Mutation of the SHP-2 Binding Site in Growth Hormone (GH) Receptor Prolongs GH-Promoted Tyrosyl Phosphorylation of GH Receptor, JAK2, and STAT5B

Mary R. Stofega, James Herrington, Nils Billestrup and Christin Carter-Su

Department of Microbiology and Immunology (M.R.S.) Department of Physiology (J.H., C.C.-S.) University of Michigan Medical School Ann Arbor, Michigan 48109
Hagedorn Research Institute (N.B.) DK-2820 Gentofte, Denmark


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Binding of GH to GH receptor (GHR) rapidly and transiently activates multiple signal transduction pathways that contribute to the growth-promoting and metabolic effects of GH. While the events that initiate GH signal transduction, such as activation of the Janus tyrosine kinase JAK2, are beginning to be understood, the signaling events that terminate GH signaling, such as dephosphorylation of tyrosyl-phosphorylated signaling molecules, are poorly understood. In this report, we examine the role of the SH2 (Src homology-2) domain-containing protein tyrosine phosphatase SHP-2 in GH signaling. We demonstrate that the SH2 domains of SHP-2 bind directly to tyrosyl phosphorylated GHR from GH-treated cells. Tyrosine-to-phenylalanine mutation of tyrosine 595 of rat GHR greatly diminishes association of the SH2 domains of SHP-2 with GHR, and tyrosine-to-phenylalanine mutation of tyrosine 487 partially reduces association of the SH2 domains of SHP-2 with GHR. Mutation of tyrosine 595 dramatically prolongs the duration of tyrosyl phosphorylation of the signal transducer and activator of transcription STAT5B in response to GH, while mutation of tyrosine 487 moderately prolongs the duration of STAT5B tyrosyl phosphorylation. Consistent with the effects on STAT5B phosphorylation, tyrosine-to-phenylalanine mutation of tyrosine 595 prolongs the duration of tyrosyl phosphorylation of GHRand JAK2. These data suggest that tyrosine 595 is a major site of interaction of GHR with SHP-2,and that GHR-bound SHP-2 negatively regulates GHR/JAK2 and STAT5B signaling.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH has long been known to be an important regulator of body growth and metabolism, but the molecular mechanism by which GH exerts these physiological effects is just beginning to be understood. The cloning of rat GH receptor (GHR) (1), the subsequent classification of GHR as a member of the cytokine receptor superfamily (2), and the identification of JAK2 as the GHR-associated tyrosine kinase (3) have greatly facilitated our understanding of GH signaling. The critical GH signaling event after GH binding to GHR is the activation of JAK2, an event that requires autophosphorylation of the kinase domain of JAK2. Activated JAK2 then phosphorylates itself on additional tyrosines and tyrosines within the cytoplasmic domain of GHR (3). The phosphorylated tyrosines in JAK2 and GHR form binding sites for SH2 (Src homology-2) domain and other phosphotyrosine binding domain-containing signaling molecules (4). Multiple signaling molecules are rapidly and transiently tyrosyl phosphorylated and/or activated after GH stimulation, including GHR, JAK2, IRS-1/2, ERK 1/2, STATs 1, 3, 5A, and 5B (reviewed in Ref. 5), SH2-Bß (6), SHP-2 (7, 8), SIRP{alpha} (7), FAK, paxillin, tensin (9), p130cas, CrkII, c-Src, c-Fyn, c-Cbl, and Nck (10). Although JAK2 is required to tyrosyl phosphorylate and/or activate many of these signaling molecules, the factor(s) that dephosphorylate these signaling molecules and terminate GH signaling are poorly understood.

One possible mechanism for the termination of GH signaling is the activation and/or recruitment of a protein tyrosine phosphatase to GHR/JAK2 signaling complexes. This phosphatase would dephosphorylate GHR and/or JAK2 and result in down-regulation of GH signaling. Dephosphorylation of the critical activating tyrosine within the kinase domain of JAK2 would be expected to deactivate JAK2, whereas dephosphorylation of GHR and other tyrosines within JAK2 would be expected to remove binding sites for various signaling proteins. Two phosphatases that could potentially negatively regulate GH signaling are the SH2 domain-containing phosphatases SHP-1 and SHP-2 (11). These phosphatases are thought to be activated as a consequence of binding to phosphorylated tyrosines (12). SHP-1 has been demonstrated to regulate negatively JAK/STAT signaling in hematopoietic cells mediated by the receptors for erythropoietin (EPO), interleukin-4 (IL-4), interferon {alpha} (IFN{alpha}), or interleukin 3 (IL-3), members of the cytokine receptor superfamily that bind Janus tyrosine kinases (JAKs) (13, 14, 15, 16). Recently, it has been suggested that SHP-1 plays a role in the dephosphorylation of JAK2 in liver in response to GH (17). However, SHP-1 is not expressed in all GH-responsive tissues and cell lines (18), including the highly responsive 3T3-F442A cells (M. Stofega and C. Carter-Su, unpublished observation). Furthermore, a fusion protein encoding the N- and C-terminal SH2 domains of SHP-1 does not associate with GHR from GH-treated 3T3-F442A cells (8). These characteristics suggest that SHP-1 may not be a general negative regulator of GHR signaling in all GH-responsive tissues and cell lines.

We therefore investigated the role of the ubiquitously expressed protein tyrosine phosphatase SHP-2 (11, 19) in GH signaling. Both GHR and JAK2 have potential binding sites for the SH2 domains of SHP-2, and SHP-2 is a signaling molecule for GH, based on the tyrosyl phosphorylation of SHP-2 in response to GH (7, 8). Although SHP-2 has been generally regarded as a positive regulator of growth factor signaling and has been implicated as a positive regulator of signaling by the cytokine receptor ligands IFN{alpha} and PRL (14, 20), as well as for GH (8), recent studies indicate that SHP-2 is a negative regulator of signaling by leptin, ciliary neurotropic factor (CNTF), IFN{alpha}, IFN{gamma}, and insulin (21, 22, 23, 24, 25). Here, we demonstrate the direct association of the SH2 domains of SHP-2 with tyrosyl-phosphorylated GHR, but not JAK2, from GH-treated cells. Tyrosine 595 of rat GHR and to a lesser extent tyrosine 487, mediates the association of the SH2 domains of SHP-2 with GHR. To assess the effects of SHP-2 on GH signaling, stable cell lines were created that express tyrosine-to-phenylalanine mutations of tyrosine 595 or 487. Tyrosine-to-phenylalanine mutation of tyrosine 595, and to a lesser extent tyrosine 487, prolongs the tyrosyl phosphorylation of STAT5B in response to GH, consistent with the effects of these mutations on the level of SHP-2 binding to GHR. The increased duration of STAT5B tyrosyl phosphorylation in cells expressing Y595F GHR correlates with prolonged tyrosyl phosphorylation of GHR and JAK2. These results suggest that SHP-2 binding to specific tyrosines in GHR negatively regulates GH signaling.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH Induces the Association of GHR with the SH2 Domains of SHP-2
We and others have previously demonstrated that in GH-treated cells, SHP-2 associates with proteins with migration properties of GHR, JAK2, as well as SIRP{alpha} (7, 8). Using glutathione-S-transferase (GST) fusion proteins, we have also shown that SIRP{alpha} from GH-treated cells binds to the SH2 domains of SHP-2 (M. Stofega and C. Carter-Su, unpublished data). Kim et al. (8) have shown a similar GH-dependent association of GHR/JAK2 complexes with the SH2 domains of SHP-2. To confirm that the SH2 domains of SHP-2 mediate its association with GHR and that GH stimulates the association of the SH2 domains of SHP-2 with GHR, cell lysates from untreated or GH-treated 3T3-F442A cells were incubated with immobilized GST fusion protein fused to the N- and C-terminal SH2 domains of SHP-2. Bound proteins were eluted and immunoblotted with antibody to GHR. As shown in Fig. 1Go, lanes 3 and 4, GH stimulates the association of GHR with the SH2 domains of SHP-2. No GHR was detectable in cell lysates precipitated with GST alone (Fig. 1Go, lanes 5 and 6). Interestingly, the GHR coprecipitating with the SH2 domains of SHP-2 comigrates with the upper portion of the GHR coimmunoprecipitated by {alpha}GH (Fig. 1Go, lane 2), suggesting that the SH2 domains of SHP-2 associate with a slower migrating subset of GHR. This subset is presumably the tyrosyl-phosphorylated form of GHR, because phosphorylation of proteins often decreases the mobility of proteins in SDS-PAGE gels, and the SH2 domains of SHP-2 would be expected to bind only to the tyrosyl-phosphorylated form of GHR. The results of Fig. 1Go therefore demonstrate that SHP-2 associates with GH-activated GHR via the SH2 domains of SHP-2.



View larger version (35K):
[in this window]
[in a new window]
 
Figure 1. GH Induces the Association of the SH2 Domains of SHP-2 with GHR

3T3-F442A cells were untreated (-) (lanes 1, 3, and 5) or treated (+) (lanes 2, 4, and 6) with 500 ng/ml of hGH for 5 min. Solubilized proteins were either immunoprecipitated with {alpha}GH, precipitated with immobilized GST fused to the N- and C-terminal SH2 domains of SHP-2, or precipitated with immobilized GST alone. The blot was probed with {alpha}GHR. The migration of a mol wt standard (x10-3) is shown on the right and the position of GHR is shown on the left.

 
The SH2 Domains of SHP-2 Directly Associate with GHR
Because both GHR and JAK2 contain potential binding sites for the SH2 domains of SHP-2 (26), and GHR and JAK2 form a tight complex in response to GH (3), it was unclear in this or previous studies (8) whether the SH2 domains of SHP-2 were binding directly to GHR or indirectly via JAK2 that was associated with GHR. To determine whether the SH2 domains of SHP-2 associate directly with GHR or with JAK2, proteins in {alpha}GHR and {alpha}JAK2 immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose, and incubated first with GST fused to the N- and C-terminal SH2 domains of SHP-2 followed by {alpha}SHP-2 (far Western blot). The blot was stripped and reprobed with antiphosphotyrosine antibody ({alpha}PY) to show the migrations and relative amounts of tyrosyl-phosphorylated JAK2 and GHR in the {alpha}JAK2 and {alpha}GHR immunoprecipitates. In {alpha}GHR immunoprecipitates, GHR migrates as a broad tyrosyl-phosphorylated band, with a small amount of tyrosyl-phosphorylated JAK2 migrating slightly slower than GHR (Fig. 2Go, lane 2). In {alpha}JAK2 immunoprecipitates, the ratio between tyrosyl-phosphorylated GHR and JAK2 is reversed. There is a smaller amount of tyrosyl-phosphorylated GHR migrating slightly faster than a larger amount of tyrosyl-phosphorylated JAK2 (Fig. 2Go, lane 4). Far Western blotting with the SH2 domains of SHP-2 and {alpha}SHP-2 reveals that the SH2 domains of SHP-2 bind to proteins that comigrate with GHR and not JAK2 in both the {alpha}GHR and {alpha}JAK2 immunoprecipitates (Fig. 2Go, lanes 6 and 8). Further, the relative amounts of SH2 domain binding to proteins in the {alpha}JAK2 and {alpha}GHR immunoprecipitates correspond to the relative amounts of tyrosyl-phosphorylated GHR in those immunoprecipitates. These results therefore indicate that the SH2 domains of SHP-2 bind directly to tyrosyl-phosphorylated GHR in response to GH.



View larger version (27K):
[in this window]
[in a new window]
 
Figure 2. The SH2 Domains of SHP-2 Directly Associate with Tyrosyl-Phosphorylated GHR from GH-Treated Cells

3T3-F442A cells were untreated (-) (lanes 1, 3, 5, and 7) or treated (+) (lanes 2, 4, 6, and 8) with 500 ng/ml hGH for 5 min. Solubilized proteins were immunoprecipitated with {alpha}GHR or {alpha}JAK2 as indicated and incubated with GST fusion protein encoding the SH2 domains of SHP-2, followed by immunoblotting with {alpha}SHP-2 (lanes 5–8). The blot was stripped and reprobed with {alpha}PY (lanes 1–4).

 
Mutation of Tyrosine 487 or 595 of GHR Impairs Association of GHR with the SH2 Domains of SHP-2
Tyrosines 487 (YAQV) and 595 (YTTV) of GHR are within consensus binding sites (Y-hydrophobic to -X-hydrophobic) for the SH2 domains of SHP-2 (26). To determine whether tyrosines 487 or 595 mediate the association of GHR with the SH2 domains of SHP-2, COS cells were transfected with cDNAs encoding wild-type GHR, Y487F GHR, Y595F GHR, or Y627F GHR alone or cotransfected with JAK2 cDNA, to obtain JAK2-dependent tyrosyl phosphorylation of GHR. Overexpression of JAK2 in COS cells results in constitutive activation and tyrosyl phosphorylation of JAK2, presumably due to transphosphorylation of the overexpressed JAK2. Cell lysates were incubated with the N- and C-terminal SH2 domains of SHP-2 fused to GST, and bound proteins were eluted and immunoblotted with {alpha}PY. As expected, tyrosyl phosphorylated, wild-type GHR associates with the SH2 domains of SHP-2 (Fig. 3Go, lane 4). The association of Y487F GHR with the SH2 domains of SHP-2 is reduced compared with that of wild-type GHR, suggesting that Y487 may be a binding site for the SH2 domains of SHP-2. Notably, the association of the SH2 domains of SHP-2 with tyrosyl-phosphorylated Y595F GHR is undetectable (Fig. 3Go, compare lanes 4 and 8), suggesting that Y595 is a major site of interaction of GHR with the SH2 domains of SHP-2. To ensure that the reduced association of SHP-2 with Y487F or Y595F GHR was due to loss of a specific SHP-2 interaction site, and not simply due to tyrosine-to-phenylalanine mutation of GHR, the interaction of the SH2 domains of SHP-2 with Y627F GHR, which retains the SHP-2 interaction motifs, was examined. As shown in Fig. 3Go, lane 10, mutation of Y627 to phenylalanine does not impair association of SHP-2 with tyrosyl-phosphorylated GHR. The differing abilities of GHR to bind to the SH2 domains of SHP-2 were not due to different levels of GHR expression, since the levels of wild-type GHR, Y595F GHR, Y487F GHR, and Y627F GHR were similar, as judged by immunoprecipitation with {alpha}GHR and immunoblotting with {alpha}GHR (data not shown). The SH2 domains of SHP-2 do not associate with tyrosyl-phosphorylated JAK2 expressed in the absence of GHR (Fig. 3Go, lane 2), in agreement with previous results (27). The results of Fig. 3Go suggest that tyrosine 595 of GHR and, to a lesser extent, tyrosine 487 of GHR mediate the association of the SH2 domains of SHP-2 with tyrosyl-phosphorylated GHR.



View larger version (34K):
[in this window]
[in a new window]
 
Figure 3. The SH2 Domains of SHP-2 Bind to Y595 of GHR

COS-7 cells were transfected with the indicated cDNAs by calcium phosphate precipitation. Forty eight hours posttransfection, solubilized proteins were precipitated with immobilized GST fused to the N- and C-terminal SH2 domains of SHP-2 and immunoblotted with {alpha}PY. The position of GHR is shown on the left and a mol wt standard ( x10-3) on the right.

 
Mutation of Tyrosines 487 or 595 Prolongs Tyrosyl Phosphorylation of STAT5B in Response to GH
To examine the effects of SHP-2 on GH signaling, CHO cell lines stably expressing Y487F GHR or Y595F GHR were created. Expression of mutated GHR in the cell lines was monitored by 125I-labeled GH binding assays, and the correct size of the expressed GHR was verified by cross-linking of [125I]hGH to GHR as described previously (28). CHO cells generally expressed 2 and 3 times the amount of Y487F GHR and Y595F GHR, respectively, as compared with wild-type GHR. We verified by far Western blotting experiments, as described for Fig. 2Go, that the SH2 domains of SHP-2 directly associate with wild-type GHR from GH-treated CHO-GHR cells. In preliminary experiments, mutating tyrosine 487 of GHR and tyrosine 595 of GHR reduced the binding of the SH2 domains of SHP-2 to GHR to about 35% and 10%, respectively, of that of wild-type GHR (data not shown), consistent with the COS cell experiment (Fig. 3Go). We next examined the effect of disruption of SHP-2 binding to GHR on GH signaling. The time course of tyrosyl phosphorylation of STAT5B in response to GH in CHO cells expressing wild-type GHR, Y487F GHR, and Y595F GHR was investigated. Tyrosyl phosphorylation of STAT5B is required for it to bind DNA and transactivate target genes (29). CHO cells were stimulated with GH for the indicated times, and STAT5B was immunoprecipitated with {alpha}STAT5B, immunoblotted with {alpha}PY (Fig. 4AGo, upper panel) and reprobed with {alpha}STAT5B (Fig. 4AGo, lower panel). The amount of tyrosyl-phosphorylated STAT5B over time was quantified for multiple (10, 11, 12, 13) experiments and graphed as a percent of maximum tyrosyl phosphorylation vs. time of incubation with GH in Fig. 4BGo. Tyrosyl phosphorylation of STAT5B in wild-type GHR cells was reduced to 30% maximum after 1 h and 19% maximum after 4 h of GH treatment (Fig. 4BGo). In contrast, tyrosyl phosphorylation of STAT5B in Y487F GHR cells decreased to only 62% maximal after 1 h and 50% maximal after 4 h of GH treatment. In cells expressing Y595F GHR, the levels of tyrosyl phosphorylation of STAT5B only decreased to 95% of maximum after 1 h of GH treatment and were 64% of maximum after 4 h of GH treatment (Fig. 4BGo). Notably, basal levels of tyrosyl-phosphorylated STAT5B in Y595F GHR or Y487F GHR cells were not elevated, as compared with wild-type GHR cells nor did maximal levels of tyrosyl-phosphorylated STAT5B differ significantly between cell lines (Fig. 4AGo). Further, the experimental results in Fig. 4Go were not different in those instances in which the levels of 125I binding in CHO cells stably expressing wild-type GHR, Y487F GHR, or Y595F GHR were similar. These data indicate that mutation of Y487F or Y595F of GHR prolongs the duration of tyrosyl phosphorylation of STAT5B in response to GH, with Y595F GHR exhibiting the most dramatic prolongation.



View larger version (40K):
[in this window]
[in a new window]
 
Figure 4. Mutation to Phenylalanine of Tyrosine 595 or 487 of Rat GHR Prolongs GH-Promoted Tyrosyl Phosphorylation of STAT5B

A, CHO cells expressing wild-type GHR, Y487F GHR, or Y595F GHR were stimulated with 500 ng/ml hGH for the indicated times. Solubilized proteins were immunoprecipitated (IP) with {alpha}STAT5B, immunoblotted (IB) with {alpha}PY, and reprobed with {alpha}STAT5B. B, The amount of tyrosyl-phosphorylated STAT5B was quantified and expressed as % maximum tyrosyl phosphorylation vs. time of GH incubation. The data are expressed as mean ± SE for Y487F GHR (n = 10), wild-type GHR (n = 13), and Y595F GHR (n = 13). An asterisk denotes that the mean is statistically different from that of wild-type GHR at the 95% confidence level.

 
Mutation of Y595 of GHR to Phenylalanine Prolongs Tyrosyl Phosphorylation of JAK2
Because JAK2 is thought to tyrosyl phosphorylate STAT5B, we examined the possibility that the prolonged time course of tyrosyl phosphorylation of STAT5B in cells expressing mutant GHR might be a consequence of prolonged tyrosyl phosphorylation and thus activation, of JAK2. CHO cells expressing wild-type GHR, Y487F GHR, or Y595F GHR were stimulated for the indicated times with GH, and JAK2 was immunoprecipitated with {alpha}JAK2 and immunoblotted with {alpha}PY (Fig. 5AGo). The level of tyrosyl phosphorylation of JAK2 was quantified and was graphed as percent maximum vs. time of GH stimulation in Fig. 5BGo. Tyrosyl phosphorylation of JAK2 in wild-type GHR cells was maximal at 5 min and reduced to an average 19% maximum after 15 min, 24% after 1 h, and 11% after 4 h of GH treatment (Fig. 5BGo). The level of tyrosyl phosphorylation of JAK2 was significantly prolonged in cells expressing Y595F GHR, compared with cells expressing wild-type GHR. Phosphorylation was maximal at 5 min in three of four experiments and delayed until 15 min in the fourth experiment. It was reduced minimally to an average 89% maximal by 15 min GH treatment. After 1 h and 4 h GH treatment, it was reduced to 80% and 24%, respectively (Fig. 5BGo). Tyrosyl phosphorylation of JAK2 in Y487F GHR cells was not prolonged, although there was a trend toward higher tyrosyl phosphorylation of JAK2 at 15 min GH in Y487F GHR compared with wild-type GHR cells. No tyrosyl phosphorylation of JAK2 was observed in the absence of GH in any of the cell lines, and maximal levels of tyrosyl phosphorylation of JAK2 detected at either 5 or 15 min were similar in all cell lines (Fig. 5AGo). The latter suggests that mutating tyrosines 487 and 595 in GHR did not significantly alter the ability of GHR to bind and activate JAK2. Levels of JAK2 protein did not change with incubation time with GH nor did they differ reproducibly between cell lines (data not shown). These data indicate that mutation of Y595F of GHR prolongs the duration of tyrosyl phosphorylation of JAK2 in response to GH.



View larger version (28K):
[in this window]
[in a new window]
 
Figure 5. Mutation to Phenylalanine of Tyrosine 595 of Rat GHR Prolongs GH-Promoted Tyrosyl Phosphorylation of JAK2

A, CHO cells expressing wild-type GHR, Y487F GHR, or Y595F GHR were stimulated with 500 ng/ml hGH for the indicated times. Solubilized proteins were immunoprecipitated (IP) with {alpha}JAK2 and immunoblotted (IB) with {alpha}PY. B, The amount of tyrosyl-phosphorylated JAK2 was quantified and expressed as % maximum tyrosyl phosphorylation vs. time of GH incubation. The data are expressed as mean ± SE for n = 4. An asterisk denotes that the mean is statistically different from that of wild-type GHR at the 95% confidence level.

 
Mutation of Y595 to Phenylalanine Prolongs Tyrosyl Phosphorylation of GHR in Response to GH
We next examined the effects of mutation of tyrosine 487 or 595 on tyrosyl phosphorylation of GHR. Tyrosyl phosphorylation of GHR is critical for the association of GHR with STAT5B and subsequent tyrosyl phosphorylation of STAT5B (30, 31). Wild-type GHR cells, Y487F GHR cells, or Y595F GHR cells were stimulated with GH for the indicated times, and GHR was immunoprecipitated with {alpha}GHR and immunoblotted with {alpha}PY (Fig. 6AGo). The level of tyrosyl phosphorylation of GHR was quantified for several experiments and was graphed as percent maximum vs. time of GH stimulation in Fig. 6BGo. The time course of tyrosyl phosphorylation of GHR is prolonged in cells expressing Y595F GHR, but not in cells expressing Y487F GHR. After 15 min of GH treatment, the level of tyrosyl phosphorylation of GHR in cells expressing wild-type GHR or Y487F GHR was 47% and 41% maximal, respectively (Fig. 6BGo). By 1 h of GH treatment, the levels of tyrosyl-phosphorylated GHR diminished to less than 10% maximal in both cell lines. In contrast, in cells expressing Y595F GHR, the level of tyrosyl phosphorylation of GHR remains near 100% of the maximal level after 15 min and only diminishes to 60% of maximal after 1 h of GH stimulation. In all three cell lines, the level of tyrosyl-phosphorylated GHR is reduced to less than 10% maximal after 4 h of GH treatment, the longest time point examined. Reprobing these membranes with {alpha}GHR revealed that the levels of wild-type or mutant GHR protein progressively diminish with time of GH treatment (Fig. 6AGo, bottom panel), with the amount of receptor protein paralleling the amount of tyrosyl phosphorylation of GHR. These data therefore indicate that mutation of Y595 prolongs the time course of GHR tyrosyl phosphorylation and delays degradation of GHR in response to GH. This prolongation of both tyrosine phosphorylation of GHR and GHR number is consistent with the hypothesis of Gebert et al. (32) that dephosphorylation of GHR signals its internalization and degradation.



View larger version (37K):
[in this window]
[in a new window]
 
Figure 6. Mutation to Phenylalanine of Tyrosine 595 of Rat GHR Prolongs GH-Promoted Tyrosyl Phosphorylation of GHR

A, CHO cells expressing wild-type GHR, Y487F GHR, or Y595F GHR were stimulated with 500 ng/ml hGH for the indicated times. Solubilized proteins were immunoprecipitated (IP) with {alpha}GHR, immunoblotted (IB) with {alpha}PY (upper panel), and reprobed with {alpha}GHR (lower panel). The data for Y487F GHR is a slightly longer exposure than for wild-type or Y595F GHR, to allow better visualization of the decline in GHR amount. The lower level of GHR for Y487F GHR was not reproducible. B, The amount of tyrosyl-phosphorylated GHR was quantified and expressed as % maximum tyrosyl phosphorylation vs. time of GH incubation. The data are expressed as mean ± SE for n = 3. An asterisk denotes that the mean is statistically different from wild-type GHR at the 95% confidence level.

 
Cells Expressing Y595F GHR Have Enhanced GH-Dependent Transcription of a STAT5B-Responsive Promoter
To assess the functional importance of prolonged tyrosyl phosphorylation of STAT5B, we measured GH-dependent activity of a STAT5B-responsive promoter region of the spi 2.1 gene. CHO cells stably expressing wild-type or mutant GHR were transfected with Spi tkluc (33) and rous sarcoma virus (RSV)-ß-gal. Luciferase and ß-galactosidase activity were measured and analyzed as described in Materials and Methods. GH increased Spi tkluc activity 4.2 ± 1.0 times relative to control (n = 3) in cells expressing wild-type GHR. To assess whether GH-dependent activity in CHO cells expressing mutant GHRs was different than activity in cells with wild-type GHR, we normalized GH-stimulated luciferase activity to that seen with wild-type GHR expressing cells. GH-induced Spi tkluc activity in cells expressing Y595F GHR was 53 ± 7% (n = 3, P < 0.01) higher than that observed in CHO cells expressing wild-type GHR (Fig. 7Go). In CHO cells expressing Y487F GHR, GH induction of Spi tkluc activity was on the average greater than the GH-induced activity in cells with wild-type GHR (increase of 26 ± 32%, n = 3; P >= 0.1); however the increase did not achieve statistical significance. These results are consistent with prolonged tyrosyl phosphorylation of GHR, JAK2, and STAT5B in cells expressing Y595F GHR, resulting in enhanced STAT5B-mediated transcription.



View larger version (19K):
[in this window]
[in a new window]
 
Figure 7. Mutation to Phenylalanine of Tyrosine 595 of Rat GHR Enhances GH-Induced Transcription of a STAT5B-Responsive Promoter

CHO cells expressing wild-type GHR, Y487F GHR, or Y595F GHR were transiently transfected with Spi tkluc and RSV-ß-gal, serum deprived overnight, and treated with 500 ng/ml hGH for 24 h or left untreated. Luciferase and ß-galactosidase activity were measured and analyzed as described in Materials and Methods. In each experiment, the GH-induced luciferase activity in cells expressing Y487F GHR or Y595F GHR was normalized to activity in cells expressing wild-type GHR. The bars represent the mean ± SE relative luciferase activity (n = 3). The asterisk denotes that the mean is statistically different from wild-type GHR at the 95% confidence level.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SHP-2 Associates Directly with GHR in Response to GH
In this report we demonstrate that the N- and C-terminal SH2 domains of SHP-2 directly associate with tyrosyl-phosphorylated GHR from GH-activated cells. This conclusion is based upon both far Western blotting and binding of JAK2-phosphorylated GHR but not JAK2 alone to the SH2 domains of SHP-2 fused to GST. All species of GHR cloned to date contain two cytoplasmic tyrosines corresponding to amino acids 487 and 595 in rat GHR that lie within the binding motifs for the SH2 domains of SHP-2 (4, 26). This makes these tyrosines the most likely candidates for the SHP-2 binding sites. Tyrosine-to-phenylalanine mutation of Y595 greatly diminishes association of the N- and C-terminal SH2 domains of SHP-2 with GHR that is tyrosyl phosphorylated by JAK2 while tyrosine-to-phenylalanine mutation of tyrosine 487 impairs association of the SH2 domains of SHP-2 with tyrosyl-phosphorylated GHR. These results suggest that either the N- or C-terminal SH2 domain, or both SH2 domains of SHP-2, have a much higher affinity for tyrosine 595 of GHR as compared with tyrosine 487 of GHR. This is the first demonstration that specific tyrosines in GHR phosphorylated by JAK2 mediate the association of GHR with SHP-2. Consistent with tyrosine 595 mediating the association of SHP-2 with tyrosyl-phosphorylated GHR, SHP-2 binds GHR-GST fusion proteins encoding amino acids 485–620 of human GHR that are tyrosyl phosphorylated in Escherichia coli by Elk tyrosine kinase (8).

Mutation of Tyrosine 595 of GHR Prolongs GH-Promoted Tyrosyl Phosphorylation of GHR and JAK2
To gain insight into the role of SHP-2 in GH signaling, we examined the effect of mutating the SHP-2 binding sites in GHR on tyrosyl phosphorylation of GH signaling molecules. Blocking the binding of SHP-2 to GHR by a point mutation of GHR has the advantage over overexpressing phosphatase-inactive SHP-2 of allowing us to examine the consequences of removing only that SHP-2 that binds to GHR. The results are not complicated by the effects of removing any SHP-2 that binds to SIRP{alpha}, IRS-1/2, STAT5, or any other cellular protein in response to GH or other modulators of cell function. Our results indicate that tyrosine-to-phenylalanine mutation of tyrosine 595 of GHR dramatically prolongs tyrosyl phosphorylation of GHR in response to GH. Consistent with prolonged tyrosyl phosphorylation of GHR resulting from mutation of the SHP-2 binding site in GHR, mutation of the SHP-2 binding site in the gp130 subunit of the leukemia-inhibitory factor (LIF) receptor prolongs tyrosyl phosphorylation of gp130 in response to ligand (34). These results suggest a model in which GH binds to a dimer of GHR and activates JAK2. The activated JAK2 phosphorylates tyrosine 595 in GHR. The two phosphorylated tyrosines at 595 on the GHR monomers simultaneously bind to the N- and C-terminal SH2 domains of SHP-2 and activate the phosphatase activity of SHP-2 (12, 35, 36). The activated phosphatase then dephosphorylates the SHP-2-associated GHR. In support of this, tandem phosphopeptides corresponding to the binding site for the SH2 domain of SHP-2 in platelet-derived growth factor receptor potently stimulate SHP-2 phosphatase activity in vitro, and SHP-2 dephosphorylates platelet-derived growth factor receptor in vivo (12, 37).

Our results indicate that mutation of the SHP-2 binding site at Y595 of GHR also prolongs tyrosyl phosphorylation of JAK2. SHP-2 bound to Y595 could potentially dephosphorylate the GHR-associated JAK2, in addition to GHR. Depending upon the tyrosine being dephosphorylated, dephosphorylation of JAK2 could inactivate it or remove the binding site(s) for specific signaling molecules. Prolonged tyrosyl phosphorylation of JAK2 has also been reported in cells expressing mutated EPO receptor lacking the tyrosine that binds the related tyrosine phosphatase SHP-1 (13). Even if JAK2 is a substrate of the GHR-associated SHP-2, it seems unlikely that GHR-associated SHP-2 is the only determinant of the phosphorylation and activation state of JAK2 because the tyrosine phosphatase inhibitor orthovanadate prolongs JAK2 phosphorylation to a much greater extent (L. Argetsinger and C. Carter-Su, unpublished observation). This suggests that other phosphatases may contribute to the dephosphorylation of JAK2. One candidate for such a phosphatase is SHP-2 that binds directly to JAK2. Although SHP-2 has been shown to bind JAK2 (27), our data predict that it does not interact via its SH2 domains and therefore would not be activated. In support of this, coexpression of SHP-2 and JAK2 in COS or Sf9 cells does not result in dephosphorylation of JAK2 (13, 27). SHP-1, which is expressed in the CHO cell lines used in these experiments, is another candidate for a JAK2-binding phosphatase. SHP-1 has been demonstrated to dephosphorylate JAK2 (38) and to associate with JAK2 in response to GH (17). Consistent with SHP-1 dephosphorylating JAK2 in response to GH, the time course of JAK2 tyrosyl phosphorylation in mutant liver cells, which lack functional SHP-1, is prolonged (17). However, SHP-1 is reported to bind to and be modestly activated by JAK2 independent of the SH2 domains of SHP-1 (38).

Other factors, including other phosphatases or members of the SOCS/CIS family of cytokine-inducible proteins (39, 40, 41), may also be involved in terminating GH activation of JAK2. GH induces expression of several members of the SOCS family (42, 43), which inhibit GH signaling by inhibiting the kinase activity of JAK2 either directly (e.g. SOCS-1) or via binding to the tyrosine-phosphorylated GHR (e.g. SOCS-3 and CIS) (44, 45). The regions of GHR involved in binding SOCS-3 and CIS in response to GH are unclear. Using portions of GHR phosphorylated in bacteria by a tyrosine kinase other than JAK2, binding of SOCS-3 to membrane-proximal phosphotyrosine residues 333/338 (45) or to phosphotyrosines in the C terminus (amino acids 455–638) (44) of GHR has been reported. CIS is thought to bind to the C-terminal portion of GHR (45). Hence, it is possible that some of the prolongation in GH signaling we observe with Y487F GHR and Y595F GHR, especially at longer periods of GH stimulation, may result from an inability of SOCS proteins to bind to GHR. However, studies with phosphatase inhibitors have demonstrated a clear role for tyrosyl dephosphorylation in the decay of GH signaling (32, 47).

In the above model, mutating Y595 to phenylalanine is hypothesized to prevent binding of SHP-2, thereby prolonging tyrosyl phosphorylation of GHR. Because GHR number decreases at a rate similar to the decrease in tyrosyl phosphorylation of GHR, this model would predict that dephosphorylation of GHR precedes GHR degradation as hypothesized by Gebert et al. (32). While we think it less likely, we cannot rule out the alternative possibility that mutation of Y595 in GHR directly interferes with GH-induced internalization and/or degradation of GHR. Arguing against tyrosines 487 and 595 being required for normal rates of internalization is the finding that COS cells expressing truncated GHR that lack tyrosines 487 and 595 exhibit normal rates of internalization of 125I-hGH (46). Whether the Y595F mutation of GHR affects GH-induced degradation is not known.

Mutation of Tyrosine 595 Prolongs GH-Promoted Tyrosyl Phosphorylation of STAT5B
To gain additional insight into the role of SHP-2 in GH signaling, we examined the effects of mutation of the SHP-2 binding sites in GHR on tyrosyl phosphorylation of STAT5B. STAT5B is rapidly and transiently tyrosyl phosphorylated in a variety of cell lines and tissues in response to GH, but how STAT5B or any other member of the STAT family is dephosphorylated and inactivated in response to ligand is still unclear (4, 47). Our data indicate that mutation of Y595 of GHR substantially prolongs GH-promoted tyrosyl phosphorylation of STAT5B and enhances STAT5B-dependent activation of a spi 2.1 reporter gene. One model consistent with our data is the following. After GH treatment, tyrosyl-phosphorylated STAT5B translocates to the nucleus, binds DNA, transactivates genes, is dephosphorylated, and then returns to the cytosol. The dephosphorylated STAT5B is recruited again to tyrosyl-phosphorylated GHR and retyrosyl phosphorylated by JAK2. A second cycle of STAT5B tyrosyl phosphorylation and activation of target genes is thereby initiated. In this model, mutating Y595 to phenylalanine would prolong the tyrosyl phosphorylation and the half-life of GHR, and the phosphorylation and activation of JAK2, allowing for more cycles of activation of STAT5B. Consistent with JAK2 initiating multiple STAT5B activation cycles before deactivation of GHR/JAK2, the lifetime of the activated GHR/JAK2 complex in liver-derived cells is longer than that of activated STAT5B (32). Furthermore, addition of genistein, an inhibitor of JAK2, hastens the decline in tyrosyl phosphorylation of STAT5B in wild-type and Y595F GHR cells (data not shown), the predicted result if STAT5B is continuously being dephosphorylated, recycling back to GHR/JAK2 complexes, and being rephosphorylated by activated JAK2/GHR complexes. The tyrosine phosphatase that dephosphorylates cytokine-activated STAT5B has not been definitively identified although both SHP-1 and SHP-2 have been suggested (48, 49). It is conceivable that dephosphorylation of STAT5B involves in part a phosphatase whose association with or ability to dephosphorylate STAT5B is dependent upon Y595.

While the stimulatory effects of Y595F on the tyrosyl phosphorylation of GHR, JAK2, and STAT5B are pronounced and consistent with the inability of Y595F GHR to bind SHP-2, statistically significant effects of mutating Y487F GHR on GHR and JAK2 phosphorylation were not detected. It may be that only a small amount of binding of SHP-2 to Y487F GHR is sufficient to prolong the tyrosyl phosphorylation of STAT5B but is insufficient to detect substantial prolongation of the tyrosyl phosphorylation of GHR or JAK2. Alternatively, Y487F GHR may cause prolonged tyrosyl phosphorylation of STAT5B by a different mechanism than Y595F. For example, mutating Y595 to phenylalanine may prolong tyrosyl phosphorylation of STAT5B primarily by preventing the binding of SHP-2 to GHR. In contrast, mutating Y487 to phenylalanine may prolong tyrosyl phosphorylation of STAT5B primarily by preventing the binding of a phosphatase or a protein required for recruiting a STAT5B phosphatase.

Roles of SHP-2 in Signaling by GH and Other Cytokine Receptors
Our results suggest that SHP-2 negatively regulates GH signaling, because mutation of the SHP-2 binding site in GHR substantially prolongs tyrosyl phosphorylation of GHR, JAK2, and STAT5B in response to GH. These data are the first indications that a single tyrosine in GHR regulates the duration of tyrosyl phosphorylation of signaling molecules for GH. Our results are consistent with previous studies, which demonstrate that mutation of the SHP-2 binding site in the receptor for CNTF or leptin enhances STAT3 DNA binding or reporter gene activity in response to ligand (21, 22, 23, 34). Taken together, these results suggest that SHP-2 may negatively regulate STAT activation in response to ligands for multiple members of the cytokine receptor superfamily.

However, other studies have implicated SHP-2 as a positive regulator of GH, PRL, and IFN{alpha} signaling, based on the ability of a catalytically inactive SHP-2 mutant to diminish c-fos, ß-casein, or IFN-stimulated response element reporter gene activity in response to ligand (20, 23, 50). Classification of SHP-2 as a positive or negative regulator of signaling may oversimplify the role(s) of SHP-2 in signaling by GH, as well as in signaling by ligands for other members of the cytokine receptor superfamily. In fact, SHP-2 has been shown to have dual roles in CNTF signaling. Overexpression of SHP-2 diminishes DNA binding of STAT3 and STAT3-dependent gene expression in response to CNTF, while SHP-2 is required for full activation of c-fos in response to ligand (23). Thus, SHP-2 may have distinct roles for activation of mitogen-activated protein kinase (MAPK)-dependent (such as c-fos expression) vs. JAK/STAT-dependent signaling pathways for cytokine receptor superfamily members, including GHR. The ability of SHP-2 to bind and dephosphorylate multiple signaling molecules, such as cytokine receptors, SIRP{alpha}1, STATs, or JAKs may also provide the basis for the diverse effects of SHP-2 on signaling by multiple cytokine receptors that utilize JAK2.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Recombinant 22,000-Da human GH was a kind gift from Eli Lilly & Co. (Indianapolis, IN). The expression vector prk5 encoding murine JAK2 was kindly provided by J. Ihle (St. Jude Children’s Research Hospital, Memphis. TN). The expression vector pLM108 encoding wild-type rat GHR was a kind gift of G. Norstedt (Karolinska Institute, Huddinge, Sweden). Endoglycosidase F/N-Glycosidase F, Triton-X 100, leupeptin, and aprotinin were purchased from Roche Molecular Biochemicals (Indianapolis, IN). Recombinant protein A agarose was from Repligen (Cambridge, MA). The enhanced chemiluminescence (ECL) detection system, antimouse, antirabbit, and antiprotein A IgG conjugated to horseradish peroxidase were from Amersham Pharmacia Biotech (Arlington Heights, IL). Prestained mol wt standards were purchased from Life Technologies, Inc. (Gaithersburg, MD). Recombinant GST fusion protein encoding the two N-terminal SH2 domains of SHP-2 (amino acids 6–213) of human SHP-2 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Antisera
Antiphosphotyrosine antibody 4G10 ({alpha}PY) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY) and was used at 1:7500 for Western blotting. Antibody to GHR ({alpha}GHR), raised against recombinant rat GH-binding protein, was kindly provided by W. Baumbach (American Cyanamid, Princeton, NJ) and was used at dilutions of 1:500 and 1:7500 for immunoprecipitations and immunoblotting, respectively. Antibody to JAK2 ({alpha}JAK2), raised against a peptide corresponding to amino acids 758–776 of murine JAK2, was used at a dilution of 1:100 for immunoprecipitations and 1:15,000 for Western blotting. Antibody to STAT5B ({alpha}STAT5B), raised against amino acids 763–779 of human STAT5B, was purchased from Santa Cruz Biotechnology, Inc.

Transfection and Cell Culture
The stock of 3T3-F442A cells was kindly provided by H. Green (Harvard University, Boston, MA). Mouse 3T3-F442A cells were cultured in DMEM supplemented with 100 U of penicillin per ml, 100 µg of streptomycin per ml, 0.25 µg amphotericin (Life Technologies, Gaithersburg, MD) per ml and 10% calf serum as described previously (51). The stock of CHO cells stably expressing full-length rat GHR has been described previously (52). CHO cells were cultured in F-12 medium supplemented with 1 mM L-glutamine, 100 U of penicillin per ml, 100 µg of streptomycin per ml, 0.25 µg of amphotericin per ml, and 10% FBS. The expression vectors encoding rat GHR in which tyrosine 487, 595, or 627 is mutated to phenylalanine were generated by PCR as described previously (53). All mutations were confirmed by DNA sequencing. Stable CHO cell lines expressing GHR Y487F or GHR Y595F were created as described previously (54). CHO cells were transfected with the plasmid pRSV neo encoding resistance for G418 and pLM108 encoding either GHR Y487F or GHR Y595F at a 1:4 ratio using Lipofectin as described by the manufacturer. Transfected cells were subjected to selection in medium containing 1 mg/ml of G418, and individual clones were isolated by limiting dilution. Expression of mutated GHR in the clonal cell lines was monitored by 125I labeled-GH binding assays and the correct size verified by cross-linking of 125I hGH to GHR as described previously (28). COS cells were maintained in DMEM supplemented with antibiotics, 1 mM L-glutamine, and 10% FBS. COS cells were transfected with expression vectors encoding the indicated cDNAs by calcium phosphate precipitation. After 24 h, cells were washed twice with DMEM and incubated for an additional 24 h with DMEM containing 10% FBS, antibiotics, and L-glutamine.

Immunoprecipitation and Western Blotting
Confluent 3T3-F442A fibroblasts or CHO cells stably transfected with rat GHR were incubated in serum-free medium overnight as described previously (55). 3T3-F442A cells or CHO cells expressing GHR were incubated at 37 C with GH for the indicated times at the indicated concentrations. CHO cells, COS cells, or 3T3-F442A cells were washed twice with ice-cold PBSV (10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 1 mM Na3VO4) and solubilized in lysis buffer (50 mM Tris, pH 7.5, 0.1% Triton X-100, 150 mM NaCl, 2 mM EGTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Cell lysates were centrifuged at 12,000 x g for 10 min, and the supernatants were incubated on ice for 2 h with the indicated antibody. Immune complexes were collected with protein A agarose for 1 h at 8 C and were washed three times with 50 mM Tris, pH 7.5, 0.1% Triton X-100, 137 mM NaCl, 2 mM EGTA) and boiled for 5 min in a mixture of lysis buffer and 5 x SDS-PAGE sample buffer (250 mM Tris, pH 6.8, 10% SDS, 10% ß-mercaptoethanol, and 40% glycerol). Samples were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies using the ECL detection system (56). As indicated, blots were either directly reprobed with antibody or stripped in stripping buffer (100 mM ß-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl) at 55 C for 30 min and then reprobed with the indicated antibody. To quantify the levels of tyrosyl phosphorylation of proteins, autoradiographs were scanned using an Agfa scanner and Fotolook SA software and quantified using the Molecular Analyst image software from Bio-Rad Laboratories, Inc. (Richmond, CA). Means and SE values were calculated. The one-tailed paired Student’s t test was used to determine the statistical significance of differences in protein tyrosyl phosphorylation in multiple independent experiments.

In Vitro Interaction Assay with GST Fusion Proteins and Far Western Blotting.
For GST fusion protein interaction assays, immobilized fusion protein containing the SH2 domains of human SHP-2 was incubated with 3T3-F442A cell lysates or transfected COS cell lysates for 2 h at 4 C, washed with lysis buffer, and boiled for 5 min in 5x SDS-PAGE sample buffer. Samples were analyzed by Western blotting as described above. For far Western blotting experiments, 3T3-F442A cell lysates were immunoprecipitated with {alpha}GHR or {alpha}JAK2 and analyzed by SDS-PAGE as described above. The membrane was incubated with GST fusion protein encoding the two SH2 domains of SHP-2 (N + C SH2) at 1.5 µg/ml overnight at 4 C. After extensive washing, the membrane was immunoblotted with {alpha}SHP-2, stripped, and reprobed with {alpha}PY.

Luciferase Assay
CHO cells stably expressing wild-type or mutant GHR were transfected with a GH-responsive luciferase reporter (Spi tkluc, 0.5 µg) and 0.1 µg RSV-ß-gal using Fugene (Stratagene, La Jolla, CA) according to the protocol suggested by the manufacturer. Spi tkluc contains eight copies of the spi 2.1 promoter (-147/-102) upstream of a luciferase gene (33). Cells were serum-deprived overnight, treated with GH for 24 h, or left untreated, and cell lysates prepared in reporter lysis buffer (100 mM potassium phosphate, pH 7.8, 0.2% Triton X-100, 1 mM dithiothreitol). Luciferase and ß-galactosidase activity were measured in triplicate using a MGM Opticomp luminometer. In each sample, luciferase activity was normalized to ß-galactosidase activity and the mean fold change in luciferase activity in response to GH for each cell line was calculated. Data from three independent experiments expressed as the ratio of fold GH-dependent luciferase activity in cells expressing mutant GHR compared with activity in cells with wild-type receptor were analyzed by one-tailed paired Student’s t test. Differences were considered to be statistically significant at P < 0.05.


    ACKNOWLEDGMENTS
 
We thank Dr. L. Argetsinger for helpful suggestions. We thank X. Wang for technical assistance and B. Hawkins for help in preparation of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Christin Carter-Su, Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622.

This work was supported by NIH Grants DK-48283 and DK-34171 (to C.C.-S.).

Received for publication April 7, 1999. Revision received May 9, 2000. Accepted for publication May 15, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Henzel WJ, Barnard R, Waters MJ, Wood WI 1987 Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330:537–543[CrossRef][Medline]
  2. Bazan, JF 1989 A novel family of growth factor receptors: a common binding domain in the growth hormone, prolactin, erythropoietin and IL-6 receptors, and the p75 IL-2 receptor ß-chain. Biochem Biophys Res Commun 164:788–795[Medline]
  3. Argetsinger LS, Campbell GS, Yang X, Witthuhn BA, Silvennoinen O, Ihle JN, Carter-Su C 1993 Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase. Cell 74:237–244[Medline]
  4. Argetsinger LS, Carter-Su C 1996 Mechanism of signaling by growth hormone receptor. Physiol Rev 76:1089–1107[Abstract/Free Full Text]
  5. Smit LS, Meyer DJ, Argetsinger LS, Schwartz J, Carter-Su C 1999 In: Kostyo JL (ed) Handbook of Physiology. Oxford University Press, New York, vol 5:445–480
  6. Rui, L, Mathews, LS, Hotta, K, Gustafson, TA, Carter-Su, C 1997 Identification of SH2-Bß as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling. Mol Cell Biol 17:6633–6644[Abstract]
  7. Stofega MR, Wang H, Ullrich A, Carter-Su C 1998 Growth hormone regulation of SIRP and SHP-2 tyrosyl phosphorylation and association. J Biol Chem 273:7112–7117[Abstract/Free Full Text]
  8. Kim SO, Jiang J, Yi W, Feng GS, Frank SJ 1998 Involvement of the Src homology 2-containing tyrosine phosphatase SHP-2 in growth hormone signaling. J Biol Chem 273:2344–2354[Abstract/Free Full Text]
  9. Zhu T, Goh EL, Lobie PE 1998 Growth hormone stimulates the tyrosine phosphorylation and association of p125 focal adhesion kinase (FAK) with JAK2. Fak is not required for stat-mediated transcription. J Biol Chem 273:10682–10689[Abstract/Free Full Text]
  10. Zhu T, Goh ELK, LeRoith D, Lobie PE 1998 Growth hormone stimulates the formation of a multiprotein signaling complex involving p130Cas and CrkII. Resultant activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). J Biol Chem 273:33864–33875[Abstract/Free Full Text]
  11. Feng GS, Hui CC, Pawson T 1993 SH2-containing phosphotyrosine phosphatase as a target of protein-tyrosine kinases. Science 259:1607–1611[Medline]
  12. Eck, MJ, Pluskey, S, Trub, T, Harrison, SC, Shoelson, SE 1996 Spatial constraints on the recognition of phosphoproteins by the tandem SH2 domains of the phosphatase SH-PTP2. Nature 379:277–280[CrossRef][Medline]
  13. Klingmuller, U, Lorenz, U, Cantley, LC, Neel, BG, Lodish, HF 1995 Specific recruitment of SH-PTP1 to the erythropoietin receptor causes inactivation of JAK2 and termination of proliferative signals. Cell 80:729–738[Medline]
  14. David, M, Zhou, G, Pine, R, Dixon, JE, Larner, AC 1996 The SH2 domain-containing tyrosine phosphatase PTP1D is required for interferon {alpha}/ß-induced gene expression. J Biol Chem 271:15862–15865[Abstract/Free Full Text]
  15. Yi, T, Mui, AL-F, Krystal, G, Ihle, JN 1993 Hematopoietic cell phosphatase associates with the IL-3 receptor ß chain and down-regulates IL-3 induced tyrosine phosphorylation and mitogenesis. Mol Cell Biol 13:7577–7586[Abstract]
  16. Haque, SJ, Harbor, P, Tabrizi, M, Yi, T, Williams, BRG 1998 Protein-tyrosine phosphatase Shp-1 is a negative regulator of IL-4- and IL-13-dependent signal transduction. J Biol Chem 273:33893–33896[Abstract/Free Full Text]
  17. Hackett RH, Wang YD, Sweitzer S, Feldman G, Wood WI, Larner AC 1997 Mapping of a cytoplasmic domain of the human growth hormone receptor that regulates rates of inactivation of Jak2 and Stat proteins. J Biol Chem 272:11128–11132[Abstract/Free Full Text]
  18. Yi T, Cleveland JL, Ihle JN 1992 Protein tyrosine phosphatase containing SH2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12–p13. Mol Cell Biol 12:836–846[Abstract]
  19. Vogel W, Lammers R, Huang J, Ullrich A 1993 Activation of a phosphotyrosine phosphatase by tyrosine phosphorylation. Science 259:1611–1614[Medline]
  20. Ali S, Chen Z, Lebrun JJ, Vogel W, Kharitonenkov A, Kelly PA, Ullrich A 1996 PTP1D is a positive regulator of the prolactin signal leading to ß-casein promoter activation. EMBO J 15:135–142[Abstract]
  21. Symes A, Stahl N, Reeves SA, Farruggella T, Servidei T, Gearan T, Yancopoulos G, Fink JS 1997 The protein tyrosine phosphatase SHP-2 negatively regulates ciliary neurotrophic factor induction of gene expression. Curr Biol 7:697–700[Medline]
  22. Carpenter LR, Farruggella TJ, Symes A, Karow ML, Yancopoulos GD, Stahl N 1998 Enhancing leptin response by preventing SH2-containing phosphatase 2 interaction with Ob receptor. Proc Natl Acad Sci USA 95:6061–6066[Abstract/Free Full Text]
  23. Servidei T, Aoki Y, Lewis SE, Symes A, Fink JS, Reeves SA 1998 Coordinate regulation of STAT signaling and c-fos expression by the tyrosine phosphatase SHP-2. J Biol Chem 273:6233–6241[Abstract/Free Full Text]
  24. You M, Yu D, Feng G 1999 Shp-2 Tyrosine phosphatase functions as a negative regulator of the interferon-stimulated Jak/STAT pathway. Mol Cell Biol 19:2416–2424[Abstract/Free Full Text]
  25. Myers Jr MG, Mendez R, Shi P, Pierce JH, Rhoads R, White MF 1998 The COOH-terminal tyrosine phosphorylation sites on IRS-1 bind SHP-2 and negatively regulate insulin signaling. J Biol Chem 273:26908–26914[Abstract/Free Full Text]
  26. Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, Neel BG, Birge RB, Fajardo JE, Chou MM, Hanafusa H, Schaffhausen B, Cantley LC 1993 SH2 domains recognize specific phosphopeptide sequences. Cell 72:767–778[Medline]
  27. Yin T, Shen R, Feng GS, Yang YC 1997 Molecular characterization of specific interactions between SHP-2 phosphatase and JAK tyrosine kinases. J Biol Chem 272:1032–1037[Abstract/Free Full Text]
  28. VanderKuur JA, Wang X, Zhang L, Campbell GS, Allevato G, Billestrup N, Norstedt G, Carter-Su C 1994 Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase. J Biol Chem 269:21709–21717[Abstract/Free Full Text]
  29. Lin JX, Mietz J, Modi WS, John S, Leonard WJ 1996 Cloning of human Stat5B. Reconstitution of interleukin-2-induced Stat5A and Stat5B DNA binding activity in COS-7 cells. J Biol Chem 271:10738–10744[Abstract/Free Full Text]
  30. Wang X, Darus CJ, Xu BC, Kopchick JJ 1996 Identification of growth hormone receptor (GHR) tyrosine residues required for GHR phosphorylation and JAK2 and STAT5 activation. Mol Endocrinol 10:1249–1260[Abstract]
  31. Hansen LH, Wang X, Kopchick JJ, Bouchelouche P, Nielsen JH, Galsgaard ED, Billestrup N 1996 Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation. J Biol Chem 271:12669–12673[Abstract/Free Full Text]
  32. Gebert CA, Park S-H, Waxman DJ 1999 Termination of growth hormone pulse-induced STAT5b signaling. Mol Endocrinol 13:38–56[Abstract/Free Full Text]
  33. Hodge C, Liao J, Stofega M, Guan K, Carter-Su C, Schwartz J 1998 Growth hormone stimulates phosphorylation and activation of Elk-1 and expression of c-fos, egr-1 and junB through activation of extracellular signal-regulated kinases 1 and 2. J Biol Chem 273:31327–31336[Abstract/Free Full Text]
  34. Kim H, Hawley TS, Hawley RG, Baumann H 1998 Protein tyrosine phosphatase 2 (SHP-2) moderates signaling by gp130 but is not required for the induction of acute-phase plasma protein genes in hepatic cells. Mol Cell Biol 18:1525–1533[Abstract/Free Full Text]
  35. Cunningham BC, Ultsch M, deVos AM, Mulkerrin MG, Clauser KR, Wells JA 1991 Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. Science 254:821–825[Medline]
  36. deVos AM, Ultsch M, Kossiakoff AA 1992 Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255:306–312[Medline]
  37. Klinghoffer RA, Kazlauskas A 1995 Identification of a putative Syp substrate, the PDGF ß receptor. J Biol Chem 270:22208–22217[Abstract/Free Full Text]
  38. Jiao H, Berrada K, Yang W, Tabrizi M, Platanias LC, Yi T 1996 Direct association with and dephosphorylation of Jak2 kinase by the SH2-domain-containing protein tyrosine phosphatase SHP-1. Mol Cell Biol 16:6985–6992[Abstract]
  39. Starr R, Willson TA, Viney EM, Murray LJ, Rayner JR, Jenkins BJ, Gonda TJ, Alexander WS, Metcalf D, Nicola NA, Hilton DJ 1997 A family of cytokine-inducible inhibitors of signalling. Nature 387:917–921[CrossRef][Medline]
  40. Endo TA, Masuhara M, Yokouchi M, Suzuki R, Sakamoto H, Mitsui K, Matsumoto A, Tanimura S, Ohtsubo M, Misawa H, Miyazaki T, Leonor N, Taniguchi T, Fujita T, Kanakura Y, Komiya S, Yoshimura A 1997 A new protein containing an SH2 domain that inhibits JAK kinases. Nature 387:921–924[CrossRef][Medline]
  41. Naka T, Narazaki M, Hirata M, Matsumoto T, Minamoto S, Aono A, Nishimoto N, Kajita T, Taga T, Yoshizaki K, Akira S, Kishimoto T 1997 Structure and function of a new STAT-induced STAT inhibitor. Nature 387:924–929[CrossRef][Medline]
  42. Adams TE, Hansen JA, Starr R, Nicola NA, Hilton DJ, Billestrup N 1998 Growth hormone preferentially induces the rapid, transient expression of SOCS-3, a novel inhibitor of cytokine receptor signaling. J Biol Chem 273:1285–1287[Abstract/Free Full Text]
  43. Tollet-Egnell P, Flores-Morales A, Stavreus-Evers A, Sahlin L, Norstedt G 1999 Growth hormone regulation of SOCS-2, SOCS-3, and CIS messenger ribonucleic acid expression in the rat. Endocrinology 140:3693–3704[Abstract/Free Full Text]
  44. Hansen JA, Lindberg K, Hilton DJ, Nielsen JH, Billestrup N 1999 Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins. Mol Endocrinol 13:1832–1843[Abstract/Free Full Text]
  45. Ram PA, Waxman DJ 1999 SOCS/CIS protein inhibition of growth hormone-stimulated STAT5 signaling by multiple mechanisms. J Biol Chem 274:35553–35561[Abstract/Free Full Text]
  46. Allevato G, Billestrup N, Goujon L, Galsgaard ED, Norstedt G, Postel-Vinay M-C, Kelly PA, Nielsen JH 1995 Identification of phenylalanine 346 in the rat growth hormone receptor as being critical for ligand mediated internalization and down-regulation. J Biol Chem 270:17210–17214[Abstract/Free Full Text]
  47. Gebert CA, Park SH, Waxman DJ 1997 Regulation of signal transducer and activator of transcription (STAT) 5b activation by the temporal pattern of growth hormone stimulation. Mol Endocrinol 11:400–414[Abstract/Free Full Text]
  48. Ram, PA, Waxman, DJ 1997 Interaction of growth hormone-activated STATs with SH2-containing phosphotyrosine phosphatase SHP-1 and nuclear JAK2 tyrosine kinase. J Biol Chem 272:17694–17702[Abstract/Free Full Text]
  49. Yu C-L, Jin Y-J, Burakoff SJ 2000 Cytosolic tyrosine dephosphorylation of STAT5. J Biol Chem 275:599–604[Abstract/Free Full Text]
  50. David M, Chen HE, Goelz S, Larner AC, Neel BG 1995 Differential regulation of the alpha/beta interferon-stimulated Jak/Stat pathway by the SH2 domain-containing tyrosine phosphatase SHPTP1. Mol Cell Biol 15:7050–7058[Abstract]
  51. Foster CM, Shafer JA, Rozsa FW, Wang X, Lewis SD, Renken DA, Natale JE, Schwartz J, Carter-Su C 1988 Growth hormone promoted tyrosyl phosphorylation of growth hormone receptors in murine 3T3–F442A fibroblasts and adipocytes. Biochemistry 27:326–334[Medline]
  52. Wang, X, Uhler, MD, Billestrup, N, Norstedt, G, Talamantes, F, Nielsen, JH, Carter-Su, C 1992 Evidence for association of the cloned liver growth hormone receptor with a tyrosine kinase. J Biol Chem 267:17390–17396[Abstract/Free Full Text]
  53. Moldrup, A, Billestrup, N, Dryberg, T, Nielsen, JH 1991 Growth hormone action in rat insulinoma cells expressing truncated growth hormone receptors. J Biol Chem 266:17441–17445[Abstract/Free Full Text]
  54. Moller, C, Hansson, A, Enberg, B, Lobie, PE, Norstedt, G 1992 Growth hormone induction of tyrosine phosphorylation and activation of mitogen activated protein kinases in cells transfected with rat GH receptor cDNA. J Biol Chem 267:23403–23408[Abstract/Free Full Text]
  55. Campbell, GS, Christian, LJ, Carter-Su, C 1993 Evidence for involvement of the growth hormone receptor-associated tyrosine kinase in actions of growth hormone. J Biol Chem 268:7427–7434[Abstract/Free Full Text]