Prolactin Stimulates Activation of c-jun N-Terminal Kinase (JNK)
Kathryn L. Schwertfeger,
Seija Hunter,
Lynn E. Heasley,
Valerie Levresse,
Ronald P. Leon,
James DeGregori and
Steven M. Anderson
Departments of Pathology (K.L.S., S.H., S.M.A.), Medicine (L.E.H.,
V.L.), Biochemistry (R.P.L., J.D.), Pediatrics (J.D.), and Program
in Molecular Biology (K.L.S., J.D., S.M.A.) University of Colorado
Health Sciences Center Denver, Colorado 80262
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ABSTRACT
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In recent years the mitogen-activated protein
(MAP) kinase family has expanded to include both c-jun
N-terminal kinases (JNKs), and the p38/HOG1 family in addition to the
extracellular regulated kinase (ERK) family. These kinases are
activated by a variety of growth factors, as well as extra- and
intracellular insults such as osmotic stress, UV light, and
chemotherapeutic agents. Stimulation of the PRL-dependent Nb2 cell line
with PRL results in the rapid activation of JNK as determined by
the glutathione-S-transferase (GST)-jun kinase assay.
Activation was maximal 30 min after stimulation with 50
nM rat PRL (rPRL) and decreased after that
time. Dose response studies indicated that concentrations as low as 10
nM rPRL resulted in maximal activation. The
interleukin-3 (IL-3)-dependent myeloid progenitor cell line 32Dcl3 was
transfected with the long, Nb2, and short forms of the rat PRL receptor
(rPRLR), as well as the long form of the human PRLR (hPRLR). The long
and Nb2 forms of the PRLR were able to stimulate activation of JNK;
however, the short form of the rPRLR was not. This corresponds with the
inability of the short form of the rPRLR to stimulate proliferation of
32Dcl3 cells. Activation of JNK in 32Dcl3 cells expressing the long
form of the hPRLR was maximal at 30 min after stimulation with 100
nM ovine PRL (oPRL) and declined after that
time. Dose response studies indicated that activation of JNK was
maximal after 30 min at a concentration of 10
nM, and the amount of activated JNK declined at
the highest concentration of oPRL, 100 nM.
Immunoblot analysis with an antibody that recognizes the activated
(phosphorylated) forms of JNK1 and JNK2 indicated that both JNK1 and
JNK2 isoforms were activated in 32D/hPRLR cells stimulated with oPRL. A
recombinant human adenovirus expressing a kinase-inactive mutant of
JNK1 (APF mutant) was used to determine the biological effect of
blocking JNK activity in Nb2 cells. Expression of the JNK1-APF mutant
inhibited cellular proliferation and induced DNA fragmentation typical
of cells undergoing apoptosis. These data suggest that activation of
JNKs may be important in mitogenic signaling and/or suppression of
apoptosis in Nb2 cells.
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INTRODUCTION
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Mitogen-activated protein (MAP) kinases were initially discovered
as low mol wt protein kinases that were rapidly activated after
stimulation of cells with a variety of mitogens. In recent years this
family of serine/threonine-specific protein kinases has expanded
dramatically and now includes protein kinases that are activated by a
variety of cellular stresses in addition to mitogenic stimulation. The
original members of this family are alternatively known as MAP
kinase-1and MAP kinase-2, or extracellular related kinase 1 and 2
(ERK-1 and ERK-2) (1). These two kinases are the prototypes for all MAP
kinases, and they share structural features with two more recently
discovered families: the JNK/SAPK (c-jun N-terminal
kinase/stress-activated protein kinase) family (2, 3, 4, 5, 6, 7), and the p38
family (8, 9). All MAP kinases are proline-directed serine/threonine
protein kinases that are activated by the phosphorylation of both a
threonine and a tyrosine residue in a Thr-X-Tyr motif in the activation
loop that lies in close proximity to the ATP- and substrate-binding
sites. Although MAP kinases are thought to have a similar structure,
they differ in the size of the activation loop, and the amino acid
present in the second position of the Thr-X-Tyr motif; ERKs have a Glu,
JNKs have a Pro, and p38 family members have a Gly in the second
"X" position.
ERK-1 and ERK-2 have been demonstrated to be critical in mitogenic
signaling and oncogenesis (10, 11). After the phosphorylation of these
two kinases, some of the ERK-1 and ERK-2 translocate to the
nucleus (12), where they have been shown to phosphorylate
transcription factors such as ternary complex factor (13). These
transcription factors are required for the transcription of the
immediate early response gene c-fos (14). Thus, MAP kinases
tie signals initiated at the plasma membrane by growth factor receptors
to the induction of immediate early response genes in the nucleus,
which are required for progression through the cell cycle. Activation
of ERK-1 and ERK-2 is also important in stimulating differentiation of
some cell types. Although it is not currently understood what
distinguishes a proliferative signal from one that induces
differentiation, some investigators have suggested that the length of
time MAP kinase remains activated is important (15). There is also
evidence that activation of ERKs stimulates protection against factors
that mediate apoptosis (16, 17).
The JNK family is defined based upon the ability of these kinases to
phosphorylate serine residues 63 and 73 in the N-terminal region of
c-jun (18), an immediate early gene product that is a
component of the AP1 transcription factor complex (19).
c-jun is preferentially phosphorylated over either junB or
junD, because c-jun contains a docking site for JNKs (19).
JNKs have also been described as SAPKs since they were activated by a
variety of cellular stresses including UV light, heat, hyperosmotic
shock, reactive oxygen species, antioxidants, protein synthesis
inhibitors, and inflammatory cytokines (interleukin-1 and tumor
necrosis factor) (2, 8, 20, 21, 22, 23, 24). There are currently three members of
the JNK/SAPK family: JNK-1, JNK-2, and JNK-3 which correspond to
SAPK-
, -
, and -ß, respectively (2, 4). Some investigators have
argued that JNK1 and JNK2, but not ERK-1 and ERK-2, are required for
induction of c-fos and c-jun (24); however, other
studies support a role for ERKs in this process. JNKs are activated by
stimulation of cells with mitogenic cytokines, although it is not clear
whether their activation is required for mitogenesis (25, 26, 27).
The PRLR is a member of the cytokine receptor superfamily. The ability
of several cytokines, including interleukin-3 (IL-3),
granulocyte-macrophage colony-stimulating factor, and erythropoietin,
to activate JNKs stimulated us to ask whether JNKs represented another
class of signaling molecules activated by the PRLR. In this study we
describe the ability of PRL to induce activation of JNKs. A time- and
dose-dependent activation of JNK was observed in the PRL-responsive
T cell line Nb2, and in 32Dcl3 cells transfected with the long and Nb2
forms of the rPRLR. Expression of a kinase-inactive mutant of JNK1 in
Nb2 cells inhibited cellular proliferation and induced apoptosis as
indicated by the induction of DNA fragmentation typical of apoptotic
cells. These data suggest that JNKs represent another signaling family
activated in response to PRL stimulation that may contribute to
mitogenic signaling and/or the suppression of apoptosis.
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RESULTS
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PRL Stimulates Activation of JNK
PRL has been reported to activate ERK1 and ERK2, two members of
the MAP kinase family (28, 29, 30). Numerous studies have indicated that
mitogenic growth factors activate another member of the MAP kinase
family, the c-jun N-terminal kinases or JNKs, in addition to
ERK1 and ERK2; these studies have included mitogenic cytokines
that stimulate proliferation of hematopoietic cells (25, 26, 27). These
observations prompted us to determine whether PRL might also
stimulate activation of JNK family members. This question was first
examined in Nb2 cells, a PRL-dependent rat T cell line. Stimulation
of Nb2 cells with 50 nM rPRL for 060 min
resulted in the rapid activation of JNK as determined by the
glutathione S-transferase (GST)-jun kinase assay (Fig. 1A
). Although a basal level of JNK
activity was detectable in unstimulated Nb2 cells, PRL stimulation
resulted in an increase in JNK activity within 5 min. JNK activity was
maximal 30 min after stimulation of Nb2 cells and declined after that
until 60 min when it was at or below the level detected in unstimulated
cells (Fig. 1A
). A dose response curve indicated that concentrations of
as low as 5 nM rPRL resulted in JNK activation,
and that higher concentrations of PRL did not substantially increase
JNK activation after 15 min as measured by the GST-jun kinase assay
(Fig. 1B
). These results clearly indicate that PRL can activate
JNK.

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Figure 1. PRL Induces Activation of JNK in Nb2 Cells
A, Nb2 cells were cultured overnight in medium supplemented with 10%
horse serum and were then concentrated and stimulated with 50
nM rPRL for 0 to 60 min. The cells were then lysed and
processed as described to examine the activation of JNK as determined
by the GST-jun kinase assay. The time in minutes is shown at the
top of the panel. B, Nb2 cells were stimulated for 15
min with 0100 nM rPRL, after which the cells were
processed as described above. The concentration of rPRL used to
stimulate the cells is shown at the top of each lane.
The arrowhead on the right side of each
panel indicates the position of the GST-jun fusion protein.
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To demonstrate the physiological relevance of PRL-induced JNK
activation in a cell type that is responsive to PRL, we examined the
activation of PRL in the human breast cancer cell line T47D.
Stimulation of T47D cells with PRL has been shown to result in the
activation of the JAK2 tyrosine kinase and the phosphorylation of STAT
molecules (31). Stimulation of T47D cells with 50 nM ovine
PRL (oPRL) resulted in the activation of JNK as determined by the
GST-jun kinase assay (Fig. 2
, lanes
16). Maximal levels of JNK activity were evident 30 min post
stimulation and declined after that time. A dose response study
indicated that 5 nM oPRL resulted in maximal activation of
JNK and higher concentrations of oPRL did not stimulate either an
increase or decrease in JNK activity (Fig. 2
, lanes 712).
Immunoblotting with phospho-JNK antibodies indicated that both JNK1 and
JNK2 were activated (data not shown).

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Figure 2. PRL Induces Activation of JNK in T47D Cells
T47D cells were cultured overnight in serum-free medium and then
stimulated with 50 nM oPRL for 060 min (lanes 16). The
cells were then lysed and processed as described to examine the
activation of JNK as determined by the GST-jun kinase assay. The time
in minutes is shown at the top of each lane. In lanes
712, T47D cells were treated in an identical manner and then
stimulated for 30 min with 0100 nM oPRL, and processed as
described above. The concentration of rPRL used to stimulate the cells
is shown at the top of each lane. Lane number is
indicated at the bottom of each lane. The
arrowhead on the right side of the panel
indicates the position of the GST-jun fusion protein.
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Only the Long and Nb2 Forms of the rPRLR Are Able to Activate
JNK
The PRLR is expressed in multiple forms that result from
differential splicing (32, 33, 34, 35). A unique form of the PRLR has been
identified in the Nb2 cell line; this form is transcribed from a PRLR
gene unique to this cell line that has undergone an internal deletion
(36). ONeal and Yu-Lee (37) have reported that both the long and Nb2
forms of the rPRLR are able to induce PRL-dependent proliferation when
introduced into the IL-3-dependent murine myeloid cell line FDCP-1. In
contrast, the short form of the rPRLR was not able to induce
proliferation in their system (37). Likewise we have observed that the
long and Nb2 forms of the rPRLR, but not the short form, are able to
induce PRL-dependent proliferation when introduced into 32Dcl3 cells
(data not shown). We were therefore interested in determining whether
each of these three forms of the rPRLR were able to induce JNK
activation. We generated a panel of 32Dcl3 cells expressing a single
form of the rPRLR which was tagged with the FLAG epitope tag such that
we could demonstrate expression of the different forms of the PRLR.
Consistent with the results of previous investigators (38, 39), the
short, Nb2, and long forms of the rPRLR were all able to induce
activation of JAK2 as assessed by immunoprecipitation of JAK2
from stimulated cells followed by immunoblotting with
antiphosphotyrosine (data not shown). Phosphorylation of
tyrosine1007 in the activation loop of JAK2 is
required for the catalytic activation of JAK2 (40) and is detected by
antiphosphotyrosine immunoblotting. The different transfected cell
lines, as well as untransfected control cells, were cultured overnight
in medium supplemented with 2% charcoal-stripped serum to allow
signaling molecules to return to their basal level. Only the long and
Nb2 forms of the PRLR stimulated activation of JNK as determined by the
GST-jun kinase assay (Fig. 3A
). No
PRL-induced activation of JNK was observed in the untransfected 32Dcl3
cells or in the 32D/short cells (Fig. 3A
). Expression of the
FLAG-tagged versions of the rPRLR was detected in the different
transfected cells (Fig. 3
, BD). We cannot comment on the relative
amounts of the different receptors in the clones of the transfected
cells since different approaches were used to detect expression of the
protein molecules in the different clones, although other clones
prepared in a similar manner in our laboratory express
3,0005,000 receptors per cell. ONeal and Yu-Lee (37) observed that
the Nb2 form of the rPRLR stimulated a more robust proliferation of
FDCP-1 cells than the long form of the rPRLR. We have performed
dose-response studies that indicate that our 32D/Nb2 cells respond to
lower concentrations of PRL and exhibit a greater extent of
proliferation as measured by an MTT [3-(4, 5-dimethyl
thiazolyl-2)-2,5-diphenyl tetrazolium bromide] assay when compared
with 32D/long cells (data not shown). However, as noted above, we
cannot directly compare the amount of protein for the long and Nb2
forms of the PRLR in the transfected cells; therefore, we cannot
eliminate the possibility that there is simply more Nb2 protein in
cells transfected with that form of the PRLR.

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Figure 3. Activation of JNK in 32Dcl3 Cells Expressing
Different Forms of the rPRLR
A, The 32Dcl3 cell line was transfected with the long, Nb2, or the
short form of the rPRLR, and single cell-derived clones were
isolated. Parental 32Dcl3 cells (lanes 14), 32D/long (lanes 58),
32D/Nb2 (lanes 912), and 32D/short cells were stimulated with 10
nM rPRL for 060 min, the cells were lysed, and the
GST-jun kinase assay was used to examine the activation of JNKs. The
time of stimulation in minutes is indicated at the top
of each lane. The arrowhead on the right
side of the panel indicates the position of the GST-jun fusion protein.
B, 32Dcl3 and 32D/long cells were lysed and membrane fractions were
prepared. Total protein (100 µg) was resolved on a 7.5%
polyacrylamide gel. The immunoblot was probed with anti-Flag M2
antibody. C, 32Dcl3 and 32D/Nb2 cells were lysed and 100 µg total
cellular protein were resolved on a 7.5% polyacrylamide gel. The
immunoblot was probed with the anti-Flag M2 antibody. D, 32Dcl3 and
32D/short cells were lysed, immunoprecipitated with the anti-Flag M2
antibody, and resolved on a 10% polyacrylamide gel. The immunoblot was
probed with anti-M2 antibody.
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The long form of the human PRLR was also transfected into 32Dcl3 cells.
Stimulation of the 32D/hPRLR cells with 100 nM oPRL
resulted in the rapid activation of JNK (Fig. 4A
). Although JNK
activation could be detected after 5 min, it was more apparent at 15
min post stimulation. Activation was maximal at 30 min post stimulation
and declined afterward until reaching basal levels at 1 h (Fig. 4A
, lanes 16). A dose response study
with 32D/hPRLR cells yielded slightly different results than obtained
in Fig. 1
. JNK activation could be detected with 5 nM oPRL,
and the maximal activation was observed at 10 nM oPRL (Fig. 4A
, lanes 712). Concentrations higher than 10 nM actually
were observed to result in lower levels of JNK activation than detected
with 10 nM (Fig. 4A
). This result is consistent with
dose-response curves for PRL-induced mitogenesis as described
previously, and results from the fact that PRL binds to the PRLR as a
monomer through two different binding sites and at high concentrations
of PRL, receptor dimerization is actually inhibited (41). Clearly the
bell-shaped dose-response curve can be influenced by many factors
including ligand affinity for both sites on the receptor, the type and
species of the cell studied, the exact response being investigated, the
number of receptors upon the cell, and the sensitivity of the bioassay
used. As a basis of comparison, the 32D/hPRLR cells were also
stimulated with 100 U/ml IL-3, a concentration 5-fold greater than that
needed to stimulate maximal proliferation of 32Dcl3 cells, and
activation of JNK was examined. Consistent with previous results by
other investigators (25, 26), IL-3 stimulated the activation of JNK as
determined by the GST-jun kinase assay (Fig. 4B
). In contrast to the
results obtained with PRL-stimulated activation of JNK in Figs. 1
, 3
, and 4A
, IL-3 appeared to stimulate JNK activation for a longer period
of time (Fig. 4B
); however, the physiological significance of this is
not clear.

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Figure 4. PRL Induces Activation of JNK in 32Dcl3 Cells
Transfected with the Human PRL Receptor
A, 32D/hPRLR cells were cultured overnight in medium supplemented with
2% charcoal-stripped serum and then concentrated and stimulated with
100 nM oPRL for 060 min (lanes 16) or stimulated with
0100 nM oPRL for 10 min (lanes 712). The cells were
then lysed and processed as described to examine the activation of JNK
as determined by the GST-jun kinase assay. The time in minutes is shown
at the top of lanes 16, and the concentration of oPRL
is indicated at the top of lanes 712. Lane numbers are
indicated at the bottom of the panel. B, 32D/hPRLR cells
were stimulated for 060 min with 100 U/ml recombinant murine IL-3,
and the cells were then processed as described above. The time of
stimulation in minutes is indicated at the top of each
lane. The arrowhead on the right side of
each panel indicates the position of the GST-jun fusion protein.
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Activation of JNKs and ERKs Occurs with Similar Kinetics
Activation of ERKs has been described as being critical in
signaling pathways that regulate cellular proliferation. We therefore
were interested in comparing the kinetics of ERK and JNK activation in
32Dcl3 cells expressing the long form of the hPRLR. These cells were
stimulated with either 10 nM oPRL or 100 U/ml IL-3 for
060 min, and activation of both JNKs and ERKs was examined by
immunoblotting with antibodies that specifically recognize the
activated forms of these enzymes (Fig. 5
). Activation of JNKs was maximal at
1530 min post stimulation with either oPRL or IL-3 (Fig. 5A
).
Some activated JNK was observed in unstimulated cell control for the
IL-3 study (Fig. 5A
, lane 7) which was surprising since they were
treated in a manner identical to the control cell for PRL stimulation
(Fig. 5A
, lane 1). We do not routinely detect a basal level of
activated JNK in 32Dcl3 cells. Bands corresponding to both p46 JNK1 and
p54 JNK2 were observed in cells stimulated with either oPRL or IL-3.
Minor bands that migrated below and above the Mr
46,000 band were observed after 15 min stimulation with either oPRL or
IL-3. These bands may correspond to minor splice variants of different
JNK family members. Reprobing the immunoblot with an antibody that
recognizes both JNK1 and JNK2, although primarily the latter, revealed
the presence of equal amounts of JNK1 and JNK2 in all samples examined.
Similar studies have been conducted with an antibody that recognizes
primarily JNK1, demonstrating no changes in the amount of JNK1 protein
over the time period investigated in this study (data not shown).

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Figure 5. Comparison of the Activation of Both JNK and ERK by
Either PRL or IL-3
32Dcl3 cells expressing the long form of the human PRLR were stimulated
with either 10 nM oPRL (lanes 16), or 100 U/ml IL-3
(lanes 712) for 060 min. Cell lysates were prepared and 50 µg
total cellular protein were resolved on an 10% polyacrylamide gel. The
immunoblot was probed with anti-phospho-JNK, which recognizes activated
JNK1 and JNK2 (panel A); anti-JNK2, which cross-reacts with JNK1 (panel
B); anti-ACTIVE ERK2, which cross-reacts with ERK1 (panel C); and
anti-ERK which detects both ERK1 and ERK2 (panel D). The time in
minutes is shown at the top of each lane. The
arrowheads on the right side of the panel
indicate the positions of JNK-1, JNK-2, ERK-1, and ERK-2.
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The same approach was used to examine the activation of both ERK1 and
ERK2 after stimulation with either oPRL or IL-3. PRL stimulation
resulted in the activation of both ERK1 and ERK2 as early as 5 min
after stimulation (Fig. 5C
, lanes 16). PRL stimulated maximal
activation of ERK1 and ERK2 after 15 min, after which the amount of
activated ERK1 and ERK2 declined (Fig. 5C
, lanes 16). Stimulation
with IL-3 resulted in a similar pattern of ERK activation with maximal
activation evident at 15 min post stimulation (Fig. 5C
, lanes 712).
Equal amounts of ERK1 and ERK2 were demonstrated by probing the
immunoblot with an antibody that recognizes both ERK1 and ERK2 (Fig. 5D
). Based upon this method of analysis, there does not appear to be
any major difference in the activation of JNKs and ERKs by either PRL
or IL-3.
A Kinase-Inactive Mutant of JNK1 Inhibits PRL-Induced
Proliferation
The data presented above indicate that PRL induces activation of
JNK1 and JNK2; however, these studies do not provide any indication of
the biological importance of JNK activation. We have chosen to use a
kinase-inactive mutant of JNK1 to address this point. As noted above,
activation of JNK family members requires the phosphorylation of both
tyrosine and threonine residues in the activation loop of these kinases
(42). Mutation of these two residues to nonphosphorylatable amino acids
results in the generation of nonactivatable kinases that are able to
suppress the activation of the endogenous JNKs (20, 21). Such dominant
negative mutants have been used to address the biological importance of
JNKs in biological processes: Butterfield et al. (20) used
the JNK1-APF and JNK2-APF mutants to demonstrate the importance of JNKs
in regulation of UV-induced apoptosis in small cell lung cancer cell
lines. The JNK1-APF mutant cDNA was cloned into a recombinant human
adenovirus by overlap recombination (43), to generate a virus capable
of transducing the dominant negative JNK1 mutant into a variety of
cells. Nb2 cells, like other T lymphocyte cells, cannot be infected
with human adenoviruses due to the absence of the viral receptor
protein coxsackie-adenovirus receptor (CAR) (44). Therefore, a recently
described deletion mutant of CAR, CAR
1 (44), was introduced into Nb2
cells to allow transduction of these cells with recombinant human
adenoviruses. Transduction of Nb2/CAR
1 cells with a recombinant
human adenovirus expressing green fluorescent protein (Adeno-GFP)
results in >95% of the cells expressing GFP (data not shown).
The effect of the JNK1-APF mutant upon the proliferation of Nb2 cells
was examined by transducing Nb2/CAR
1 and control Nb2 cells with
varying amounts of the virus ranging from a multiplicity of infection
(MOI) of 05. As shown in Fig. 6
, transduction of Nb2/CAR
1 cells with Adeno-JNK1-APF virus caused a
dramatic decrease in the number of viable cells over a 3-day period.
There were consistently fewer cells in cultures of Nb2/CAR
1 cells
transduced with the JNK1-APF virus at all time points examined at both
MOIs of 2.5 or 5. This decrease occurred in a dose-dependent manner
with fewer cells being present in cultures transduced at a MOI of 5
compared with those transduced at a MOI of 2.5. There was no
significant difference in the number of Nb2/CAR
1 cells transduced
with Adeno-GFP compared with normal control cells. These studies were
done on cells cultured in the presence of 200 ng/ml (10 nM)
PRL; however, identical results were obtained when the cells were
cultured in complete media containing FCS (data not shown).
To demonstrate the effect of the JNK1-APF mutant on the activation of
the endogenous JNK1, Nb2 and Nb2/CAR
1 cells were transduced at MOIs
of 0, 0.5, and 5 with either the Adeno-JNK1-APF or the Adeno-GFP.
Transduction of Nb2/CAR
1 cells with the JNK1-APF mutant at a MOI of
5 suppressed the level of activated JNK1 after PRL stimulation as
determined by immunoblotting with anti-phospho-JNK antibody (Fig. 7A
, lane 9). There did not appear to be a
decrease in the activation of JNK1 in cells transduced with the
JNK1-APF virus at a MOI of 0.5 compared with mock infected cells (Fig. 7A
, lanes 7 and 8), nor did transduction of Nb2/CAR
1 cells with
Adeno-GFP suppress PRL-induced JNK1 activation (Fig. 7A
, lanes 1012).
Likewise, transduction of Nb2 cells with either the Adeno-JNK1-APF or
Adeno-GFP viruses had no effect upon the activation of JNK1 (Fig. 7A
, lanes 16). Expression of the hemagglutinin (HA)-tagged JNK1 protein
was only detected in cells transduced with the JNK1-APF mutant at a MOI
of 5 (Fig. 7B
, lane 9). An endogenous background band is seen in all
lanes migrating just above the HA-tagged JNK1-APF protein. This band is
seen in all cells, including the mock-transduced control cells (Fig. 7B
, lanes 1, 4, 7, and 10). We interpret these data to indicate that
transduction of Nb2/CAR
1 cells with the Adeno-JNK1-APF virus
suppresses the PRL-induced activation of JNK1 and suppresses the
PRL-induced proliferation of Nb2/CAR
1 cells.

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Figure 7. Kinase Inactive JNK1-APF Mutant Blocks PRL-Induced
JNK Activation in Nb2/CAR 1, but Not Nb2 Cells
Nb2 (lanes 16) and Nb2/CAR 1 (lanes 712) were transduced with the
recombinant human adenovirus encoding the JNK1-APF mutant (lanes 13
and 79) or Adeno-GFP (lanes 46 and 1012), at a MOI of 0
(lanes 1, 4, 7, and 10), MOI of 0.5 (lanes 2, 5, 8, and 11), or a MOI
of 5 (lanes 3, 6, 9, and 12) for 24 h in Fishers medium
supplemented with 10% horse serum and 200 ng/ml oPRL. The cells were
lysed and clarified, and the protein concentration was determined as
described above. A 50-µg sample of each lysate was resolved on a 10%
SDS polyacrylamide gel, and the proteins were transferred to a nylon
membrane and then immunoblotted with anti-phospho-JNK1 (panel A), or
anti-HA monoclonal antibody (panel B). The positions of phospho-JNK1
and the HA-tagged JNK1-APF proteins are indicated on the
right side of the panel. The virus and MOI are indicated
at the top of each lane.
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Kinase-Inactive Mutant of JNK1 Induces Apoptosis of Nb2 Cells
The observed decrease in cellular proliferation described in Fig. 6
above could result from a decrease in mitogenic signaling or an
increase in apoptosis. To determine whether there was an increase in
apoptotic cells, we looked for whether there was an increase in DNA
fragmentation typical of cells undergoing apoptosis. Nb2 and
Nb2/CAR
1 cells were transduced with the Adeno-JNK1-APF virus at MOIs
varying from 05. After 24 h the cells were harvested from the
cultures, the DNA isolated, and the fragmentation of the DNA assessed
by resolving a 10-µg sample of each DNA on a 1.5% agarose gel. As
can be seen in Fig. 8
, transduction of
the Nb2/CAR
1 cells, but not the Nb2 cells, resulted in the induction
of a DNA ladder typical of cells undergoing apoptosis. Transduction of
Nb2/CAR
1 cells with recombinant human adenovirus encoding GFP at the
same MOI did not induce DNA fragmentation.

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Figure 8. Kinase-Inactive JNK1-APF Mutant Induces DNA
Fragmentation in Nb2/CAR 1, but Not Nb2 Cells, Grown in the Presence
of PRL
Nb2/CAR 1 cells were left untransduced (lanes 2 and 5),
transduced with the recombinant human adenovirus encoding the JNK1-APF
mutant (lanes 3 and 4), or transduced with Adeno-GFP (lanes 6 and 7). A
MOI of 2.5 was used in lanes 3 and 6, and a MOI of 5 was used in lanes
4 and 7. After transduction the cells were cultured for 24 h in
the presence of 200 ng/ml oPRL before being lysed as described. After
purification and quantitation of the amount of DNA in each sample, a 10
µg amount of DNA was resolved on a 1.5% agarose gel. Lane 1 contains
size markers.
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DISCUSSION
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In recent years the MAP kinase family has grown to include three
subfamilies: the ERK family, the JNK family, and most recently the
p38/HOG1 family. Current investigations are focused upon
determining the role of each of these different kinases in mitogenesis,
response to cellular stress, apoptosis, differentiation, and oncogenic
transformation. The activation of JNK family members was first
demonstrated to occur in response to stresses such as TNF-
(4), heat
shock (4), protein synthesis inhibitors (4, 24), UV light (2, 7, 21),
-irradiation (21, 45), nitric oxide and hydrogen peroxide (23), and
osmotic stress (2). A variety of proliferative signals have also been
demonstrated to induce JNK activation, such as the BCR-ABL oncogene
(46), and several hematopoietic growth factors, including stem cell
factor, IL-3, granulocyte macrophage-colony stimulating factor,
erythropoietin, and thrombopoietin (25, 26, 27).
In this study we have demonstrated that PRL induces activation of JNK
in a time- and dose-dependent manner. This was observed in a number of
different cell lines including the PRL-dependent Nb2 cell line, and an
IL-3-dependent murine myeloid cell line stably expressing different
forms of the PRLR, and the T47D cell line. Our data suggest that only
the long and Nb2 forms of the rPRLR are able to stimulate activation of
JNKs, as well as induce proliferation of transfected 32Dcl3 cells.
Although other investigators have reported that the short form of the
PRLR can induce proliferation of NIH3T3 cells (47), we have not
observed any biological effect of this form of the PRLR.
A role for JNK activation in mitogenesis is suggested by the fact that
the two forms of the rPRLR that are able to stimulate proliferation
(37) are both able to induce activation of JNK. The effect of
expression of the JNK1-APF mutant in Nb2 cells also suggests that JNK1
may provide an important mitogenic signal downstream of the PRLR. The
fact that the JNK1-APF mutant appears to induce apoptosis suggests that
either apoptosis is the default pathway in these cells and in the
absence of a mitogenic signal the cells undergo apoptosis, or that JNK1
is important in suppressing apoptosis. Our current studies do not
distinguish between these two possibilities. Clearly, JNK inhibition
results in a dominant phenotype resulting in cellular death. It would
be interesting to determine whether a constitutively activated form of
JNK1 would suppress apoptosis or induce growth factor-independent
proliferation.
The JNK family members are encoded by three different genes (JNK1,
JNK2, and JNK3), and the transcripts of each gene are able to undergo
alternative splicing, resulting in the production of multiple protein
products (48). Our immunoblotting studies indicate that both JNK1 and
JNK2 are activated by PRL, and that there are additional minor splice
variants that can be detected by immunoblotting. We have not detected
the activation of JNK3 in these cells, which may not be surprising
since current evidence suggests that expression of JNK3 may be highest
in neuronal tissues (48, 49).
The activation of ERK1 and ERK2 can either stimulate cellular
proliferation or differentiation. Although it is not known what
biochemical events distinguish a proliferative signal generated by ERKs
from one that induces differentiation, it has been suggested that the
duration of ERK activity may be involved. Transient activation of ERKs
has been suggested to mediate a proliferative signal (15). Depending
upon the stimulus, JNK activation may be involved in either
mitogenesis, oncogenic transformation, differentiation, or induction of
apoptosis. It has been suggested that the transient induction of JNK
activity by activation of the T cell receptor may lead to cell
proliferation, whereas prolonged induction by certain apoptotic signals
leads to apoptosis (21). PRL induces a transient activation of JNK,
peaking at 1530 min and then decreases in all the cell types we
examined. Our data indicate that both JNK and ERK are activated with
approximately the same rate in several different cell types. It is not
clear at this time whether the activation of both JNKs and ERKs is
required for PRL-induced mitogenesis or whether each kinase has a
unique function. Our data suggest that JNKs represent another class of
signaling molecules that lie downstream of the PRLR that may be
required for mitogenesis by this receptor. Understanding the role that
JNKs play in signaling, and the identification of mechanisms that
regulate activation of JNK in response to PRL, is fundamental to our
understanding of signaling events that regulate cellular proliferation,
differentiation, and cell death.
 |
MATERIALS AND METHODS
|
---|
Cells and Cell Culture
The Nb211 cell line was obtained from Dr. Arthur Buckley
(University of Cincinnati School of Medicine, Cincinnati, OH).
The cells were maintained in Fishers medium supplemented with
10% FCS, 10% horse serum, 1 mM L-glutamine,
100 U/ml penicillin, 100 µg/ml streptomycin, and
10-4 M ß-mercaptoethanol.
The 32Dcl3 cell line was obtained from Dr. Joel Greenberger (University
of Pittsburgh, Pittsburgh, PA). Characterization of 32Dcl3 cells with
the human and Nb2 forms of the PRLR were described previously (50).
Cultivation of 32Dcl3 cells has been described recently (51).
Charcoal-stripped FCS was obtained from HyClone Laboratories, Inc. (Logan, UT), and FCS was from Summit Biotechnology (Fort Collins, CO). All other media components
were from Life Technologies, Inc./BRL (Gaithersburg, MD).
Human (lot AFP-3855A), rat (lot APF-6452B), and ovine (lot AFP-10677C)
PRL were obtained from the National Hormone and Pituitary Program
(Rockville, MD). Recombinant murine IL-3 was obtained from
Collaborative Biomedical Products (Bedford, MA).
Kinase Assay for JNK Activity
Cells to be analyzed were cultured overnight in media
supplemented with charcoal-stripped serum. Cells (2 x
107) were resuspended in 1 ml serum-free tissue
culture medium supplemented with the indicated concentration of either
PRL or IL-3 for the indicated times. The cells were pelleted in a
microfuge and lysed in JNK lysis buffer [25 mM HEPES, pH
7.7, 20 mM ß-glycerophosphate, 0.1 mM sodium
orthovanadate, 0.1% Triton X-100, 0.3 M NaCl, 1.5
mM MgCl2, 0.2 mM EDTA,
0.5 mM dithiothreitol, 4 µg/ml Aprotinin
(Calbiochem, La Jolla, CA)] and allowed to sit on ice for
30 min. The lysates were clarified by spinning at 10,000 rpm for 5 min
in a refrigerated Savant SRF13K microfuge. Protein concentrations of
the clarified lysate were determined by the Pierce Chemical Co. Coomassie Plus protein assay (Rockford, IL), and 300 µg
total cellular protein were used in each assay. A 100 µl volume of a
10% suspension of GST-c-jun (179) was added to 300 µg
total cellular protein in a final volume of 1 ml and incubated for
2 h at 4 C. The beads were then washed three times with 20
mM HEPES, pH 7.7, 50 mM
NaCl, 2.5 mM MgCl2, 0.1
mM EDTA, 0.05% Triton X-100. The washed beads
were then incubated for 20 min at 30 C, in 40 µl 50
mM ß-glycerophosphate, pH 7.6, 0.1
mM sodium orthovanadate, 10
mM MgCl2, 20
µM ATP (5,000 cpm/pmol). The reactions were
terminated by the addition of 10 µl 5 x SDS sample buffer and
boiled, and the reaction products were resolved on a 10% SDS
polyacrylamide gel. The position of GST-jun was determined by staining
the gel, and the extent of GST-jun phosphorylation was determined by
autoradiography.
The GST-c-jun (179) expression vector and purification of
the fusion proteins have been described previously (20). A 5 ml
overnight culture of the bacterial was diluted to 500 ml and allowed to
grow until an OD600 of 0.6. IPTG
was added to a final concentration of 0.4 mM for
3 h, after which the bacteria were collected by centrifugation and
the pellets frozen at -20 C. The pellets were thawed, resuspended in
10 ml NETN [20 mM Tris, pH 7.6, 100
mM NaCl, 1 mM EDTA, 2
mM dithiothreitol, 0.5% NP-40, 100 U/ml
Aprotinin (Calbiochem)], and 200 µl of 1 mg/ml lysozyme
were added. After 1 h on ice, the bacteria were sonicated for 2
min, the lysates clarified by centrifugation at 10,000 rpm for 10 min,
and the supernatant fluid transferred to a new tube. A 50% slurry of
glutathione agarose (1.5 ml) (Pharmacia Biotech,
Piscataway, NJ) was added, and the tube was placed on a rocking
platform for 1 h at 4 C. The beads were washed three times with
NETN buffer and resuspended in JNK lysis buffer to a
concentration of 10%.
Immunoblotting
Cells were lysed in JNK lysis buffer and the lysates clarified
by spinning at 10,000 rpm in a Savant RCF13K refrigerated microfuge for
30 min. Protein concentrations were determined as described above. Each
cell lysate (5075 µg) was resolved on a 10% gel, transferred to an
Immobilon membrane, and immunoblotted with the desired antibody as
described previously (51, 52). Immunoprecipitation with the
anti-Flag antibody and preparation of membrane fractions were
performed as described previously (53).
Anti-JNK1 and anti-JNK2 and an anti-MAP kinase antibody that
cross-reacts with both ERK1 and ERK2 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-ACTIVE ERK2
antibody was obtained from Promega Corp. (Madison, WI).
Anti-phospho-JNK antibody and control anti-JNK antibody were obtained
from New England Biolabs, Inc. (Beverly, MA). The anti-HA
monoclonal antibody was obtained from Roche Molecular Biochemicals (Indianapolis, IN). The anti-Flag monoclonal
antibody M2 was obtained from Sigma (St. Louis, MO).
Generation of Recombinant Human Adenovirus Encoding Kinase
Inactivate Mutants of JNK1 and Infection of Nb2 Cells
Recombinant human adenovirus encoding the JNK1-APF mutant was
generated by overlap recombination (43). A fragment encoding the
JNK1-APF isoform cDNA was ligated between the HindIII and
XbaI sites in the pACCMV#95, which encodes the left end of
the adenovirus chromosome with the E1A gene and the 5'-half of the E1B
gene replaced by the cytomegalovirus major immediate early promoter, a
multiple cloning site, intron, and polyadenylation sequences from SV40
to yield the plasmid pACCMV#95-JNK1-APF. The plasmid (5 µg) was mixed
with BstBI-digested
Ad5dl327Bstß-Gal-TP complex.
The mixture was precipitated with calcium phosphate for 1 min in a
total volume of 400 µl and then overlaid on 293 cells that were at
50% confluency. After 816 h, fresh DMEM supplemented with 10% FCS
was added, and the transfected cells were incubated for 7 days. The
transfected cells were lysed by three cycles of freezing and thawing,
and dilutions of the freeze-thaw lysates were used to infect fresh 293
cells to plaque purify the virus. The infected 293 cells were overlaid
with DMEM containing 10% FCS and 1% Noble agar, fed with the same
medium 4 days later, and then stained with
5-bromo-4-chloro-3-indoylb-D-galactopyranoside
and neutral red. Clear plaques, which are derived from recombinant
adenovirus that have lost the LacZ gene present in the parental viral
chromosome, were picked, and the DNA purified. The viral DNA was
amplified by PCR for the presence of the JNK1-APF cDNA.
Human adenoviruses do not readily infect hematopoietic cells of human
or rodent origin, apparently due to the lack of the cellular adenovirus
and coxsackievirus receptor (CAR) (44). CAR was introduced into Nb2
cells by infecting the cells with the LXSN retrovirus encoding CAR
1
(44), and infected cells were selected using 0.5 mg/ml G418 since the
LXSN vector contains the neomycin-resistance gene. After selection of
G418-resistant cells, the mixed population of cells was infected with
recombinant human adenovirus encoding GFP, and 24 h after
transduction, GFP-positive cells were selected separated with a
fluorescent cell sorter, and separated into 96-well dishes. The sorted
cells were expanded and screened again for their ability to be
transduced with the GFP-encoding recombinant adenovirus. Clones with
the highest rate of infection were chosen for use in these studies.
Typically, >95% of the Nb2/CAR
1 cells could be transduced with the
GFP-adenovirus (data not shown).
Nb2/CAR
1 cells were transduced with varying multiplicities of
infection (MOI) ranging from 15 virus particles per cell with either
Adeno-GFP or Adeno-JNK1-APF in Fishers media lacking horse
serum for 1 h, after which complete Fishers media supplemented
with 10% horse serum and 200 ng/ml oPRL was added. Controls for these
studies included normal Nb2 cells that could not be infected with
recombinant human adenoviruses (data not shown). Expression of the
JNK1-APF protein was confirmed by immunoblotting with anti-HA
monoclonal antibody.
DNA Fragmentation Assay
Nb2 and Nb2/CAR
1 were transduced with the adeno-JNK1-APF
virus as described above and then cultured for 048 h in Fishers
media supplemented with 10% horse serum and 200 ng/ml oPRL. At 0, 24,
and 48 h, the cells were lysed in DNA lysis buffer (50
mM Tris, pH 7.5, 10 mM EDTA, 0.5% SDS, 100
µg RNase A, 4 µg/ml Proteinase K) and incubated at 37 C for 6
h. The DNA was extracted using 400 µl phenol- chloroform-isoamyl
alcohol (25:24:1). 0.25 M sodium acetate was added, and the
DNA was precipitated using 100% ethanol. The DNA was pelleted and
resuspended in TE with 20 µg/ml RNase. The DNA was quantitated
spectroscopically, and 10 µg of DNA were analyzed for fragmentation
on a 1.5% agarose gel.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Karen Helm of the University of Colorado
Cancer Center FACS Core for her assistance in isolating the Nb2/CAR
1
cells. We also thank Drs. Elizabeth Burton, Arthur Gutierrez-Hartmann,
and Mary Reyland for their comments on the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Steven M. Anderson, Department of Pathology, Box B-216, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262. E-mail:
steve.anderson{at}uchsc.edu
This research was supported by NIH Grant DK-53858 (to S.M.A.), and a
University of Colorado Cancer Center "Wines for Life" Seed Grant.
The University of Colorado Cancer Center FACS Core is supported by a
Cancer Center grant (CA-46934) from the National Cancer Institute.
{hd2}Note Added in Proof
{texf}While this manuscript was under review, Olazabal et al.
(54 ) demonstrated that PRL stimulation of bovine mammary gland
epithelial cells transfected with the long from of the PRLR stimulated
JNK activation. Furthermore, treatment of these cells with
dexamethasone inhibited JNK activated. Although these authors
demonstrated that PRL can induce proliferation and AP-1 activation,
they do not demonstrate that dominant negative JNK mutants block either
of these effects.
Received for publication November 17, 1999.
Revision received June 20, 2000.
Accepted for publication June 28, 2000.
 |
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