Down-Regulation of Liver JAK2-STAT5b Signaling by the Female Plasma Pattern of Continuous Growth Hormone Stimulation

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
 
The suppression of male-specific, GH pulse-induced, liver transcription in adult female rats has been linked to the down-regulation of STAT5b activation by the female plasma pattern of near-continuous GH exposure. The mechanism underlying this down-regulation was studied in the rat liver cell line CWSV-1, where continuous GH suppressed the level of activated (tyrosine- phosphorylated) STAT5b to approximately 10–20% of the maximal GH pulse-induced STAT5b signal within 3 h. In contrast to the robust JAK2 kinase-dependent STAT5b activation loop that is established by a GH pulse, JAK2 kinase signaling to individual STAT5b molecules was found to be short lived in cells treated with GH continuously. Moreover, maintenance of the low-level STAT5b signal required ongoing protein synthesis and persisted for at least 7 days provided that GH was present in the culture continuously. Increased STAT5b DNA-binding activity was observed in cells treated with the proteasome inhibitor MG132, suggesting that at least one component of the GH receptor (GHR)-JAK2-STAT5b signaling pathway becomes labile in response to continuous GH treatment. The phosphotyrosine phosphatase inhibitor pervanadate fully reversed the down-regulation of STAT5b DNA-binding activity in continuous GH-treated cells by a mechanism that involves both increased STAT5b activation and decreased STAT5b dephosphorylation. Moreover, the requirement for ongoing GH stimulation and active protein synthesis to maintain STAT5b activity in continuous GH-treated cells were both eliminated by pervanadate treatment, suggesting that phosphotyrosine dephosphorylation may be an obligatory first step in the internalization/degradation pathway for the GHR-JAK2 complex. Finally, the sustaining effect of the serine kinase inhibitor H7 on GH pulse-induced JAK2 signaling to STAT5b was not observed in continuous GH-treated cells. These findings suggest a model where continuous GH exposure of liver cells down-regulates the STAT5b pathway by a mechanism that involves enhanced dephosphorylation of both STAT5b and GHR-JAK2, with the latter step leading to increased internalization/degradation of the receptor-kinase complex.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cytokine and growth factor-induced cell surface receptor aggregation is coupled to the activation of transcriptional events in the nucleus by several interdependent signaling pathways, including the mitogen-activated protein (MAP) kinase (1, 2) and JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathways (3, 4). Recent studies on cytokine-induced signaling have focused on the biochemical events that occur in ligand-naïve cells shortly after ligand stimulation. These events are particularly relevant to situations characterized by a pulsatile plasma profile for the cytokine or growth factor, as occurs during interleukin release in response to acute stress (5, 6) or PRL release in response to suckling (7, 8). In other cases, including many therapeutic applications, stimulation of a cytokine receptor may be continuous (9, 10). The polypeptide hormone GH is a unique example of a cytokine/growth factor that circulates in the blood and stimulates target tissues in either of two, temporally distinct plasma profiles regulated by hypothalamic stimulatory and inhibitory release factors. In the rat, GH is released from the pituitary gland in a pulsatile manner in males and in a near-continuous fashion in females (11, 12). Plasma GH pulses in male rats are approximately 1 h in duration with peak blood plasma concentration of 200 ng/ml, followed by an approximately 2.5-h period where GH is undetectable in plasma (<2 ng/ml). By contrast, circulating GH levels in female rats are more continuous, and typically range from about 20–40 ng/ml. These sex-dependent plasma GH patterns lead to distinct, sex-dependent patterns of gene expression in the liver, a major target organ of GH action, as exemplified by the patterns of transcription of the sex-dependent steroid hydroxylase P450 genes CYP2C11 (male-specific expression) and CYP2C12 (female-specific expression) (13, 14, 15). This implies that GH can activate two distinct intracellular signaling pathways via the plasma membrane-bound GH receptor (GHR) and in a manner that is dependent on the temporal pattern of plasma GH stimulation.

In the adult male rat, GH pulses stimulate an intracellular signaling pathway that is dependent on the tyrosine kinase JAK2 and the signaling molecule and transcriptional activator STAT5b. This pathway involves the repeated activation of STAT5b by sequential plasma GH pulses, a process that occurs in male but not female rat liver (16) and is required for the sexual dimorphism of liver gene expression and of whole body growth rates (17). A continuous GH-activated nuclear factor, termed GHNF, which is distinct from the GH-activated STATs and whose DNA-binding activity is high in female, as compared with male, liver nuclear extracts, has also been described (18). However, the intracellular signaling pathway(s) and events that are stimulated in hepatocytes in response to continuous GH exposure (19) and that may activate this or other factors or contribute to the suppression of STAT5b signaling (16) are poorly understood.

In vivo studies in hypophysectomized rats have established that the time interval between plasma GH pulses is a critical factor in the ability of liver cells to reset their signaling systems to respond to subsequent GH pulses. Physiological GH dose replacement at six pulses per 24-h period leads to restoration of a normal male pattern of liver gene expression, whereas no such restoration occurs when the GH pulse interval is shortened slightly, by increasing the GH frequency to seven pulses/24 h (20). Continuous GH exposure both suppresses the male transcriptional response and at the same time induces (or activates) the GH-activated nuclear factor GHNF in association with the transcriptional activation of female-expressed liver genes (14, 18). Suppression of the male transcriptional response in continuous GH-treated male rats (14, 15) appears to be due to the loss of activated, nuclear STAT5b, which first becomes evident within several hours of continuous GH treatment in vivo, but which requires several days to be complete (16). The cellular mechanism underlying this down-regulation of the STAT5b pathway in liver cells exposed to GH continuously is presently unknown and is the subject of the present study, carried out in the GH-responsive rat liver cell line CWSV-1 (21, 22, 23). These cells respond to a pulse of GH with a rapid increase in the cellular level of tyrosine-phosphorylated, activated STAT5b molecules (<=10 min), which subsequently undergo phosphorylation on serine or threonine, dimerization, nuclear translocation (24), and DNA binding (22). Evidence is provided in support of a model where continuous GH exposure down-regulates GH signaling to STAT5b through the action of phosphotyrosine phosphatase(s) that deactivate both STAT5b and the GHR-JAK2 signaling complex. The latter step is proposed to lead to enhanced degradation of the receptor-kinase complex, necessitating ongoing protein synthesis and the continual generation of fresh GHR-JAK2 complexes to maintain a low level of activated STAT5b in continuous GH-treated liver cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Continuous GH Exposure Stimulates Low Levels of Activated STAT5b
GH given as a continuous infusion to hypophysectomized rats suppresses STAT5b activation over a period of hours to days (16). To determine whether the cultured liver cell model CWSV-1 also responds to continuous GH with a decrease in STAT5b activation, cells were treated with GH for periods up to 7 days. Electrophoretic mobility shift assay (EMSA) revealed that activated STAT5b levels fall from their peak level at approximately 45 min to about 10–20% of the peak level within about 2 h of continuous GH treatment (Fig. 1AGo). This decrease of STAT5b EMSA activity to a lower steady-state level in continuous GH-treated cells is termed phase 2 STAT5b signaling to distinguish it from the initial GH pulse activation event, which is termed phase 1 STAT5b signaling (23). The low STAT5b EMSA activity stimulated by continuous GH could be maintained for at least 7 days at both 500 ng/ml GH (Fig. 1AGo) and at 50 ng/ml (cf., female plasma GH level of ~20–40 ng/ml GH). This low-level STAT5b EMSA activity was reduced by about half at a continuous GH concentration of 5 ng/ml (data not shown), in accord with the reported dissociation constant (Kd) of GHR for GH of about 2 ng/ml (25).



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Figure 1. Down-Regulation of STAT5b Signaling in Phase 2, Continuous GH-Treated CWSV-1 Cells

Panel A, CWSV-1 cells were treated continuously with 500 ng/mL GH for the indicated times. Culture media was changed and fresh GH was added every other day (i.e. on days 2, 4, and 6, in the case of the 7-day continuous GH samples, and on day 2 in the case of the 4-day GH sample). In the case of cells treated with GH for 7 days, cells were trypsinized and then passaged on day 4 at a 1:4 split into media containing fresh GH. Cell extracts were prepared and then analyzed by EMSA using the STAT5-binding ß-casein probe. Shown is a graph displaying EMSA data combined from several experiments. In each case, the STAT5b EMSA signal intensity is graphed as a percentage of the signal obtained 45 min after initiation of a GH pulse, which corresponds to the maximum intensity signal during GH pulse-induced phase 1 signaling. The basal level of phase 2 STAT5b EMSA activity seen in these samples corresponds to approximately 20% of the maximal STAT5b signal at 45 min. The basal level was similar in experiments carried out at 50 ng/ml GH, while at 5 ng/ml GH the STAT5b signal was reduced to about 10%. In experiments carried out with later passages of CWSV-1 cells, continuous GH at 50 or 500 ng/ml induced a STAT5b response that corresponded to about 10% of the 45-min peak (cf. Fig. 5DGo and STAT5b Western blot shown in Fig. 1BGo). Inset, EMSA of extracts from cells treated with GH for the times indicated. In data not shown, supershift analysis using STAT5 form-specific anti-COOH terminal region antibodies (Santa Cruz Biotechnology; STAT5a antibody sc-1081 and STAT5b antibody sc-835) confirmed that the GH-induced gel shift complex contains STAT5b but not other GH-activatable liver STATs (22 31 ). Panel B, Anti-STAT5b Western blot of CWSV-1 extracts, comparing relative amounts and STAT5b SDS-PAGE banding pattern in response to an initial GH pulse (lane 9) and longer-term, continuous GH phase 2 signaling (lanes 10–12). Also shown is the same blot reprobed with anti-JAK2 antibody (lanes 13–17). The multiple STAT5b bands correspond to a pair of (unresolved) unphosphorylated bands (bands 0), tyrosine-phosphorylated STAT5b (band 1), which comigrates in the Western blot shown with the constitutively serine (or threonine)-phosphorylated STAT5b (band 1a), and tyrosine + serine/threonine-diphosphorylated STAT5b (band 2), as characterized and described in detail elsewhere (22 ). STAT5b bands 1 and 2 are both active when assayed by EMSA using the ß-casein DNA probe, as shown by phosphatase treatment (31 ), and by using H7 to inhibit the serine/threonine phosphorylation associated with conversion of band 1 to band 2 (22 ). Band 2 is absent from lane 8 and is just barely visible in lanes 10–12. Panel C, Anti-STAT5b Western blot of extracts from CWSV-1 treated with 500 ng/ml GH for times ranging up to 8 h. Lanes 21–25 show immunoprecipitation of cell extract (150 µg) using agarose-conjugated antiphosphotyrosine monoclonal antibody PY99. Lanes 18–20, direct STAT5b Western blot analysis of samples shown in lanes 21–23 (20 µg cell extract/lane).

 
Continuous GH-treated CWSV-1 cell extracts were analyzed by Western blotting to determine whether continuous GH exposure decreases the cellular level of STAT5b or JAK2 protein. No significant change in the abundance of either protein was seen after GH treatment (Fig. 1BGo, lanes 10–12 vs. 8; lanes 15–17 vs. 13). Moreover, the low-level STAT5b EMSA activity during phase 2 is associated with a correspondingly low level of tyrosine + serine (threonine) diphosphorylated STAT5b, detected on Western blots of continuous GH-treated cell extracts (Fig. 1BGo, STAT5b band 2, lanes 10–12 vs. 9). Antiphosphotyrosine immunoprecipitation using monoclonal antibody PY99 followed by STAT5b Western blotting confirmed the presence of tyrosine-phosphorylated STAT5b (band 1) and tyrosine + serine/threonine diphosphorylated STAT5b (band 2) in the continuous GH-treated cells at relative ratios similar to those induced by a pulse of GH (Fig. 1CGo, lanes 23–25 vs. 22).

The Low Level of STAT5b EMSA Activity during Phase 2 Is Dependent on Continuous Signaling by JAK2 Kinase
The low level of STAT5b EMSA activity seen in CWSV-1 cells exposed to GH continuously could reflect 1) ongoing signaling by JAK2 kinase, but at a lower level than after an initial GH pulse, or 2) residual activated STAT5b molecules that remain from the phase 1 pulse but are not deactivated by phosphotyrosine phosphatases or degraded. To distinguish between these two possibilities, we examined the effects of GH removal and of tyrosine kinase inhibition on STAT5b EMSA activity during phase 2. In the first experiment, CWSV-1 cells were incubated with GH for 24 h (Fig. 2AGo, lanes 1–5), after which GH was removed from the culture medium. GH removal led to a total loss of STAT5b EMSA activity within 30 min (lane 6 vs. 5). Thus, GH must be continually present to maintain a phase 2 STAT5b EMSA signal. Readdition of GH after a GH-free interval of 30 min, 1 h, or 3 h led to reactivation of STAT5b, albeit only to the level seen during phase 2 signaling (lanes 7, 9, and 11 vs. lane 5). Thus, during phase 2, CWSV-1 cells respond to GH with weak activation of STAT5b compared with that seen when cells are treated with a GH pulse. This suggests that the cells’ responsiveness to GH addition has been intrinsically altered by continuous GH exposure.



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Figure 2. Continued GHR-JAK2 Signaling Is Required to Maintain Low-Level STAT5b EMSA Activity during Phase 2

Panel A, CWSV-1 cells were treated with 500 ng/ml GH for times ranging from 45 min up to 24 h (lanes 2–5). Twenty four hours after GH exposure, the cells were washed with PBS, fresh medium without GH was added, and the cells were incubated for an additional 30 min, 1 h, or 3 h (lanes 6, 8, and 10), as shown in the step diagram below panel A. GH was added after each of these time intervals (X hr) and EMSA activity was assayed 45 min later (lanes 7, 9, and 11). Cell extracts were prepared and analyzed by EMSA. Note total loss of STAT5b EMSA signal upon GH removal (lanes 6, 8, and 10). Panel B, Cells were treated with 500 ng/ml GH for 3 h (lane 13), at which point 500 µM genistein or 2 µM staurosporin was added and the cells incubated for an additional 15 min with GH + the indicated kinase inhibitor (lanes 14 and 15). Cell extracts were analyzed by EMSA. The STAT5b EMSA signal shown in lane 13 is already low (since it corresponds to a phase 2 signal), which in the reproduction shown may make it difficult to compare with the signal in lanes 14 and 15, which is undetectable. Panel C, JAK2 immunoprecipitation followed by Western blot detection with antiphosphotyrosine antibody 4G10 of samples from the same experiment shown in Fig. 1CGo. JAK2 is seen to be tyrosine phosphorylated in continuous GH-treated cells, albeit at a much lower level than after the initial GH pulse (lanes 18–20 vs. 17) (upper gel). Also shown is a reprobing of the same gel with anti-JAK2 (lower gel). Small differences in JAK2 immunoprecipitation efficiency and in JAK2 protein mobility between lanes are apparent in this experiment.

 
In a separate experiment, CWSV-1 cells were incubated with GH for 3 h, long enough to establish phase 2 signaling, and then were treated with either the tyrosine kinase inhibitor genistein or the general kinase inhibitor staurosporin. EMSA of STAT5b activity demonstrated that the phase 2 STAT5b signal was lost within 15 min (Fig. 2BGo, lanes 14 and 15 vs. 13). We conclude that the low STAT5b EMSA activity in continuous GH-treated cells is dependent on continued signaling via a GH-activated tyrosine kinase. Since genistein and staurosporin both cause a complete loss of STAT5b EMSA activity within 15 min, the lifespan of an activated STAT5b molecule during phase 2 may be shorter than during phase 1, which we have estimated to be 25–30 min long (i.e. three decay half-lives at t1/2 = 8.8 min) (23).

Immunoprecipitation of JAK2 tyrosine kinase, followed by antiphosphotyrosine 4G10 Western blotting verified that continuous GH activates (phosphorylates) JAK2, albeit at a much reduced level compared with that achieved after a GH pulse (Fig. 2CGo, lanes 18–20 vs. 17). Thus, JAK2 is likely to be the tyrosine kinase involved in the ongoing phase 2 GH signaling to STAT5b. The maintenance of a low level of phosphorylated JAK2 for at least 8 h in continuous GH-treated cells (Fig. 2CGo, lane 20) contrasts with the transient phosphorylation of JAK2 after a GH pulse (23, 24) and is consistent with our conclusion that STAT5b EMSA activity is maintained at a low level during GH phase 2 as a consequence of an ongoing, low level signaling by JAK2 kinase to STAT5b.

Phase 2 Is Characterized by Increased Phosphotyrosine Phosphatase Action
We next investigated whether the low level of STAT5b EMSA activity in continuous GH-treated cells is the result of elevated phosphotyrosine phosphatase activity associated with dephosphorylation, and thus deactivation, of either GHR, JAK2, or STAT5b. To test this hypothesis, continuous GH-treated cells were incubated with the phosphotyrosine phosphatase inhibitor pervanadate. Figure 3AGo shows that pervanadate dramatically increased STAT5b EMSA activity within 30 min, fully restoring it to the maximum level seen after a GH pulse (i.e. during phase 1 signaling). By contrast, pervanadate had no effect on the rate of STAT5b activation at low GH concentrations or on the maximal STAT5b EMSA signal induced by a pulse of GH (22). In addition, pervanadate alone added to GH-naive CWSV-1 induced only a marginal activation of STAT5b (cf. Fig. 8, lane 1, of Ref. 22). The reversal by pervanadate of the down-regulated STAT5b signal seen in continuous GH-treated cells therefore suggests that phase 2 signaling is associated with increased phosphotyrosine phosphatase action on the GHR/JAK2/STAT5b pathway, leading to a significant decrease (80–90%) in the steady-state level of activated STAT5b.



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Figure 3. Influence of Pervanadate and Genistein on Phase 2 GH Signaling

CWSV-1 cells, incubated with 500 ng/ml GH for 3 h, were treated with 60 µM pervanadate either in the absence or presence of 500 µM genistein. Extracts prepared at the indicated time points after pervanadate addition were analyzed by EMSA. Panel A, PhosphorImager (Molecular Dynamics, Sunnyvale, CA) quantitation of STAT5b activities obtained by EMSA. Genistein was added to the GH-containing culture dishes 5 min before pervanadate, 5 or 15 min after pervanadate, or not at all, as indicated. Individual plates were analyzed for STAT5b EMSA activity at times ranging from 5 to 30 min after pervanadate addition, as shown. Pervanadate only (’no gen’) and GH only (no pervanadate or genistein) sets of samples (’control’) were also analyzed. GH was present throughout the course of the experiment. Panel B, Shown are EMSA gels from a set of experiments similar to the one shown in panel A, except that GH was incubated with the cells continuously for 3 h, and then was removed from the culture medium 5 min after pervanadate addition (’pervan at 2:55’; panel B2). In panel B3, pervanadate was added as in panel B2 and genistein was added 5 min later, at the time when GH was withdrawn. In the absence of GH, the STAT5b EMSA signal is lost within 15 min (panel B1, lanes 3 vs. 2) (cf. Fig. 2Go), whereas the STAT5b signal increases approximately 5-fold in intensity in the presence of pervanadate (panel B2) or about 2- to 2.5-fold in the presence of genistein + pervanadate (panel B3).

 
The stimulatory effect of pervanadate on STAT5b activity seen in these experiments could be due to the inhibition by pervanadate of the phosphatase(s) that dephosphorylate JAK2 and/or GHR, leading to sustained activation of the GHR-JAK2 signaling complex. Alternatively, it could be due to a more direct inhibition of STAT5b dephosphorylation. To determine the relative contributions of these two mechanisms, continuous GH-treated cells were incubated with pervanadate in combination with the JAK2 tyrosine kinase inhibitor genistein. Since the rates of cellular uptake of these two inhibitors might differ, genistein was added to the cells either 5 min before or 5 min after the addition of pervanadate, as shown in the schematic below Fig. 3AGo. In both cases, genistein reduced the pervanadate-stimulated increase in activated STAT5b from 5-fold to only 2–2.5 fold, as seen in the EMSAs quantitated in Fig. 3AGo. Addition of genistein 15 min after pervanadate (at which time the STAT5b EMSA signal was already increased ~3.8-fold above its low basal level) blocked further increases in the accumulation of activated STAT5b (Fig. 3AGo). Since genistein fully blocks new tyrosine kinase signaling to STAT5b (22), our finding that genistein reduced the pervanadate-stimulated increase by about half (Fig. 3AGo) suggests that approximately 50% of the pervanadate-stimulatory effect results from pervanadate inhibition of JAK2 or GHR dephosphorylation, and that the balance of the pervanadate effect is due to its inhibition of STAT5b dephosphorylation. Thus, the major suppression of STAT5b signaling seen in continuous GH-treated liver cells reflects an increase in pervanadate-sensitive phosphotyrosine phosphatase activity toward both GHR-JAK2 and STAT5b.

Interestingly, if GH was removed from the culture medium 5 min after pervanadate addition, then the 5-fold increase in STAT5b EMSA signal was still observed (Fig. 3Go, panel B2 vs. B1). Again, genistein was able to reduce the pervanadate-stimulated increase in STAT5b activity to only about 2- to 2.5-fold (panel B3). This suggests that pervanadate is able to effect its dramatic stimulation of phase 2 STAT5b activity, restoring a normal phase 1 GH pulse response, even without the recruitment by GH of new GHR-JAK2 complexes.

STAT5b Signaling during Phase 2 Differs from Phase 1 Signaling in its Response to the Serine/Threonine Kinase Inhibitor H7
H7 and other serine kinase inhibitors can prolong STAT5b activity in CWSV-1 cells after a GH pulse (22, 23). In contrast, H7 did not prolong phase 2 signaling after removal of the continuous GH stimulus. Figure 4AGo presents an experiment where CWSV-1 cells were incubated with GH for 3 h, to establish STAT5b phase 2 signaling, at which time H7 was added. GH was removed from the culture medium 30 min later. EMSA showed the STAT5b signal was lost within 30 min of GH removal (Fig. 4AGo, lanes 6–8 vs. lanes 4, 5), just as in the absence of H7 (cf. Fig. 2AGo, lane 6). By contrast, H7 could prolong a phase 1 STAT5b signal at approximately 60–65% of its initial level for a full 1.5 h after termination of a GH pulse (Fig. 4BGo, lanes 12–14). Thus, the requirement of an H7-sensitive serine/threonine kinase activity for termination of GH-induced STAT5b EMSA activity seen in GH pulse-treated cells (22, 23) is abolished once phase 2 becomes established.



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Figure 4. Serine/Threonine Kinase Inhibitor H7 Does Not Prolong Phase 2 Signaling

Panel A, CWSV-1 cells were incubated with GH for 2.5 h, at which time 200 µM H7 was added to the culture medium. GH was removed 30 min later, but H7 was retained in the medium. Samples were taken for EMSA at 3 h, i.e. just before GH withdrawal (lane 5), and at several times points thereafter (lanes 6–8), as illustrated in the step diagram. Extracts were analyzed for STAT5b DNA-binding by EMSA. Panel B, CWSV-1 cells were incubated with 200 µM H7 30 min before GH addition and maintained in culture medium after the withdrawal of GH at 1 h. Extracts taken at the times indicated were analyzed by EMSA. Panel C, CWSV-1 cells were incubated with GH for 3 h, at which time 200 µM H7 was added to the medium. Samples were taken at times ranging up to 4 h later and analyzed by EMSA. Note slow increase in STAT5b activity with prolonged H7 treatment.

 
Interestingly, when continuous GH-treated cells were incubated in the presence of H7 for several hours, the activated STAT5b signal increased several-fold (Fig. 4CGo, lanes 19–21 vs. lanes 17 and 18). This suggests that a serine/threonine kinase-activated mechanism may contribute to the lower level of STAT5b EMSA activity during phase 2. Prolonged H7 treatment did not, however, increase STAT5b EMSA activity to the same extent as did pervanadate (cf. Fig. 3Go). This suggests that this serine/threoine kinase mechanism contributes to down-regulation of STAT5b activity in a minor way, compared with the effects of phosphotyrosine phosphatases.

Addition of 20 µM PD98059, a MEK kinase inhibitor that blocks GH-induced signaling to MAP kinase (26), also did not block the loss of STAT5b EMSA activity upon GH removal during phase 2, nor did it inhibit STAT5b activation during phase 2 when added to cells incubated in the presence of continuous GH (data not shown). Thus, the MAP kinase pathway, which can be activated by GH (27, 28), does not contribute to the activation or deactivation of STAT5b during phase 2 GH signaling.

Proteasome Inhibitor MG132 Elevates Activated STAT5b Levels during Phase 2 Signaling
When CWSV-1 cells were incubated with GH for 3 h to establish phase 2 signaling, and then were treated with the proteasome inhibitor MG132, STAT5b EMSA activity increased approximately 2-fold within 1 h, and then slowly declined (Fig. 5AGo, lanes 4–7 vs. 3). This effect was less dramatic than, but additive with, the pervanadate-stimulatory effect (panel B, lanes 11–22 and panel D). This suggests that proteasome degradation of one or more factors required for STAT5b activation occurs during phase 2 GH signaling, thereby giving a higher steady-state level of STAT5b EMSA activity when MG132 is added to the cells. Figure 5CGo shows, however, that STAT5b protein (bands 0, 1, 1a, and 2, collectively) and JAK2 protein are not significantly increased by MG132 treatment (lanes 27–30 vs. lanes 25, 26). Finally, when GH was removed 5 min after MG132 addition, the activated STAT5b EMSA signal disappeared (data not shown). Since GH must be continuously present to observe MG132’s stimulatory effect, MG132 most likely exerts its effect on GHR-JAK signaling to STAT5b, and not at the level of STAT5b dephosphorylation.



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Figure 5. Effect of Proteasome Inhibitor MG132 on STAT5b Activation

Panel A, CWSV-1 cells were incubated with GH for times up to 3 h (lanes 1–3), at which time 50 µM MG132 was added to the culture medium. Extracts were analyzed by EMSA at times ranging up to 2.5 h after MG132 addition (lanes 4–7). Shown is an EMSA of STAT5b activity. Panel B, Shown is the EMSA of an experiment similar to that in panel A, except that the 3-h continuous GH-treated cells were treated with MG132 alone (lanes 11–14), with 60 µM pervanadate alone (lanes 15–18), or with MG132 + pervanadate (lanes 19–22). Note that panel B presents a lighter exposure than the one shown in panel A. Panel C, Western blot of samples from the same treatments shown in panel B probed with a mixture of anti-JAK2 and anti-STAT5b antibodies. The multiple, differentially phosphorylated STAT5b bands, labeled 0–2, are identified in the legend to Fig. 1BGo. The two unphosphorylated STAT5b forms, bands 0, partially resolve in this gel. Of these, the lower band is preferentially lost in the pervanadate-treated samples in association with the conversion of STAT5b to the active, diphosphorylated band 2 (lanes 31–34 vs. duplicate controls shown in lanes 25 and 26). MG132 slightly increases the intensity of band 2, which migrates just below the lower of the two nonspecific (n.s.) bands, which originate from the anti-JAK2 antibody. MG132 does not have a significant effect on the total JAK2 or STAT5b immunoreactivity. Panel D, STAT5b EMSA bands from panel B were integrated using PhosphoImager and graphed as a percentage of the STAT5b EMSA activity at 45 min. The error bar on the 45-min sample point represents the SD of three data points. Note that the phase 2 level of STAT5b activity in this experiment, and in the one shown in Fig. 6Go, is approximately 10% of the initial GH pulse signal (vs. ~20% in Fig. 1AGo). UT, No inhibitors added at 3 h; VO, addition of pervanadate at 3 h; MG, addition of MG132 at 3 h (vertical arrow).

 
Cycloheximide Eliminates the STAT5b Signal during Phase 2
Cycloheximide inhibition experiments have shown that new protein synthesis is not required for CWSV-1 cells to respond repeatedly to sequential, physiological pulses of GH under conditions where STAT5b is quantitatively converted to its nuclear, tyrosine-phosphorylated form (22). This implies that STAT5b is recycled from the nucleus back to the cytosol after termination of a GH pulse, and that the other components required for phase 1 signaling (GHR and JAK2) are either present in the cell in excess or are effectively reutilized. In contrast, cycloheximide treatment of CWSV-1 cells already in phase 2 completely eliminated STAT5b EMSA activity within 1.5 h (Fig. 6AGo, lanes 5–8 vs. lane 4). This indicates that at least one essential component of the GHR/JAK2/STAT5b pathway is labile during phase 2 GH signaling. Western blot analysis showed, however, that the cycloheximide-sensitive protein component is neither JAK2 nor STAT5b (Fig. 6CGo, lanes 27–29 vs. lanes 25 and 26).



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Figure 6. Loss of STAT5b Phase 2 EMSA Activity upon Cycloheximide Treatment and Reversal by Pervanadate

Panel A, CWSV-1 cells were treated with GH for up to 3 h (lanes 1–4), at which time 10 µg/ml cycloheximide was added to the culture medium for an additional 1–2.5 h (lanes 5–8). Shown is an EMSA indicating the loss of STAT5b activity by 1.5–2 h after cycloheximide addition (lanes 5–8 vs. control in lane 4). The strong initial GH pulse/phase 1 signal is seen in lanes 2 and 3. Panel B, An experiment similar to that in panel A was performed except that continuous GH-treated samples were additionally treated with cycloheximide (lanes 12–14), pervanadate (lanes 15–18), or pervanadate + cycloheximide (lanes 19–22). Samples were analyzed for STAT5b activity by EMSA. Panel C, Western blot of samples shown in panel B probed with a mixture of anti-JAK2 and anti-STAT5b antibodies, as detailed in the legend to Fig. 5CGo. The loss of STAT5b activity with >=1.5 h cycloheximide treatment seen in panels A, B, and D is not accompanied by any decrease in total JAK2 or STAT5b immunoreactivity. Panel D, EMSA bands shown in panel C were integrated using a PhosphoImager and graphed as a percentage of the STAT5b activity at 45 min. The pervanadate-only (VO) and the untreated data points are from the same samples shown in Fig. 5Go.

 
Our working hypothesis to explain both the effects of cycloheximide during phase 2 and the pervanadate-stimulatory response (Figs. 3Go and 5Go) is that dephosphorylation of the GHR-JAK2 signaling complex occurs rapidly during phase 2 signaling and is followed by GHR internalization and degradation (see Fig. 7Go, below). A clear prediction of this model is that blockage of the initial dephosphorylation step by pervanadate treatment will eliminate the protein synthesis requirement for continuous GH signaling to STAT5b. Indeed, while cycloheximide eliminated the phase 2 STAT5b EMSA signal within 2 h (Fig. 6BGo, lanes 12–14 vs. 11), it had no significant effect in pervanadate-treated cells (Fig. 6BGo, lanes 19–22 vs. 15–18). Pervanadate treatment fully restored the maximal STAT5b activity seen after a GH pulse (Fig. 6DGo). This effect of pervanadate is associated with the formation of activated STAT5b, band 2 (tyrosine + serine/threonine diphosphorylated STAT5b) (Fig. 6CGo, lanes 30–32).



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Figure 7. Model for Down-Regulation of GHR-JAK2 Cycle and STAT5b Activation Loop in Continuous GH-Treated Liver Cells

GH-induced assembly of GHR-JAK2 signaling complexes is proposed to establish a STAT5b activation loop, whereby multiple STAT5b molecules become activated by phosphorylation of Tyr699, followed by serine or threonine phosphorylation (not shown) and nuclear translocation. Continuous GH exposure is proposed to down-regulate the overall pathway of STAT5b activation through enhanced phosphotyrosine dephosphorylation of activated STAT5b (step A) and of activated GHR-JAK2 signaling complexes (step B) (thick arrows). The latter step is proposed to lead to GHR internalization and degradation (step C). This gives rise to the observed requirement for new protein synthesis, which can be blocked by cycloheximide (step D), and to a continual need for GH to be present to stimulate assembly of new GHR-JAK2 signaling complexes (step E).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The activation of liver STAT5b in response to plasma GH pulses in adult male rats is an essential component in the GH pulse-induced expression of a male pattern of liver gene expression (16, 17). By contrast, in female rat liver, which is exposed to plasma GH nearly continuously, STAT5b is activated only at a very low level (16), an observation that helps to explain the near absence, in female liver, of GH pulse-activated, STAT5b-inducible gene products. Earlier studies found that GHR (29, 30), JAK2, and cytoplasmic STAT5b (24, 31) are expressed in female rat liver at levels similar to (JAK2, STAT5b) or higher than (GHR) those of males. This suggests that a deficiency in one of these components does not account for the lack of a robust liver STAT5b response to GH in the female. The present study investigates the mechanism that underlies this apparent down-regulation of the STAT5b signaling pathway in continuous GH-treated liver cells. These studies were carried out in the GH-responsive rat liver cell line CWSV-1 (21), which responds to physiological GH pulses with a repeated pattern of JAK2-catalyzed STAT5b tyrosine phosphorylation, followed by serine/threonine phosphorylation, nuclear translocation, and then phosphotyrosine dephosphorylation (22, 23, 24). Continuous GH treatment of CWSV-1 cells is presently shown to result in a low level of activated STAT5b (~10–20% of the maximal EMSA signal observed after a GH pulse) that can persist for at least 7 days. This low-level STAT5b EMSA activity results from ongoing, low-level signaling from JAK2 tyrosine kinase to STAT5b and is termed phase 2 GH signaling. This signaling pathway, like that which characterizes phase 1 signaling in response to a GH pulse, is likely to involve dimerization of GHR by GH and recruitment of JAK2 to the receptor complex, followed by entry into an activation loop, in which the GHR-JAK2 complex recruits, activates, and then releases tyrosine-phosphorylated STAT5b (Fig. 7Go). In contrast to the STAT5b activation loop that characterizes phase 1 GH pulse signaling (23), however, the GHR-JAK2 complexes formed during phase 2 are proposed to retain their activity and remain in the activation loop for a much shorter period of time, thereby explaining the apparent cessation of GHR-JAK2 signaling that results in STAT5b deactivation within 15–30 min of GH removal or tyrosine kinase inhibition (Fig. 2Go).

Lower Steady-State Level of Activated STAT5b in Continuous GH-Treated Cells
The much lower level of STAT5b EMSA activity that is maintained by continuous GH (phase 2), as compared with that which is induced by pulsatile GH (phase 1), may be explained by one or more of the following: 1) one of the factors required for formation of a functional GHR-JAK2-STAT5b activation loop becomes limiting at the cytoplasmic face of the plasma membrane, where STAT activation is believed to occur; 2) increased phosphotyrosine phosphatase activity shortens the half-life of tyrosine-phosphorylated STAT5b molecules, decreasing the steady-state pool size of activated STAT5b (Fig. 7Go, step A); and 3) increased phosphotyrosine phosphatase activity shifts the equilibrium from phosphorylated (active) to dephosphorylated (inactive) GHR-JAK2 complexes (step B). In this latter situation, fewer GHR-JAK2 complexes would signal to STAT5b at any given point in time, and fewer STAT5b activation cycles would occur per GHR dimerization event. If dephosphorylation of the GHR-JAK2 signaling complex leads to its dissociation and/or internalization and degradation (step C), this could explain several of the requirements and characteristics of phase 2 signaling identified in this study. These include a need for: 1) ongoing protein synthesis, necessary to replenish degraded components (step D), and 2) a continuous GH presence, necessary to dimerize and activate fresh GHR-JAK2 complexes (step E). More complicated models can be envisaged. These include 1) continuous GH induction of a signal-inhibitory protein belonging to the recently described SOCS/CIS family (32, 33, 34) (see below) and 2) continuous GH activation of a SIRP family protein (35, 36) which recruits a phosphotyrosine phosphatase such as SHP2 to the GHR-JAK2 complex.

Cell surface GHR levels are more abundant in liver cells exposed to GH continuously (female rat liver) as compared with intermittently (male rat liver) (29, 30). This sex difference in GHR levels reflects the stimulation of GHR expression by continuous GH in females, a response also seen in cultured CWSV-1 cells (21), coupled with the GH pulse-induced internalization of cell surface GHR that occurs in males (37, 38). Thus, there appears to be an abundance, rather than a shortage, of cell surface GHR on CWSV-1 cells treated with GH continuously compared with cells exposed to GH pulses. The other two protein components, JAK2 and STAT5b, are also present in CWSV-1 cells during phase 2 GH signaling at a level similar to untreated cells or cells in phase 1 signaling (Fig. 1BGo), so these proteins are also not likely to be limiting in continuous GH-treated cells. However, GHR (39), JAK2 (24, 40), and STAT5b can also be found in intracellular compartments, such as the nucleus, so that Western blot measurements of total cellular levels of these components might not necessarily be indicative of their functional availability for phase 2/continuous GH signaling to STAT5b. For example, GHR-JAK2 signaling complex components may become sequestered or otherwise inhibited from forming a functional complex after continuous GH exposure. Further studies of the effects of continuous GH on the intracellular localization of each of these components will be required to address this point.

One potential inhibitory mechanism for STAT5b activity in continuous GH-treated cells, noted above, could involve the cytokine-inducible suppressor CIS. This STAT5-inducible negative feedback regulator binds to the tyrosine- phosphorylated erythropoietin and interleukin 3 receptors to inhibit STAT5 phosphorylation and downstream STAT5-dependent transcriptional responses (32, 33), perhaps by a mechanism that involves enhanced receptor degradation (41). Recent studies identify a family of CIS-related proteins, also termed SOCS, JAB, or SSI proteins, which are rapidly transcribed in a variety of cell types in response to cytokines and growth factors, and which function as negative regulators of cytokine signaling, probably by a range of mechanisms, including binding to, and inhibition of JAK kinases as well as cytokine/growth factor receptors (42, 43, 44, 45). Conceivably, one or more of these cytokine signaling-inhibitory proteins could be induced by continuous GH and contribute to the observed down-regulation of the STAT5b activation pathway in female rat liver [cf. recent report that GH can induce SOCS3 mRNA in mouse liver (34)]. Since SOCS/CIS protein expression is transcriptionally induced by STATs, and given that the transcriptional activity of STATs is modulated by serine phosphorylation (46, 47), the increase in STAT5b EMSA activity that is observed upon longer-term H7 treatment (Fig. 4CGo) could conceivably reflect an inhibition of GH-induced CIS/SOCS transcription.

Experiments using the phosphotyrosine phosphatase inhibitor pervanadate and the JAK2 kinase inhibitor genistein indicated that enhanced phosphotyrosine phosphatase activity toward both JAK2 and STAT5b makes an important contribution to the approximately 80–90% decrease in STAT5b activation seen when CWSV-1 cells are treated with GH continuously. By contrast, phosphotyrosine phosphatase activity appears to be low during the first 20 min of a GH pulse, as judged from the absence of an effect of the inhibitor pervanadate on the net rate of accumulation of tyrosine-phosphorylated STAT5b in cells treated with low concentrations of GH (22). Distinct phosphotyrosine phosphatases are probably responsible for dephosphorylation of STAT5b and dephosphorylation of the GHR-JAK2 complex, since these deactivations occur during distinct periods of time. STAT5b dephosphorylation occurs at a steady rate from 20–90 min after GH pulse stimulation, while dephosphorylation of JAK2 only begins after about 45 min (23). Moreover, STAT5b, but probably not GHR-JAK2, may be dephosphorylated while still in the nucleus (P. A. Ram and D. J. Waxman, unpublished experiments). The SH2 domain-containing phosphotyrosine phosphatase SHP-1 is rapidly activated approximately 3- to 4-fold by a pulse of GH and can bind to GH-activated STAT5b (24) and to JAK2 kinase (48), suggesting that it could be involved in one of these dephosphorylation events. SHP-1 translocates from the cytosol to the nucleus in response to a pulse of GH, suggesting that STAT5b may be directly dephosphorylated in the nucleus by SHP-1 to terminate a phase 1 GH signal (24); however, further studies are required to establish whether SHP-1 plays a direct catalytic role in STAT5b dephosphorylation, either after a GH pulse or in continuous GH-treated cells.

GH must be present continuously to sustain the low-level signaling to STAT5b during phase 2 (Fig. 2Go), suggesting that there is an ongoing requirement to recruit and activate fresh GHR-JAK2 complexes. Nevertheless, activation of STAT5b could proceed even in the absence of GH once pervanadate was added to the cells (Fig. 3BGo). This suggests, as a working hypothesis, that exit of the GHR-JAK2 complex from the STAT5b activation loop requires phosphotyrosine phosphatase action (Fig. 7Go, step B). According to this proposal, when phosphatase activity is inhibited by pervanadate, this loop continues to activate STAT5b molecules unabated, even if GH-stimulated entry of new GHR-JAK2 complexes ceases. Our proposal that GHR-JAK2 dephosphorylation is an obligatory first step along the pathway leading to receptor degradation (step C) is further supported by the observation that the need for ongoing protein synthesis to sustain a phase 2 STAT5b EMSA signal (Fig. 6AGo) (cf. Fig. 7Go, step D) is eliminated in pervanadate-treated cells (Figs. 6BGo, 6CGo). This proposal is also consistent with the finding that GHR molecules that undergo GH-induced ubiquitination, which has been linked to receptor internalization (49), are not tyrosine phosphorylated (50).

Destruction of the GHR-JAK2 Signaling Complex
Treatment of CWSV-1 cells with a pulse of GH is proposed to induce a STAT5b activation loop, where each activated GHR-JAK2 complex can sequentially recruit, phosphorylate, and release multiple STAT5b molecules (23). If JAK2 becomes dephosphorylated and deactivated, then its intrinsic autophosphorylation activity might, perhaps, return it directly to the activation loop. Similarly, if GHR were to be deactivated by dephosphorylation of its cytoplasmic phosphotyrosine-binding sites for STAT5b (51, 52, 53), then so long as the receptor remains in a dimeric complex associated with JAK2, JAK2 may rephosphorylate and thereby reactivate the receptor’s STAT5b-binding sites (Fig. 7Go, upper arrow of ’GHR-JAK2 cycle’). A high phosphotyrosine phosphatase level might therefore be expected to inhibit, but not totally eliminate, reentry of GHR-JAK2 complexes into the STAT5b activation loop. Our finding that the low steady-state level of STAT5b EMSA activity during phase 2 GH signaling can in large part be ascribed to elevated phosphotyrosine phosphatase activity is consistent with this model. However, the situation is likely to be more complicated, as indicated by our finding that phase 2 GH signaling to STAT5b requires the constant presence of GH, that it depends on new protein synthesis, and that it can proceed at an approximately 2-fold higher level when proteasome degradation is blocked. Together, these observations suggest that, in addition to the down-regulatory effects of phosphotyrosine phosphatase(s), the GHR-JAK2 complex is actively disassembled or degraded in continuous GH-treated cells. This degradation likely proceeds via ubiquitination leading to receptor internalization and degradation (49). Dissociation leading to degradation of the inactivated GHR-JAK2 complex may rapidly follow its dephosphorylation, as proposed above, since when pervanadate was added to CWSV-1 cells just 30 sec after GH removal, the dramatic increase in activated STAT5b levels in response to pervanadate (Fig. 3AGo) was not observed (data not shown).

The protein synthesis inhibitor cycloheximide eliminated the continuous GH-activated STAT5b EMSA signal within 90 min (Fig. 6Go). Since neither STAT5b protein nor JAK2 is depleted from the cells under these circumstances (Fig. 6CGo), GHR may be the component that is down-regulated and degraded during phase 2, thus explaining the requirement for continued protein synthesis to maintain signaling to STAT5b (Fig. 7Go, step D). The inhibitory effect of cycloheximide on GH signaling to STAT5b during phase 2 contrasts with its protective effect on signaling to STAT5b at the conclusion of phase 1 (23), which apparently blocks transition of the cells from GH phase 1 to phase 2 signaling. Furthermore, the absence of a protein synthesis requirement for reactivation of STAT5b by repeated GH pulses given to CWSV-1 cells (22) indicates that all of the components of the receptor-kinase activation pathway are stable molecules during phase 1, or that they are present in sufficient excess, such that they are not depleted by just two GH pulses. By contrast, the GHR-JAK2 complex appears to become more labile in cells treated with GH continuously than in GH pulse-treated cells, thus contributing to the down-regulation of JAK2-STAT5b signaling, which characterizes female rat liver.

The serine/threonine kinase inhibitor H7 prolongs the activated STAT5b signal induced by a GH pulse (22, 23) but did not prolong the activated STAT5b signal in continuous GH-treated CWSV-1 cells once GH was removed (Fig. 4Go). In the case of a GH pulse, H7 apparently can both prevent the inhibition of new GHR-JAK2 complex assembly and sustain JAK2 signaling to STAT5b. Neither of these effects is seen when H7 is added to CWSV-1 cells at the conclusion of a GH pulse (23). Our finding that H7 has no immediate effect when added to CWSV-1 cells during phase 2 continuous GH signaling (Fig. 4AGo) is consistent with the suggestion that both H7-sensitive inhibitory mechanisms are initiated early after exposure of the cells to GH. For H7 to have an effect when added to cells after the initial GH treatment, it needs to be incubated with the cells for times longer than the half-lives of any inhibitory molecule(s) that may have already been induced by the initial GH treatment. Indeed, with prolonged H7 incubation, an elevation of activated STAT5b levels was observed (Fig. 4CGo). This effect of prolonged H7 treatment could occur at the level of the phosphatase responsible for the low level STAT5b signal, e.g. by inhibiting its ongoing transcription, or perhaps it may occur via H7’s effects on the assembly of new GHR-JAK2 complexes. Further study will be required to address this and other questions regarding the mechanisms whereby continuous GH down-regulates GHR-JAK2 signaling via the STAT5b pathway.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CWSV-1 Cell Culture and GH Treatment
Culture of CWSV-1 cells, treatment with GH, alone or in combination with inhibitors, and preparation of cell extracts were performed using the general methods described elsewhere (22). 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 specified otherwise. GH was removed from the cells 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.

General Experimental Design
GH was added to CWSV-1 cell culture medium and the dishes were typically incubated for 3 h, sufficient time to achieve the low level ’phase 2' STAT5b signaling (see Results). At that time, inhibitors were added to the culture medium as detailed in the figure legends. Except where indicated, GH was maintained in the culture medium during the entire course of the experiment. Data points shown in each experimental set represent separate culture dishes that had been treated identically up to the point where the cells were harvested and cell extracts were prepared. The experimental design for each study is illustrated by a step diagram, where large steps represent addition or removal of GH, and small steps represent addition or removal of inhibitors. Times at which individual cell culture dishes were harvested for analysis of STAT5b activity by EMSA are represented in the step diagram by vertical arrows. Time intervals shown in the EMSA and Western blot data are related to the time of GH addition to the culture medium, or to the time of inhibitor addition, as noted in each figure. For example, in the EMSA gel shown in Fig. 2BGo, lanes marked 3h and 15' (lanes 2 and 3) correspond to samples taken 3 h after the initiation of GH treatment (lane 2) and 3 h after GH treatment followed by a 15 min further incubation with GH + genistein (lane 3). Data shown are representative of results obtained in at least three independent experiments.

Inhibitors
Inhibitors were obtained from Sigma Chemical Co.( St. Louis, MO), except for MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), which was from Peptides International (Louisville, KY). Inhibitors were prepared as 100x stock solutions in dimethylsulfoxide (genistein, staurosporin 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), which in control experiments not presented did not alter the intensity of STAT5b EMSA activity in response to a GH pulse or after continuous GH exposure (phase 2 STAT5b signal). Pervanadate stock solution was prepared by incubating equal volumes of 12 mM sodium vanadate (freshly dissolved in water) and 12 mM hydrogen peroxide at room temperature for 20 min before use.

Other Methods
Biochemical analyses, including EMSA, Western blotting, and immunoprecipitation, were carried out as previously described (22). Antiphosphotyrosine monoclonal antibody PY99 conjugated to agarose beads, used for immunoprecipitation, and an anti-STAT5b-specific antibody (sc-835) used for Western blotting were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antiphosphotyrosine antibody 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY), and anti-JAK2 antibody used for Western blotting (QCB, Inc., Hopkinton, MA) were from the indicated sources. Immunoprecipitation of JAK2 was carried out using antibody generously provided by Dr. C. Carter-Su (University of Michigan).


    FOOTNOTES
 
Address requests for reprints to: Dr. David J. Waxman, Department of Biology, Boston University, 5 Cummington Street, Boston, Massachusetts 02215. email: djw@bio.bu.edu

Supported in part by NIH Grant DK-33765 (to D.J.W.).

Received for publication September 11, 1998. Revision received November 3, 1998. Accepted for publication November 4, 1998.


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 MATERIALS AND METHODS
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