Regions of IL-2R important for the
association of Jak1 and Jak3. The extracellular, transmembrane
(TM), and cytoplasmic domains are shown. In the cytoplasmic
domain, the Box B1, Box B2, S region, and A region are shown on the
left. On the right are shown the regions and
residues important for the association of Jak1 and Jak3.
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Fig. 9.
Schematic model showing an important role for
Jak3 association with IL-2R for IL-2-induced Stat5 activation in
both transfected 293T+ cells (A) and
IL-2-dependent cells (B). A, in
293T+ cells, Jak3 associates with both IL-2R and
c in order to achieve full Stat5 activation upon IL-2
stimulation (shown as ++++ on the left). When the truncated
c, which cannot bind Jak3, was present instead of
wild-type c, Jak3 associates with IL-2R ; in this
setting, there is still Stat5 activation, albeit decreased (shown as ++
in the middle). Finally, in the absence of Jak3, there is
very little Stat5 activation (shown as +/ on the right).
B, in IL-2-dependent cells, IL-2R and
c form a heterodimer after IL-2 stimulation. Jak3 binds
both IL-2R and c, perhaps stabilizing the receptor
complex, allowing for potent downstream signaling.
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We have now delineated regions on IL-2R
that are important for the
interaction of Jak1 and Jak3. We show that four point mutations in the
Box B1/Box B2 region of IL-2R
that diminished proliferation also
decreased the binding of both Jak1 and Jak3. This is consistent with
the important role of this region of a number of type I cytokine
receptors for Jak interaction (14, 29-32). Interestingly, however,
analysis of a series of deletion and truncation mutants not only
demonstrated differences in the regions of IL-2R
that mediate
recruitment of Jak1 versus Jak3, but unexpectedly also
provided evidence that regions more distal than previously suspected
play major roles in the recruitment of the Jak kinases (see Fig. 8). To
our knowledge, these data represent the most detailed mapping on a
cytokine receptor of the region/residues involved in Jak kinase
association. Previously, for all cytokine receptors studied, including
IL-2R
, only the membrane proximal and Box1/Box2 regions have been
shown to be important for the association of Jak kinases; thus, our
findings have implications regarding the interaction sites of Jak
kinases for other type I cytokine receptors as well. Although some
receptor chains, such as
c, appear to be uniquely
associated with a single Jak, the gp130 signal transducing receptor
component that is shared by the receptors for IL-6, IL-11, leukemia
inhibitory factor, ciliary neurotrophic factor, oncostatin M, and
cardiotrophin-1, can associate with more than one Jak. gp130 has been
reported to associate with Jak1, Jak2, and Tyk2 (35, 36), but it
remains unknown whether these three Jak family kinases serve completely distinctive roles and how they associate with gp130. Our data therefore
provide the first example wherein more than one Jak (Jak1 and Jak3) can
independently interact with a single receptor molecule (IL-2R
) via
overlapping but non-identical regions.