Termination of Growth Hormone Pulse-Induced STAT5b Signaling

Carol A. Gebert, Soo-Hee Park and David J. Waxman

Division of Cell and Molecular Biology Department of Biology Boston University Boston, Massachusetts 02215


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
STAT5b (signal transducer and activator of transcription 5b) is a key mediator of the effects of plasma GH pulses on male-specific liver gene expression. STAT5b is activated in liver cells in vivo by physiological pulses of GH and then is rapidly deactivated. Investigation of the cellular events involved in this activation/deactivation cycle using the rat liver cell line CWSV-1 established that a brief exposure to GH and the associated activation of JAK2 (Janus kinase 2) tyrosine kinase activity are both necessary and sufficient to initiate all of the downstream steps associated with STAT5b activation by tyrosine phosphorylation and the subsequent deactivation of both JAK2 kinase and STAT5b. JAK2 signaling to STAT5b at the conclusion of a GH pulse could be sustained by the protein synthesis inhibitor cycloheximide or by the proteasome inhibitor MG132, indicating that termination of this JAK2-catalyzed STAT activation loop requires synthesis of a labile or GH-inducible protein factor and is facilitated by the proteasome pathway. This factor may be a phosphotyrosine phosphatase, since the phosphatase inhibitor pervanadate both sustained GH pulse-induced JAK2 signaling to STAT5b and blocked the rapid deactivation of phosphorylated STAT5b (t1/2 = 8.8 ± 0.9 min) seen in its absence. Finally, the serine kinase inhibitor H7 blocked down-regulation of JAK2 signaling to STAT5b in a manner that enabled cells to respond to a subsequent GH pulse without the need for the ~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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The polypeptide hormone GH signals to hepatocytes and other target cells via its plasma membrane-bound receptor, GH receptor (GHR), a member of the cytokine/growth factor receptor superfamily (1, 2). GH plays a major role in the transcriptional control of many liver-specific genes, including steroid hydroxylase genes belonging to the cytochrome P450 (CYP) superfamily, which can be regulated by GH in a sex-specific manner (3). Two such examples are the P450 genes CYP2C11 and CYP2C12, which are transcriptionally activated and respectively expressed in an exclusive manner in the livers of male and female rats (4, 5). These CYP genes are regulated by the temporal profile of circulating GH, which is sharply delineated by gender in rodents. Adult male rats are characterized by plasma GH pulses of ~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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH Activation of STAT5b: Phase 1 and Phase 2 STAT5b Signaling
Treatment of CWSV-1 cells with GH activates STAT5b by tyrosine phosphorylation (17) and activates its DNA-binding activity, as shown by electrophoretic mobility shift analysis (EMSA) using a DNA probe containing a STAT5 response element derived from the ß-casein gene (Fig. 1Go). 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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Figure 1. GH Pulse-Induced STAT5b Activation and Deactivation Profile

CWSV-1 cells were incubated with 500 ng/ml of GH for either 10 min or 60 min. The culture medium was then aspirated, the cells were washed, and fresh medium without GH was added. Individual culture dishes were used to prepare total cell extracts at each of the indicated time points relative to the time of GH addition. Panel A, Cell extracts comparing 60-min GH pulse (left panel) and 10-min GH pulse (right panel) were analyzed by EMSA using the STAT5-binding rat ß-casein promoter probe. Panel B, EMSA band signals shown in panel A were detected using a PhosphorImager and integrated using ImageQuant software. Data are graphed as a percentage of the maximal level of STAT5b activity measured 30 min after GH stimulation. Experimental design is shown in the step graph below panel A, with arrows indicating data points taken for EMSA.

 
In contrast to the intermittent activation of liver STAT5b that occurs in adult male rats, the near continuous plasma GH pattern in adult female rats is associated with a persistent, low level of activated STAT5b. This STAT5b signal is barely detectable on Western blots of female rat liver nuclear extracts (7) and corresponds to approximately 5–10% of the peak EMSA signal seen in the case of adult male rat liver (H. K. Choi and D. J. Waxman, unpublished). When the female plasma GH pattern is modeled by treating CWSV-1 cells with GH in a continuous manner, the initial phase 1 response is followed by a drop in the level of activated STAT5b, to approximately 10–20% of its former peak (17). This long-term, low level response of STAT5b to continuous GH corresponds to the low level of activated STAT5b in female rat liver and is presently termed phase 2 STAT5b signaling.

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. 1Go). 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. 1AGo, 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. 1BGo). 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. 1Go) 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. 2AGo) 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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Figure 2. Duration of Phosphotyrosyl-JAK2 and JAK2 Signaling to STAT5b and Half-Life of Activated STAT5b

CWSV-1 cells were treated with a 20-min GH pulse at 50 ng/ml. GH was then removed and the cells incubated for times up to a total of 120 min. In some samples, 500 µM genistein was added to the culture medium at 20, 30, 40, or 50 min after GH. Panel A, Shown are Western blots of anti-JAK2 immunoprecipitated cell extracts. Extracts were prepared from CWSV-1 cells treated with GH (UT; lanes 1–14), or with GH followed by genistein added at the indicated times (lanes 15–18). Samples (150 µg protein) were immunoprecipitated with anti-JAK2 and analyzed on an SDS-PAGE gel followed by antiphosphotyrosine Western blotting using monoclonal antibody 4G10 (lanes 8–18). Reprobing of the blot shown in lanes 8–14 with anti-JAK2 antibody confirmed the consistency of JAK2 immunoprecipitation (lanes 1–7). n.s., Nonspecific bands commonly detected with anti-JAK2 antibody. Panel B, Extracts prepared from GH-stimulated cells were analyzed for activated STAT5b by EMSA, as in Fig. 1AGo. Shown is a linear plot of the PhosphorImager quantitation of EMSA band intensities as a percentage of the maximal STAT5b activity measured at 30 min. Solid squares correspond to the time course of STAT5b activation and deactivation in GH-induced samples without genistein treatment. Vertical arrows at t = 20, 30, and 40 min correspond to the times at which genistein was added to parallel sets of dishes. The more rapid decay of STAT5b EMSA activity in these sets of genistein-treated samples is shown by the corresponding three sets of dashed curves. Panel C, Data shown in panel B are graphed on a semilog plot, where 100% represents the activated STAT5b level at the time of genistein addition. Included in panel C are data from a separate but identical experiment where genistein was added at 50 min. Also shown is the corresponding STAT5b activity decay curve with no genistein treatment, based on the 60-, 75-, and 90-min data points shown in panel B (UT curve; solid squares). The 20-, 30-, and 40- min data points without genistein treatment were not included in this latter decay curve because JAK2 is still active at these time points (panel A, lanes 9–11) and, consequently, new tyrosine-phosphorylated STAT5b molecules are still being formed (panel B, solid line vs. dashed curves). Linear regression analysis yielded half-life values for activated STAT5b of 8.1, 8.1, 10.3, 9.4, and 8.1 min, respectively, for the five curves shown, corresponding to genistein added at t = 20, 30, 40, and 50 min, and no genistein addition.

 
The tyrosine kinase inhibitor genistein fully blocks JAK2-catalyzed STAT5b tyrosine phosphorylation induced by GH in CWSV-1 cells (17). To investigate how quickly individual STAT5b molecules deactivate, genistein was added to GH-treated cells at various times after the beginning of a 20-min GH pulse to halt further STAT5b activation. In the absence of genistein, STAT5b EMSA activity peaked at 30 min and deactivation proceeded over the next 60 min (Fig. 2BGo, solid black squares). Genistein addition at 20, 30, and 40 min (Fig. 2BGo, arrows) or 50 min (Fig. 2CGo) 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. 2AGo, lanes 9–11). Semilog plots of the decline in activated STAT5b after genistein addition (Fig. 2CGo) 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. 2CGo), 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. 2BGo, 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. 2AGo, 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. 3AGo, 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. 3BGo). 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. 3AGo, 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. 3BGo, 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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Figure 3. Freezing of GHR-JAK2 Signaling Complex by Genistein

Panel A, GH (500 ng/ml) was added to CWSV-1 cells, followed 30 sec later by 500 µM genistein (GEN). After 1 h, the cells were placed in fresh medium without GH or genistein. At the times indicated, cell extracts were prepared and analyzed by EMSA for STAT5b activity. Lanes 1–4, Time course for GH pulse stimulation; lanes 5 and 6, decay in STAT5b signal at the indicated times after GH removal; lanes 7–9, GH + genistein treatment; lanes 10–13, time points after GH removal. Panel B, STAT5b activities obtained in panel A were quantitated by PhosphorImager and graphed as a percentage of the activity measured at 45 min in the absence of genistein. Panel C, Select samples from panel A were immunoprecipitated with anti-JAK2 antibody, run on an SDS-PAGE gel, and analyzed on a Western blot probed with antiphosphotyrosine antibody 4G10. Lanes 14–18 correspond to the same sample set in lanes 1 and lanes 3–6; lanes 19–23 correspond to the sample set in lanes 7–9, 11, and 12.

 
JAK2 phosphotyrosine analysis revealed a robust phosphorylated JAK2 signal at 45 min in cells treated with GH in the absence of genistein. JAK2 phosphorylation declined as early as 10 min after GH removal (Fig. 3CGo, 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. 4AGo, 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. 4AGo). 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. 4DGo). 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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Figure 4. Cycloheximide Prolongs JAK2 Signaling to STAT5b

CWSV-1 cells were preincubated for 30 min with 10 µg/ml cycloheximide (CHX) before GH addition (500 ng/ml). When GH was removed after a 60-min pulse, fresh medium containing cycloheximide was added to ensure continued inhibition of protein synthesis. At the times indicated, cell extracts were prepared. Panel A, EMSA of STAT5b activity. Lanes 1–7, Sample set treated with GH for 60 min in the absence of cycloheximide (lanes 1–4); lanes 5–7, time points after GH removal. Lanes 8–14, sample set corresponding to treatments of lanes 1–7, except that cycloheximide was included for the duration of the experiment. Panel B, EMSA for a cycloheximide experiment similar to panel A, lanes 8–14, but including an additional set of samples treated with 500 µM genistein (GEN) added at the time when GH was removed. Lanes 15–17, GH pulse time course; lanes 18–20, time points after GH removal; lanes 21–23, time points after GH removal for cycloheximide cells treated with genistein. The quantitated band intensities from the experiment shown in panel B are graphed in panel D as a percentage of the activity measured at 10 min in the absence of inhibitors. Panel C, Select samples from the experiment in panel B were immunoprecipitated with anti-JAK2 antibody and analyzed on a Western blot probed with antiphosphotyrosine antibody 4G10 (upper gel in panel C). The blot was then reprobed with anti-JAK2 antibody to verify the uniformity of JAK2 immunoprecipitation (lower gel). Lanes 24–26, Time course of GH treatment; lane 27, 30 min after GH removal; lanes 28–30, GH + cycloheximide treatment; lane 31, 30 min after GH removal for cycloheximide-treated samples. The apparent variability in JAK2 protein levels between samples shown in panel C reflects intersample differences in the efficiency of JAK2 imunoprecipitation.

 
We next determined whether the prolonged STAT5b EMSA activity in cycloheximide-treated cells results from prolonged JAK2 signaling to STAT5b, or alternatively, whether it involves a decrease in the rate of STAT5b deactivation. Cycloheximide-pretreated cells were stimulated with GH for 1 h, at which time genistein was added to block further JAK2-dependent STAT5b activation. Figure 4BGo 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. 4Go, 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 5Go 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. 5AGo, lanes 18–21, and Fig. 5CGo). 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. 5BGo, 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. 5DGo, 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. 5CGo; also see Fig. 8AGo, 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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Figure 5. Impact of H7 Pretreatment on JAK2 Signaling to STAT5b

CWSV-1 cells were treated with 200 µM H7 beginning 30 min before GH treatment (500 ng/ml) for 1 h. Control cells were treated with GH without any H7 pretreatment. Where indicated, the H7-treated samples were treated with 500 µM genistein (GEN) added 45 min after the initial GH addition. Cells were then incubated in the continued presence of H7 or H7 + genistein for times up to 2 h after GH removal. Panel A, EMSA of STAT5b activity. Lanes 1–3, 8–10 and 15–17 correspond to 0–60 min GH treatment, with or without H7 and genistein; lanes 4–7, 11–14, and 18–21 correspond to times from 15 min to 2 h following GH removal. Panel B, Samples from the experiment shown in panel A, lanes 1, 2, and 5 (no H7) and lanes 8, 9, and 12 (+H7), respectively, were immunoprecipitated with anti-JAK2 antibody and blotted with antiphosphotyrosine antibody 4G10 (top gel) or reprobed with anti-JAK2 antibody (lower gel). Panel C, Graph of the integrated STAT5b band intensities for the samples shown in panel A as a percentage of the activity measured at 45 min in the absence of inhibitors. Panel D, In a separate experimental set, H7 was added to CWSV-1 cells either 30 min before a 1-h GH pulse, as in panel A (lanes 5–8), or at the conclusion of that 1-h GH pulse (lanes 9–12). Samples were analyzed by EMSA for STAT5b activity. For comparison, lanes 1–4 show GH-treated controls without H7 treatment for the same experimental set.

 


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Figure 8. Impact of H7 on Reactivation of GHR-JAK2 Signaling

Panel A, Two sets of CWSV-1 cells were treated with 200 µM H7 beginning 30 min before GH addition. In one set, cells were stimulated with GH (500 ng/ml) for 60 min (middle curve), and in the other, GH was present continuously, both for the initial 60 min, and for up to an additional 3 h, as shown on the x-axis (upper curve). As a control, cells not treated with H7 were treated with GH for 60 min followed by incubation for up to an additional 3 h without GH (UT; lower curve). Samples were taken for STAT5b EMSA at 45 min after GH addition, corresponding to the maximum GH pulse response (=100%), and then again at 30 min, 1 h, and 3 h after the initial 1-h GH treatment period. STAT5b activity levels, determined by EMSA and quantitated by PhosphorImager, are graphed as a percentage of the STAT5b activity level at 45 min. Data shown for the H7-treated samples are averaged from three to four separate experiments. Panel B, CWSV-1 cells were treated with 200 µM H7 beginning 30 min before GH addition (H7, right panel) or were untreated (UT, left panel). Cells were then treated with 500 ng/ml GH for 1 h, followed by a GH-free period (interpulse interval) of either 1, 1.5, or 2 h, as indicated on the x-axis. One set of samples for each time point was assayed for STAT5b activity by EMSA at the end of the GH interpulse interval (bars marked ‘before P2’), while a second set of samples was assayed 45 min after addition to the cells of a second GH pulse (bars marked ‘P2’), as indicated by vertical arrows in the step diagram. Shown are STAT5b activities assayed by EMSA and graphed as a percentage of the STAT5b activity measured 45 min after the initial GH pulse addition. Data shown are from two separate experiments.

 
Pervanadate Prolongs the Activated STAT5b Signal by Inhibiting both STAT5b Dephosphorylation and JAK2 Dephosphorylation
The phosphotyrosine phosphatase inhibitor pervanadate (31) can sustain high levels of STAT5b EMSA activity after the usual end point of GH-induced phase 1 signaling (17). Pervanadate could indirectly activate STAT5b by blocking dephosphorylation of GHR and/or JAK2. Alternatively, pervanadate might directly inhibit STAT5b dephosphorylation. We examined the importance of these two potential mechanisms by using genistein to block further contributions of JAK2 kinase activity to the observed STAT5b EMSA signal. A low concentration of GH was used in this experiment (5 ng/ml) to ensure that there would still remain a pool of non-tyrosine-phosphorylated (i.e. inactive, cytoplasmic) STAT5b molecules at the time when pervanadate was added to the cells (c.f. Ref. 17). This would enable us to detect any further increases in STAT5b activation that might be stimulated by pervanadate. Figure 6AGo 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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Figure 6. Stabilization of Activated JAK2 Kinase and Activated STAT5b by Pervanadate

Panel A, EMSA of extracts of CWSV-1 cells incubated with 5 ng/ml GH for 60 min (top gel). At 45 min, 60 µM pervanadate was added, either without (middle gel) or with 500 µM genistein (lower gel). After GH removal, the cells were incubated for a further 15, 30, or 60 min, as indicated (lanes 4, 8–10, and 14–16). Note the presence of a higher mobility EMSA band in the pervanadate-only samples (lanes 9 and 10), identified as STAT1 by anti-STAT1 supershift analysis (not shown). Panel B, CWSV-1 cells were treated with 50 ng/ml GH for 20 min and then incubated without inhibitors up to t = 120 min (solid squares; UT). At 30, 40, or 50 min, 60 µM pervanadate (closed symbols) or 60 µM pervanadate + 500 µM genistein (open symbols and dashed lines) was added to the culture medium, as indicated by the vertical arrows. Samples were taken at times up to 120 min after the initial GH addition, and analyzed by EMSA, and the integrated STAT5b band intensities were graphed as a percentage of values measured at 20 min in the absence of inhibitors. The pervanadate + genistein data were averaged based on either five or six data points to give the dashed lines shown; for clarity, the average value of each data set is plotted.

 
In a follow-up experiment, we compared the effects of pervanadate to those of pervanadate + genistein added at various times after a 20-min GH pulse (Fig. 6BGo; 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. 1Go). 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. 6BGo, 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. 6AGo, 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 7Go 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. 7Go, 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. 4Go and 5Go). Genistein abolished this effect of MG132, demonstrating that MG132 treatment prolongs signaling from JAK2 to STAT5b (Fig. 7AGo, lanes 7–9 vs. 4–6, and Fig. 7CGo). JAK2 phosphotyrosine analysis confirmed this conclusion, as judged by the prolonged phosphotyrosyl-JAK2 signal seen in MG132-treated cells (Fig. 7BGo, 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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Figure 7. Proteasome Inhibitor MG132 Blocks Down-Regulation of JAK2 Signaling to STAT5b

Panel A, CWSV-1 cells were incubated with or without 50 µM MG132 for 30 min before GH addition (500 ng/ml). Lanes 1–3, Samples taken at the indicated times after GH addition. After 1 h, GH was removed, and fresh medium containing MG132 was added. Cells were incubated for a further 30 min, 1 h, or 2 h (lanes 4–6). Some of the MG132-treated samples were given 500 µM genistein at the same time as GH removal (lanes 7–9). Cell extracts were analyzed for STAT5b activity by EMSA. Panel B, Samples shown in lanes 1–4 of panel A were immunoprecipitated with anti-JAK2 antibody and analyzed by antiphosphotyrosine antibody 4G10 Western blotting (lanes 14–17) (upper gel). Corresponding controls not treated with MG132 (carried out as part of the experiment shown in panel A, but not shown in panel A; see panel C) were immunoprecipitated with anti-JAK2 and are shown in lanes 10–13. The membrane was reprobed with anti-JAK2 to confirm the consistency of JAK2 immunoprecipitation (lower gel). MG132 did not have any discernable effect on JAK2 protein levels. Panel C, EMSA bands shown in panel A were quantitated and graphed as a percentage of the activity measured at 10 min in the absence of inhibitors.

 
H7 Abolishes the Requirement of an Interpulse Interval for Full Reactivation of STAT5b
As described above, GH activation of JAK2 in CWSV-1 cells ceases by about 30 min after GH addition (c.f. Figs. 1Go and 2Go). 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. 4DGo and 7CGo). 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. 8AGo). 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. 1Go and 2Go), 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. 8BGo, right panel). Cells treated with a second GH pulse but without any H7 pretreatment served as controls (Fig. 8BGo, 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. 8BGo). 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. 5Go). 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).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GH-responsive, rat liver cell line CWSV-1 was used to investigate the intracellular signaling pathways that are activated in liver cells by a physiological pulse of GH, including the events that regulate activation and deactivation of the GHR-JAK2 signaling complex and its STAT5b substrate. A brief exposure to a GH pulse was sufficient to initiate all of the downstream cellular events required for GH pulse-induced STAT5b signaling, including recruitment of STAT5b to the GHR-JAK2 complex, activation and deactivation of STAT5b, and deactivation of the GHR-JAK2 signaling complex (Fig. 9Go). 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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Figure 9. GHR-JAK2 Cycle and STAT5b Activation Loop: Proposed Pathways for Termination of GH Pulse-Induced STAT5b Signaling

Shown are steps at which GH pulse-induced signaling through STAT5b is hypothesized to be down-regulated at the conclusion of a male plasma GH pulse. These steps include: dephosphorylation of the activated GHR-JAK2 signaling complex A by a pervanadate-sensitive phosphotyrosine phosphatase (step B); removal of GHR-JAK2 from the STAT5b activation loop by binding of a GH pulse-induced SOCS or CIS inhibitory protein to phosphotyrosine residues on GHR or JAK2 (step C); and ubiquitination of dephosphorylated GHR, leading to receptor internalization (step D) followed by either recycling of GHR back to the cell surface or protein degradation. In addition, dephosphorylation of activated STAT5b molecules, catalyzed by a nuclear, pervanadate-sensitive phosphotyrosine phosphatase [perhaps SHP1 (26 )] is proposed to regenerate the cytoplasmic pool of inactive, monomeric STAT5b molecules (step E). The tyrosine kinase inhibitor genistein is proposed to block the STAT5b activation loop by inhibiting GHR-JAK2-catalyzed tyrosine phosphorylation of STAT5b. The serine/threonine kinase inhibitor H7 sustains GHR-JAK2 signaling, probably at multiple steps. These may include inhibition of the down-regulation of JAK2 signaling to STAT5b (step B) by inhibition of GH-induced synthesis of a phosphotyrosine phosphatase or a SOCS/CIS-inhibitory protein, or enhancement of the recycling of internalized GHR molecules, leading to their greater availability for new GHR-JAK2 complex assembly (step D). Steps that lead to activation of the GHR-JAK2 complex and cycling through the STAT5b activation loop are shown using thick arrows to designate the high activity of these steps during the first ~40 min following GH pulse stimulation.

 
GH Phase 1 Signaling: Activation and Deactivation Events
The initial recruitment and activation of STAT5b in response to a GH pulse is presently shown to be followed by two distinct down-regulatory events, deactivation of JAK2 kinase (and hence deactivation of the GHR-JAK2 signaling complex) and deactivation of STAT5b. Both steps are necessary to terminate the effects of a GH pulse on the hepatocyte and to effect the GH off period that is required for GH pulses to induce a male pattern of liver gene expression (33). Conceivably, deactivation of the STAT5b pathway could have been initiated by a GHR-dependent signaling cascade that is independent of tyrosine kinase activity, such as has been described for the GH-induced Ca2+ oscillations (34) and ubiquitination of GHR (35). However, deactivation of JAK2 and STAT5b were both found in the present study to be dependent on tyrosine kinase activity. The tyrosine kinase requirement for these downstream signaling events could result from a direct activation by JAK2 kinase of signal-inhibitory molecules, such as phosphotyrosine phosphatases. Alternatively, as suggested by the delay in these down-regulatory events in cycloheximide-treated cells (Fig. 4Go), 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. 3Go) 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. 9Go). 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. 1Go and 3Go) 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. 2AGo), and the continued activation of STAT5b molecules even in the absence of GH (Fig. 2BGo) 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. 9Go). 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. 9Go); 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. 6BGo). 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. 2AGo). 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. 4Go). 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. 6BGo. 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. 4Go.

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. 9Go. 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. 4Go) 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. 4Go). 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. 7Go) 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. 9Go (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. 2AGo) 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. 9Go). 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. 2CGo). 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. 4Go, 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 {gamma}-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. 5Go), H7 decreased (and perhaps eliminated) the requirement for a ~3 h GH interpulse interval for efficient reactivation of STAT5b (Fig. 8BGo). 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. 9Go, step D). Since H7 must be added to the cells before the initial GH pulse to prolong JAK2 signaling (Fig. 5DGo), 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. 1Go). 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. 8AGo). This suggests that, in addition to its inhibition of the down-regulation of JAK2 signaling (Fig. 9Go, 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. 9Go, 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Inhibitors
Inhibitors used in the present study were obtained from Sigma Chemical Co. (St. Louis, MO), except for MG132, which was from Peptides International (Louisville, KY). With the exception of pervanadate, inhibitors were prepared as 100x stock solutions in dimethylsulfoxide (genistein, H7, MG132) or in water (cycloheximide, pervanadate) and kept frozen in aliquots at -20 C. Final concentrations were: genistein (500 µM), pervanadate (60 µM), H7 (200 µM), cycloheximide (10 µg/ml), and MG132 (50 µM). Where used, final dimethylsulfoxide concentrations were generally 1% (vol/vol). Pervanadate (6 mM stock) was prepared fresh by incubating equal volumes of 12 mM sodium vanadate (freshly dissolved in water) and 12 mM hydrogen peroxide for 20 min at room temperature before use.

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.


    FOOTNOTES
 
Address requests for reprints to: Dr. David J. Waxman, Department of Biology, Boston University, 5 Cummington Street, Boston, Massachusetts 02215. E-mail: djw{at}bio.bu.edu

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.


    REFERENCES
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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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