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Division of Cell and Molecular Biology Department of Biology Boston University Boston, Massachusetts 02215
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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3-h interpulse interval normally required for full recovery of GH
pulse responsiveness. Termination of GH pulse-induced STAT5b signaling
is thus a complex process that involves multiple biochemical events.
These are proposed to include the down-regulation of JAK2 signaling to
STAT5b via a cycloheximide- and H7-sensitive step, proteasome-dependent
degradation of a key component or regulatory factor, and
dephosphorylation leading to deactivation of the receptor-kinase
signaling complex and its STAT5b substrate via the action of a
phosphotyrosine phosphatase. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 h duration every 3.5–4 h, with a typical peak
plasma GH concentration of 200 ng/ml, while females exhibit more
continuous plasma GH levels of about 20–40 ng/ml (6). Recent studies
have identified the transcription factor STAT5b (signal transducer and
activator of transcription 5b) as a key signaling molecule that
responds to GH in male but not female rat liver and can transduce the
male GH pulse signal from the plasma membrane-bound GHR to
male-expressed target genes within the nucleus (7). STAT1 and STAT3 can
also be activated by GH in rat liver (8, 9, 10) but not in a sex-specific
manner (10). Targeted disruption of STAT5b leads to a
selective loss of the male-specific pattern of liver gene expression,
as well as the loss of GH pulse-induced male whole body growth rates
(11), indicating that STAT5b may be a direct mediator of a
broad range of physiological responses to GH pulses. Indeed, several GH
pulse-activated, male-expressed liver genes have functional binding
sites for STAT5b in their 5'-flank (12) (S.-H. Park and D. J. Waxman,
unpublished). STAT5b can be repeatedly activated by physiological GH pulses by tyrosine phosphorylation (7) catalyzed by JAK2 kinase (Janus kinase 2) (13, 14). STAT5b is then deactivated by a dephosphorylation reaction catalyzed by a phosphotyrosine phosphatase and then is reactivated by tyrosine rephosphorylation in response to a subsequent plasma GH pulse. This repeat activation of STAT5b occurs both in intact rat liver in vivo (7) and in CWSV-1 cells, a cultured rat hepatocyte line that is responsive to GH (15, 16) and contains an intact GHR-JAK2-STAT5b signaling pathway (17). In order for a GH pulse to induce a full cycle of STAT5b activation and deactivation, several upstream signaling events need to occur. First, GH must bind to and dimerize its receptor (18), after which JAK2 is recruited (13, 14), phosphorylating itself and GHR on multiple tyrosine residues (19, 20). STAT5b is subsequently recruited to the GHR-JAK2 signaling complex (21, 22) and then is activated by JAK2-catalyzed phosphorylation on tyrosine 699 (23). GH-activated STAT5b also undergoes an H7-sensitive secondary phosphorylation on serine (or threonine) (10, 17) that may modulate its transcriptional activity. This process is analogous to the secondary phosphorylation of other STATs on serine and is associated with enhanced transcriptional responses (24, 25). GH-activated STAT5b dimerizes via SH2 domain interactions and is then transported to the nucleus (26), where it binds directly to DNA response elements upstream of GH target genes (12, 27, 28, 29). The repeated activation in vivo of STAT5b by physiological GH pulses (7) requires that this JAK-STAT signaling pathway not only respond rapidly to the stimulatory effect of a GH pulse, but also that it deactivate between GH pulses. The activation of STAT5b by a second GH pulse does not require new protein synthesis, provided there is an interpulse interval of at least 2.5–3 h (17), corresponding to the physiological spacing of GH pulses in adult male rats (6). Since this process is independent of protein synthesis under conditions where cellular STAT5b is quantitatively converted to its tyrosine phosphorylated, nuclear form (17), GH-activated STAT5b molecules must dephosphorylate and recycle from the nucleus back to the cytosol, rather than be degraded. Further details of the STAT5b deactivation pathway are unknown.
In the present study, we investigate the cellular events
associated with the physiologically important down-regulation of STAT5b
activation that needs to occur at the conclusion of each plasma GH
pulse. The use of selective inhibitors to perturb normal functioning of
the JAK-STAT pathway has enabled us to develop testable models for
GH-stimulated activation and termination of a receptor-kinase cycle and
STAT activation loop. Evidence is presented for two distinct
biochemical events that contribute to the termination of GH-activated
JAK2-STAT5 signaling. We also show that the reset mechanism, which
requires a GH interpulse interval of 3 h to restore full STAT5b
responsiveness, can be circumvented by inhibition of serine/threonine
kinase activity.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). This complex was shown to
contain STAT5b, but not two other GH-activated STATs (STATs 1 and 3) by
supershift analysis using anti-STAT5b antibody (17). The activation of
STAT5b in GH-treated cells lasts 1 h, after which STAT5b activity
declines rapidly. The rapid, high-level activation of STAT5b by a pulse
of GH, followed by the decline in the STAT5b signal, is presently
termed phase 1 STAT5b signaling. This phase 1 signal corresponds
to the pattern of STAT5b response to pulsatile GH that occurs in adult
male rat liver (7).
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STAT5b Deactivation Is Initiated Early during a GH Pulse and in a
Tyrosine Kinase-Dependent Manner
We first examined how long GH must be maintained in contact
with the cells to initiate a full cycle of STAT5b activation and
deactivation. CWSV-1 cells were incubated with GH for either 10 or 60
min, after which the cells were washed and placed in GH-free medium.
EMSA of total cell extracts for STAT5b activity revealed that in both
cases the activated STAT5b signal was near maximal by 10 min (Fig. 1
).
Small differences were observed in the overall length of the STAT5b
cycle, which lasted approximately 75 min in the case of a 60-min GH
pulse (Fig. 1A, lanes 2–6) or a 30 min pulse (data not shown) as
compared with approximately 60 min for a 10-min GH pulse (lanes 9–12)
(also see Fig. 1B). All of the elements required for a cycle of STAT5b
activation and deactivation are therefore initiated early during the GH
pulse, and it is not necessary for GH to be present for the full 1 h
of a physiological plasma GH pulse to achieve a complete cycle of phase
1 STAT5b signaling. However, since the duration of STAT5b activity was
found to be the same after either a 30-min or a 60-min pulse, new
STAT5b activation probably does not continue beyond 30 min. This could
result from a block in further assembly of ligand, receptor, and kinase
into functional GHR-JAK2 complexes, perhaps by depletion of monomeric
GHR or JAK2 molecules from the cell surface. Alternatively, the initial
GH pulse may induce a factor that blocks further GHR-JAK2 complex
assembly.
Lifespan of GH Pulse-Activated JAK2 and STAT5b
The duration of the activated STAT5b signal measured by EMSA (Fig. 1) depends both on the duration of JAK2-catalyzed STAT5b activation and
on the half-life of individual activated STAT5b molecules. To establish
the time period that JAK2 remains active and capable of phosphorylating
STAT5b, cells were given a 20-min GH pulse and, at various times
thereafter, cell extracts were immunoprecipitated with anti-JAK2
antibody and then probed for phosphotyrosine content using
monoclonal antibody 4G10. Western blot analysis (Fig. 2A) indicated that JAK2 remains in its
tyrosine phosphorylated and presumably activated state through
approximately 40 min (lanes 9–11), and then declines at 60 min (lane
12) to the point where little or no signal is visible 75 min after the
addition of GH (lane 13). Cellular JAK2 protein levels were not
significantly changed during this time period (lanes 1–7). That JAK2
remains maximally phosphorylated for 40 min, well after the time of GH
removal (20 min in this experiment) implies that the activated GHR-JAK2
complex is not rapidly disassembled or deactivated once excess GH
ligand is removed.
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, solid black
squares). Genistein addition at 20, 30, and 40 min (Fig. 2B, arrows) or 50 min (Fig. 2C) led to a premature decline in
activated STAT5b. This demonstrates that, in the absence of the
tyrosine kinase inhibitor genistein, JAK2 signaling to new STAT5b
molecules continues even 50 min after the onset of a GH pulse. This is
consistent with our finding that JAK2 remains in its active,
phosphorylated state for about this length of time (Fig. 2A, lanes
9–11). Semilog plots of the decline in activated STAT5b after
genistein addition (Fig. 2C) yielded parallel lines for each time point
of genistein addition, with slopes corresponding to a half-life of
8.8 ± 0.9 min. The rate of STAT5b deactivation determined from
these data is essentially constant between 20 and 90 min after GH
addition. Moreover, the same half-life was calculated from measurements
of the decay in STAT5b EMSA activity after 60 min in the absence of any
inhibitors (UT curve, Fig. 2C), consistent with our conclusion that
there is little or no new signaling by JAK2 to STAT5b at this
point.
Although genistein added either 30 or 40 min after GH addition
effectively blocked the subsequent activation of STAT5b by JAK2 (Fig. 2B, dashed curves), genistein prolonged the phosphorylated
JAK2 signal compared with samples treated with GH alone, as revealed by
JAK2 immunoprecipitation followed by phosphotyrosine analysis (Fig. 2A, lanes 15–18 vs. 11–13). One possible explanation for this
observation is that the binding of genistein to JAK2 may block access
of the phosphatase to the JAK2 phosphotyrosine residue. Alternatively,
tyrosine kinase activity may be required to activate the phosphatase
that dephosphorylates JAK2.
Intrinsic Stability of Activated GHR-JAK2 Complex
Since the JAK2 kinase inhibitor genistein fully inhibits
GH-induced STAT5b activation (17), we used genistein as an inhibitory
probe to freeze GH-activated JAK2 in a catalytically inactive state.
This enabled us to test directly whether GH itself must continue to be
present in the culture medium for phase 1 STAT5b signaling to proceed.
CWSV-1 cells were treated with GH for 30 sec, at which point genistein
was added and the incubation continued for 1 h. EMSA revealed a
very low level of active (tyrosine phosphorylated) STAT5b during the
1-h GH pulse (Fig. 3A, lanes 8 and 9
vs. genistein-free controls in lanes 2–4), which
corresponds to approximately 85–90% inhibition of JAK2-dependent
STAT5b activation (Fig. 3B). Removal of both genistein and GH from the
cells at t = 1 h released JAK2 from genistein inhibition, and
resulted in a dramatic increase in the level of activated STAT5b (Fig. 3A, lanes 10 and 11). The activation of STAT5b after genistein removal
closely mirrors a normal GH pulse-induced phase 1 STAT5b EMSA profile,
both with respect to the time course and the maximal level of STAT5b
activation and deactivation (Fig. 3B, solid circles). A
similar experiment where genistein was added to the culture medium 15
min before GH gave an identical STAT5b activation and deactivation
profile. These data suggest that the cellular events required for
STAT5b activation and deactivation are both initiated by GH at the same
time point relative to the onset of JAK2 tyrosine kinase activity.
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, lanes 15–17). In contrast, the genistein-freeze samples showed a
robust phosphorylated JAK2 band through the 60-min period of GH +
genistein treatment (lanes 20 and 21) which remained for at least 45
min after removal of GH and genistein (lane 22). The decline in JAK2
phosphorylation by 75 min after genistein removal (lane 23) proceeded
with a time course similar to the corresponding decline in
genistein-free samples by 70 min after GH addition (i.e.
lane 17). Thus, genistein treatment freezes JAK2 in a tyrosine
phosphorylated form that is proposed to remain inactive but stable
until the tyrosine kinase inhibitor is removed.
Cycloheximide Sustains Activated STAT5b by Prolonging JAK2
Signaling
CWSV-1 cells respond to sequential GH pulses with the repeated
activation of essentially the entire cellular pool of STAT5b without
the need for new protein synthesis (17). Thus, the bulk of STAT5b
molecules deactivate at the end of a GH pulse without undergoing
protein degradation; they then recycle from the nucleus back to the
cytosol, where they can subsequently be activated by an incoming GH
pulse. Further experiments revealed, however, that the protein
synthesis inhibitor cycloheximide markedly slows down the decline in
STAT5b EMSA activity at the end of a GH pulse (Fig. 4A, lanes 12–14 vs. 5–7).
This effect of cycloheximide is seen both when GH continues to be
present after 1 h (data not shown) and when GH is removed at
1 h (Fig. 4A). This indicates that the prolonged STAT5b EMSA
signal does not result from the assembly of new GHR-JAK2 complexes.
Ultimately, however, the level of STAT5b EMSA activity declines back to
baseline by 2 h after GH removal (Fig. 4D). This protein
synthesis-independent regeneration of an unactivated pool of STAT5b
molecules is consistent with our earlier finding that ongoing protein
synthesis is not required for STAT5b to respond fully to a second GH
pulse when it is applied 3 h after termination of the first pulse
(17).
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shows that the
protective effect of cycloheximide on STAT5b EMSA activity normally
seen 30 min and 1 h after GH removal was abolished (lanes 21 and
22 vs. 18 and 19; panel D). Moreover, phosphorylated JAK2
levels declined more slowly in cycloheximide-treated cells compared
with cells treated with GH alone (cf. lane 31 vs.
27). Thus, cycloheximide prolongs STAT5b EMSA activity by prolonging
the time during which JAK2 signals to STAT5b, rather than by protecting
activated STAT5b molecules from dephosphorylation. Protein synthesis is
thus required for the rapid deactivation of JAK2 that occurs within
1 h after the initiation of GH treatment. The observation that
JAK2 signaling to STAT5b ultimately declines in the presence of
cycloheximide (cf. 2 h data point; Fig. 4, B and D)
indicates, however, that an additional, slower deactivation mechanism
may proceed even in the absence of new protein synthesis.
H7 Prolongs the STAT5b EMSA Signal by Prolonging JAK2 Signaling to
STAT5b
An H7-sensitive kinase can contribute to the cytokine/growth
factor-Induced secondary phosphorylation of STATs on serine (or
threonine) (17, 30). In view of this, as well as our earlier
observation that the GH-activated STAT5b signal can be prolonged in the
presence of H7 and other serine/threonine kinase inhibitors (17), we
sought to clarify whether H7 blocks JAK2 deactivation or whether it
inhibits STAT5b dephosphorylation. Figure 5 shows that H7 pretreatment sustains the
GH pulse-activated STAT5b EMSA signal compared with control (panel A,
lanes 11–14 vs. 4–7). However, when genistein was added 45
min after GH addition to block further JAK2 phosphorylation of STAT5b,
the sustaining effect of H7 was significantly reduced (Fig. 5A, lanes
18–21, and Fig. 5C). Thus, H7 prolongs JAK2 signaling to STAT5b,
rather than inhibits STAT5b deactivation. JAK2 phosphotyrosine analysis
confirmed the ability of H7 to maintain JAK2 in its active,
tyrosine-phosphorylated state after GH removal (Fig. 5B, lane 6
vs. 3). The possibility that H7 may act at the same step as
cycloheximide, i.e. by blocking synthesis of a protein that
inhibits or deactivates JAK2, is supported by the inability of H7 to
prolong JAK2 signaling to STAT5b when it is added at the end of a 1-h
GH pulse, i.e. at a time when H7-sensitive, STAT5b
transcriptional responses would have already been initiated (Fig. 5D, lanes 11 and 12 vs. 7 and 8). STAT5b activity in H7-treated
CWSV-1 cells eventually declined, even in the absence of genistein
(Fig. 5C; also see Fig. 8A, below), suggesting the existence of two
pathways for cessation of JAK2 signaling, only one of which is blocked
by H7. Alternatively, the effects of H7 may be incomplete, since H7
slows down, but does not fully block GH-induced STAT5b phosphorylation
on serine (or threonine) (17).
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shows that pervanadate can sustain STAT5b EMSA activity for at least
1 h after GH removal (lanes 8–10). Prolonged STAT5b activity was
also observed when genistein was added in combination with pervanadate,
albeit at a somewhat lower level than was seen with pervanadate alone
(lanes 14–16 vs. 8–10). Pervanadate is thus proposed to
prolong STAT5b EMSA activity by two mechanisms: 1) by inhibiting STAT5b
dephosphorylation, and 2) by inhibiting the deactivation of JAK2
kinase-dependent STAT5b activation, as indicated by the somewhat lower
STAT5b activity seen in pervanadate + genistein treated cells compared
with pervanadate alone.
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; vertical arrows). A short GH pulse was
chosen for this experiment to minimize contributions of new GHR-JAK2
complex assembly to STAT5b activation (c.f. Fig. 1).
Addition of pervanadate, to stabilize activated STAT5b molecules
already formed, together with genistein, to block JAK2-dependent
phosphorylation of additional STAT5b molecules, resulted in STAT5b EMSA
activity being maintained at a constant level, independent of whether
the inhibitors were added 30, 40, or 50 min after initiation of the GH
pulse (Fig. 6B, dashed lines). By contrast, pervanadate
alone induced an increase in STAT5b activity at all three time points
of addition. In each case, the increase in STAT5b EMSA activity leveled
off at a point that corresponds to depletion of the higher mobility,
unphosphorylated STAT5b, band 0, as seen on Western blots
(c.f. 17 (data not shown). This maximum level of STAT5b
EMSA activity in pervanadate-treated cells thus corresponds to the
point where the cell’s entire STAT5b substrate pool has been
activated.
Pervanadate treatment in combination with GH was associated with the
formation of an additional, higher mobility protein-DNA complex (Fig. 6A, lanes 9, 10) that was identified as containing STAT1 (but not
STAT5b) by anti-STAT EMSA supershift studies (10) (data not shown).
Formation of this STAT1 complex was blocked by genistein in the
pervanadate-treated cells (lanes 15 and 16), supporting the
interpretation that pervanadate does enable a tyrosine kinase (probably
JAK2) to retain activity after GH removal. GH-induced STAT1
phosphorylation is weak in CWSV-1 cells, as it is in rat liver in
vivo at physiological GH doses (10).
Inhibition of Proteasome Action Sustains Phase 1 STAT5b
Signaling
We next investigated whether proteasome activity contributes to
termination of GH pulse-induced STAT5b signaling. Figure 7 shows that the proteasome inhibitor
MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal) prolongs GH-induced
STAT5b EMSA activity for at least 2 h after removal of GH (Fig. 7, A and C) (c.f. total, or near-total loss of STAT5b signal in
the absence of MG132 within 30 min after the end of a 1-h GH pulse;
Figs. 4 and 5). Genistein abolished this effect of MG132, demonstrating
that MG132 treatment prolongs signaling from JAK2 to STAT5b (Fig. 7A, lanes 7–9 vs. 4–6, and Fig. 7C). JAK2 phosphotyrosine
analysis confirmed this conclusion, as judged by the prolonged
phosphotyrosyl-JAK2 signal seen in MG132-treated cells (Fig. 7B, lane
13 vs. lane 17). Since new protein synthesis is not required
for CWSV-1 cells to respond to a second GH pulse (17), GH is unlikely
to stimulate extensive degradation of GHR or JAK2. The stabilizing
effect of MG132 on phospho-JAK2 and on JAK2 signaling to STAT5b
reported here may thus result from an indirect stabilizing effect of
MG132 on the receptor-kinase complex.
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and 2). We
therefore investigated whether this cessation of JAK2 activation
contributes to the unresponsiveness of liver cells to a second GH pulse
early during the GH interpulse interval (17). We first examined whether
any of the inhibitors that sustain JAK2 signaling to STAT5, namely H7,
cycloheximide, and MG132, can prolong the time period during which
GH-induced JAK2 activation, and thus STAT5b activation, is dependent on
the continued presence of exogenous GH. To test this hypothesis, CWSV-1
cells were preincubated with each of the inhibitors, and then were
treated with GH either for a 1-h pulse, or continuously. STAT5b EMSA
activity was measured and expressed as a percentage of the peak
activity level observed 45 min after GH addition. For cycloheximide and
MG132, STAT5b EMSA activity that was measured at 1 or 2 h after
the initial GH pulse had decayed to the same level in samples
maintained in the continued presence of GH (data not shown) as in
samples where GH was withdrawn after 60 min (c.f. Figs. 4D and 7C). With H7, however, STAT5b EMSA activity could be maintained,
for a full 3 h beyond the initial 60-min GH treatment, at a
distinctly higher level in cells cultured in the continued presence of
GH compared with when GH was removed after the initial hormone pulse
(Fig. 8A). The requirement that GH
continue to be present to manifest this more complete, positive effect
of H7 on JAK2 signaling to STAT5b suggests that, in H7-treated cells,
GH can continue to activate JAK2, even after 1 h. Since, in the
absence of H7, formation of new GHR-JAK2 complexes apparently ceases by
30 min (c.f. Figs. 1 and 2), an H7-inhibitable kinase may
contribute to this cessation in new receptor-kinase complex
formation.
We next investigated whether a block in new GHR-JAK2 complex assembly
might, in part, underlie the 3-h interpulse time requirement for
regain of GH pulse responsiveness (17). GH pulse reactivation
experiments were carried out in cells pretreated with H7 and then given
a 1-h GH pulse. GH was then removed and the cells continued to incubate
in the presence of H7. After GH-free intervals of 1, 1.5, or 2 h,
the cells were treated with a second GH pulse (Fig. 8B, right
panel). Cells treated with a second GH pulse but without any H7
pretreatment served as controls (Fig. 8B, left panel). In
the absence of H7, a GH interpulse interval of either 1, 1.5, or 2
h resulted in only 10–15% reactivation of STAT5b, as seen
previously (17). In H7-treated cells, however, close to full
reactivation of STAT5b was achieved by the second GH pulse, regardless
of the length of the interpulse interval (Fig. 8B). This effect is seen
most clearly in the 1.5-h and 2-h interpulse interval samples (compare
bars marked ‘before P2’ to ‘P2’ samples). In the 1-h
interpulse interval samples, only a small increase in STAT5b EMSA
activity could be stimulated by the second GH pulse (shaded
bars) owing to the high basal STAT5b EMSA activity before GH pulse
2 addition (speckled bars). This high basal signal reflects
the fact that H7 itself prolongs the time during which JAK2 catalyzes
ongoing STAT5b activation residual from GH pulse 1 (Fig. 5). In
separate experiments, the preservation of GH pulse responsiveness did
not occur if H7 was added after initiation of the first GH pulse (data
not shown). This suggests that the events leading to loss of GH pulse
responsiveness are initiated early in the GH pulse, and therefore can
only be blocked by H7 at an early step. By contrast, cycloheximide was
unable to shorten the GH interpulse interval requirement for efficient
STAT5b reactivation (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Deactivation of the JAK2-containing
signaling complex could be slowed down by the protein synthesis
inhibitor cycloheximide, the proteasome inhibitor MG132, and the
serine/threonine kinase inhibitor H7, each of which stabilized the
complex or inhibited JAK2 deactivation. By contrast, the
phosphotyrosine phosphatase inhibitor pervanadate sustained a STAT5b
signal at two steps: by inhibiting JAK2 deactivation and by blocking
STAT5b dephosphorylation. The cycle of STAT5b activation and
deactivation in response to a GH pulse, termed phase 1 signaling,
exhibits several features that distinguish it from the low level phase
2 signaling to STAT5b that becomes established in liver cells after
continuous GH treatment (32). Important differences between these two
GH-activated pathways include: 1) the transient nature of GH
pulse-induced phase 1 signaling (45–60 min) vs. the
persistence of phase 2 signaling, even after 7 days of continuous GH
exposure; 2) the apparent increase in phosphotyrosine phosphatase
activity, which is a key factor in the low steady-state level of
activated JAK2 and activated STAT5b during phase 2 signaling; and 3)
the dependence of phase 2, but not phase 1, GH signaling on ongoing
protein synthesis (32).
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), it may involve signaling that is
secondary to the JAK2-catalyzed formation of phosphotyrosine docking
site on the GHR cytoplasmic tail and the ensuing stimulation of
STAT5b-dependent transcription or other signaling events.
The ability of the JAK2 inhibitor genistein to delay for at least
1 h the onset of a phase 1 STAT5b-signaling cascade (Fig. 3)
suggests that genistein is able to freeze JAK2, most likely in the form
of a functional, cell surface-bound, GH-dimerized GHR-JAK2 signaling
complex (labeled A in Fig. 9). This complex apparently remains inactive
but stable until the JAK2 inhibitor is removed. The fact that GH need
not be present in the culture medium for the GH-(GHR-JAK2)2
complex to proceed through a full cycle of STAT5b activation and
deactivation (Figs. 1 and 3) further suggests that the receptor-kinase
complex is stable until the point where it is dephosphorylated,
internalized, and degraded or otherwise deactivated or disassembled.
This finding challenges the general presumption that ligand-receptor
interactions are dynamic on/off systems of short duration and is in
accord with reports showing some ligand-receptor complexes to be
long-lived and resistant to dissociation (36, 37). The maintenance of
JAK2 in its active, tyrosine-phosphorylated state for at least 40 min
after the onset of GH treatment, i.e. 30–35 min after
initiation of the STAT5b signal (Fig. 2A), and the continued activation
of STAT5b molecules even in the absence of GH (Fig. 2B) further
indicate that the GHR-JAK2 complex is not degraded after release of its
activated STAT5b substrate, but rather, that it remains functionally
active and able to recruit additional STAT5b molecules. On the basis of
these findings, we propose that the GHR-JAK2 complex enters a STAT5b
activation loop in which multiple STAT5b molecules are sequentially
activated by each individual receptor-kinase complex (Fig. 9).
Deactivation of the GHR-JAK2 signaling complex would, in turn,
terminate the STAT5b activation loop. Potential mechanisms for
termination of GHR-JAK2 signaling are discussed below. They include: 1)
dephosphorylation of the GHR-JAK2 complex through the action of a
phosphotyrosine phosphatase (exit route labeled B in Fig. 9); 2)
binding of SOCS/CIS inhibitory molecules that block further signaling
(pathway C); and 3) endocytosis of the complex, probably after its
dephosphorylation and ubiquitination (see below), leading to its
deactivation, dissociation, or degradation (pathway D).
Phosphatase Action
Phosphotyrosine phosphatases such as SHP1 and SHP2 can associate
with receptor-JAK kinase complexes via SH2 domain interactions and in
some cases may serve as negative regulators of cytokine-activated
receptor activity (38, 39). In the case of the GH pulse-activated
GHR-JAK2 complex, phosphotyrosine phosphatase activity contributes in a
significant manner to the down-regulation of receptor-kinase activity
beginning approximately 40 min after GH stimulation, as judged from the
ability of genistein to block the increase in STAT5b EMSA activity seen
in pervanadate-treated cells (Fig. 6B). That the rate of STAT5b
activation upon addition of pervanadate at 50 min was constant until 90
min, at which point the unphosphorylated STAT5b substrate pool was
depleted, suggests that pervanadate stabilizes the GHR-JAK2 complex and
blocks the decline in receptor-kinase activity that otherwise occurs
during this time period (c.f., decrease in
phosphotyrosyl-JAK2; Fig. 2A). Although pervanadate may interact with
other cellular proteins, it has been shown to inhibit phosphotyrosine
phosphatases most strongly (31). Accordingly, phosphotyrosine
dephosphorylation is likely to be the primary mechanism for the
deactivation of GHR-JAK2 that becomes operative beginning at
approximately 40 min, with no other exit pathway preceding this
deactivation step. The phosphatase that catalyzes this
dephosphorylation step may be induced by new protein synthesis, as
suggested by our finding that the protein synthesis inhibitor
cycloheximide prolongs JAK2 signaling to STAT5b (Fig. 4). Although we
cannot rule out the possibility that pervanadate might directly
activate JAK2, we have shown previously that pervanadate treatment
alone results in very little STAT5b activation in CWSV-1 cells (17).
Phosphotyrosine phosphatases are of lesser significance during the
first 20 min of GH treatment, as indicated by the lack of effect of
pervanadate on the net rate of STAT5b activation during that time
period (17). While phosphotyrosine phosphatases such as SHP1 and SHP2
may become associated with JAK2 or the cytoplasmic domain of GHR in
GH-treated cells (40, 41), this association has not been shown to lead
to dephosphorylation of JAK2 or GHR. In addition, dephosphorylation of
GHR-JAK2 by a bound phosphatase could be followed by reactivation of
the receptor-kinase complex if tyrosine rephosphorylation of JAK2 and
GHR were to occur within the confines of a stable GHR-JAK2 complex.
This type of reactivation mechanism could, in fact, contribute to the
robust STAT5b activation rate that is stimulated by pervanadate added
at 50 min in the experiment shown in Fig. 6B. Further studies are
required to identify the specific phosphatase(s) that contribute to
JAK2 deactivation and whether this enzyme corresponds to the
cycloheximide-sensitive inhibitory factor identified in Fig. 4.
Interaction of GHR-JAK2 with Signal-Inhibitory Molecules
Phosphorylated GHR-JAK2 complexes that become unable to recruit or
activate new STAT5b molecules, e.g. due to the binding of a
competitor or inhibitory molecule at the receptor’s STAT5b docking
site or at JAK2’s catalytic site, could deactivate and thereby exit
the activation loop as shown in step C in Fig. 9. Such an inhibitory
molecule would either need to be activated or its synthesis rapidly
induced by GH for this mechanism to contribute to GH pulse-induced
down-regulation of receptor-kinase activity. The requirement of ongoing
protein synthesis for rapid deactivation of JAK2 signaling to STAT5b
(Fig. 4) supports the latter possibility. Further support is provided
by the finding that JAK2 deactivation can be blocked by the
serine/threonine kinase inhibitor H7, which is known to block
STAT-induced transcriptional responses (25). Precedent for such a model
is provided by the rapid synthesis of receptor-kinase inhibitory
molecules induced by a variety of cytokines. These inhibitory
molecules, which are synthesized via STAT-dependent transcriptional
mechanisms, include the cytokine signaling-inhibitory proteins SOCS1
(JAB) and SOCS3 (CIS3) (42, 43, 44, 45, 46, 47). Indeed, GH was recently reported to
induce SOCS3, which can inhibit GH-induced trans-activation
of the Spi 2.1 promoter in transfection assays (48). The half-lives of
some of these SOCS mRNAs are rather short (
1 h), suggesting that one
or more SOCS proteins could correspond to labile,
cycloheximide-sensitive inhibitory factors (c.f. Fig. 4).
However, the present findings suggest that, in CWSV-1 liver cells, such
an inhibitory mechanism would need to operate in the context of
phosphotyrosine phosphatase active on the receptor-kinase complex.
Proteasome Degradation
A third possible mechanism for GHR-JAK deactivation involves
receptor polyubiquitination, which is induced by GH and occurs on
multiple lysine residues of GHR soon after the onset of receptor
dimerization. GHR ubiquitination has been linked to ligand-induced GHR
internalization (49), which ultimately leads to lysosomal degradation
of the receptor (50). Our finding that JAK2 signaling to STAT5b is
prolonged by the proteasome inhibitor MG132 (Fig. 7) is consistent with
this mechanism. However, since new protein synthesis is not required
for CWSV-1 cells to respond to a second GH pulse (17), proteasome
degradation of GHR or JAK2 after a single GH pulse is most likely
limited to only a portion of the cellular GHR and JAK2 pool.
Accordingly, the protective effects of MG132 on JAK2 activity are
probably indirect. Since, as discussed above, phosphotyrosine
phosphatases catalyze a key initial step in the JAK2 deactivation
pathway, MG132 may stabilize the active, phosphorylated signaling
complex by blocking proteasome degradation of a hypothetical
phosphotyrosine phosphatase inhibitor. Prolonged signaling from
interleukin 2-activated JAK1 and JAK3 to STAT5 has also been observed
in cells treated with proteasome inhibitor, where it is also speculated
that a phosphotyrosine phosphatase inhibitor is the target of
proteasome degradation (51).
An examination of ubiquitinated GHR molecules in GH-treated cells
reveals that these receptor molecules are not tyrosine phosphorylated
(35). This suggests that tyrosine-phosphorylated GHR molecules are
first deactivated by phosphotyrosine dephosphorylation and only then
become targeted for internalization, as depicted in Fig. 9 (step B
followed by step D). Phosphotyrosine dephosphorylation of GHR would
thus deactivate the receptor and also facilitate its subsequent
polyubiquitination and internalization. This, in turn, would prevent
further signaling to STAT5b, even in cells treated with a fresh GH
pulse, until the time that GHR reappears on the cell surface. This
latter event could involve both recycling of existing GHR and
replacement of degraded receptors via new protein synthesis. While our
earlier cycloheximide experiments imply that the cellular GHR pool is
not degraded to a significant extent after a GH pulse (17), this
prediction is difficult to test, due to the unavailability of suitable
anti-GHR antibodies. However, it is clear that neither JAK2 (Fig. 2A)
nor STAT5b (17) is depleted from CWSV-1 cells after a GH pulse,
consistent with the lack of a protein synthesis requirement for
responsiveness to a second hormone pulse.
STAT5b Activation and Deactivation
The net rate of STAT5b activation at any given point in time is a
function of the concentration of activated GHR-JAK2 complexes, the
concentration of non-tyrosine-phosphorylated cytoplasmic STAT5b
substrate, and the rate of STAT5b deactivation by phosphotyrosine
dephosphorylation. During the first 20 min after GH treatment,
pervanadate has no effect on the net rate of STAT5b activation (17),
implying that phosphatase activity on STAT5b (and on GHR-JAK2) is of
little consequence during this time period. Subsequently, the rate of
formation and the maximal level of active, phosphorylated STAT5b
becomes limited by the availability of STAT5b substrate. The size of
the STAT5b substrate pool is a function both of the absolute abundance
of STAT5b protein and its rate of dephosphorylation and
recycling back from the nucleus (step E, Fig. 9).
Tyrosine-phosphorylated STAT5b was shown to exhibit a half-life of
about 8.8 min and to deactivate by dephosphorylation at a rate that was
constant from 20 to 90 min after GH addition (Fig. 2C). Although this
dephosphorylation might be catalyzed by a constitutively active,
nuclear phosphatase, it is alternatively possible that a GH-activated
phosphatase carries out this reaction. New protein synthesis is not
required for STAT5b deactivation, since STAT5b EMSA activity is rapidly
lost in cycloheximide-treated cells when further JAK2 signaling is
blocked by genistein (Fig. 4, B and D). One candidate for the
phosphotyrosine phosphatase active on STAT5b is SHP1, which is rapidly
activated in CWSV-1 cells by a pulse of GH; GH also stimulates SHP1
nuclear translocation and SHP1 binding to tyrosine-phosphorylated
STAT5b (26).
Requirement of an Interpulse Interval
The ability of liver cells to respond to repeated GH pulse-induced
STAT5b activation requires that all components of the signaling system
be present and available for assembly. STAT5b can be reactivated by
repeated GH pulses in the absence of new protein synthesis provided
that the culture medium is depleted of GH for a 2.5–3 h minimum
recovery period (17). Given the near-quantitative conversion of STAT5b
to its phosphorylated forms under the conditions of those experiments,
STAT5b must be recycled, rather than degraded and resynthesized, after
a GH pulse. Recycling of interferon 
-activated STAT1 back to the
cytoplasm has also been observed (52). In addition to STAT5b, GHR and
JAK2 would also need to be recycled to the cell surface, unless they
are in such abundance that a single GH pulse does not significantly
deplete the available cell surface pool. In this context, it is
interesting to note that in addition to H7’s effect in slowing down
the down-regulation of GHR-JAK2 activity (Fig. 5), H7 decreased (and
perhaps eliminated) the requirement for a 3 h GH interpulse interval
for efficient reactivation of STAT5b (Fig. 8B). Thus, H7 may block the
switch that otherwise turns off the cell’s responsiveness to
GH-induced GHR-JAK2 signaling (perhaps via H7’s effects on new
GHR-JAK2 complex assembly; see below), and that normally requires a
GH-free interval of about 3 h to reset. Phorbol esters and other
activators of protein kinase C induce rapid internalization of GHR
(t1/2 = 15 min) (53, 54), suggesting that this effect of H7
could, in part, be due to its inhibition of this protein kinase
C-dependent receptor internalization step (Fig. 9, step D). Since H7
must be added to the cells before the initial GH pulse to prolong JAK2
signaling (Fig. 5D), the effects of H7, whether by modulation of the
phosphorylation state of a preexisting protein or by inhibition of the
transcription of a regulatory protein, must be effected early in the GH
pulse.
Cessation of GHR-JAK2 Complex Assembly
The STAT5b activation profile generated by a 10-min GH pulse was
only moderately lengthened when the GH pulse was extended to 30 or 60
min (Fig. 1). This suggests that GH activation of new GHR-JAK2
complexes ceases by 30 min GH exposure, at least at a GH concentration
of 500 ng/ml. Assembly of new receptor-kinase complexes does not cease
altogether, however, since the continuous presence of GH leads to
ongoing STAT5b activation at a low rate, termed phase 2 signaling (32).
The near-cessation of GHR-JAK2 complex assembly early during a GH pulse
may reflect depletion of the cellular pool of GHR and/or JAK2.
Alternatively, it may be the result of a specific, GH-induced mechanism
that blocks further complex assembly. Although GHR internalization can
lead to receptor degradation (50), recycling of GHR back to the cell
surface has also been observed (55). If cell surface depletion of GHR
(or JAK2) does contribute to the apparent cessation of GHR-JAK2 complex
assembly, then this recycling would help explain our earlier finding
that STAT5b reactivation can be achieved in CWSV-1 cells in response to
a second GH pulse even in the absence of new protein synthesis
(17).
In H7-treated cells, JAK2 signaling to STAT5b persists for at least
4 h, provided that GH continues to be present (Fig. 8A). This
suggests that, in addition to its inhibition of the down-regulation of
JAK2 signaling (Fig. 9, step B), H7 may block the cessation of
GH-inducible GHR-JAK2 complex formation that otherwise occurs within 30
min of the initial GH pulse. The cessation of new assembly of GHR-JAK2
complexes in the absence of H7 may thus proceed via a serine/threonine
kinase-dependent mechanism. We speculate that H7 decreases the
internalization and degradation of GHR and/or JAK2 after complex
disassembly, perhaps by inhibiting protein kinase C-induced
down-regulation of cell surface GHR, as noted above. Alternatively, H7
may stimulate GHR synthesis, or perhaps increase the GHR-JAK2
complex’s recycle-to-degradation ratio, thus increasing the cell
surface pool of GHR and/or JAK2 (Fig. 9, events following step D).
Further study will be required to elucidate the mechanisms that
underlie these interesting effects of H7 on GHR signaling.
|
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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General Experimental Design
Growth and passage of CWSV-1 cells was carried out as described
(17). GH was added to CWSV-1 cells, which were then incubated for times
ranging from 10 min to 1 h, as indicated in each experiment. GH
was removed by aspiration of the medium (1.5 ml), after which the cells
were washed with PBS (3 ml) and then incubated with fresh medium (1.5
ml) in the absence of GH. In control experiments, this washing
procedure was shown to be fully effective in removing GH, as judged by
the inability of media removed from the washed cells to induce any
detectable STAT5b activation (EMSA activity) when added to GH-naive
cells (data not shown). Inhibitors were generally added to the culture
medium before GH, at times and concentrations detailed in each figure.
Individual data points shown in each experiment represent separate
culture dishes treated identically until the time the cells were
harvested. The experimental design is illustrated in each figure by a
step diagram, where large steps represent addition or removal of GH,
small steps represent addition or removal of inhibitors, and individual
data points are marked by vertical arrows. Time zero
generally corresponds to the time of GH addition to the cells. In some
experiments, data points were taken at time points measured relative to
the time of GH removal. Data shown are representative of findings
confirmed in at least three independent experiments. Variations in the
shapes of the time courses between experiments generally precluded the
combination of data from separate experiments for formal statistical
analysis.
GH Treatment
For GH and/or inhibitor treatments, cells were incubated in 1.5
ml of fresh medium per 60-mm dish on the day of the experiment. Rat GH
(dissolved in PBS containing 0.1% BSA and kept on ice until use) and
inhibitors were added in volumes of 15 µl. Rat GH was a hormonally
pure preparation obtained from Dr. A. F. Parlow, National Hormone
and Pituitary Program, NIDDK (rGH-B-14-SIAFP, BIO), and was added to
the cells to give a final concentration of 500 ng/ml unless indicated
otherwise.
Cell Extracts and EMSAs
Total extracts of GH-stimulated CWSV-1 cells were prepared as
described (17). EMSA of STAT5b DNA-binding activity using a ß-casein
STAT5 response element probe was carried out as described previously
(17). Gels were exposed to PhosphorImager plates for 16 h for
quantitation using Molecular Dynamics (Sunnyvale, CA) equipment and
ImageQuant software. Typically, between 1 x 106 and
4 x 106 PhosphorImager counts were obtained in the
STAT5b EMSA band 20–45 min after GH pulse stimulation. This value was
set to 100% as indicated in each figure legend. Background signals
3% of this value were subtracted from each sample. The absolute
number of STAT5b activity units obtained in any particular EMSA
experiment was dependent on the time of exposure to the PhosphorImager
plate and the EMSA probe’s specific activity.
Immunoprecipitation and Western Blot Analysis
Total CWSV-1 cell extract protein (150 µg) was preimmune
cleared for 1 h at 4 C in a total volume of 200 µl containing 20
µl of 50% Protein A-Sepharose beads and 1 µg of rabbit antimouse
IgG. Protein A-Sepharose beads were removed by centrifugation, and
anti-JAK2 antiserum (1 µl) was added to the preimmune cleared cell
lysates and incubated for 3 h on ice. Rabbit antibody to JAK2 used
in these experiments was a gift of Dr. C. Carter-Su (University of
Michigan) and was raised against a synthetic peptide corresponding to
the hinge region between domain 1 and 2 of murine JAK2 (amino acids
758–776). Immunocomplexes were collected by centrifugation after a
further 1-h incubation with 20 µl of 50% Protein A-Sepharose beads
at 4 C. Samples were washed three times with 50 mM
Tris-HCl, pH 7.5, 0.1% Triton X-100, 150 mM NaCl, and 2
mM EGTA, and then boiled for 5 min in 20 µl SDS-PAGE
sample buffer (125 mM Tris-HCl, pH 6.7, 1% SDS, 7.5%
2-mercaptoethanol, and 15% glycerol). Immunocomplexes were
electrophoresed on 10% Laemmli SDS gels, electrotransferred onto
nitrocellulose membranes, and then probed with antiphosphotyrosine
antibody 4G10 (Upstate Biotechnology Inc., Lake Placid, NY;
1:3000 dilution). Nitrocellulose membranes were blocked for 1 h at
37 C with 4% BSA in TST buffer (10 mM Tris-HCl, pH 7.5, 50
mM NaCl, 0.1% Tween 20). Antibody binding was visualized
on x-ray film by enhanced chemiluminescence using the ECL kit from
Amersham Corp. (Arlington Heights, IL). To reprobe with anti-JAK2
antibody (diluted to 1 µg/ml) (QCB Inc., Hopkinton, MA), the
nitrocellulose membranes were first heated in stripping buffer (62.5
mM Tris-HCl, pH 7.6, 2% SDS, 50 mM
2-mercaptoethanol) for 20 min at 50 C and then reblocked with 4% BSA
in TST buffer, as described above. Results are presented in figures
prepared from grayscale scans of portions of the x-ray films of each
blot. Scans were obtained using a Cannon IX-4015 scanner (Cannon
Instrument Company, State College, PA) outfitted with Ofoto
scanning software.
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FOOTNOTES |
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This work was supported in part by NIH Grant DK-33765 (to D.J.W.).
Received for publication May 19, 1998. Revision received September 25, 1998. Accepted for publication October 30, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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