Mechanism of Inhibition of Growth Hormone Receptor Signaling by Suppressor of Cytokine Signaling Proteins

Johnny A. Hansen, Karen Lindberg, Douglas J. Hilton, Jens H. Nielsen and Nils Billestrup

Hagedorn Research Institute (J.A.H., K.L., J.H.N., N.B.) DK-2820 Gentofte, Denmark
The Walter and Eliza Hall Institute for Medical Research (D.J.H.) Parkville, Victoria 3052, Australia


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study we have investigated the role of suppressor of cytokine signaling (SOCS) proteins in GH receptor-mediated signaling. GH-induced transcription was inhibited by SOCS-1 and SOCS-3, while SOCS-2 and cytokine inducible SH2-containing protein (CIS) had no effect. By using chimeric SOCS proteins it was found that the ability of SOCS proteins to inhibit GH-mediated transcription was located in the amino-terminal 40–80 amino acids. In SOCS-3, 46 amino acids C-terminal to the SH2 domain were required for the inhibitory activity, while a truncated SOCS-1 having only 2 amino acids C-terminal to the SH2 domain was able to inhibit GH-mediated transcription. Both SOCS-1 and SOCS-3 were able to inhibit GH-induced STAT5 (signal transducer and activator of transcription) activation. SOCS-1 inhibited the tyrosine kinase activity of Janus kinase 2 (JAK2) directly, while SOCS-3 only inhibited JAK2 when stimulated by the GH receptor. All four SOCS proteins were able to bind to a tyrosine-phosphorylated glutathione-S-transferase-GH receptor fusion protein, and SOCS-3 required the same 46 C-terminal amino acids for GH receptor binding as it did for inhibition of GH-mediated transcription and STAT5 activation. These data suggest that SOCS-1 and -3 can suppress GH-induced transcriptional activity, presumably by inhibiting the kinase activity of JAK2 either directly in the case of SOCS-1 or via binding to the tyrosine-phosphorylated GH receptor in the case of SOCS-3.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
While much progress has been made toward understanding cytokine receptor stimulation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, much less is known about how these signals are turned off. Recently a family of proteins involved in the suppression of cytokine signaling has been identified (1, 2, 3). The SOCS family of proteins consists of eight members: CIS and SOCS-1 through 7. CIS was originally identified in 1995 as an immediate early gene induced by interleukin 3 (IL-3) and erythropoietin (Epo) in BaF3 cells (4). CIS was found to be able to bind to the phosphorylated Epo and IL-3 receptors and to suppress the proliferation of hematopoietic cells (BaF3 and FDCP-1) in response to IL-3 (4). SOCS-1 was subsequently identified as a factor capable of inhibiting IL-6-induced differentiation of monocytic leukemic M1 cells into macrophages (2), and at the same time a factor termed JAB was cloned based on its ability to interact with the kinase domain of the JAK2 kinase (5) and SSI-1 was cloned based on homology to the SH-2 domain of STAT3 (6). Analysis of the sequences of SOCS-1, JAB, and SSI-1 revealed that these three factors were identical.

The induction of SOCS gene expression by cytokines has been reported in a number of different cell types both in vitro and in vivo. Stimulation of CIS and SOCS-1, -2, and -3 mRNA expression in bone marrow cells by Epo, thrombopoietin, granulocyte-colony stimulating factor (G-CSF), leukemia-inhibiting factor (LIF), granulocyte macrophage-colony stimulating factor (GM-CSF), IL-1, -2, -3, -4, -6, -7, -12, -13, and interferon-{gamma} (IFN-{gamma}) has been reported (2). In this study great variation in inducibility was observed with some cytokines inducing the expression of all four SOCS mRNA (GM-CSF and IFN{gamma}) while others induced only a subset of the SOCS genes. In BaF3 cells, IL-2 and IL-3 induced the expression of CIS (4, 7), and in M1 cells both IL-6 and LIF induced the induction of SOCS-1 and CIS while no induction of SOCS-2 and -3 was observed. In 3T3-F442A preadipocytes, GH induced a rapid and transient expression of SOCS-3 and, to a lesser extent, SOCS-1 (8). In the hypothalamus, leptin has been reported to induce the expression of SOCS-3 mRNA, and increased levels of SOCS-3 mRNA were observed in the hypothalamus of the obese lethal yellow (Ay/a) mouse, which is known to exhibit leptin resistance (9).

SOCS-1 was originally identified by its ability to inhibit IL-6-induced M1 cell differentiation (2) and by its binding to the kinase domain of JAK2 (5), indicating an inhibitory action in IL-6 signaling at the level of the JAK2 kinase. Subsequently it was demonstrated that SOCS-1 inhibited the intrinsic kinase activity of all four JAK family members when overexpressed in COS or 293 cells (5). In accordance with this observation, it was reported that SOCS-1 inhibited IL-6-induced tyrosine phosphorylation of STAT3, the IL-6 receptor-signaling subunit gp130 and JAK2 (10). In contrast, it was found that SOCS-3 and CIS were unable to inhibit the intrinsic kinase activity of JAK2, and it was proposed that these SOCS proteins inhibit cytokine signaling at a step distal to JAK activation. CIS has been shown to interact directly with the phosphorylated Epo and GM-CSF receptors, and it was suggested that this binding might prevent the recruitment of STAT factors to the receptor/JAK complex.

The SOCS proteins can be divided into three domains: the N terminus exhibiting little sequence identity among the SOCS proteins, the centrally located SH2 domain, and the SOCS box located in the C terminus (2). Recently it has been found that both the N terminus and SH2 domain of SOCS-1 were required for suppression of IL-6 and LIF signaling and inhibition of JAK activity (11). The SH2 domain and an additional N-terminal 12 amino acids of SOCS-1 were found to be required for interaction with tyrosine 1007 in the activation loop of JAK2 and thus inhibition of kinase activity (12). The SOCS box of SOCS-1 is not required for its inhibitory activity but rather seems to be involved in protein stability (13), possibly by interacting with elongins B and C (14).

GH preferentially induces the expression of SOCS-3 (8), and since it has been observed that SOCS-3, in contrast to SOCS-1, could not inhibit the intrinsic kinase activity of JAK2, we have in this study investigated the mechanism by which SOCS-1 and SOCS-3 inhibit signaling by the GH receptor. We have identified specific domains of SOCS-1 and SOCS-3 involved in the suppression of GH-mediated transcription, STAT5 activation, and JAK2 activity, and binding to the tyrosine-phosphorylated GH receptor.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The N-Terminal Domain of SOCS-1 and SOCS-3 Confers Specificity for the Inhibition of GH-Induced Transcription
The effect of SOCS expression on GH-induced transcription was analyzed by transient transfections of CHO cells with a GH-responsive STAT5-dependent reporter. In cells not transfected with SOCS expression plasmids, GH induced a 4-fold increase in reporter activity (Fig. 1Go). Coexpression of either SOCS-1 or SOCS-3 inhibited GH-induced transcription in a dose-dependent manner, while coexpression of CIS did not affect GH induction. Expression of SOCS-2 resulted in a slightly higher induction by GH as compared with control.



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Figure 1. Effect of SOCS on GH-Stimulated Transactivation of the Spi 2.1 Promoter

CHO cells were transiently transfected with the Spi 2.1 promoter/CAT construct, a GH receptor expression vector, together with the indicated amount of SOCS expression vectors. After transfection, extracts were prepared from cells cultured in the absence or presence of 20 nM GH. CAT activity was determined and normalized to ß-galactosidase activity to control for transfection efficiency. The level of CAT activity, in the absence of GH and SOCS expression vectors, was given the value of 1. The results presented correspond to the mean values of three independent experiments, with the error bars representing the mean ± SD.

 
To identify the domains of the SOCS proteins that impart the specificity in the ability of SOCS-1 and SOCS-3 to inhibit GH-induced transcription, we generated chimeric SOCS expression vectors by swapping of the N-terminal domain and the SOCS box as shown in Fig. 2Go, B and C. The construction of chimeric SOCS proteins was facilitated by the introduction of specific restriction sites between the N-terminal domain and the SH2 domain and between the SH2 domain and the SOCS box. The activity of the SOCS proteins containing these introduced restriction sites was found to be identical to that of the wild-type SOCS proteins (Fig. 2AGo vs. Fig. 1Go). Analyzing the chimeric SOCS proteins revealed that the SOCS box was interchangeable and did not confer specificity, since SOCS proteins having the SOCS box of CIS and the N-terminal and SH2 domains of SOCS-1 or SOCS-3 were able to inhibit GH-induced transcription (Fig. 2BGo). In contrast, the N-terminal domain was able to confer specificity as shown by the inhibitory effect of chimeric SOCS proteins having the N-terminal domain of SOCS-1 or SOCS-3 and the SH2 domain and the SOCS box from CIS (Fig. 2CGo).



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Figure 2. The Role of SOCS Protein Domains in the Inhibition of GH-Stimulated Transactivation of the Spi 2.1 Promoter

CHO cells were transiently transfected with the Spi 2.1 promoter/CAT construct, together with plasmids expressing the GH receptor, ß-galactosidase, alone or together with expression vectors (1 µg) encoding mutated or chimeric SOCS proteins. The mutated and chimeric SOCS proteins are illustrated schematically to the left (A, B, and C). The black box represents the linker domain. CAT activity was determined and normalized to the activity observed in the absence of hGH. The results presented are the mean values of three independent experiments, with the error bars representing the mean ± SD.

 
SOCS-3, but Not SOCS-1, Requires the Linker Domain between the SH2 Domain and the SOCS Box for Functional Activity
The functional role of the SOCS box and the linker domain between the SOCS box and the SH2 domain was investigated by generating truncated versions of SOCS-1 and SOCS-3 as shown schematically in Fig. 3Go. The linker domain in SOCS-1 consists of only 5 amino acids while that of SOCS-3 has 47 amino acids (Fig. 3CGo). The activity of SOCS-1 was not altered by removing the SOCS box and 3 amino acids of the linker domain, leaving only 2 amino acids C-terminal to the SH2 domain, whereas in SOCS-3 46 amino acids of the linker domain were required for function. Thus, in both SOCS proteins, the SOCS box was dispensable, but SOCS-3 requires amino acids located between positions 177 and 184.



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Figure 3. The Role of the C-Terminal Domain of SOCS-1 and SOCS-3 in the Inhibition of GH-Stimulated Transactivation of the Spi 2.1 Promoter

CHO cells were transiently transfected with the Spi 2.1 promoter/CAT construct, together with plasmids expressing the GH receptor, ß-galactosidase, alone or together with expression vectors (1 µg) encoding either the wild-type or truncated versions of SOCS-1 or SOCS-3 as illustrated schematically in panel A (SOCS-3) and panel B (SOCS-1). CAT activity was determined and normalized to the activity observed in the absence of GH. The results presented are the mean values of three independent experiments, with the error bars representing the mean ± SD. C, The amino acid sequence of the region between the SH2 domain and the SOCS-box in the murine SOCS-3 and SOCS-1 protein.

 
SOCS-1 and SOCS-3 Inhibit STAT5 DNA Binding Activity
Since GH activation of reporter activity is mediated by STAT5, we investigated the ability of SOCS proteins to inhibit GH-induced STAT5 activation by measuring the DNA binding activity of STAT5 by electrophoretic mobility shift assay. Nuclear extracts from 293 cells transfected with the GH receptor and STAT5, with or without SOCS expression vectors, were analyzed using the STAT5 binding element from the Spi 2.1 promoter. In the absence of SOCS expression or in cells expressing CIS and SOCS-2, STAT5 DNA binding was observed (Fig. 4Go). In cells transfected with SOCS-1 or SOCS-3, no STAT5 DNA binding could be detected. Western blot analysis using total cell extracts revealed that STAT5 was expressed at comparable levels in all cell extracts (Fig. 4BGo). The level of SOCS protein expression was highest in cells transfected with CIS and lowest in SOCS-3-transfected cells (Fig. 4CGo).



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Figure 4. The Role of SOCS Proteins in the Activation of STAT5 DNA Binding Activity Induced by GH

A, Electrophoretic mobility shift assay using the STAT5 binding element from the Spi 2.1 promoter as a probe and nuclear extracts made from 293 cells transiently transfected with plasmids expressing the GH receptor and HA-STAT5, alone (lanes 1–2) or together with an expression vector encoding the indicated FLAG-SOCS protein (lanes 3–10). Serum-starved cells were cultured in the absence or presence of 20 nM GH for 5 min before nuclear extract preparation. B, Western blot analysis was performed on total cell lysates using anti-HA antibodies to detect STAT5; C, anti-FLAG antibodies were used to detect SOCS proteins. The results shown are representative of three independent experiments.

 
We also investigated the abilities of selected chimeric and truncated SOCS proteins to inhibit STAT5 DNA binding (Figs. 5Go and 6Go). It was demonstrated that the same chimeric SOCS proteins that could inhibit GH-induced transcription also inhibited STAT5 activity. Furthermore, we found that the region of the linker domain in SOCS-3 required for inhibition of transcription was also required for inhibition of STAT5 activity.



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Figure 5. The Role of SOCS Domains in the Inhibition of GH-Induced STAT5 Activation

A, Electrophoretic mobility shift assay using the STAT5 binding element from Spi 2.1 promoter as a probe and nuclear extracts made from 293 cells transiently transfected with plasmids expressing the GH receptor and HA-STAT5, alone (lanes 1–2) or together with an expression vector encoding the indicated chimeric FLAG-SOCS proteins (lanes 3–10). Serum-starved cells were cultured in the absence or presence of 20 nM GH for 5 min before nuclear extract preparation. B, Western blot analysis was performed on total cell lysates using anti-HA antibodies to detect STAT5; C, anti-FLAG antibodies were used to detect SOCS proteins. The results shown are representative of three independent experiments.

 


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Figure 6. The Role of the C-Terminal Part of SOCS-3 in the Inhibition of GH-Induced STAT5 Activation

A, Electrophoretic mobility shift assay using the STAT5 binding element from the Spi 2.1 promoter as a probe and nuclear extracts made from 293 cells transiently transfected with plasmids expressing the GH receptor and HA-STAT5, alone (lanes 1–2) or together with an expression vector encoding the indicated truncated FLAG-SOCS-3 proteins (lanes 3–12). Serum-starved cells were cultured in the absence or presence of 20 nM GH for 5 min before nuclear extract preparation. B, Western blot analysis was performed on total cell lysates using anti-HA antibodies to detect STAT5; C, anti-FLAG antibodies were used to detect SOCS proteins. The results shown are representative of three independent experiments.

 
SOCS-1 Inhibits the Intrinsic Activity of JAK2
Previous work has demonstrated that the interaction of SOCS-1 with JAK kinases markedly reduces their kinase activity (5). To investigate the possibility that SOCS-1 and SOCS-3 could influence STAT5 activation by inhibition of the JAK2 kinase activity, JAK2 was transiently expressed in 293 cells, alone or together with different SOCS-encoding plasmids. Overexpression of the JAK2 protein in these cells resulted in a constitutive phosphorylation of the kinase, presumably because the high level of expression allows dimerization and trans-phosphorylation to take place in the absence of GH and GH receptor (Fig. 7Go). By immunoprecipitation of JAK2 followed by Western blot analysis using antiphosphotyrosine antibodies, we observed that only SOCS-1 and not SOCS-2, SOCS-3, or CIS was able to inhibit tyrosine phosphorylation of the kinase (Fig. 7Go).



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Figure 7. The Role of SOCS Proteins in JAK2 Activation

HEK 293 cells were transiently transfected with a JAK2- encoding plasmid (1 µg) alone or together with a SOCS expression vector (10 µg). After transfection, cell lysates were used for immunoprecipitation (IP) using {alpha}JAK2 antibodies. A, Immunoprecipitated JAK2 was separated by SDS-PAGE followed by Western blot analysis using antiphosphotyrosine antibodies ({alpha}PY). B, The blot shown in panel A was stripped and reprobed with anti-JAK2 antibodies. C, Cell lysates were analyzed by Western blot analysis using anti-FLAG antibodies.

 
SOCS Proteins Bind to the Tyrosine-Phosphorylated GH Receptor
The fact that both SOCS-1 and SOCS-3 were found to inhibit GH-induced transcription and STAT5 activation but that only SOCS-1 was able to inhibit the intrinsic activity of JAK2, suggested to us that SOCS-3 might require the presence of the GH receptor to exhibit its inhibitory action on GH signaling. Previously, CIS has been demonstrated to associate with tyrosine-phosphorylated IL-3ß and Epo receptors (4). Therefore, we analyzed the ability of SOCS proteins to bind tyrosine-phosphorylated glutathione-S-transferase (GST)-GH receptor fusion protein in a GST-binding assay. Human 293 cells were transiently transfected with the individual SOCS-encoding plasmids, and cell lysates were used for binding to GST-GH receptor fusion proteins. By this procedure, it was demonstrated that all four SOCS proteins were able to associate with GST-GH receptor 455–638 fusion protein only when the fusion protein was tyrosine phosphorylated (Fig. 8Go). The amount of SOCS protein applied in each reaction was evaluated by including a sample of cell lysate from nontransfected cells and from each of the cell lysates used for the binding reactions (lanes 7–9 and 16–18).



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Figure 8. Binding of SOCS Proteins to Tyrosine-Phosphorylated GST-GH Receptor 455–638 Fusion Protein

Cell lysates made from 293 cells transiently transfected with expression plasmids encoding FLAG-tagged SOCS protein were incubated together with the indicated GST fusion protein coupled to glutathione-Sepharose beads. The bound proteins were resolved by SDS-PAGE and analyzed by Western blot analysis using anti-FLAG antibodies. Cell lysates from either nontransfected cells (lanes 7 and 16) or from cells transiently transfected with the different FLAG-SOCS encoding plasmids (lanes 8, 9, 17, and 18) were included for comparison. The results shown are representative of three independent experiments.

 
We have previously shown that STAT5 can bind to three phosphotyrosines in the GH receptor and that these three tyrosines (534, 566, and 627) are required for GH activation of transcription and STAT5 activation (15, 16). To test whether SOCS-3 could bind to the same three phosphotyrosines as STAT5, we tested the ability of specific phosphopeptides derived from the GH receptor sequence to inhibit the binding of SOCS-3 to the tyrosine-phosphorylated GST-GHR fusion protein. We found that a phosphopeptide containing tyrosine 487 was the most effective in competing for SOCS-3 binding. Phosphopeptides containing tyrosines 534, 595, and 627 also competed for SOCS-3 binding, while phosphopeptide 566 only inhibited slightly (Fig. 9Go). None of the nonphosphorylated peptides affected SOCS-3 binding to the GST-GH receptor fusion protein.



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Figure 9. Inhibition of SOCS-3 Binding to the Tyrosine-Phosphorylated GST-GH Receptor 455–638 Fusion Protein by Phosphopeptides Derived from the GH Receptor

Cell lysates from 293 cells transiently transfected with the FLAG-SOCS-3 encoding plasmid were incubated together with tyrosine-phosphorylated GST-GH receptor 455–638 fusion protein coupled to glutathione-Sepharose beads in the presence of the indicated peptide in a concentration of 300 µM. Precipitated proteins were subjected to SDS-PAGE and analyzed by Western blot analysis using {alpha}FLAG antibodies. The results shown are representative of three independent experiments.

 
To test whether SOCS-3 could directly compete for STAT5 binding to the tyrosine-phosphorylated GST-GH receptor fusion protein, we included increasing amounts of SOCS proteins in a GST-GH receptor STAT5 binding assay (Fig. 10Go). From this experiment it was observed that none of the SOCS proteins could inhibit STAT5 binding to the receptor, even though an approximately 50-to 250-fold molar excess of SOCS protein compared with STAT5 was used. This finding suggests that SOCS-3 does not inhibit STAT5 binding to the GH receptor and that the inhibition of STAT5 by SOCS-3 cannot be explained by competition for binding sites within the GH receptor.



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Figure 10. The Effect of SOCS Proteins on STAT5 Binding to the Tyrosine-Phosphorylated GST-GH Receptor 455–638 Fusion Protein

A, Cell lysates from STAT5-transfected 293 cells were incubated together with the GST-GH receptor 455–638 TKX1 fusion protein coupled to glutathione-Sepharose beads. Increasing amounts (50- and 200-fold molar excess) of cell lysate from 293 cells transiently transfected with the indicated SOCS-encoding plasmid was included in the reaction. Precipitated proteins were resolved by SDS-PAGE and analyzed by Western blot analysis using anti-HA antibodies. Lanes 8 and 9 contain cell lysates from nontransfected or STAT5-transfected 293 cells, respectively. B, Cell lysates from either nontransfected cells (lane 10) or cells transiently transfected with the indicated SOCS expression plasmid are included for comparison.

 
To analyze the functional domain of SOCS-3 involved in the inhibition of GH-induced transcription and STAT5 activation, we used GST-GH receptor binding to examine the truncated forms of SOCS-3. The same region of the linker domain that was required for inhibition of GH-induced transcription and STAT5 activation was also required for binding to the tyrosine-phosphorylated GST-GH receptor fusion protein (Fig. 11Go). These observations indicate that SOCS-3 binding to the GH receptor plays an important role in the mechanism by which it suppresses GH-mediated signaling.



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Figure 11. Binding of Truncated SOCS-3 to the Tyrosine-Phosphorylated GST-GH Receptor 455–638 Fusion Protein

Cell lysates made from 293 cells transiently transfected with different expression plasmids encoding either wild-type or truncated versions of FLAG-tagged SOCS-3 protein were incubated together with the tyrosine-phosphorylated GST-GH receptor 455–638 fusion protein coupled to glutathione-Sepharose beads. The proteins were resolved by SDS-PAGE and analyzed by Western blot analysis using anti-FLAG antibodies. Lysates from either nontransfected cells (lane 6) or from cells transiently transfected with the different expression plasmids encoding either wild-type or truncated versions of FLAG-tagged SOCS-3 protein (lanes 7–11) are included.

 
SOCS-3 Inhibits JAK2 Activity by a GH Receptor-Dependent Mechanism
To investigate the hypothesis that SOCS-3 can inhibit GH signaling by inhibiting JAK2 kinase activity in a GH receptor-dependent manner, we transiently transfected 293 cells with JAK2 alone or together with a SOCS-3 encoding plasmid in combination with an increasing amount of a GH receptor-encoding plasmid. In contrast to the experiment with overexpression of the JAK2 protein in the 293 cells, which resulted in a constitutive tyrosine phosphorylation of the kinase (Fig. 7Go), a reduced amount of JAK2-encoding plasmid was used in this experiment, resulting in a GH-inducible and GH receptor-dependent tyrosine phosphorylation of JAK2. By immunoprecipitation of JAK2 followed by Western blot analysis using antiphosphotyrosine antibodies, we observed that SOCS-3 was able to inhibit GH-induced tyrosine phosphorylation of JAK2 (Fig. 12Go). Inhibition of this tyrosine phosphorylation of the kinase was observed using 3 µg GH receptor expression plasmid, and further inhibition of the GH-induced tyrosine phosphorylation of JAK2 was observed using 10 µg GH receptor expression plasmid. This finding suggests that SOCS-3 can inhibit JAK2 tyrosine phosphorylation by a mechanism dependent on the GH receptor.



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Figure 12. Inhibition of GH-Stimulated JAK2 by SOCS-3

HEK 293 cells were transiently transfected with JAK2 (50 ng) and SOCS-3 (20 µg) and the indicated amount of GH receptor. After transfection, cells were cultured in the absence or presence of 20 nM GH for 5 min. Cell lysates were used for immunoprecipitation (IP) using anti-JAK2 antibodies. A, Immunoprecipitates were analyzed by SDS-PAGE followed by Western blot analysis using antiphosphotyrosine antibodies ({alpha}-PY). B, An identical amount of immunoprecipitate was analyzed by Westen blotting using anti-JAK2 antibodies. C, Cell lysates were analyzed by SDS-PAGE followed by Western blot analysis using anti-FLAG antibodies.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have previously shown that SOCS-3 is induced transiently by GH both in vivo and in vitro, while the expression of SOCS-1, SOCS-2, and CIS is induced only marginally by GH (8). In this study we examine the mechanism by which SOCS proteins inhibit GH-induced transcription of the STAT5-responsive Spi 2.1 promoter. Both SOCS-1 and SOCS-3 were found to inhibit GH-induced transcription of the Spi 2.1 promoter. The generation of chimeric SOCS proteins showed that the N-terminal domain dictates the specificity by which SOCS proteins inhibit GH-mediated signaling. Previously, the N-terminal domain has been found to be essential for SOCS-1 inhibition of IL-6 signaling and the ability to inhibit JAK activity (13), and the N-terminal domains of SOCS-1 and SOCS-3 were found to be functionally interchangeable for inhibition of IL-6 and LIF signaling (11). However, only the SH2 domain of SOCS-1 was found to be required for binding to JAK. Together these data indicate that the N-terminal domain is required for the inhibitory action of SOCS proteins on JAK activity and that some degree of specificity is encoded within this domain since the N-terminal domains of SOCS-1 and SOCS-3 are able to inhibit GH signaling when placed in conjunction with the SH2 domain and SOCS box from CIS. In agreement with this it was recently reported that SOCS-1 and, to a minor extent, SOCS-3 were the only SOCS proteins that interacted directly with the kinase domain of JAK2 both in vitro and in vivo (10) and that amino acids N-terminal to the SH2 domain were required for the interaction with JAK2 (12).

The C-terminal 40-amino acid region of the SOCS proteins has been termed the SOCS box, and this domain exhibits approximately 50% identity among the SOCS family members. The functional role of this domain is not known as it has been reported to be not required for inhibition of IL-6-induced transcription or JAK kinase activation. In this study we found that the SOCS box is not important for inhibition of GH-induced transcription by SOCS-1 and SOCS-3. However, the linker region between the SH2 domain and the SOCS box of SOCS-3 was required for SOCS-3 to inhibit GH signaling. This linker region is considerably longer in SOCS-3 compared with SOCS-1 (Fig. 3Go). Inhibitory activity was observed when only two amino acids of the linker region were present in SOCS-1, whereas almost the entire linker region (46 amino acids) was required for SOCS-3 inhibition of GH-induced transcription. The same 46 amino acids were also required for inhibition of GH-induced activation of STAT5.

Previous studies have shown that CIS is able to bind to the tyrosine-phosphorylated erythropoietin and IL-3 receptors (4), and it was suggested that CIS binding to these receptors reduces the interaction between STAT5 and the receptor, resulting in inhibition of STAT5 activation. Using GST-GH receptor fusion proteins, we could show that all four SOCS proteins were able to bind specifically to the tyrosine-phosphorylated GH receptor. It was also demonstrated that binding of SOCS-3 to the GST-GH receptor fusion protein was inhibited by a phosphopeptide containing tyrosine 487, whereas phosphopeptides containing either tyrosine 534, 595, or 627 were less inhibitory and phosphopeptide 566 inhibited only slightly. Since we previously identified three of these tyrosine residues (Y534, Y566, and Y627) as STAT5 binding sites (15, 16) in the GH receptor, we tested whether SOCS-3 could compete with STAT5 for binding to the GH receptor. However, no competition could be observed, suggesting that the inhibitory mechanism of SOCS-3 in GH signaling was not by competing for STAT5 binding sites in the GH receptor. The fact that Y566 did not bind SOCS-3 but is able to bind STAT5 might explain the lack of competition between these two factors. The observation that all four SOCS members are able to bind to the GH receptor also suggests that binding to the receptor is not sufficient for inhibition of GH signaling. The role of SOCS binding to the GH receptor is not known at present; however, we found that the same region of the linker domain in SOCS-3 that was required for inhibition of GH-induced signaling was also required for binding to the GH receptor. This finding was surprising since the SH2 domain is believed to be able to bind phosphotyrosines by itself, but in the case of SOCS-3 an additional 46 amino acids C-terminal to the SH2 domain seem to be required for this binding.

Thus, these data in combination with the observations that only SOCS-1 was able to inhibit tyrosine phosphorylation of overexpressed JAK2, whereas SOCS-3 was able to inhibit GH-stimulated tyrosine phosphorylation of JAK2 only in the presence of the GH receptor, indicate that SOCS-1 and SOCS-3 inhibit GH signaling by two different mechanisms. It is tempting to suggest that SOCS-1 directly binds to the JAK2 kinase and thereby inhibits the kinase activity, whereas SOCS-3 only inhibits JAK2 kinase activity after binding to the GH receptor. This hypothesis furthermore predicts that two functional domains are present in the SOCS proteins: one, which includes the SH2 domain, is involved in binding of the SOCS protein to phosphotyrosines in cytokine receptors or in JAK kinases, and the other, which includes the N-terminal domain, is involved in the actual inhibition of kinase activity. Interestingly, a similar difference in the mechanism by which GH activates STAT1 and STAT5 was previously reported (17). STAT1 is activated by a mechanism that does not require GH receptor tyrosine phosphorylation (18), whereas STAT5 activation by GH is dependent upon phosphorylation of at least one of three tyrosines in the intracellular domain of the GH receptor (16, 19).

The physiological role of SOCS proteins in GH signaling is not known at present; however, since SOCS-3 is the major SOCS protein induced by GH both in vitro and in vivo, this factor is presumed to be the main regulator of GH signaling. The transient nature of SOCS-3 mRNA induction by GH and the relative short half-life of SOCS-3 protein suggests that SOCS-3 acts in a classical negative feed-back loop, suppressing GH signaling for a limited time period. Interestingly GH is secreted in a pulsatile manner in most species with a frequency of 3 to 4 h between each peak. This time period is in good agreement with the time required for SOCS-3 levels to return to basal levels after a GH pulse, and the role of SOCS-3 might be to protect against overstimulation by GH or alternatively to restrict the time in which a cell is responsive to GH stimulation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
Flag epitope-tagged SOCS-1, SOCS-2, SOCS-3, and CIS expression vectors were constructed in the pEF-BOS plasmid as described previously (2). The rat GH receptor expression vector pLM 108 was constructed as described (20). The hemagglutinin-epitope-tagged STAT5 expression vector was obtained from Dr. Groner (Georg-Speyer-Haus, Institute for Biomedical Research, Frankfurt, Germany) (21), and the JAK2 expression vector was obtained from Dr. Schlessinger (New York University, New York).

Cell Culture
Chinese hamster ovary (CHO) cells were cultured in Ham’s F-12 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were transfected at 90% confluency in 60-mm cell culture dishes. Twenty-four hours before transfection, cells were washed twice and incubated with 3 ml serum free GC3 medium (1:1 mixture of DMEM and Ham’s F-12 (Life Technologies, Inc.), adjusted to pH 7.1 with 7.5% NaHCO3 solution, supplemented with 10 µg/ml transferrin, 160 mU/ml insulin, 2 mM L-glutamine, 2 mM nonessential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin). Cells were transiently transfected by calcium phosphate precipitation as described previously (15). Each dish was transfected with 0.01–1 µg SOCS expression vector, 3 µg pCH110 ß-galactosidase expression vector, 1.5 µg Spi 2.1/chloramphenicol acetyltransferase (CAT) plasmid, and 1.5 µg GH receptor expression plasmid, and cultured in the absence or presence of 20 nM hGH (Novo Nordisk, Bagsvaerd, Denmark) overnight. CAT assay was performed on total cellular extracts as described (22) using ß-galactosidase as an internal control. Human kidney 293 cells were cultured in DMEM supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Cells were seeded in 100-mm cell culture dishes with 10 ml culture medium and transiently transfected the next day (50% confluency) by calcium phosphate precipitation.

Peptides
Synthetic peptides were purchased from Affinity Research Products Ltd. (Mamhead, UK) either nonphosphorylated or tyrosine phosphorylated. Five 13 amino acid long peptides derived from the GH receptor were synthesized; LANIDFYAQVSDI (peptide Y487), FIMDNAYFCEADA (peptide Y534), FNQEDIYITTESL (peptide Y566), EMPVPDYTSIHIV (peptide Y595), and FLSSCGYVSTDQL (peptide Y627). The peptides were purified by HPLC and their composition verified by mass spectrometry.

GST-GHR Binding Assay
Cell extracts were isolated by addition of lysis buffer (50 mM HEPES, pH 7.2, 250 mM NaCl, 10% glycerol, 2 mM EDTA, 2 mM EGTA, 0.1% NP-40, 1 mM 4-(2-aminoethyl) benzene sulfonyl fluoride (AEBSF), 1 µg/ml aprotinin, 1 µg/ml leupeptin, 10 µg/ml herbimycin, and 1 mM sodium orthovanadate) to the cells followed by 20 min incubation on ice. After centrifugation at 10,000 x g for 10 min at 4 C, the supernatants were used for GST binding assays. Varying amounts (indicated in each individual experiment) of the extracts were added to 50 µl (50%) glutathione-Sepharose 4B beads to which 25 µg of GST fusion protein were bound and incubated for 12–14 h at 4 C with rotation (when competition with GH receptor peptides was performed, the peptide was added before the cell lysate). The Sepharose pellets were then washed five times with ice-cold lysis buffer and analyzed by Western blot analysis.

GST Fusion Proteins
The cDNA encoding the membrane-proximal region or the carboxy-terminal region of the rat GH receptor was amplified by PCR from the GH receptor plasmid pLM 108. PCR products were ligated into the GST fusion vector pGEX-5X-3 (Pharmacia Biotech, Piscataway, NJ). The resulting plasmids were sequenced to verify the fidelity of PCR and to confirm proper, in-frame cloning. Induction and affinity purification of GST proteins and GST-GH receptor fusion proteins were performed as recommended by the manufacturer (Pharmacia Biotech). In addition, GST proteins and tyrosine-phosphorylated GST-GH receptor fusion proteins were induced and purified from the Escherichia coli TKX1 strain that harbors a plasmid-encoded inducible tyrosine kinase gene as recommended by the manufacturer (Stratagene, La Jolla, CA).

Immunoprecipitation
Protein G-Sepharose (Pharmacia Biotech) and 1–10 µl of the respective antibody were added to the cell lysate (prepared in IP buffer: 50 mM Tris/HCl, pH 7.5, 0.1 M Triton X-100, 137 mM NaCl, 2 mM EGTA, 1 mM AEBSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM sodium orthovanadate), and samples were incubated 12–14 h at 4 C. Samples were washed three times with IP buffer, and the pellets were then washed five times with ice-cold lysis buffer and analyzed by SDS-PAGE and Western blot analysis.

Nuclear Extracts and Electrophoretic Mobility Shift Assay (EMSA)
Cells were cultured with or without GH (20 nM) for 5 min, washed twice with ice-cold PBS, and lysed in buffer A (20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM AEBSF, 1 mM sodium orthovanadate, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 20% glycerol) containing 0.5% Triton X-100. After 5 min of incubation on ice, the nuclei were collected by centrifugation at 2500 x g for 7 min at 4 C. The nuclei were resuspended in 5 volumes of a hypertonic buffer (buffer A containing 400 mM NaCl) and incubated on a rocking platform for 30 min at 4 C. The supernatant was collected after centrifugation at 20,000 x g for 30 min at 4 C. The double-stranded Spi 2.1 GLE1 oligonucleotide (5'-agctATGTTCTGAGAAAATC-3' and 5'-agctGATTTTCTCAGAACAT-3') was 32P-labeled in a fill-in reaction using [{alpha}-32P]dCTP and DNA polymerase (Klenow fragment). Approximately 20 fmol probe were used per reaction with 10 µg nuclear extract in EMSA buffer (100 mM HEPES, pH 7.9, 10 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 10% glycerol) containing 0.1 µg/µl double-stranded poly dI/dC (polydeoxyinosinic-deoxycytidylic acid). EMSA reactions were preincubated for 30 min at 30 C before separation on a 5% polyacrylamide gel containing 2% glycerol and 0.25% TBE (25 mM Tris/HCl, 25 mM boric acid, and 0.25 mM EDTA, pH 7.9). The gel was dried and exposed to x-ray film.

Construction of SOCS Mutants
SOCS chimera expression vectors were generated by introduction of ClaI sites (between the N-terminal domain and the SH2 domain) and NotI sites (between the SH2 domain and the SOCS-box) in the various SOCS-encoding plasmids using the Quickchange site-directed mutagenesis kit (Stratagene). The plasmids were digested with XbaI/ClaI, and cDNA fragments encoding the following amino acid sequences were purified: CIS (1–79 and 80–257); SOCS-1 (1–77 and 78–212); and SOCS-3 (1–43 and 44–225). The plasmids were also digested with XbaI/NotI, and cDNA fragments encoding the following amino acid sequences were purified: CIS (1–210 and 211–257); SOCS-1 (1–174 and 175–212); and SOCS-3 (1–184 and 185–225). The various SOCS domain swap mutants were generated by cross-ligation of appropriate fragments, e.g. (fragments 1, 3, and 3) encoding SOCS-1 (1–77) fused to SOCS-3 (44–225), etc. The different SOCS mutants encoding truncated forms of SOCS-1 and SOCS-3 were generated by introducing stop codons using the Quickchange site-directed mutagenesis kit (Stratagene).

Western Blot Analysis
Proteins were resolved by SDS-PAGE (4% stacking gel, 7.5%, 10%, or 12% running gel) and transferred by electroblotting to ECL nitrocellulose membranes (Amersham Pharmacia Biotech, Arlington Heights, IL). Membranes were blocked for 1 h in TBST buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk. Primary antibody diluted in TBST was added, and the blot was incubated for 1 h at room temperature. After three successive 20-min washes with TBST, the secondary antibody was added and membranes were incubated for 1 additional hour, and the proteins were visualized by the ECL detection system according to the manufacturer’s instructions (Amersham Pharmacia Biotech).


    ACKNOWLEDGMENTS
 
We thank Jannie Rosendahl Christensen for technical assistance and Dr. Erica Nishimura for critical review of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Nils Billestrup, Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark.

Johnny A. Hansen and Karen Lindberg are supported by the Danish Research Academy. Part of this work was supported by The National Health and Medical Research Council, Canberra, Australia, The NIH, Bethesda, Maryland (Grant CA-22556), and the Australian Federal Government’s Cooperative Research Centre Program.

Received for publication April 29, 1999. Revision received July 16, 1999. Accepted for publication July 20, 1999.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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