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,
,,§
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Virginia Mason Research Center, Seattle, WA 98101;
Fred Hutchinson Cancer Research Center, Seattle, WA 98104; and Departments of
Immunology and
§
Medicine, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-2Rß, and 
c chains (1),
with the consequent heterodimerization of IL-2Rß and c inducing
the proliferative signal (2, 3). c contributes to
proliferative signaling by recruiting the tyrosine kinase
Jak3(4, 5, 6, 7, 8), the activation of which also requires a
membrane-proximal S region of IL-2Rß (9, 10).
Additionally, at least one of three cytoplasmic tyrosine
phosphorylation sites (Tyr338, Tyr393, or
Tyr510) distal to the S region on IL-2Rß must be present
for proliferation, suggesting that obligate mitogenic signaling
molecules interact with these phosphotyrosines (11, 12).
Indeed, the adapter molecule Shc interacts with Tyr338
(11, 13) and can deliver a proliferative signal
(14). The other two tyrosines, Tyr510 and
Tyr393, provide docking sites for the transcription factor
Stat5 (11), which has led to the hypothesis that Stat5
mediates a proliferative signal parallel to the one involving Shc.
However, Shc and Stat5 have very different biochemical properties,
which raises the question of whether they could indeed generate
redundant proliferative signals from IL-2Rß. In addition, the fact
that a single phosphotyrosine on a receptor chain can interact with
multiple signaling molecules, as exemplified by studies of the
platelet-derived growth factor receptor (15), raises the
possibility that a factor other than Stat5 mediates the proliferative
signal from Tyr393 and/or Tyr510. Stat5 refers to either of two highly homologous members of the larger Stat family of signal-transducing activators of transcription, Stat5a and Stat5b (16, 17, 18). Stat activation conventionally commences with selective binding of a Stat SH2 domain to a phosphorylated tyrosine motif on a ligated receptor (19, 20, 21, 22), approximating the Stat molecule with a receptor-associated, activated Janus kinase which phosphorylates a key tyrosine residue on the Stat molecule (23, 24). These Stat phosphotyrosines preferentially bind to Stat SH2 domains (21), causing Stat molecules to dimerize, release from receptors, enter the nucleus, and bind palindromic DNA sequences (22) in certain promoters (25, 26, 27), inducing gene transcription via a C-terminal trans-activation domain (TAD)3 (28, 29, 30, 31). Some members of the Stat family also demonstrate less conventional activities. Stat3, for example, serves as an adapter molecule between PI3 kinase and type I IFN receptors (32). Furthermore, Stat1 and, in cooperation with the glucocorticoid receptor, Stat5 mediate the expression of certain genes through a TAD-independent mechanism (33, 34, 35). Thus, not all signaling functions of Stat molecules involve the conventional trans-activation domain.
A critical role for Stat5 in IL-2-mediated mitogenesis has been suggested by the failure of TCR-stimulated T cells from mice lacking both the Stat5a and Stat5b genes to proliferate in response to IL-2 (36). However, the IL-2R can mediate proliferation in the absence of detectable Stat5 activation through alternative mitogenic signaling molecules, such as Shc (11, 14). Indeed, IL-2Rß mutants that fail to activate Stat5 mediate proliferation in cultured cell lines (11, 37, 38), and primary T cells (39). Thus, the inability of Stat5-null T cells to proliferate may reflect a recently described essential role for Stat5 in TCR signaling (40) rather than a requisite role in IL-2 signaling.
This report assesses the role of Stat5 in signals derived from the IL-2R in cell lines that do not require TCR stimulation for proliferation. A receptor mutagenesis and rescue strategy was used to study signaling by Stat5 in the absence of any redundant signals from Shc or other molecules potentially associating with IL-2Rß. We demonstrate that Tyr510 of IL-2Rß mediates proliferation through the activation of Stat5 and that either the Stat5a or the Stat5b isoform can transduce the mitogenic signal. Furthermore, proliferative signaling by Stat5 is critically dependent on its TAD, indicating that Stat5a mediates proliferation through its conventional role as a transcription factor. Indeed, IL-2-induced transcription of the proliferative and survival genes c-myc, bcl-2, and bcl-x is mediated by Stat5a through the TAD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression vectors encoding the chimeric - and
ßß-chains (formerly denoted GM/2 and GMß/2ß) under the
control of the human ß-actin promoter have been described previously
(2, 9, 14, 41). Mutants of ßß were generated by
annealing sense and antisense oligonucleotides encoding novel
C-terminal sequences for ßß and/or premature stop codons, and
cloning overhanging ends of these annealed primers between a unique
AflII site in the cytoplasmic domain of ßß and a unique
XbaI site immediately 3' to the stop codon of ßß. For
analyses in BA/FG cells, the full length, nonchimeric human IL-2Rß
cDNA was cloned into the expression vector used above, and the C
terminus of this chain, between the AflII site and a unique
3' ScaI site, was then replaced with the respective mutated
sequences. The 
713 mutant of Stat5a was generated by PCR with an
antisense oligonucleotide encoding residues 708 to 712 of Stat5a
followed by the flag epitope, a stop codon, and an EcoRV
site. This PCR product was then cut with XhoI and
EcoRV enzymes and ligated between these same sites in a
vector containing murine Stat5a, described previously
(29). 713, as well as C-terminally flag epitope-tagged
wild-type (wt) murine Stat5a and Stat5b, were next excised with
EcoRI and HindIII and cloned between
SalI and HindIII sites in the ß-actin
promoter-driven plasmid used above for receptor expression. All mutated
Stat5a and IL-2Rß regions were sequenced with the ABI Prism dye
terminator cycle sequencing kit (Perkin-Elmer, Norwalk, CT).
Cell culture and transfection
The murine T cell line CTLL-2 was obtained from the American
Type Culture Collection (Manassas, VA) and maintained as described
previously (14). BA/F3 pro-B cells were obtained from
Immunex (Seattle, WA) and maintained in RPMI supplemented with 10%
FCS, 2 mM L-glutamine, 50U/ml penicillin, and 50 mg/ml
streptomycin, as well as 10% WEHI3-conditioned medium as a source of
murine IL-3. To grow BA/F3 cells in the absence of Stat5 activation,
the cells were transfected with a chimeric G-CSFR/gp130 receptor chain
described previously (42) and maintained with 100 ng/ml
recombinant human G-CSF (Amgen, Thousand Oaks, CA) instead of IL-3. The
resultant cell line is referred to hereafter as BA/FG. Linearized
plasmids were introduced into cells by electroporation, and
transfectants were selected for resistance to G418 (Life Technologies,
Gaithersburg, MD) in 96-well plates at limiting dilution to isolate
independent subclones. Receptor expression was assessed by flow
cytometry with Abs to human GM-CSFR or ßc (Santa Cruz
Biotechnology, Santa Cruz, CA), or to IL-2Rß (PharMingen, San Diego,
CA). Stat5 expression was assessed by Western blot with an Ab to Stat5
(Transduction Labs, Lexington, KY), or by flow cytometry of cells
stained intracellularly with an Ab to the flag epitope (Sigma, St.
Louis, MO). Subclones with comparable receptor and/or Stat5 expression
were chosen for further analyses.
Proliferative assays
Thymidine incorporation assays were conducted in triplicate
wells with 104 cells/well exposed to the
indicated doses of GM-CSF, IL-2, G-CSF, or IL-3 for 24 h, with
[3H]thymidine (2.5 µCi/well) present during
the last 4 h. Cells were harvested onto glass fiber filters, and
DNA synthesis was quantified by liquid scintillation counting. The data
presented in
Figs. 1–4
used 100 ng/ml GM-CSF or 3000 U/ml IL-2, which
were found to elicit a maximal response from all functional receptor
mutants in CTLL-2 or BA/F3 cells, respectively.
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Jak1 and Jak3 were immunoprecipitated and subjected to kinase assays as described previously (9). Whole cell lysates were generated by boiling cells in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM DTT, and 0.1% bromphenol blue. Lysates or immunoprecipitates were electrophoresed on acrylamide gels and transferred to nitrocellulose. Nitrocellulose blots were blocked with 0.1 M Tris base (pH 7.5), 0.9% sodium chloride, 0.05% Tween 20 (TTBS) containing 1% BSA (for antiphosphotyrosine probes) or 5% powdered skim milk (Carnation, Glendale, CA), and probed with rabbit antisera recognizing Jak1 (Santa Cruz Biotechnology) or Jak3 (Upstate Biotechnology, Lake Placid, NY) or murine Abs recognizing phosphotyrosine (4G10; Upstate Biotechnology) or Stat5 (Transduction Laboratories, Lexington, KY). Blots were then washed with TTBS, probed with peroxidase-conjugated goat anti-rabbit or anti-mouse Abs (Life Technologies), and washed again with TTBS. Bound Abs were detected by enhanced chemiluminescence (DuPont NEN, Boston, MA). Blots were stripped between probings with a 30-min, 50°C incubation in 62.6 mM Tris-HCl (pH 6.7), 0.1 M ß-mercaptoethanol, and 2% SDS.
Electrophoretic mobility shift assays
Cells stimulated as indicated were washed once with buffer H (20
mM HEPES (pH 7.9), 1 mM EDTA, 0.1 mM EGTA, 2 mM magnesium chloride, 1
mM sodium o-vanadate, 20 mM sodium fluoride, 1 mM DTT, 0.1
mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 1 mg/ml leupeptin) and
lysed in buffer H plus 0.2% Nonidet P-40 at 0°C. Nuclei were
pelleted by centrifugation and extracted with buffer K (buffer H plus
0.42 M sodium chloride and 20% v/v glycerol). To generate a probe for
Stat activity, incompletely overlapping oligonucleotides corresponding
to the sense and antisense strands of the Stat-responsive DNA element
from the FcR1 promoter were annealed, radiolabeled with
[-32P]dCTP by an end-filling T4 polymerase
reaction, and purified with a MicroSpin G-25 column (Pharmacia,
Piscataway, NJ). The probe was added to nuclear extracts in 50 mM
potassium chloride, 10 mM HEPES (pH 7.9), 10% glycerol, 1 mM DTT, and
87.5 mg/ml dITP/dCTP at room temperature for 30 min in the presence or
absence of either an Ab recognizing the flag epitope (Sigma) or a
combination of Abs recognizing Stat5a and Stat5b (Santa Cruz
Biotechnology). Reaction mixtures were electrophoresed on a nonreducing
0.25x Tris-buffered EDTA-acrylamide gel which was then dried and
subjected to autoradiography.
Northern blots
Cells stimulated as indicated were pelleted by centrifugation
and flash-frozen in a dry ice-ethanol bath. RNA was harvested from
thawed pellets with the RNA Stat 60 kit (Tel Test, Friendswood, TX);
denatured for 10 min at 65°C in 20 mM MOPS, 5 mM sodium acetate, 0.5
mM EDTA, 2.4 M formaldehyde, and 50% formamide; and run on a 1.2%
agarose gel (containing 20 mM MOPS, 5 mM sodium acetate, 0.5 mM EDTA,
and 1.1 M formaldehyde) in 20 mM MOPS, 5 mM sodium acetate, and 0.5 mM
EDTA at pH 7.0. RNA was passively transferred to Zetabind membranes
(Cuno, Meriden, CT) with 10x SSC (1.5 M sodium chloride, 0.15 M sodium
citrate, pH 7.0), and UV cross-linked. Blots were prehybridized at
43°C in hybridization buffer (1 M sodium phosphate (pH 7.1), 2 mM
EDTA, 2% BSA, 10% SDS, 50% formamide, and 0.16 mg/ml yeast tRNA or
herring sperm DNA). To generate nucleic acid probes, a 0.4-kb
PstI fragment of murine c-myc, a 0.9-kb
PstI fragment of murine bcl-2, a 1-kb
EcoRI fragment of murine bcl-x, and a 1.2-kb
PstI fragment of murine GAPDH cDNA were
radiolabeled with [-32P]dCTP using a
random-primed labeling kit (Boehringer Mannheim, Indianapolis, IN) and
purified with Centri-Sep spin columns (Princeton Separations, Adelphia,
NJ). Probes were boiled for 5 min and added to blots in hybridization
buffer. After overnight incubation at 43°C, blots were washed 2–3
times with 2x SSC, 0.1% SDS; once with 0.2x SSC, 0.1% SDS at room
temperature; and 0–2 times with 0.2x SSC, 0.1% SDS at 55°C before
autoradiography. Blots were stripped between probings with a 2-min
immersion in boiling water.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Structure/function analyses of the cytoplasmic domain of IL-2Rß
were performed in the IL-2-dependent murine T cell line CTLL-2. To
avoid activation of the endogenous IL-2R, we introduced a previously
described chimeric GM-CSF/IL-2 receptor consisting of two chains,
and ßß, containing the extracellular domains of the human
GM-CSF receptor - and ß-chains fused, respectively, to the
transmembrane and intracellular regions of c and IL-2Rß. When
coexpressed, and ßß deliver in response to human GM-CSF a
signal that is biochemically and physiologically indistinguishable from
that induced in the same cell by the wt IL-2 receptor (2, 9, 14, 41). A CTLL-2 clone demonstrating stable high expression of
was generated and transfected with wt or mutated derivatives of
ßß (Figs. 1
A and
2B). Subclones of transfectants were analyzed for receptor
expression by flow cytometry (data not shown), and those expressing
both and ßß-chains at comparable levels were chosen for
further study. The wt version of ßß delivered a proliferative
signal in response to GM-CSF similar to that mediated by the endogenous
IL-2R. By contrast, a version of ßß from which all cytoplasmic
tyrosines had been removed by introduction of a premature stop codon at
codon 325 (relative to the human IL-2Rß sequence) (ßß325 (Fig. 1A)) failed to mediate proliferation (Fig. 1B),
in accordance with prior reports (11, 12, 14). However,
both ßßwt and ßß325 induced phosphorylation and activation
of Jak1 and Jak3 (Fig. 1C), confirming that Janus kinase
activation, whereas critical for Stat activation, is not sufficient for
proliferation.
To investigate the role of Stat5 in proliferation, 13 amino acids from
the C terminus of IL-2Rß that encompass Tyr510, the most
efficient Stat5-activating tyrosine on IL-2Rß (11), were
attached to the C terminus of the ßß325 receptor (ßß325 +
Tyr510 (Fig. 1A)). Like ßßwt and the
endogenous IL-2R, this receptor induced strong Stat5 activation (Fig. 1D), as well as cell proliferation (Fig. 1B).
Thymidine incorporation was 30–40% less than that induced by ßßwt
or the endogenous IL-2R, likely due to the absence of a concurrent
proliferative signal mediated through Shc (11, 14).
Nonetheless, ßß325 + Tyr510 supported the long term
proliferation and survival of CTLL-2 cells in culture (Fig. 3, D and E; data not shown). However, if
Tyr510 was mutated to phenylalanine in this receptor
context (ßß325 + Phe510 (Fig. 1A)), neither Stat5 activation nor proliferation was induced
(Fig. 1, B and D), indicating both events were
dependent on Tyr510.
Like previous reports (11, 43), these data show a
correlation between Stat5 activation and proliferation but do not
exclude the participation of an undefined molecule interacting with
Tyr510. Indeed, such a molecule has been previously
proposed to explain how Tyr510 mediates IL-2R expression
(44). If a molecule other than Stat5 mediates the
proliferative signal from Tyr510, specific disruption of
the interaction between Stat5 and ßß325 + Tyr510
should not eliminate mitogenesis. To test this possibility, point
mutations were introduced near Tyr510 in ßß325 +
Tyr510 to specifically disrupt the consensus Stat5-binding
motif without eliminating Tyr510. Analysis of Stat5-binding
sites in several cytokine receptors suggested that the SH2 domain of
Stat5 preferentially associates with a phosphorylated tyrosine followed
by hydrophobic amino acids one and three positions C-terminal to the
tyrosine (i.e., YLSL in the case of IL-2Rß) (Fig. 2A). These residues, in the
context of ßß325 + Tyr510, were point mutated
individually or concomitantly to arginine or glutamic acid to generate
four mutant receptors (Fig. 2B). Compared with ßß325
+ Tyr510, these receptors demonstrated a spectrum of Stat5
activation potencies (Fig. 2C) which correlated directly
with proliferation (Fig. 2D).
IL-2 delivers a proliferative signal through Stat5a or Stat5b
Although designed to specifically disrupt Stat5 binding, these
mutations near Tyr510 could also disrupt the binding of
other putative mitogenic molecules. Therefore, to directly test whether
Stat5 delivers the proliferative signal from Tyr510, the
effect of overexpressing Stat5 on the marginal Stat5-activating
potential of ßß325 + YRSL was assessed. By mass action,
overexpression of Stat5 was expected to overcome the affinity barrier
between Stat5 and Tyr510 created by the mutation of
Leu511 to arginine. A flag epitope-tagged version
of murine Stat5a was cotransfected with ßß325 + YRSL into CTLL-2
cells bearing , and stable subclones that coexpressed ,
ßß, and flag-Stat5a were identified by flow cytometry (data not
shown). Expression levels of transfected Stat5a were estimated by
Western blot to be 5- to 10-fold higher than endogenous Stat5 (Fig. 3A). As intended,
overexpression of Stat5a restored the ability of ßß325 + YRSL to
induce Stat5 DNA-binding activity in response to GM-CSF to levels equal
to or greater than that mediated by ßß325 + Tyr510
(Fig. 3B). Rescue of Stat5 activation in turn restored the
ability of ßß325 + YRSL to induce thymidine incorporation (Fig. 3C), and cell proliferation (Fig. 3D) and
maintain cell viability (Fig. 3E). Thus, the proliferative
signal from Tyr510 of IL-2Rß can be mediated by Stat5a.
Overexpression of Stat5a also enhanced the level of Stat5 DNA-binding
activity induced by the endogenous IL-2R (Fig. 3B) but had
no observable effect on IL-2-mediated proliferation or viability of
CTLL-2 cells (Fig. 3, D and E). This suggests
that the degree of Stat5 activation normally induced by the IL-2R is
saturating with respect to cell proliferation.
Functional differences have been described between the DNA-binding
activities of Stat5a and Stat5b (45, 46). Additionally,
mice lacking either Stat5a or Stat5b genes have distinct phenotypic
abnormalities (26, 47, 48, 49, 50). Thus, Stat5a and Stat5b may
serve nonredundant functions. To determine whether Stat5b can also
mediate a proliferative signal from Tyr510, a flag
epitope-tagged version of Stat5b was cotransfected with ßß325 +
YRSL, as above. Although we were unable to overexpress Stat5b to the
levels achieved with Stat5a (Fig. 3A), Stat5b overexpression
nevertheless enhanced the ability of ßß325 + YRSL to activate
Stat5 (Fig. 3B) induce thymidine incorporation (Fig. 3C), support cell proliferation (Fig. 3D), and
inhibit cell death (Fig. 3E).
The TAD of Stat5 is critical for Stat5 to mediate proliferation
Stat molecules conventionally require both a conserved tyrosine phosphorylation site and a C-terminal TAD to mediate transcription (23, 24, 29). However, the TAD of Stat5 has been reported to be dispensable for some transcriptional events, such as ß-casein induction mediated by Stat5 in cooperation with the glucocorticoid receptor (34, 35). Similarly, the TAD of Stat5 appears dispensable for induction of c-myc by IL-3 (51) and for proliferation of the promyeloid line 32D in response to the Stat5-activating cytokines Epo and IL-3 (29).
To test the requirement for the TAD of Stat5 in IL-2R signaling, we
assessed whether overexpression of a TAD-deficient version of Stat5
would enhance or inhibit the proliferative signal mediated by
Tyr510 of IL-2Rß. 713 is a naturally occurring isoform
of Stat5a that lacks the TAD (Fig. 4A) but retains all other
functional domains and therefore can become tyrosine phosphorylated and
bind DNA in response to cytokine stimulation (29). If
overexpressed in CTLL2 cells, 713 could potentially exert a dominant
negative blockade on some Stat5-mediated transcriptional events
(29, 31, 51) and impose undesirable selective pressure
before analysis. Therefore, experiments with 713 were performed in a
cytokine-dependent lymphocyte line that can grow in the absence of any
Stat5 activity. The IL-3-dependent pro-B cell line BA/F3 was
transfected with a chimeric receptor containing the extracellular
domain of the human G-CSF receptor and the transmembrane and
cytoplasmic domains of the gp130 receptor chain (42), to
produce a cell line termed BA/FG. gp130 activates Stat3, but not Stat5
(52), so this chimeric receptor promoted the proliferation
of BA/F3-derived cells in response to human G-CSF (42)
without activation of Stat5 (data not shown). BA/FG cells express an
endogenous c chain but do not respond to IL-2 due to the absence of
IL-2Rß, which made it unnecessary to use the chimeric and
ßß receptor chains used in CTLL-2 cells. Instead, IL-2Rß
mutants were tested using the normal ectodomain of
IL-2Rß. Stable transfectants of BA/FG cells were generated that
expressed the different IL-2Rß mutants at similar levels, as assessed
by flow cytometry (data not shown). Experiments were performed with a
high dose of IL-2 (3000 U/ml) sufficient to saturate IL-2Rß and c
irrespective of IL-2R expression, in that the latter is dependent on
Stat5 activity (27).
We first confirmed that BA/FG cells, like CTLL2 T cells, were dependent
on Stat5 to mediate the proliferative signal from Tyr510 of
IL-2Rß. As expected, BA/FG expressing 325 + Tyr510
activated Stat5 and proliferated in response to IL-2, unless
Tyr510 was point mutated to phenylalanine (325 +
Phe510 (Fig. 4)). Moreover, BA/FG cells
expressing 325 + YRSL demonstrated dramatically impaired Stat5
activation and proliferation, and both defects could be rescued by
overexpression of wtStat5a (Fig. 4, B and C).
To test the role of the Stat5 TAD in signaling, stable BA/FG clones
coexpressing 325 + Tyr510 and the 713 version of
Stat5a were generated. Expression of 713 did not appear to impose
adverse selective pressure on BA/FG cells, inasmuch as 1) cells
expressing 713 and wtStat5a were generated with similar cloning
efficiencies, 2) the two versions of Stat5 were expressed at similar
levels as revealed by flow cytometry (data not shown), and 3) 713
had no effect on the proliferative response of BA/FG cells to G-CSF
(Fig. 4D). Similar to wtStat5a, overexpression of 713
dramatically enhanced Stat5 DNA-binding activity induced by 325 +
Tyr510, as well as by the endogenous IL-3 receptor (Fig. 4B), confirming that the TAD is not required for
receptor-mediated nuclear translocation or DNA binding by
Stat5(29). Despite the capacity to inducibly bind DNA,
713 severely reduced the ability of either 325 +
Tyr510 or the IL-3 receptor to induce BA/FG proliferation
(Fig. 4, C and D). Thus, the transcriptional
activation domain is essential for Stat5 to mediate proliferative
signals from the Tyr510 site of IL-2Rß and the full
length IL-3 receptor.
Stat5 mediates induction of c-myc, bcl-2, and bcl-x through its TAD
The obligate role for the TAD in Stat5 mitogenic signaling suggests that Stat5 may mediate proliferation through induction of genes critical for cell cycle progression and/or survival. c-myc, a protooncogene induced by IL-2 (53), is essential for T cells to enter S phase in response to stimulation (54). bcl-2 and bcl-xL, also induced by IL-2 (55), have been shown to support lymphocyte survival (56, 57). Constitutive expression of both c-myc and bcl-2 is sufficient to support factor-independent growth of the lymphoid cell line BAF/B03 (58).
To assess the role of Stat5 in c-myc, bcl-2, and
bcl-x induction, Northern blot analyses were performed with
the different BA/FG transfectants described above. The CIS
gene was used as an internal control for Stat5 activity
(26). 325 + YRSL, which induces minimal Stat5
activation, induced c-myc to a moderate level and only
weakly induced CIS, bcl-2, and
bcl-x in comparison with the endogenous IL-3
receptor (Fig. 5A).
Overexpression of Stat5a with 325 + YRSL enhanced induction not only
of CIS but also of c-myc, bcl-2, and
bcl-x (Fig. 5A), thereby implicating Stat5 in the
regulation of all four genes. To test the role of the Stat5 TAD in the
induction of these genes, the effects of 713 were assessed. Although
expression of 713 enhanced the ability of 325 +
Tyr510 to promote Stat5 DNA-binding activity (Fig. 4B), it prevented induction of the CIS gene by
this receptor, as well as the IL-3 receptor (Fig. 5B). Thus,
713 exerted dominant negative inhibition of conventional Stat5
transcriptional activity, consistent with prior assessment of this
construct (29). In so doing, 713 also inhibited the
ability of 325 + Tyr510, as well as the IL-3 receptor,
to induce c-myc, bcl-2, and bcl-x
(Fig. 5B), indicating that Stat5 regulates expression of
these genes by a TAD-dependent mechanism.
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325 +
Tyr510 signaling were assessed in the presence of the
protein synthesis inhibitor cycloheximide. Although cycloheximide
enhanced the basal expression of genes in the absence of cytokine as
reported for c-myc (59, 60), IL-2 and IL-3
still induced expression of c-myc and bcl-x above
this basal level (Fig. 5C). By contrast, induction of
bcl-2, which occurred with slower kinetics than seen with
c-myc and bcl-x (Fig. 5, A and
B), was blocked by cycloheximide treatment (Fig. 5C). Thus, whereas bcl-2 induction requires de
novo protein synthesis, c-myc and bcl-x are
induced by Stat5 in an immediate-early fashion, suggesting that Stat5
may interact directly with the promoters of these genes rather than via
an intermediary transcription factor. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies of the role of Stat5 in proliferative signaling have
yielded somewhat contradictory results. Mui et al. (51)
reported that a dominant-negative mutant of Stat5 significantly
inhibited the proliferation of the pro-B cell line BA/F3 in response to
IL-3, another cytokine which activates Stat5. This occurred in the face
of normal c-myc induction, suggesting that Stat5 was
critical for some, but not all, aspects of the proliferative signal. By
contrast, Wang et al. (29) reported that dominant-negative
inhibition of Stat5 activity in the promyeloid cell line 32D had no
impact on proliferative responses to IL-3 and Epo. The residual
proliferative responses seen in these two studies may have been
mediated by one or more redundant proliferative pathways operating in
parallel with Stat5, such as the Shc pathway (14, 61).
Indeed, the Stat5-binding sites on IL-2Rß (Tyr510 and
Tyr393) are only critical for proliferation when Shc
activity is abrogated by mutation of its binding site at
Tyr338 (11). Consequently, 713 or other
dominant negative versions of Stat5 would not be expected to block
proliferation mediated by full length IL-2Rß. For this reason, the
experiments in the present study utilized a mutated version of IL-2Rß
that lacked the Shc-binding site and contained only the Stat5
activation site at Tyr510. By eliminating potentially
redundant pathways, a definitive role for Stat5 in proliferative
signaling was revealed.
Although both Stat5 and Shc are clearly able to mediate cell
proliferation, the extent to which each contributes to the IL-2-induced
proliferation of primary T cells remains controversial. Fujii et al.
(39) reconstituted IL-2Rß-deficient mice with a mutated
version of IL-2Rß lacking the ability to detectably activate Stat5,
but containing an intact Shc-binding site. They found that this mutated
receptor promoted a normal proliferative response to IL-2 in primary T
cells (39), consistent with prior studies in cell lines
(11, 37, 38, 62). By contrast, Moriggl et al. reported
that T cells from mice lacking both the Stat5A and Stat5B genes show no
proliferative response to IL-2 (36), which led them to
conclude that Stat5 is absolutely essential for IL-2-induced
proliferation in primary T cells. It is unclear why no
Stat5-independent proliferative pathway was operational in T cells from
the latter mice, as it apparently was in the cells studied by Fujii et
al. (39). One possible explanation for the discrepant
results is that the mutant form of IL-2Rß used by Fujii et al. might
have been able to activate Stat5 to a physiologically relevant but
experimentally undetectable level. However, this seems an unlikely
explanation, in that we show here that even detectable, low level Stat5
activity is insufficient for the high level of T cell proliferation
observed by Fujii et al. (39) (Figs. 2 and 3,
B--E). An alternative explanation is that
deletion of both the Stat5a and Stat5b genes by Moriggl et al. had
unanticipated detrimental effects on the expression or function of one
or more essential components of the IL-2R complex, such as Jak3,
rendering the receptor globally dysfunctional in the total absence of
Stat5. There is precedent for such a mechanism, because the basal
activity of Stat1 has recently been shown to be required for expression
of caspases, which in turn are required to sensitize cells to
TNF--mediated apoptosis (33). Finally, it is also
possible that the severe proliferative defect seen in
Stat5a/Stat5b-deficient T cells in part reflects impaired signaling
through the TCR, which has recently been show to induce transient
activation of Stat5 (40).
The molecular mechanism by which Stat5 mediates proliferative signaling has also remained obscure. A truncated version of Stat5, which was unable to dimerize and hence bind DNA, inhibited IL-3-mediated proliferation by a dominant negative mechanism (51), thus implicating Stat5 in the IL-3 proliferative signal. However, in a second study by Wang et al. (29), a less severely truncated version of Stat5 lacking only the TAD had no impact on the IL-3-proliferative signal. Together, these studies suggested that Stat5 may mediate proliferative signaling by a TAD-independent mechanism. Consistent with this notion, a version of Stat5 capable of binding DNA, but lacking its TAD, can nevertheless interact with the glucocorticoid receptor to induce transcription of the ß-casein gene (34, 35), thereby demonstrating that Stat5 is capable of TAD-independent transcriptional regulation. However, our results define a clear requirement for the C-terminal TAD of Stat5 for the proliferative response of lymphocytes to both IL-2 and IL-3. This indicates that, despite its capacity for TAD-independent signaling, Stat5 induces cell proliferation through the conventional TAD at its C terminus. The discrepancy between our findings and those of Wang et al. may reflect the fact that their cells were cultured with IL-3 in the face of constitutive dominant negative Stat5 expression, which would be expected to impose a strong selective pressure in favor of Stat5-independent signaling mechanisms. In the present study, we avoided this potential problem by creating a cell line, BA/FG, that does not use Stat5 for growth and instead responds to a Stat3-dependent proliferative signal delivered by the gp130 receptor subunit. This allowed for expression of the TAD-deficient form of Stat5 at a level sufficient for complete dominant negative activity, which likely accounts for the more pronounced impact on IL-2- and IL-3-mediated proliferation observed in this study compared with Wang et al. (29) and Mui et al. (51).
In addition to cell proliferation, Stat5 was also found to induce
expression of the growth-related genes c-myc,
bcl-2, and bcl-x through a TAD-dependent
mechanism (Fig. 5, A and B). Induction of the
c-myc and bcl-x genes by Stat5 did not require de
novo protein synthesis (Fig. 5C), suggesting that Stat5a
interacts directly with the promoters of these genes. Although no Stat5
binding sites have yet been functionally defined in these promoters,
other Stat family members have been shown to regulate these genes.
Stat1 can interact with a TTCGGAGAA motif in the human bcl-x
promoter, and this site is involved in the regulation of
bcl-x expression (63). Stat3 has also been
proposed to regulate bcl-x expression (64) and
can associate with and trans-activate the c-myc
promoter (65). However, the site in the c-myc
promoter with which Stat3 interacts is not appreciably bound by
Stat5(65) (J.D.L., unpublished observations), suggesting
that Stat3 and Stat5 mediate c-myc induction by binding
distinct promoter sites.
In contrast to c-myc and bcl-x, induction of
bcl-2 by Stat5 appears to be a delayed early event, because
it occurs with slower kinetics and requires de novo protein synthesis
(Fig. 5). This suggests that Stat5 induces expression of one or more
secondary transcription factors that in turn bind to and
trans-activate the bcl-2 promoter. Such a model,
in which Stat5 initiates a cascade of gene activation events, would
explain how a single transcription factor could promote the diverse
array of cellular changes that are necessary for cell cycle
progression, including protooncogene induction, up-regulation of cell
metabolism, and activation of the cell cycle machinery.
Although the 325 + Tyr510 receptor was able to mediate
Stat5 activation at least as potently as ßßwt and the endogenous wt
IL-2 receptor (Fig. 1D), it was 
40% weaker in its
ability to promote cell proliferation (Fig. 1B). This
suggests that Stat5 is not sufficient to mediate all aspects of the
IL-2 proliferative signal. Indeed, activation of the
ras/receptor-activated factor/mitogen-activated protein
kinase signaling pathway, culminating in the induction of
c-fos, c-jun, and other genes, is mediated
exclusively by Shc through a Stat5-independent mechanism (14, 66, 67, 68). Conversely, Stat5 uniquely induces expression of the
CIS and CD25 genes (26, 27, 44) and
mediates a bcl-2-independent survival signal that cannot be
delivered through Shc (38). Perhaps STAT5 acts
cooperatively with Shc, or potentially other proliferative signals from
the IL-2R, to deliver a signal of maximum mitogenic potential. In vivo,
where receptor and/or cytokine concentrations may be limiting, synergy
between Stat5- and Shc-mediated mitogenic signals from the IL-2R may be
essential for rapid T cell expansion, and hence the generation of an
effective immune response to an invading pathogen.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Brad Nelson, Virginia Mason Research Center, 1201 9th Avenue, Seattle, WA 98101-2795.
3 Abbreviations used in this paper: TAD, trans-activation domain; wt, wild type.
Received for publication September 3, 1999. Accepted for publication December 10, 1999.
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References |
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, the same clones
cultured without cytokine. Data for each clone are expressed as a
percentage of the thymidine incorporated after stimulation with 100
U/ml human IL-2. C, ßß
), or no cytokine (