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Originally published In Press as doi:10.1074/jbc.M702838200 on August 28, 2007

J. Biol. Chem., Vol. 282, Issue 42, 31019-31027, October 19, 2007
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The Long Form of the Leptin Receptor Regulates STAT5 and Ribosomal Protein S6 via Alternate Mechanisms*

Yusong Gong{ddagger}1, Ryoko Ishida-Takahashi{ddagger}1, Eneida C. Villanueva{ddagger}§, Diane C. Fingar, Heike Münzberg{ddagger}, and Martin G. Myers, Jr.{ddagger}§2

From the {ddagger}Division of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, and the Departments of §Molecular and Integrative Physiology and Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109

Received for publication, April 3, 2007 , and in revised form, August 7, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The action of leptin via the long form of its receptor (LepRb) is central to the control of body energy homeostasis and neuroendocrine function, but the mechanisms by which LepRb regulates intracellular signaling have remained incompletely understood. Here we demonstrate that leptin stimulates the phosphorylation of STAT5 and ribosomal protein S6 in the hypothalamic arcuate nucleus in mice. In cultured cells, we investigate the mechanisms by which leptin regulates each of these pathways. Our analysis reveals a dominant role for LepRb Tyr1077 (which we demonstrate to be phosphorylated during receptor activation) and a secondary role for LepRb Tyr1138 in the acute phosphorylation of STAT5a and STAT5b. Tyr1138 and STAT3 attenuate STAT5-dependent transcription over the long-term, however. In contrast, Tyr985 (the LepRb phosphorylation site required for ERK activation) mediates the phosphorylation of the ribosomal S6 kinase (RSK) and S6, as well as cap-dependent translation. Thus, these data demonstrate the phosphorylation of Tyr1077 on LepRb during receptor activation, substantiate the hypothalamic regulation of STAT5 and S6 by leptin, and define the alternate LepRb signaling pathways that mediate each of these signals and their effects in cultured cells. Dissecting the contributions of these individual pathways to leptin action will be important for our ultimate understanding of the processes that regulate energy balance in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The prevalence of obesity continues to increase at alarming rates throughout the world, fostering the rise in obesity-related comorbidities, such as diabetes and cardiovascular disease (1-3). Whereas body energy homeostasis is tightly regulated, only recently have we begun to understand the physiologic mechanisms that regulate feeding and body weight to effect this balance (4, 5). One important mediator of body energy homeostasis is leptin, which is produced by adipocytes as a signal of the repletion of body energy (fat) stores (6, 7). Leptin acts in the central nervous system to promote satiety and enable neuroendocrine energy expenditure (8-14). The lack of leptin action due to mutations in leptin (e.g. ob/ob mice) or LepRb (e.g. db/db mice) or as a consequence of lowered fat stores results in increased appetite and an energy-sparing neuroendocrine starvation response that includes infertility and growth retardation (6, 10). In ob/ob and db/db animals, hyperphagia paired with decreased energy expenditure results in morbid obesity and a propensity to Type 2 diabetes (12). Conversely, in normal leptin-sensitive animals, high leptin levels tend to reduce appetite and permit neuroendocrine energy expenditure, and leptin administration decreases feeding and body weight while preserving metabolic energy utilization (10). The failure of elevated leptin levels to mediate weight loss in common forms of human obesity suggests the attenuation of leptin action ("leptin resistance") in obese states, as with diet-induced obesity in rodents (15-17). Potential mechanisms to explain this leptin resistance include alterations in leptin signaling, among others (18, 19).

Leptin binding activates the constitutively LepRb-associated Janus kinase (Jak)3 2 tyrosine kinase to mediate tyrosine phosphorylation-dependent leptin signaling via several pathways (8, 20-23). Phosphorylated LepRb Tyr1138 recruits the latent transcription factor, signal transducer and activator of transcription (STAT) 3 to mediate its tyrosine phosphorylation (20, 22, 23). The tyrosine phosphorylation of STAT proteins, including STAT3, promotes their nuclear translocation and ability to mediate transcriptional regulation (24, 25); hence, STAT3 recruitment by LepRb Tyr1138 mediates its activation. Phosphorylated Tyr985 of LepRb binds SH2-containing tyrosine phosphatase-2 (SHP2; aka PTPN11), which participates in extracellular signal-regulated kinase (ERK) activation during leptin signaling in cultured cells (22, 26). Tyr985 additionally binds the suppressor of cytokine signaling (SOCS) 3, and contributes to the attenuation of LepRb signaling (22, 26, 27). LepRb-associated Jak2 may also mediate signals independently of LepRb tyrosine phosphorylation sites (8, 22), in addition to providing a second, lower affinity binding site for SOCS3 (28, 29).

LepRb has also been implicated in the regulation of other signaling pathways, including the activation of additional STAT proteins, such as STAT5, in cultured cells (30, 31). LepRb also controls the activation of phosphatidylinositol (PI) 3'-kinase and pathways that regulate the phosphorylation of ribosomal protein S6 in the hypothalamus, and mediates the tissue-specific regulation of the AMP-dependent protein kinase (32-36).

Ribosomal protein S6 is an evolutionarily conserved ribosomal subunit implicated in the regulation of translational initiation and protein synthesis in response to extracellular stimuli (37). Two pathways converge on S6 to mediate its phosphorylation. First, the mTOR (TORC1)-dependent and rapamycin-sensitive S6 kinase pathway (38), and second, the ERK pathway-regulated ribosomal S6 kinase (RSK) pathway (39, 40). A variety of data from cultured cells and genetic mouse models suggests that the phosphorylation of S6 (S6(P)) contributes to increased ribosomal binding to the 7-methylguanosine cap and cap-dependent translational initiation and protein synthesis (37, 40-42).

Whereas data from cultured cells have suggested the ability of LepRb to phosphorylate and activate STAT5 (30, 31), potential interactions between STAT3 and STAT5 have not been explored, and the ability of leptin to activate STAT5 in vivo remains unclear (43). Additionally, whereas the regulation of S6(P) by leptin has been shown in vivo (34), the molecular mechanisms by which leptin mediates S6(P) and the cellular effects of this regulation are not known. Here we demonstrate the regulation of STAT5 by leptin in vivo and dissect the phosphorylation events by which leptin regulates STAT5 and ribosomal protein S6 phosphorylation and action.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies, Growth Factors, and Reagents—Rabbit {alpha}Jak2 and {alpha}LepRb have been described previously (22, 44). Antibodies specific for phosphorylated Tyr985, Tyr1077, and Tyr1138 of LepRb were raised in rabbits by injection of a keyhole limpet hemocyanin-coupled synthetic 11-amino acid phosphorylated peptide centered on the phosphorylated residue in question. All site- and phospho-specific anti-sera were affinity purified on the antigen peptide coupled to a mixture of Affi-Gel 10 and 15 (Bio-Rad), followed by passage over Affi-Gel coupled to irrelevant phosphopeptides and non-phosphorylated antigen peptide to remove antibodies directed against other sites of phosphorylation and to the non-phosphorylated form of the site. Monoclonal 4G10 was used for {alpha}PY immunoblotting (Chemicon). Antibodies directed against ERK(PT202/PY204), STAT5, STAT5(PY694), STAT3(PY705), S6(PS235/236), and S6(PS240/244), RSK, and RSK(PS380) were purchased from Cell Signaling Technology (Beverly, MA). Recombinant human erythropoeitin (Epo) was purchased from Amgen. Bovine serum albumin fraction V was purchased from Sigma. Protein A-Sepharose 6MB and horseradish peroxidase-protein A were from Amersham Biosciences and secondary antibodies for immunoblotting were from Santa Cruz Biotechnology (Santa Cruz, CA).

Generation of Mutant ELR cDNAs—pcDNA3ELR, pcDNA3ELRL985, pcDNA3ELRL985/S1138, pcDNA3ELRS1138, and pcDNA3ELRL985/F1077/S1138 (aka pcDNA3ELRTriple) have been described previously (22) and were used as templates for mutagenesis using the QuikChange kit (Stratagene) to generate pcDNA3ELRL985/F1077, pcDNA3ELRF1077/S1138, and pcDNA3ELRF1077. The presence of the desired mutations and the absence of adventitious mutations were confirmed by DNA sequencing.

Preparation of Cell Lysates for Immunoprecipitation—HEK293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and maintained in a humidified atmosphere containing 5% CO2 and 95% air at 37 °C. ELR constructs in pcDNA3 were transiently transfected into subconfluent HEK293 cells using Lipofectamine (Invitrogen) as described (44). Prior to each experiment, subconfluent cells were made quiescent by overnight incubation in Dulbecco's modified Eagle's medium or Dulbecco's modified Eagle's medium containing 0.5% bovine serum albumin before stimulation with Epo for the indicated time at 37 °C. Cells were lysed in 20 mM Tris, pH 7.4, containing 137 mM NaCl, 2 mM EDTA, 10% glycerol, 50 mM beta-glycerophosphate, 50 mM NaF, 1% Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride, and 2 mM sodium orthovanadate (lysis buffer). Insoluble material was removed by centrifugation at 16,000 x g at 4 °C for 20 min. Protein concentrations of the resulting lysates were determined using the BCA protein assay kit (Pierce) and bovine serum albumin standards, and equivalent amounts of protein were added to the appropriate antibodies for immunoprecipitation or denatured in Laemmli buffer for direct resolution by 10% SDS-PAGE. For immunoprecipitates, lysates were incubated with antibody at 4 °C overnight followed by incubation with protein A-Sepharose for 60 min. Immune complexes were collected by centrifugation and washed three times in lysis buffer before denaturation in Laemmli buffer and separation by 8% SDS-PAGE. Immunoblotting was performed as previously described (44). For stripping blots, the membrane was stripped with Reblot plus (Chemicon) according to the manufacturer's instructions. Stripped membranes were blocked overnight in blocking buffer and reprobed as described above.

Analysis of Reporter Activity—HEK293 cells were transfected in triplicate with the ELR constructs and/or the cDNAs for STAT3 (27), STAT5a, or STAT5b (the latter were the generous gift of Drs. Christin Carter-Su and Lawrence Argetsinger, University of Michigan) plus Spi2.1-Luc (the generous gift of Dr. S. A. Berry) (45) and control Renilla luciferase plasmids. Cells were made quiescent overnight before stimulation with vehicle or various concentrations of Epo for the indicated times.

HEK293 cells were transiently transfected with a bicistronic luciferase reporter plasmid, pRL-5'-IRES-FL (the generous gift of Dr. John Blenis), which directs cap-dependent translation of the Renilla luciferase (RL) gene and cap-independent HCV IRES-mediated translation of the firefly (FL) gene (40). Cells were lysed and assayed for Firefly and Renilla luciferase using the Dual Luciferase reporter assay system (Promega) on a Victor3 instrument (PerkinElmer Life Sciences).

Analysis of Hypothalamic Protein—Mice were C57Bl/6 animals from our in-house breeding program at the University of Michigan or purchased from Taconic Farms with intracerebroventricular cannulae in place. Mice had ad libitum access to food and water and all experimental procedures were approved by the University Committee on the Use and Care of Animals. Hypothalami were isolated between 10 and 12 a.m., arcuate nucleus was microdissected and snap frozen. Lysis, SDS-PAGE analysis, and immunoblotting procedures were as described (17), using the antibodies indicated above.


Figure 1
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  		FIGURE 1.
Phosphorylation of STAT5 and ribosomal protein S6 in cultured cells and in vivo during signaling by the intracellular domain of ELR. A, 293 cells were transiently transfected with ELR, made quiescent overnight, and stimulated with Epo (10 units/ml) for the indicated times before lysis. B, C57Bl/6 mice were injected with 1 µg of leptin or vehicle intracerebroventricular and sacrificed for the dissection of the hypothalamic arcuate nucleus 30 min later. ARC tissues were snap frozen and stored at -70 °C until lysis. A and B, equivalent amounts of protein were resolved by SDS-PAGE and processed for immunoblotting (IB) with the indicated antibodies. The migration of detected proteins is indicated to the right of each panel. C, immunofluorescent (IF) detection of S6(P) in wild-type mice treated with leptin for 60 min before perfusion and processing for the detection of S6(P). 3V, third cerebral ventricle. Results for each figure are representative of at least three independent experiments. PBS, phosphate-buffered saline.

 
Immunofluorescence—8-10-Week-old mice were treated with recombinant mouse leptin (5 µg/g) (National Hormone & Peptide Program (NHPP), Torrance, CA) or vehicle immunoprecipitation for 60 min before being deeply anesthetized (90 mg/kg sodium pentobarbital) and perfused transcardially with 4% paraformaldehyde. Removal of the brain, post-fixation, cryoprotection, sectioning, and immunohistochemistry were as described. In brief, free-floating tissue sections were blocked in donkey serum and then incubated with {alpha}S6(PS240/244) (Cell Signaling) or with rabbit {alpha}STAT5 (Cell Signaling) and chicken {alpha}GFP (Abcam), before further processing for immunofluorescent detection. Images were captured under fluorescence or confocal microscopy with a digital camera. All images for a single figure were from sections that were processed simultaneously and obtained under identical camera settings (17).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Phosphorylation of STAT5 and S6 during LepRb Signaling in Cultured Cells and in Vivo—We initially examined the phosphorylation of STAT5 and S6 (STAT5(P) and S6(P), respectively) in cultured 293 cells expressing a chimera of the erythropoeitin (Epo) receptor extracellular domain with the intracellular domain from the long form of the mouse leptin receptor (LepRb) (ELR chimera). In this ELR chimera, the activation of LepRb-dependent signals is under the control of Epo stimulation (22). We and others have utilized ELR extensively to examine signal transduction by the intracellular domain of LepRb, because ELR faithfully recapitulates the intracellular signaling program initiated by native LepRb (22, 26, 28, 46, 47). Additionally, the shorter Epo receptor extracellular domain mediates increased cell surface expression of ELR compared with native LepRb in cultured cells, facilitating the analysis of intracellular signaling systems.

We thus transfected ELR into 293 cells, rendered them quiescent, and examined the phosphorylation of downstream signaling proteins following various times of ligand stimulation (Fig. 1A). As expected, this analysis revealed the rapid phosphorylation of endogenous STAT3 protein (STAT3(P)) on the tyrosine responsible for transcriptional activation following ELR stimulation. Epo treatment of these cells also mediated STAT5(P) and S6(P), demonstrating the ability of the intracellular domain of LepRb to mediate the activation of these signaling pathways in cultured cells. The tyrosine phosphorylation of STAT proteins, such as the phosphorylation of STAT5 on Tyr694 (detected here), promotes their nuclear translocation and ability to mediate transcriptional regulation (24, 25). S6 kinase 1/2 and the ERK/RSK pathways mediate S6 phosphorylation on several sites, including Ser235/236 and Ser240/244 to modulate protein translation (40, 48).

Others have shown the LepRb-dependent induction of STAT5(P) in cultured cells (30, 31), but it has not been clear whether leptin is able to activate this signaling pathway in the hypothalamus of intact animals (43). We therefore examined the ability of leptin to regulate STAT5(P) and S6(P) in mice by immunoblotting lysates from the microdissected arcuate nucleus of mice following intracerebroventricular injection of leptin (Fig. 1B). Whereas anti-STAT5(P) antibodies have not proved sufficiently robust to enable the immunohistochemical detection of STAT5(P) in brain, leptin treatment of wild-type mice robustly increased the immunofluorescent detection of S6(P) in the arcuate nucleus of the hypothalamus (ARC) (Fig. 1C), as previously reported for rats (34). Thus, leptin promotes the phosphorylation of STAT5 and S6 in the ARC of mice.

Requirement for LepRb Tyr1077 for the Phosphorylation of STAT5 and Phosphorylation of LepRb Tyr1077 during Receptor Activation—To examine the mechanism by which LepRb regulates STAT5, we employed a panel of ELR mutants of various intracellular tyrosine residues to examine the role for potential events to mediate STAT5(P) (Fig. 2A). As previously demonstrated (20, 22, 23, 30), this analysis revealed that Tyr1138 is required and sufficient for complete STAT3(P). In contrast, loss of no single residue sufficed to completely abrogate the phosphorylation of endogenous STAT5, although mutation of Tyr1077 alone substantially reduced STAT5(P). Furthermore, whereas Tyr985 did not contribute to the ability of ELR to mediate STAT5(P), the combination of Tyr1077 and Tyr1138 accounted for the entirety of ELR-mediated endogenous STAT5(P).

To examine interacting roles for LepRb tyrosine phosphorylation and STAT proteins, we also examined the phosphorylation of overexpressed STAT proteins by ELR mutants (Fig. 2B). As previously shown, Tyr1138 mediates the phosphorylation of overexpressed, as well as endogenous STAT3 protein.


Figure 2
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  		FIGURE 2.
Phosphorylation of STAT5 and Tyr1077 of LepRb during signaling by mutant ELR isoforms. A and B, 293 cells were transiently transfected with cDNAs encoding the indicated ELR isoforms and/or STAT proteins, made quiescent overnight, and stimulated with Epo (10 units/ml) for 15 min before lysis. Equivalent amounts of protein were immunoprecipitated (IP) with {alpha}Jak2 or were denatured and directly resolved by SDS-PAGE and processed for immunoblotting (IB) with the indicated antibodies. C, 293 cells were transiently transfected with the indicated ELR isoforms, made quiescent overnight, and stimulated with Epo (10 units/ml) for 15 min before lysis. Equivalent amounts of protein were immunoprecipitated with the indicated antibodies, resolved by SDS-PAGE, and processed for immunoblotting with the indicated antibodies. The migration of detected proteins is shown to the right of each panel. Results are representative of multiple independent experiments. WT, wild type.

 
Because the two isoforms of STAT5 (STAT5a and STAT5b) are produced by separate genes and possess some differences (49), we examined the phosphorylation of STAT5a and STAT5b by ELR isoform mutants for Tyr1077 and/or Tyr1138 to determine the roles for these two residues in the regulation of STAT5 isoforms (Fig. 2B). Overexpressed STAT5a and STAT5b were both phosphorylated during ELR activation in 293 cells, and the phosphorylation of each of these isoforms required the integrity of Tyr1077 and Tyr1138. The Tyr1138-dependent phosphorylation of endogenous STAT3 was not appreciably altered by STAT5 overexpression.

The preceding analysis suggests that, in addition to Tyr985 and Tyr1138, which are known LepRb phosphorylation sites, Tyr1077 (whereas not previously demonstrated to be phosphorylated) also represents a likely site of phosphorylation, as STAT proteins are generally phosphorylated following SH2 domain-mediated recruitment to a cognate tyrosine phosphorylation site (50). Our previous analysis of phosphorylation utilizing ELRL985/S1138 demonstrated inconsequential residual reactivity with a general {alpha}PY antibody (22), suggesting either that Tyr1077 is not phosphorylated or (more likely given our present data) that the amino acid motif containing Tyr1077 is poorly reactive with standard {alpha}PY preparations. To examine the phosphorylation of Tyr985, Tyr1077, and Tyr1138 more specifically, we thus prepared a panel of antibodies reactive with the phosphorylated form of each of these sites using phosphopeptide antigens, affinity purifying the resulting sera on the antigen peptide, and then sub-tracting nonspecific {alpha}PY reactivity from each of these preparations on an affinity column containing irrelevant tyrosyl phosphopeptides (see "Experimental Procedures"). We transfected 293 cells with ELR isoform mutants for individual tyrosine residues, treated them in the absence or presence of Epo, and then analyzed the phosphorylation of Jak2 and each site on the receptor by immunoblotting with the resulting phosphospecific antibodies (Fig. 2C). This analysis demonstrated the similar tyrosine phosphorylation of Jak2 protein by each receptor, as expected. Similarly, immunoblotting with antibody that recognizes the intracellular domain of LepRb confirmed the similar expression of each receptor mutant. Also, each antibody preparation directed at the phosphorylated version of each tyrosine residue on the intracellular domain of LepRb reacted with wild-type ELR in a ligand-dependent manner, and failed to react only to receptor containing a mutation at its cognate phosphorylation site. These data suggest that each antibody reacts against the phosphorylated form of the appropriate LepRb tyrosine residue, and that each of the three intracellular tyrosine residues on LepRb (including Tyr1077) is phosphorylated during receptor activation. We have also consistently observed that mutation of Tyr1077 attenuates the phosphorylation of Tyr985; it is not clear whether this belies some ordering of LepRb phosphorylation events or rather results from a conformational change in ELRF1077 compared with ELR.


Figure 3
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  		FIGURE 3.
Contributions of Tyr1077 and Tyr1138 to STAT3- and STAT5-dependent transcriptional regulation. 293 cells were transiently transfected with the indicated ELR isoforms and (A) vector only (V), (B) STAT3 (S3), (C) STAT5a (S5a), or (D) STAT5b (S5b), plus pRL-TK and Spi2.1-Luc. Cells were made quiescent and stimulated with the indicated concentration of Epo for 12 h, lysed, and assayed for firefly and Renilla luciferase activities. The ratio of firefly to Renilla luciferase activity is plotted for each sample (mean ± S.E.). Results for each figure are representative of multiple independent experiments. A.U., absorbance units.

 
Roles for Tyr1077 and Tyr1138 in STAT5-dependent Transcriptional Regulation—To determine roles for Tyr1077 and Tyr1138 in STAT5-dependent transcriptional regulation, we examined the ability of various ELR tyrosine mutants in transcriptional assays employing the STAT5-responsive luciferase reporter plasmid, Spi2.1-Luc (45) (Fig. 3). As for other STAT-responsive reporter plasmids, this reporter is somewhat, but not absolutely, selective for individual STAT isoforms. Mutation of Tyr985 in the ELRL985 mediated increased Spi2.1-dependent luciferase activity (Fig. 3A), consistent with the inability of Tyr985 to mediate the recruitment of either STAT protein and also consistent with the ability of this phosphorylation site to recruit the LepRb signaling inhibitor, SOCS3, to mediate feedback inhibition of LepRb signaling (27). Mutation of Tyr1138 attenuated much of the ligand-stimulated activation of the reporter, and the mutation of Tyr1077 modestly decreased reporter activity, suggesting that whereas Tyr1077-dependent STAT5 signaling activates the Spi2.1 promoter, the Tyr1138-dependent activation of STAT3 dominates the transcriptional response of this promoter in 293 cells (which appear to express a large amount of STAT3 relative to STAT5).

To more closely examine the regulation of STAT3- and STAT5-dependent transcription by Tyr1077 and Tyr1138 during ELR signaling, we examined Spi2.1-mediated luciferase activity by ELR isoform mutants for these residues during overexpression of STAT3 (Fig. 3B), STAT5a (Fig. 3C), and STAT5b (Fig. 3D). Overexpression of any of these STAT isoforms increased ligand-stimulated reporter activity compared with that in the absence of overexpressed STAT, demonstrating that this overexpression revealed the transcriptional regulation by the overexpressed isoform (Fig. 3, B-D). Indeed, as expected, the vast majority of the increased luciferase activity mediated by overexpressed STAT3 required the integrity of Tyr1138 (Fig. 3B), although, as above for STAT3(P), overexpressing STAT3 drives a minor amount of Tyr1138-independent STAT3-dependent transcription, consistent with a detectable but presumably physiologically irrelevant artifact of high level STAT3 overexpression.


Figure 4
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  		FIGURE 4.
Relative amounts of STAT3 and STAT5 in the cell govern transcriptional effects. A, 293 cells were transiently transfected with the indicated ELR isoforms and vector only, STAT3, STAT5b, or STAT3 + STAT5b, plus pRL-TK and Spi2.1-Luc. Cells were made quiescent and stimulated with the indicated concentration of Epo for 12 h, lysed, and assayed for firefly and Renilla luciferase activities. The ratio of firefly to Renilla luciferase activity is plotted for each sample (mean ± S.E.). Results are representative of multiple independent experiments. B, immunofluorescent analysis of STAT5 protein content in ARC neurons of mice with GFP expression in LepRb-containing neurons. Shown are Z-stacked confocal images for GFP (green, left), STAT5 (red, middle), and merged (right). Arrows indicate examples of: 1) GFP/LepRb neuron with high STAT5 content; 2) GFP/LepRb neuron with moderate STAT5 content; 3) GFP/LepRb neurons with undetectable STAT5; 4) STAT5 expression in non-GFP/LepRb neuron.

 
The examination of Spi2.1 luciferase activity during STAT5 isoform overexpression revealed a crucial role for Tyr1077 in the regulation of STAT5a- and STAT5b-dependent transcription, although, as above, the high level overexpression of each STAT5 isoform mediated a small amount of Tyr1077/Tyr1138-independent STAT5 activation. This analysis also revealed an inhibitory role for Tyr1138 in STAT5-dependent translation, as ELRS1138 mediated increased transcription compared with ELR, and ELRPhe/Ser mediated increased transcription compared with ELRF1077. These results suggest that although Tyr1077 and Tyr1138 both contribute to the phosphorylation of STAT5 isoforms during acute stimulation, Tyr1077 dominates in the regulation of STAT5a- and STAT5b-dependent transcription during long-term stimulation, and that Tyr1138 and/or STAT3 may mediate inhibition of Tyr1077/STAT5-dependent transcription under these conditions.


Figure 5
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  		FIGURE 5.
Phosphorylation of ribosomal protein S6 during signaling by mutant ELR isoforms. 293 cells were transiently transfected with the indicated ELR isoforms, made quiescent overnight, and stimulated with Epo (10 units/ml) for the indicated time before lysis. Equivalent amounts of protein were denatured and directly resolved by SDS-PAGE and processed for immunoblotting (IB) with the indicated antibodies. The migration of detected proteins is shown to the right of each panel. Results are representative of multiple independent experiments. WT, wild type.

 
To examine a role for STAT3-mediated attenuation of STAT5-dependent transcription more closely, we assayed the effect of co-expressing STAT3 and STAT5b on the Spi2.1-dependent reporter activity by this same set of ELR mutants (Fig. 4A). In addition to confirming the results of Fig. 3 regarding the crucial roles for Tyr1138 in the STAT3-mediated transcription and for Tyr1077 in STAT5-mediated gene expression, this analysis revealed that STAT3 overexpression attenuated STAT5b-dependent transcriptional activation. Specifically, when STAT3 was overexpressed at high levels such that a modest amount of Tyr1138-independent STAT3 activation was promoted (see STAT3 only condition in this Fig. 4 and Fig. 2B), this overexpressed STAT3 significantly attenuated the STAT5b-dependent activation of Spi2.1 promoter activity in cells expressing ELRS1138 or ELRPhe/Ser. Also, the mechanism by which STAT3 inhibits STAT5-dependent transcription is independent from competition for Tyr1138, consistent with the notion that STAT3 mediates feedback inhibition to block the long-term transcriptional effects of STAT5. These findings are in agreement with our previous demonstration that Tyr1138 and STAT3 promote SOCS3 expression to mediate the feedback inhibition/attenuation of Jak2 phosphorylation during LepRb signaling over a similar 12-24-h time course (22, 28).

These data suggest that the contributions of Tyr1077/STAT5 and Tyr1138/STAT3 to transcriptional events in vivo depend not only upon the promoters available in each cell type, but also upon the amount of each STAT isoform in a particular neuron. We thus examined the potential variation in STAT5 expression in LepRb-expressing cells of the arcuate nucleus (ARC) by examining the colocalization of STAT5 immunoreactivity with GFP in LepRbGFP mice (which express GFP in LepRb-expressing neurons) (51) (Fig. 4B). This analysis revealed a great deal of variability in STAT5 expression in neurons of the arcuate nucleus, including in LepRb-expressing neurons.


Figure 6
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  		FIGURE 6.
Tyr985 mediates ERK and RSK pathway activation and modulates cap-dependent translation in 293 cells. A and B, 293 cells were transiently transfected with the indicated ELR isoforms and made quiescent overnight. Cells in A were pretreated for 30 min with 50 µM U0126, which inhibits the upstream activation of the ERK pathway. A and B, cells were stimulated with Epo (10 units/ml) for the indicated time before lysis. Equivalent amounts of protein were denatured and directly resolved by SDS-PAGE and processed for immunoblotting with the indicated antibodies. The migration of detected proteins is shown to the right of each panel. Results are representative of multiple independent experiments. C, 293 cells were transfected with the indicated ELR isoforms plus pRL-5'-IRES-FL. Cells were made quiescent and stimulated with the indicated concentration of Epo for 12 h before lysis for the determination of firefly and Renilla luciferase activities. The ratio of Renilla to firefly luciferase activity is plotted for each sample (mean ± S.E.) (n = 3). Results are representative of multiple independent experiments. *, p < 0.01 versus all other groups; all other comparisons not significantly different.

 
Requirement for Tyr985 and ERK Pathway Signaling in S6(P) during LepRb Activation—To decipher the mechanisms by which leptin regulates S6(P), we also examined S6(P) during activation of ELR and ELR isoform mutants for individual tyrosine phosphorylation sites or for all three sites (Fig. 5). As expected, ELRTriple failed to mediate the phosphorylation of STAT3, STAT5, or ERK. In addition, this ELR mutant failed to mediate S6(P) as revealed by immunoblotting with antibodies recognizing the phosphorylated form of two separate sets of sites on S6. Whereas ELRF1077 and ELRS1138 each mediated S6(P), ELRL985 failed to mediate S6(P), although it mediated STAT3(P) and STAT5(P) at least as well as wild-type ELR. Whereas the detection of S6(P) was modestly decreased in cells expressing ELRF1077 compared with ELR; this likely reflects a combination of slightly decreased expression of the ELRF1077 in this experiment (as revealed by decreased STAT3(P)) and may also reflect some decreased phosphorylation of Tyr985 on ELRF1077, as shown in Fig. 2C and consistent with slightly attenuated ERK activation by this mutant. Thus, Tyr985 is required for the stimulation of S6(P) by the intracellular domain of LepRb in 293 cells.

Because Tyr985 recruits SHP2 to activate the ERK cascade during ELR/LepRb signaling, we postulated that this pathway might be involved in the regulation of S6(P). We thus utilized U0126, an inhibitor of MEK1, the direct upstream activator of ERK, to probe the role of the MEK1 -> ERK pathway in the regulation of S6(P) during ELR signaling (Fig. 6A). As expected, U0126 did not alter ERK-independent tyrosine phosphorylation of STAT3, but blocked MEK-dependent activating phosphorylation of the ERK kinases, and attenuated the ability of ELR to increase the phosphorylation of S6 in response to ligand. These data suggest that the regulation of S6(P) by leptin in cultured cells is mediated via the ERK pathway.

ERK signaling promotes the activation of RSK, an upstream mediator of S6(P) (40). Thus, the findings that Tyr985 and ERK mediate S6(P) suggest a potential role for RSK in the regulation of S6(P) by ELR. Indeed, whereas we were unable to detect the phosphorylation of S6 kinase by ELR in 293 cells (data not shown), the intracellular domain of LepRb mediated the phosphorylation of RSK via Tyr985 (Fig. 6B). Furthermore, we utilized a translational reporter (in which cap-dependent translation controls Renilla luciferase and cap-independent translation mediates firefly luciferase production (40, 41)) to examine the regulation of cap-dependent translation by LepRb Tyr985. This analysis revealed that stimulation of the intracellular domain of LepRb in ELR increased cap-dependent translation and that this effect required Tyr985. Thus, these data suggest that the regulation of S6(P) and cap-dependent translation by LepRb proceeds via a Tyr985/ERK/RSK-dependent pathway in 293 cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In addition to confirming the phosphorylation of STAT5 and S6 by leptin action in vivo, our present results reveal the phosphorylation of LepRb on three intracellular tyrosine residues and demonstrate roles for distinct residues in the regulation of STAT5 and S6 phosphorylation by different LepRb phosphorylation sites in cultured cells. In addition, these data reveal roles for Tyr985 in the regulation of transcription by LepRb and for STAT3 in the attenuation of STAT5 action (Fig. 7).

Whereas the physiologic activation of STAT3 by leptin in the hypothalamus has been shown previously (52), and copious data suggest an important role for STAT3 in the regulation of body energy homeostasis (53), the physiologic role for STAT5 in leptin action has been less clear (43, 52). We now demonstrate the leptin-mediated tyrosine phosphorylation of STAT5 in the hypothalamus of rodents, suggesting the potential role for STAT5 in leptin action in vivo. Whereas whole body deletion of STAT5 isoforms produces a constellation of severe phenotypes due to the crucial role played by STAT5 in signaling by numerous growth factors and cytokines (54, 55), mice with central nervous system deletion of STAT5 appear relatively normal with the exception of moderate obesity in the face of elevated leptin levels.4 Thus, STAT5 may play an important role in the regulation of energy homeostasis by leptin, and the direct examination of a role for STAT5 in the regulation of physiology by leptin will be important.


Figure 7
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  		FIGURE 7.
Intracellular signaling pathways regulated by LepRb. Ligand binding to the extracellular domain of LepRb initiates the phosphorylation and activation of the constitutively associated Jak2 tyrosine kinase. The activated Jak2 then phosphorylates the intracellular domain of LepRb on Tyr985, Tyr1077, and Tyr1138. Tyr985 plays a complex role in LepRb signaling, as it not only serves as a docking site for the inhibitory SOCS3 protein that is induced by STAT3 in the cell, but also binds SHP-2, an upstream activator of the ERK cascade that participates in the regulation of RSK, S6(P), and translational control in 293 cells. Tyr1138, well known for its binding and activation of STAT3, contributes to the acute phosphorylation of STAT5, although Tyr1077 plays a dominant role in STAT5 transcriptional activation and Tyr1138/STAT3-mediated feedback inhibition attenuates STAT5-dependent transcription during chronic receptor activation.

 
We have demonstrated the phosphorylation of Tyr1077 on the intracellular domain of LepRb and have shown that this residue contributes to downstream signaling by mediating the phosphorylation and transcriptional activation of STAT5. Although never directly demonstrated, others have suggested that Tyr1077 might represent a phosphorylation site on LepRb, because synthetic phosphopeptides or reporter constructs based upon Tyr1077 have been reported to interact with SH2 domain-containing proteins, including SOCS3 and other members of the SOCS family, STATs, and other proteins, at least in vitro or in overexpression systems (30, 56-58). Our data suggest no role for Tyr1077 in the attenuation of LepRb signaling (as for Tyr985), however, because the mutation of Tyr1077 does not increase the amplitude of any LepRb signal that we have examined, but only attenuates STAT5(P) and STAT5-dependent transcriptional events. The finding of decreased Tyr985 phosphorylation by ELRF1077 also suggests the possibility that Tyr1077 may play a minor role in regulating the phosphorylation of Tyr985.

Whereas Tyr1077 and Tyr1138 of LepRb cooperate to stimulate acute STAT5(P), the role of Tyr1138/STAT3 in (presumably SOCS3-mediated) feedback inhibition results in a net inhibitory effect of Tyr1138 on STAT5-mediated gene expression and Tyr1077 mediates the most important net activation of STAT5. This Tyr1138/STAT3-dependent feedback inhibition of STAT5 is consistent with feedback inhibition on Jak2 phosphorylation during long-term receptor activation, and also with the finding that certain leptin actions (e.g. indicators of immune function and growth) are improved in mice devoid of LepRb -> STAT3 signaling (28, 53, 59). The strength of the leptin-mediated STAT5 signal likely varies among neural cell types, as the overall and relative levels of these proteins crucially affect gene transcription, and STAT5 levels vary widely within the ARC and ARC LepRb neurons.

We have also confirmed the leptin-stimulated phosphorylation of S6 in the ARC of rodents. Whereas Cota et al. (57) demonstrated the phosphorylation of mTOR and S6 kinase as well as S6 in response to leptin and nutritional cues in the ARC of rats, we were not able to detect these phosphorylation events in response to LepRb signaling (data not shown). We showed that the intracellular domain of LepRb stimulates S6(P) via Tyr985 and upstream regulators of the ERK pathway in cultured cells. Furthermore, Tyr985 modulates cap-dependent translation and the phosphorylation of RSK, the ERK-dependent mediator of S6(P) and cap-dependent translation. Thus, these data suggest that Tyr985 of LepRb acts via the ERK/RSK pathway to control S6(P) and cap-dependent translation in 293 cells.

Clearly, the interconnected circuitry of hypothalamic LepRb-expressing neurons raises the possibility that the regulation of these signals in vivo may be more complex than that observed in 293 cells. Indeed, the regulation of AMP-dependent protein kinase by leptin differs by region of the brain (32, 60), and distinct LepRb-expressing neural populations demonstrate alternate regulation of phosphatidylinositol 3-kinase by leptin (35). Thus, it will ultimately be important to confirm the mechanisms by which LepRb regulates these signals in intact animals.

Overall, our present results reveal the leptin-induced phosphorylation of STAT5 and S6 in the hypothalamus, demonstrate the phosphorylation of Tyr1077 on LepRb, and define crucial roles for Tyr1077 and Tyr985 in the regulation of STAT5 and RSK/S6(P)/cap-dependent translation, respectively, in cultured cells. Given the known role for Tyr985 in the feedback inhibition of LepRb (in addition to its role in ERK activation) and the importance of mTOR signaling for the anorectic actions of leptin, it will be crucial to understand the physiologic interplay among these signals, as well as defining the contribution of Tyr1077 and STAT5 signaling to overall leptin action.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01 DK56731 and R01 DK57768 (to M. G. M.) and R01 DK78135 (to D. C. F.) and grants from the American Diabetes Association (to M. G. M. and D. C. F.), the American Heart Association (to H. M.), and core facilities of the Michigan Diabetes Research and Training Center supported by National Institutes of Health Grant DK20572 and Michigan Comprehensive Cancer Center National Institutes of Health Grant P30CA046592. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally. Back

2 To whom correspondence should be addressed: 5560 MSRB II/0678, 1150 W. Medical Center Dr., Ann Arbor, MI 48109. Tel.: 734-647-9515; Fax: 734-936-6684; E-mail: mgmyers{at}umich.edu.

3 The abbreviations used are: Jak, Janus kinase; STAT, signal transducers and activators of transcription; SH2, Src homology domain 2; ERK, extracellular signal-regulated kinase; SOCS3, suppressor of cytokine signaling 3; RSK, ribosomal S6 kinase; S6(P), phosphorylation of S6; GFP, green fluorescent protein; Epo, erythropoeitin; {alpha}PY, anti-phosphotyrosine antibody; ARC, arcuate nucleus of the hypothalamus. Back

4 L. Hennighausen, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Lothar Hennighausen for sharing results prior to publication, Drs. Christin Carter-Su and Lawrence Argetsinger for STAT5 constructs, and Dr. S. A. Berry for Spi2.1-Luc.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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