JAK2 and STAT5, but not JAK1 and STAT1, Are Required for Prolactin-Induced ß-Lactoglobulin Transcription
Yulong Han,
Diane Watling,
Neil C. Rogers and
George R. Stark
Department of Molecular Biology (Y.H., G.R.S.) Research
Institute The Cleveland Clinic Foundation Cleveland, Ohio
44195
Imperial Cancer Research Fund (D.W., N.C.R.)
Lincolns Inn Fields London WC2A 3PX, UK
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ABSTRACT
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Several different Janus kinases (JAKs) and
signal transducers and activation of transcription (STATs) have been
implicated in mediating the biological responses induced by PRL, based
on their ligand-dependent tyrosine phosphorylation and activation.
However, these criteria alone do not prove that a particular JAK or
STAT is essential for signal transduction. We have used mutant cell
lines defective in JAK1, JAK2, or STAT1 to examine their roles in
PRL-dependent signaling. JAK2 is absolutely required for PRL-dependent
phosphorylation of the receptor, activation of STATs, and induction of
ß-lactoglobulin. Wild type, but not kinase-negative JAK2, restores
all responses to PRL in JAK2-defective cells, suggesting that JAK2
function, not merely the protein, is required. In contrast, JAK1, which
is phosphorylated in response to PRL, is not required for any of these
functions. Although STAT1 homodimers do form in response to PRL, no
defect in PRL-dependent signaling is apparent when STAT1 is missing,
suggesting that STAT5, which is strongly activated in response to PRL,
is primarily responsible for driving the expression of PRL-responsive
genes.
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INTRODUCTION
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Janus kinases (JAKs) and signal transducers and activators of
transcription (STATs), originally identified as components of
interferon-dependent signaling pathways (1), have also been shown to
mediate many of the biological responses induced by cytokines and
growth factors (2, 3). To date, four mammalian JAKs (JAK1, JAK2, JAK3,
and TYK2) (4, 5, 6, 7, 8, 9) and seven STATs (STATs 1 to 4, 5a, 5b, and 6) have
been cloned (10, 11, 12, 13, 14, 15, 16). JAK and STAT family members have also been
identified in Drosophila (17, 18, 19, 20, 21, 22). Ligand binding leads to
tyrosine phosphorylation and activation of JAKs, which are usually
bound to the receptors. Phosphorylation of receptor subunits and STATs
then ensues. JAKs may also activate other signaling components (23),
and STATs may be activated by the intrinsic kinases of some growth
factor receptors (24) or by nonreceptor kinases (25, 26, 27, 28).
PRL, a polypeptide hormone from the anterior pituitary gland, regulates
several different physiological responses, especially milk gene
expression, and also has a role in modulating immune responses (29, 30). PRL binds to a cell-surface receptor, PRLR, which belongs to a
class of cytokine and growth factor receptors lacking intrinsic kinase
activity (31, 32). Binding of PRL stimulates rapid, transient tyrosine
phosphorylation of several intracellular proteins, including the
receptor and a subset of JAKs and STATs (14, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45), and also leads to
the transcriptional activation of PRL-responsive milk protein genes
(46, 47) and the interferon-regulatory factor 1 (IRF-1) gene (48, 49, 50).
JAK2 (35, 36, 37, 38, 39, 40, 41) and JAK1 (35) are both bound to PRLR and become
phosphorylated upon ligand binding. STATs 1, 3, and 5 are activated in
response to PRL and form homo- and heterodimers, which bind to
-interferon-activated sequence (GAS) elements in the promoters of
milk protein and IRF-1 genes (41, 42, 43, 44, 45).
Recent studies of other pathways reveal that the JAKs and STATs
activated in response to ligand binding may not always be essential for
signaling. For example, interleukin-6 (IL-6) induces the
phosphorylation of several different JAKs, with a pattern that varies
in different cells. However, only JAK1 is vital for IL-6-dependent
phosphorylation of the gp130 receptor subunit, STAT activation, and
transcriptional induction of the IRF-1 gene (51). GH also activates
both JAK1 and JAK2, but only JAK2 is required for phosphorylation and
activation of the receptor, the signal transducer Shc, and the STATs
(23). Platelet-derived growth factor induces phosphorylation of JAK1,
JAK2, and TYK2, but deletion of each of these kinases singly does not
affect STAT activation (52). Epidermal growth factor (EGF) stimulates
phosphorylation of JAK1 and STATs 1 and 3, but JAK1 is not required for
STAT activation, and neither JAK1 nor STAT1 is required for induction
of the c-fos gene by EGF (24). Furthermore, STAT1-deficient
mice lack all responses to interferons (IFNs), as expected from the
prior work with STAT1-deficient human cells (53), but the mice respond
normally to GH, EGF, and IL-10, which activate STAT1 in cell lines,
arguing against any essential role for STAT1 in these pathways
(54).
Here, by using PRLR-transfected cell lines lacking JAK1, JAK2, or
STAT1, we show that JAK2 is essential for PRL-dependent STAT
activation, receptor phosphorylation, and ß-lactoglobulin induction
and that JAK1 and STAT1 are not.
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RESULTS
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Expression of Functional PRLR
The parental cell lines 2C4 and 2fTGH (not shown) and the mutant
cell lines derived from them do not express endogenous PRLR, as
determined by use of the FACScan (Fig. 1
, top
left) and Western analyses (data not shown). Therefore, each cell
line was transfected with a cDNA encoding the long form of rabbit PRLR.
After selection for stable transfectants, cells from individual clones
were scanned to identify those that expressed high levels of PRLR.
FACScan (Fig. 1
) and Western analyses (data not shown) revealed that
the cells used in all experiments expressed similar levels of PRLR. PRL
stimulation of 2C4 cells transfected with rabbit PRLR (2C4/PRLR)
induced the phosphorylation on tyrosine of several cellular proteins
with apparent molecular masses of 90130 kDa (data not shown),
indicating that the transfected cells did express functional
receptors.

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Figure 1. Expression of PRLR in Parental and Mutant Cell
Lines
FACScan analysis (10,000 data points) of cell surface PRLR expression
in the cell lines 2C4/PRLR, U4C/PRLR, 2A/PRLR, and U3A/PRLR
(thick solid lines). Profiles of unstained 2C4/PRLR
(dotted line) and stained 2C4 (thin solid
line) cells are included for comparison.
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PRL-Dependent Activation of STATs
Previously, PRL has been shown to stimulate phosphorylation on
tyrosine of STATs 1, 3, and 5. To examine STAT phosphorylation in
response to PRL treatment of the transfected cells listed above,
immunoprecipitations with antibodies to STATs 1 and 5 were performed,
using extracts of cells treated with PRL or left untreated. Cells
treated with IFN
in parallel were used as controls. PRL induces
strong phosphorylation of STAT5 on tyrosine in 2C4/PRLR cells (Fig. 2A
, lane 2), but STAT1 phosphorylation was not detected
using the same cell extracts (Fig. 2B
, lane 2). As expected, IFN
stimulates tyrosine phosphorylation of STAT1 (Fig. 2B
, lane 3), but not
STAT5 (Fig. 2A
, lane 3). In U3A/PRLR cells, the intensity of
PRL-dependent STAT5 phosphorylation (Fig. 2A
, lane 11) was similar to
the intensity in control 2C4/PRLR cells (lane 2), suggesting that
PRL-dependent STAT5 phosphorylation is independent of STAT1. Similar
results were observed by electrophoretic mobility shift assay (EMSA)
(Fig. 3
, lanes 2 and 12). Activation of STAT3 by PRL has
been observed in some cell lines (see Discussion) but was
not detected in our cells by either immunoprecipitation (data not
shown) or EMSA (see below).

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Figure 3. EMSA Analysis of PRL-Induced Binding to a
ß-Casein GAS Probe
Extracts were prepared from the cell lines 2C4/PRLR, U4C/PRLR,
2A/PRLR, 2A/PRLR/JAK2, 2A/PRLR/JAK2KE, or U3A/PRLR, untreated
(lane 1), treated with PRL (lanes 2, 4, 6, 8, 10, 12), or treated with
IFN (lanes 3, 5, 7, 9, 11, 13) for 15 min at 37 C. The positions of
the PRL-inducible complexes A and B are indicated.
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EMSA was used to characterize the DNA-binding properties of the
phosphorylated STAT proteins induced by PRL. At least two PRL-inducible
complexes (A and B) were detected in extracts of parental 2C4/PRLR
cells with a GAS probe from the promoter of the PRL-responsive
ß-casein gene (Fig. 3
, lane 2). The intensity of complex A was at
least 10 times higher than that of complex B. Complex B migrated
similarly to the IFN
-induced complex (Fig. 3
, lane 3) and was not
observed in U3A/PRLR cells treated with PRL or IFN
(Fig. 3
, lanes 12
and 13). The formation of complex B was completely competed by various
unlabeled GAS elements, but complex A was completely competed only by
the ß-casein GAS. Unrelated DNA elements did not affect either
complex (data not shown). To analyze the STATs present in these
complexes, antibody supershift experiments were performed. Complex B
was supershifted only by anti-STAT1 (Fig. 4
, lanes 2 and
9), indicating that this complex probably consists of STAT1 homodimers
bound to the probe. STAT15 heterodimers were not observed: Complex A,
observed only in cells treated with PRL (Fig. 3
, lane 2) and not IFN
(Fig. 3
, lane 3), was supershifted only by anti-STAT5 (Fig. 4
, lane 5).
Antisera to STATs 2, 3, and 6 and preimmune serum had no effect (Fig. 4
, lanes 3, 4, 6, and 7).

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Figure 4. Antibody Supershift Analysis of PRL-Induced STAT
Binding to a ß-Casein GAS Probe
Whole-cell extracts from PRL-treated 2C4/PRLR (lanes 17) or
2A/PRLR/JAK2 cells (lanes 810) were analyzed. The STATs present in
complexes were evaluated with antisera to STAT1 (lanes 2 and 9), STAT2
(lane 3), STAT3 (lane 4), STAT5 (lanes 5 and 10), STAT6 (lane 6) or
with preimmune serum (PI, lane 7). The positions of the PRL-induced
complexes A and B, observed in both cells, are indicated.
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JAK2 Is Essential for the PRL-Dependent Activation of STATs
The PRLR lacks an intrinsic kinase domain and signals through
receptor-associated kinase(s). JAK2 is strongly phosphorylated in
response to PRL in our cells, and JAK1 is phosphorylated very weakly,
about 10% as strongly as in response to IFN
(data not shown). JAK1
has been shown by some (35) but not others (36, 37, 55) to be activated
in response to PRL in other cell types, suggesting that JAKs 1 and 2
might both be involved in PRL-induced signaling. STAT phosphorylation
and activation in response to PRL were normal in U4C/PRLR cells
(lacking JAK1, Fig. 2A
, lane 5, and Fig. 3
, lane 4) but were not
observed in
2A/PRLR cells (lacking JAK2, Fig. 2
, lane 8, and Fig. 3
lane 6). Wild type murine JAK2 (Fig. 2A
, lane 14, and Fig. 3
, lane 8),
but not kinase-negative JAK2 (Fig. 2A
, lane 17, and Fig. 3
, lane 10),
restored PRL-induced STAT phosphorylation and DNA-binding activity in
2A/PRLR cells. These results show that functional JAK2, but not
JAK1, is absolutely required for PRL-dependent STAT activation. As
expected, both JAK1 and JAK2 are required for STAT1 activation by
IFN
(Fig. 2B
, lanes 6 and 9; Fig. 3
, lanes 5 and 7).
PRL-Induced Tyrosine Phosphorylation of the PRLR Requires JAK2
Tyrosine phosphorylation of the PRLR is required for PRL-dependent
cell proliferation and gene activation (33, 34, 35, 36, 37, 38). Although both JAK1 and
JAK2 become phosphorylated, it is not clear whether both are required
for receptor phosphorylation. To examine this issue, immunoprecipitates
prepared from cell lysates of PRLR-transfected cells with anti-PRLR
antibody S46 were analyzed with antiphosphotyrosine antibodies. In
response to PRL, the receptor is phosphorylated on tyrosine in 2C4/PRLR
and U4C/PRLR cells (Fig. 5
, lanes 4 and 6), but not in
2A/PRLR cells (lane 8). (The difference in PRLR expression between
lanes 4 and 6 of Fig. 5A
and B was not seen in independent repeats of
this experiment.) Wild type murine JAK2 (lane 10), but not
kinase-negative JAK2 (lane 12), restored receptor phosphorylation in
2A/PRLR cells, to a level similar to that of parental 2C4/PRLR
cells. As expected, no signal was observed with 2C4 cells, which lack
the PRLR (lane 1) and no phosphorylated PRLR was detected in 2C4/PRLR
cells treated with IFN
. These data show that JAK2, but not JAK1, is
required for PRL-dependent receptor phosphorylation.

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Figure 5. Requirement for JAK2 in PRL-Dependent PRLR
Phosphorylation
2C4, 2C4/PRLR, U4C/PRLR, 2A/PRLR, 2A/PRLR/JAK2, or
2A/PRLR/JAK2KE cells were untreated (lanes 3, 5, 7, 9, 11) or
treated with PRL (lanes 1, 4, 6, 8, 10, 12) or IFN (lane 2) for 15
min at 37 C. Cell lysates were analyzed by immunoprecipitation with a
polyclonal antibody to rabbit PRLR, followed by Western blotting and
analysis with antiphosphotyrosine antibodies (A). The blots were
stripped and reprobed with anti-PRLR sera (B).
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JAK2-Dependent Stimulation of ß-Lactoglobulin Expression
Stimulation of responsive cells by PRL activates transcription of
several genes, including ß-lactoglobulin. Since JAK2, but not JAK1,
is required for PRL-dependent activation of STAT proteins and
phosphorylation of the PRLR, it was important to evaluate the role of
JAKs in PRL-induced transcriptional activation. ß-Lactoglobulin
promoter/chloramphenicol acetyltransferase (CAT) constructs were stably
transfected into 2C4/PRLR, U4C/PRLR, or
2A/PRLR cells. In the
absence of JAK2, CAT induction was almost absent in response to PRL
(Fig. 6
, lane 11), but in the absence of JAK1 (lane 14),
CAT induction was similar to that in parental 2C4/PRLR cells (Fig. 6
, lane 5). Wild type murine JAK2 substantially restored CAT induction by
PRL in
2A/PRLR cells in a transient transfection assay (lane 8).
Surprisingly, although IFN
induced strong STAT1 activation (Figs. 2
and 3
), it did not induce CAT activity in 2C4/PRLR cells (Fig. 6
, lane
3), showing that the STAT1 homodimer induced by IFN
cannot drive
gene expression from the ß-lactoglobulin GAS element.

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Figure 6. PRL-Dependent Activation of ß-Lactoglobulin
Expression in Mutant Cell Lines
Cells were transfected stably (2C4/PRLR, U4C/PRLR, 2A/PRLR,
U3A/PRLR) or transiently ( 2A/PRLR/JAK2) with the
ß-lactoglobulin/CAT construct pBJ23. CAT assays were performed with
extracts from serum-starved cells, untreated (lanes 1, 4, 7, 10, 13) or
treated with PRL (lanes 2, 5, 8, 11, 14) or IFN (lanes 3, 6, 9, 12,
15). Values are expressed as percent chloramphenicol conversion and as
fold induction calculated from the basal level activities
(representative of three experiments).
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DISCUSSION
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Previous observations have shown that PRL activates the rapid and
transient tyrosine phosphorylation of JAK family members, either JAK2
in Nb2 cells (36, 37, 38, 39, 40, 41) or JAK2 and JAK1 (weakly) in BAF-3 cells (35).
Nb2 cells are a rat T cell lymphoma line expressing a truncated form of
the PRLR with a deletion of 198 amino acids in the middle of the
cytoplasmic domain (56); BAF-3 and 2C4 are an IL-3-dependent pro-B
murine lymphoid line and a human fibrosarcoma line, respectively, and
both express the full-length PRLR (35). It is not yet clear whether
activation of a different set of JAKs by PRL in these cells is due to
cell type differences or to the different types of PRLRs that are
present. When we transfected the long form of PRLR into human
fibrosarcoma 2C4 cells, the pattern of JAK phosphorylation in response
to PRL was similar to that in BAF-3 cells (data not shown). The
PRL-induced phosphorylation of JAK1 has been detected only in cells
expressing the full-length receptor (35), and the activation of JAK1 by
GH requires regions of that receptors cytoplasmic domain beyond the
membrane-proximal region that usually associates with JAKs (Y. Han and
G. R. Stark, unpublished data). These results suggest that the
activation by PRL or GH of JAK1 is different from that of JAK2. In
addition, the pattern of activation of several different JAKs by some
cytokines is distinct in different cell lines (57).
We and others have demonstrated recently that a particular JAK may not
be essential for STAT-mediated signal transduction even if it becomes
phosphorylated and activated upon ligand binding (23, 24, 51, 52).
Therefore, we examined the functions of JAK1 and JAK2 in PRL signaling
in mutant cell lines lacking these kinases. Complete defects in the
PRL-dependent activation of STATs, phosphorylation of the receptor, and
induction of ß-lactoglobulin were observed in
2A/PRLR cells
lacking JAK2, but not in U4C/PRLR cells lacking JAK1, revealing that
JAK2 but not JAK1 has an obligatory role in PRL-dependent signaling.
Wild type JAK2, but not a kinase-negative mutant, restores the
response, indicating that active JAK2, and not merely the protein, is
required. However, a requirement for JAK1 in other PRL-dependent
signaling cannot be excluded insasmuch as JAKs can function in
alternative pathways (23).
STAT5 was originally identified as a PRL-activated transcription factor
involved in the expression of milk proteins in sheep (14).
Subsequently, it was shown that mice have two STAT5 homologs, 5a and 5b
(58, 59, 60). Two forms of human STAT5 have also been identified (61). Both
STATs 5a and 5b bind to a GAS element in the ß-casein gene promoter
and function in signaling in response to IL-3, IL-12, and PRL (59, 60, 61).
STATs 5a and 5b heterodimers were recently observed (62), but STAT5
heterodimers with other STATs have not been reported. Therefore, the
activated STAT5 we have observed could represent either a STAT5b
homodimer, a STAT5a-5b heterodimer, or both, inasmuch as the antibody
used is specific for STAT5b and does not cross-react with other
STATs.
In PRL-treated rat Nb2 T-lymphocytes, activation of STAT1 has been
observed by immunoprecipitation (40). STAT1 was activated much less
than STAT5 in our PRLR-transfected cells, and phosphorylated STAT1 was
detected only in the sensitive EMSA. As a control, STAT1 was strongly
activated by IFN
in the same cells. These observations reveal that
IFN
prefers to activate STAT1, and PRL prefers STAT5 (1, 2, 3, 14).
Less activation of STAT1 by PRL in 2C4/PRLR cells compared with Nb2
cells seems not to be due to a difference in the amount of the STAT1
protein because the level is high in both cases, with strong activation
by IFN
(Figs. 2
and 3
). The difference could be due to the fact that
Nb2 cells have an abnormally high response to PRL, which may be related
to their truncated form of the receptor (35, 56). Activation of STAT3
by PRL has been observed in cos-1 cells cotransfected with STAT3 and
PRLR cDNAs (42) and in 32D premyeloid cells (63), but not in our cells,
even when EMSA was performed with several different GAS elements (data
not shown). Different patterns of STAT activation in different cell
types have been observed with several cytokines and also with growth
factors (23, 24, 64), raising the possibility that selective activation
of specific STAT subsets can contribute to the regulation of specific
subsets of genes in a cell type-specific manner.
Although our understanding of the mechanism of PRL-dependent STAT
activation is incomplete, tyrosine phosphorylation of receptor subunits
is important for ligand-dependent STAT activation in other
cytokine-signaling pathways. STATs interact directly with specific
tyrosine residues of receptors, and this is crucial for STAT activation
in at least some cases (65, 66). In vitro, the direct
phosphorylation of STAT5 by JAK2 to confer specific DNA-binding
activity (34) suggests the possibility of alternative mechanisms of
STAT activation. Mutation of a single tyrosine residue in the C
terminus of the PRLR did not affect JAK2 phosphorylation, but did
prevent the induction of ß-casein (33), suggesting that STAT
activation by PRL may require STAT-receptor interaction. The sequential
activation of STATs 2 and 1 has been proposed in IFN
signaling (67).
The activation of STAT5 by PRL is normal in U3A/PRLR cells, indicating
that it is independent of STAT1. However, the sequential activation of
STAT5 and then STAT1 by PRL has not been excluded. A more detailed
investigation of PRLR, JAK2, and STAT activation using mutant cell
lines and dominant-negative STATs could contribute important new
information concerning how STATs are activated by PRL.
The promoters of the PRL-induced milk protein genes, including
ß-casein and ß-lactoglobulin, and of the IRF-1 gene have GAS
elements that can bind to homodimers of STATs 1 or 5 and various
heterodimers (14, 39, 44, 47, 68, 69). These observations suggest that
STATs 1 and 5 may be involved in the PRL-induced expression of both
milk protein and IRF-1 genes. The activation of STAT5 and the induction
of milk protein genes have been well characterized (14, 45, 46, 47), but
the role of STAT1 in activating these genes remains unclear. The
activation of STAT1 by PRL in wild type 2C4/PRLR cells raises the
possibility that it may function in this cell type. However, there was
no difference in the PRL-dependent induction of ß-lactoglobulin in
wild type and STAT1-null cell lines, and IFN
did not induce
ß-lactoglobulin transcription (Fig. 6
), even though STAT1 was
strongly activated (Figs. 2
and 3
), arguing against a requirement for
STAT1 in the activation of this gene. We cannot exclude the possibility
that STAT1 functions in activating other PRL-induced genes. However,
recent experiments with STAT1 knockout mice reveal that it is required
for IFN signaling (54) but not for apparently normal development,
consistent with our findings. In contrast, we observed activation of
the IRF-1 gene by ribonuclease (RNase) protection in cells treated with
IFN
, but not with PRL, indicating that STAT5 is not involved in
regulation of this process (data not shown).
Recent data demonstrate that, in addition to their important roles in
coupling many receptors to the STATs, JAKs may also function to couple
some receptors to other pathways (23). Furthermore, kinases other than
JAKs, such as those intrinsic to some receptors, may be involved in
STAT activation (24, 25, 26, 27, 28). Interestingly, PRL-induced phosphorylation of
mitogen-activate protein (MAP) kinase (70), ras (71), and raf-1 (72)
have been observed. Further investigation is required to determine the
role of ras-dependent signaling in the overall response to PRL and the
nature of the kinases involved in this pathway.
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MATERIALS AND METHODS
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Cell Lines, Ligands, and Antisera
We used the parental cell lines 2fTGH and 2C4 and the
IFN-unresponsive mutant lines U3A (lacking STAT1) (53, 73), U4C
(lacking JAK1), and
2A (lacking JAK2) (74). Recombinant human IFN
from Genentech (South San Francisco, CA) and ovine PRL (NIDDK-oPRL-20)
from the National Hormone and Pituitary Program (Baltimore, MD) were
used at final concentrations of 500 IU/ml or 1 µg/ml, respectively.
Anti-STAT1 and anti-STAT2 were gifts of James Darnell, Jr. (Rockefeller
University, New York, NY), anti-STAT3 and anti-STAT6 were gifts of
James Ihle (St. Judes, Memphis, TN). Anti-STAT5b (C-17), which does
not recognize STAT5a, was from Santa Cruz Biotechnology (Santa Cruz,
CA); peroxidase-conjugated goat antimouse serum was from Boehringer
Mannheim (Indianapolis, IN); fluorescein-conjugated goat antimouse
serum was from DAKO (Carpinteria, CA); antiphosphotyrosine monoclonal
antibodies PY20 and 4G10 were from Transduction Laboratories
(Lexington, KY) and Upstate Biotechnology (Lake Placid, NY),
respectively; and anti-PRLR monoclonal antibodies mAb M110 and mAb A917
and polyclonal antibody S46 (34) were from Jean Djiane (Institut
National de la Recherche Agronomique, Jouy-en-Josas, France).
Transfections and Use of the Fluorescence-Activated Cell Scanner
(FACScan)
Cells were stably transfected with the rabbit PRLR cDNA
pE-R23 (75) by the calcium phosphate method
(76). After selection, cloned cells were incubated with both anti-PRLR
monoclonal antibodies (2 µg/ml), followed by fluorescein-conjugated
goat antimouse antibody. Clones expressing high levels of PRLR,
identified by use of the FACScan, were used in all experiments.
For complementation, vectors expressing wild type murine JAK2
(pRK5/mJAK2) and a kinase-negative mutant protein (pRK5/mJAK2 K>E),
kindly provided by James Ihle (St. Judes, Memphis, TN) (9, 77), were
stably transfected into
2A/PRLR cells. The levels of JAK2 proteins
were analyzed by Western blotting. Clones expressing similar levels of
wild type or mutant JAK2 were used in all experiments.
Immunoprecipitation and Western Blot Analyses
Cells were serum-starved overnight and then treated with PRL or
IFN
at 37 C for 1015 min before cell extracts were prepared. PRLR
and STAT proteins, immunoprecipitated as described by Schindler
et al. (78), were separated by electrophoresis and adsorbed
to polyvinyldifluoridine membranes (Stratagene, La Jolla, CA). Tyrosine
phosphorylation was analyzed on Western blots, using
antiphosphotyrosine antibodies. The bands were visualized by enhanced
chemiluminescence, using Renaissance reagents (DuPont, Boston, MA).
EMSA
Whole-cell extracts, prepared as described by Sadowski et
al. (79), were assayed with a 32P-labeled
oligonucleotide (5'-AGATTTCTAGGAATTCAATCC-3') corresponding to the GAS
element of the bovine ß-casein gene (14). For supershift analyses,
the extracts were incubated with preimmune or STAT-specific antisera
for 30 min on ice before addition of the labeled probes.
CAT Assays
Cloned cell lines expressing PRLR were stably cotransfected with
the ß-lactoglobulin promoter/CAT construct pBJ23 (75) and hygromycin
resistance plasmids or transiently transfected with pBJ23 by the
calcium phosphate method (76). After selection with hygromycin, pooled
cells were serum-starved overnight and then treated with PRL or IFN
at 37 C for 20 h before cell extracts were prepared. CAT assays
were performed with [14C]-labeled chloramphenicol
(Amersham, Arlington Heights, IL). The acetylated and nonacetylated
forms of chloramphenicol were separated by TLC and quantitated by use
of a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Values are
expressed as percent chloramphenicol conversion and as fold induction
calculated from the basal activity.
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ACKNOWLEDGMENTS
|
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We thank James Darnell, Jr., for anti-STATs 1 and 2, James Ihle
for anti-STATs 3 and 6 and the JAK2 constructs, Jean Djiane for
anti-PRLR, and Jean Djiane and John Clark for pBJ23.
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FOOTNOTES
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Address requests for reprints to: George R. Stark, Department of Molecular Biology, Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195.
This work was supported by NIH Grant P01 CA-62220.
Received for publication February 26, 1997.
Accepted for publication April 7, 1997.
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