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Molecular Endocrinology, doi:10.1210/me.2007-0111
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Molecular Endocrinology 21 (9): 2270-2281
Copyright © 2007 by The Endocrine Society

SH2B1 Enhances Leptin Signaling by Both Janus Kinase 2 Tyr813 Phosphorylation-Dependent and -Independent Mechanisms

Zhiqin Li, Yingjiang Zhou, Christin Carter-Su, Martin G. Myers, Jr. and Liangyou Rui

Department of Molecular and Integrative Physiology (Z.L., Y.Z., C.C.-S., L.R.), Division of Metabolism, Endocrinology and Diabetes, and Department of Internal Medicine (M.G.M.), University of Michigan Medical School, Ann Arbor, Michigan 48109-0622

Address all correspondence and requests for reprints to: Liangyou Rui, Ph.D., Department of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622. E-mail: ruily{at}umich.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Leptin controls body weight by activating its long form receptor (LEPRb). LEPRb binds to Janus kinase 2 (JAK2), a cytoplasmic tyrosine kinase that mediates leptin signaling. We previously reported that genetic deletion of SH2B1 (previously known as SH2-B), a JAK2-binding protein, results in severe leptin-resistant and obese phenotypes, indicating that SH2B1 is a key endogenous positive regulator of leptin sensitivity. Here we show that SH2B1 regulates leptin signaling by multiple mechanisms. In the absence of leptin, SH2B1 constitutively bound, via its non-SH2 domain region(s), to non-tyrosyl-phosphorylated JAK2, and inhibited JAK2. Leptin stimulated JAK2 phosphorylation on Tyr813, which subsequently bound to the SH2 domain of SH2B1. Binding of the SH2 domain of SH2B1 to phospho-Tyr813 in JAK2 enhanced leptin induction of JAK2 activity. JAK2 was required for leptin-stimulated phosphorylation of insulin receptor substrate 1 (IRS1), an upstream activator of the phosphatidylinositol 3-kinase pathway. Overexpression of SH2B1 enhanced both JAK2- and JAK2(Y813F)-mediated tyrosine phosphorylation of IRS1 in response to leptin, even though SH2B1 did not enhance JAK2(Y813F) activation. Leptin promoted the interaction of SH2B1 with IRS1. These data suggest that constitutive SH2B1-JAK2 interaction, mediated by the non-SH2 domain region(s) of SH2B1 and the non-Tyr813 region(s) in JAK2, increases the local concentration of SH2B1 close to JAK2 and inhibits JAK2 activity. Leptin-stimulated SH2B1-JAK2 interaction, mediated by the SH2 domain of SH2B1 and phospho-Tyr813 in JAK2, promotes JAK2 activation, thus globally enhancing leptin signaling. SH2B1-IRS1 interaction facilitates IRS1 phosphorylation by recruiting IRS1 to JAK2 and/or by protecting IRS1 from dephosphorylation, thus specifically enhancing leptin stimulation of the phosphatidylinositol 3-kinase pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE HYPOTHALAMUS CONTROLS energy balance and body weight by integrating multiple signals, including neuronal, hormonal, and nutritional signals. Leptin, which is produced and secreted by adipocytes proportionally to fat mass, is a key adiposity signal for the hypothalamus. Leptin binds to and activates its long form receptor (LEPRb) in hypothalamic neurons, resulting in weight loss by both inhibiting feeding and promoting energy expenditure. Consequently, genetic deficiency of either leptin or LEPRb results in voracious overfeeding and morbid obesity (1, 2, 3).

LEPRb is associated with Janus kinase 2 (JAK2), a cytoplasmic tyrosine kinase that binds to multiple members of the cytokine receptor family and mediates cell signaling. Leptin stimulates JAK2, which phosphorylates LEPRb on multiple tyrosines, including Tyr985 and Tyr1138 (4, 5, 6). Phosphorylated Tyr985 binds to SHP2 and suppressor of cytokine signaling 3 (4, 6). SHP2 promotes the activation of the ERK1/2 pathway, whereas suppressor of cytokine signaling 3 inhibits leptin signaling (4, 6, 7, 8). Phosphorylated Tyr1138 binds to the Src homology 2 (SH2) domain of signal transducer and activator of transcription (STAT)3, allowing JAK2 to phosphorylate and activate this transcription factor (6). Genetic disruption of the STAT3 pathway results in severe leptin resistance, hyperphagia, and morbid obesity in mice, indicating that this pathway is required for maintaining normal energy balance and body weight (9, 10, 11). Leptin also promotes tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) and IRS2, which activates the phosphatidylinositol (PI) 3-kinase pathway (12, 13, 14, 15). Pharmacological inhibition of hypothalamic phosphatidylinositol 3 (PI 3)-kinase abrogates the ability of leptin to inhibit feeding, and genetic deletion of the IRS2 gene in the brain results in leptin resistance, hyperphagia, and obesity in mice (16, 17, 18, 19). These observations indicate that the IRS proteins/PI 3-kinase pathway is also required for leptin regulation of appetite and body weight.

LEPRb promotes JAK2 autophosphorylation on multiple tyrosines, including Tyr221, Tyr570, and Tyr1007 (20, 21). Tyr570 phosphorylation appears to inhibit JAK2 activity, whereas the role of Tyr221 phosphorylation remains unclear (20, 22). Tyr1007 phosphorylation is required for cytokines to activate JAK2 (21). Interestingly, GH, the receptor of which also binds to JAK2, stimulates Tyr813 phosphorylation in JAK2 (23). Phosphorylated Tyr813 binds to the SH2 domain of SH2-B (23, 24, 25). Whether leptin similarly stimulates Tyr813 phosphorylation and/or Tyr813-mediated interaction of JAK2 with SH2-B is not known.

SH2-B is a member of the SH2B family (SH2-B, APS, and Lnk), which contains a conserved N-terminal dimerization (DD), central pleckstrin homology (PH), and C-terminal SH2 domain. SH2-B, APS, and Lnk were renamed recently by HUGO Gene Nomenclature Committee as SH2B1, SH2B2, and SH2B3, respectively. The SH2B1 (SH2-B) gene encodes four isoforms ({alpha}, ß, {gamma}, and {delta}) by alternative mRNA splicing; these four forms have identical DD, PH, and SH2 domains, but differ at their C termini (26, 27). SH2B1ß was originally identified as a JAK2-binding protein in a yeast two-hybrid screen (28). In cultured cells, SH2B1ß not only is tyrosyl phosphorylated by JAK2, but also potentiates JAK2 activation, in response to GH (25, 28, 29). SH2B2 (APS) appears to be involved in insulin signaling (30, 31, 32, 33, 34, 35). The SH2B2 gene encodes two isoforms ({alpha} and ß) via alternative mRNA splicing (36). SH2B2ß, which lacks SH2 domain, binds to both SH2B1ß and SH2B2{alpha}, thereby attenuating the ability of SH2B1ß or SH2B2{alpha} to enhance JAK2 activation and insulin signaling (36). These observations suggest that SH2B2ß functions as an endogenous inhibitor of SH2B1 and SH2B2{alpha}.

The SH2B1 gene has been disrupted independently by two groups. Yoshimura’s group (37) reported that deletion of the SH2B1 gene impairs reproduction in mice. We demonstrated that genetic deletion of the SH2B1 gene results in severe leptin resistance, hyperphagia, and morbid obesity (30, 38). SH2B1-deficient mice also develop hyperlipidemia, insulin resistance, and type 2 diabetes (38, 39). Impaired reproduction and type 2 diabetes are associated with leptin resistance and obesity. Neuron-specific restoration of SH2B1 in SH2B1-deficient mice rescues leptin-resistant and obese phenotypes (40). In addition, overexpression of SH2B1 in the brain protects against high-fat diet-induced leptin resistance and obesity in SH2B1 transgenic mice (40). Together, these observations suggest that SH2B1 is a key endogenous enhancer of leptin sensitivity in animals. However, the molecular mechanisms by which SH2B1 enhances leptin signaling remain largely unknown.

In this study, we have identified Tyr813 in JAK2 as a leptin-dependent site of JAK2 phosphorylation. Leptin promoted the binding of the SH2 domain of SH2B1 to phosphorylated Tyr813, thereby enhancing JAK2 activation. SH2B1 also bound to IRS1 and promoted leptin-stimulated, JAK2-mediated tyrosine phosphorylation of IRS1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Leptin Stimulates the Phosphorylation of Tyr813 in JAK2
To determine whether leptin stimulates JAK2 phosphorylation on Tyr813, LEPRb, JAK2, and JAK2(Y813F) were stably introduced into {gamma}2A cells. {gamma}2A cells are human fibroblasts genetically deficient of JAK2 (41, 42). JAK2(Y813F) was generated by replacing Tyr813 in JAK2 with Phe. {gamma}2A cells were infected with recombinant retroviruses expressing mouse LEPRb to generate {gamma}2ALEPRb cells that stably express LEPRb. LEPRb expression was confirmed by immunoblotting with anti-LEPRb antibodies (data not shown). Similarly, {gamma}2ALEPRb cells were infected with recombinant retroviruses expressing JAK2 or JAK2(Y813F) to generate {gamma}2ALEPRb/JAK2 or {gamma}2ALEPRb/Y813F cells, respectively.

To estimate the levels of recombinant JAK2 and JAK2(Y813F), cell extract was immunoblotted with anti-JAK2 antibody ({alpha}JAK2) and anti-ß-actin ({alpha}-Actin). Parental {gamma}2A cells did not express endogenous JAK2 as expected, whereas JAK2 in {gamma}2ALEPRb/JAK2 cells and JAK2(Y813F) in {gamma}2ALEPRb/Y813F cells were expressed at a similar level (Fig. 1AGo).


Figure 1
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  		Fig. 1. Leptin Stimulates Phosphorylation of JAK2 on Tyr813

A, Cell extract was prepared from {gamma}2A, {gamma}2ALEPRb/JAK2, and {gamma}2ALEPRb/Y813F cells and immunoblotted with {alpha}JAK2 or {alpha}-actin. B and C, {gamma}2ALEPRb/JAK2 and {gamma}2ALEPRb/Y813F cells were treated with 100 ng/ml leptin or vehicle for 10 min. JAK2 in cell extract was immunoprecipitated with {alpha}JAK2 and immunoblotted with {alpha}PY (B) or {alpha}pY813 (C). The same blots were reprobed with {alpha}JAK2. D, {gamma}2ALEPRb/JAK2/SH2B1 cells were treated with 50 ng/ml leptin for 0, 5, 10, 30, or 60 min. JAK2 in cell extract was immunoprecipitated with {alpha}JAK2 and immunoblotted with {alpha}pY813. The same blot was reprobed with {alpha}JAK2. E, {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells were treated with 100 ng/ml leptin for 0, 5, 10, 30, or 60 min. JAK2 in cell extract was immunoprecipitated with {alpha}JAK2 and immunoblotted with {alpha}PY. The same blot was reprobed with {alpha}JAK2. IB, Immunoblotting; IP, immunoprecipitation.

 
To determine whether {gamma}2ALEPRb/JAK2 and {gamma}2ALEPRb/Y813F cells respond to leptin, cells were deprived of serum overnight and treated with 100 ng/ml leptin for 10 min. JAK2 in cell extract was immunoprecipitated with {alpha}JAK2 and immunoblotted with antiphosphotyrosine antibodies ({alpha}PYs). Basal tyrosine phosphorylation of JAK2 and JAK2(Y813F) was undetectable, whereas leptin robustly stimulated tyrosine phosphorylation of JAK2 in {gamma}2ALEPRb/JAK2 cells (Fig. 1BGo). Leptin also stimulated tyrosine phosphorylation of JAK2(Y813F) in {gamma}2ALEPRb/Y813F cells, but to a lesser extent (Fig. 1BGo).

To provide direct evidence that leptin stimulates Tyr813 phosphorylation, {gamma}2ALEPRb/JAK2 cells were treated with 100 ng/ml leptin for 10 min, and JAK2 was immunoprecipitated with polyclonal {alpha}JAK2 and immunoblotted with antiphospho-JAK2 [pTyr813 antibodies ({alpha}pY813)]. {alpha}pY813 has been previously characterized to recognize specifically phosphorylated Tyr813 in JAK2 (23). Leptin stimulated Tyr813 phosphorylation in {gamma}2ALEPRb/JAK2 cells (Fig. 1CGo). In contrast, {alpha}pY813 was unable to detect JAK2(Y813F) in {gamma}2ALEPRb/Y813F cells treated with or without leptin (Fig. 1CGo), even though JAK2(Y813F) was phosphorylated on other tyrosines (Fig. 1BGo). Phosphorylation of Tyr813 was detectable by 5 min and sustained for at least 60 min after leptin stimulation (Fig. 1DGo). Leptin-stimulated overall tyrosine phosphorylation of both JAK2 and JAK2(Y813F) was prolonged to a similar degree (Fig. 1EGo), suggesting that the binding of SH2B1 to phosphorylated Tyr813 does not protect JAK2 from dephosphorylation.

Phosphorylated Tyr813 in JAK2 Mediates Leptin-Stimulated Association of JAK2 with SH2B1
To determine whether phosphorylated Tyr813 mediates the interaction of JAK2 with SH2B1, {gamma}2ALEPRb/JAK2 and {gamma}2ALEPRb/Y813F cells were infected with recombinant retroviruses expressing rat SH2B1ß to generate {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells, respectively. To compare SH2B1 levels in the different cell lines, cell extract was immunoblotted with {alpha}SH2B1. Recombinant SH2B1 protein levels were similar between {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells, but much higher than the levels of endogenous SH2B1 in parental {gamma}2ALEPRb/JAK2 and {gamma}2ALEPRb/Y813F cells (Fig. 2AGo).


Figure 2
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  		Fig. 2. Phosphorylated Tyr813 in JAK2 Mediates Leptin-Stimulated Interaction of SH2B1 with JAK2

A, Cell extract was prepared from {gamma}2ALEPRb/JAK2, {gamma}2ALEPRb/Y813F, {gamma}2ALEPRb/JAK2/SH2B1, and {gamma}2ALEPRb/Y813F/SH2B1 cells and subjected to immunoblotting with {alpha}SH2B1 or {alpha}-actin. B, {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells were treated with 100 ng/ml leptin or vehicle for 10 min. SH2B1 in cell extract was immunoprecipitated with {alpha}SH2B1 and immunoblotted with {alpha}JAK2. The same blots were reprobed with {alpha}PY or {alpha}SH2B1. C, {gamma}2ALEPRb/JAK2/SH2B1 cells were treated with 100 ng/ml leptin for 10 min. JAK2 was immunoprecipitated with the monoclonal {alpha}JAK2 and immunoblotted with {alpha}SH2B1 or {alpha}JAK2. D, {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/JAK2/R555E cells were treated with 100 ng/ml leptin for 0, 10, or 60 min. SH2B1 was immunoprecipitated with {alpha}SH2B1 and immuoblotted with monoclonal {alpha}JAK2, {alpha}PY, or {alpha}SH2B1 as indicated. These experiments were performed three times with similar results. E, {gamma}2ALEPRb/K882E/R555E and {gamma}2ALEPRb/K882E cell extracts were immunoprecipitated with {alpha}SH2B1 and immuoblotted with monoclonal {alpha}JAK2 or {alpha}SH2B1. Cell extracts were also immunoblotted with {alpha}JAK2 or {alpha}SH2B1. IB, Immunoblotting; IP, immunoprecipitation.

 
To examine the interaction of SH2B1 with JAK2 or JAK2(Y813F), {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells were stimulated with 100 ng/ml leptin for 10 min, and SH2B1 in cell extract was immunoprecipitated with {alpha}SH2B1 and immunoblotted with polyclonal {alpha}JAK2. Leptin promoted coimmunoprecipitation of SH2B1 with JAK2 but not JAK2(Y813F) (Fig. 2BGo). Moreover, tyrosyl-phosphorylated JAK2, but not tyrosyl-phosphorylated JAK2(Y813F), was easily detected in {alpha}SH2B1 immunoprecipitates from leptin-treated cells (Fig. 2BGo). Interestingly, SH2B1 was undetectable in polyclonal {alpha}JAK2 immunoprecipitates (data not shown), suggesting that this polyclonal {alpha}JAK2 disrupts the interaction of JAK2 with SH2B1, and/or binds to a site in JAK2 that overlaps with SH2B1-binding site(s). To confirm that JAK2 coimmunoprecipitates with SH2B1, {gamma}2ALEPRb/JAK2/SH2B1 cells were treated with or without leptin, and JAK2 was immunoprecipitated with monoclonal {alpha}JAK2. Leptin stimulated the coimmunoprecipitation of JAK2 with SH2B1 (Fig. 2CGo).

To determine whether the SH2 domain of SH2B1 mediates leptin-induced interaction of SH2B1 with JAK2, SH2 domain-defective SH2B1(R555E), which has a replacement of the conserved Arg555 within its SH2 domain with Glu, was stably introduced into {gamma}2ALEPRb/JAK2 cells via retroviral-mediated gene transfer to generate {gamma}2ALEPRb/JAK2/R555E cells. {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/JAK2/R555E cells were treated with or without leptin, and SH2B1 and SH2B1(R555E) were immunoprecipitated with {alpha}SH2B1 and immunoblotted with monoclonal {alpha}JAK2. In the absence of leptin stimulation, SH2B1 was constitutively bound to a small amount of JAK2 that was not tyrosyl phosphorylated in {gamma}2ALEPRb/JAK2/SH2B1cells (Fig. 2DGo). Leptin markedly increased the binding of SH2B1 to JAK2 that was tyrosyl phosphorylated as expected; leptin-stimulated association of SH2B1 with JAK2 was still detectable after 60 min stimulation (Fig. 2DGo). SH2B1ß(R555E) was also constitutively bound to JAK2 in {gamma}2ALEPRb/JAK2/R555E cells; however, leptin was unable to further increase the binding of SH2B1ß(R555E) to JAK2 (Fig. 2DGo). To exclude the possibility that {alpha}SH2B1 binds to JAK2 nonspecifically, {gamma}2ALEPRb/K882E and {gamma}2ALEPRb/K882E/R555E cell lines were established via retroviral-mediated gene transfer to stably express kinase-inactive JAK2(K882E) (lacking the ATP-binding site) in {gamma}2ALEPRb/K882E cells and both JAK2(K882E) and SH2B1ß(R555E) in {gamma}2ALEPRb/K882E/R555E cells. SH2B1(R555E) was detected in {gamma}2ALEPRb/K882E/R555E but not in {gamma}2ALEPRb/K882E cells as expected; in contrast, JAK2(K882E) was expressed at a similar level between {gamma}2ALEPRb/K882E/R555E and {gamma}2ALEPRb/K882E cells (Fig. 2EGo, bottom two panels). Cell extracts were immunoprecipitated with {alpha}SH2B1 and immunoblotted with {alpha}JAK2. JAK2(K882E) was detected in {alpha}SH2B1 precipitates from {gamma}2ALEPRb/K882E/R555E but not {gamma}2ALEPRb/K882E cells (Fig. 2EGo, top two panels), indicating that {alpha}SH2B1 did not bind to JAK2 nonspecifically, and that SH2 domain-defective SH2B1(R555E) constitutively bound to kinase-inactive JAK2(K882E). Endogenous SH2B1-bound JAK2(K882E) was undetectable in {gamma}2ALEPRb/K882E cells, presumably due to very low expression of endogenous SH2B1 (Fig. 2EGo). Together, these results suggest that before leptin stimulation, SH2B1 constitutively binds to JAK2 independent of the SH2 domain of SH2B1 and phosphorylated-Tyr813 in JAK2, thereby increasing the local concentration of SH2B1 close to JAK2. Upon leptin stimulation, Tyr813 is phosphorylated and rapidly binds to the SH2 domain of SH2B1 that is preassociated with JAK2.

Interestingly, leptin did not stimulate the autophosphorylation of SH2B1ß(R555E)-bound JAK2 in {gamma}2ALEPRb/JAK2/R555E cells (Fig. 2DGo), suggesting that SH2B1ß(R555E) binds to JAK2 and inhibits leptin stimulation of JAK2. Leptin also did not stimulate the autophosphorylation of SH2B1-bound JAK2(Y813F) (Fig. 2BGo and data not shown), suggesting that SH2B1 binds to JAK2(Y813F) and inhibits leptin stimulation of JAK2(Y813F). Because both SH2B1ß(R555E)-JAK2 interaction and SH2B1-JAK2(Y813F) interaction are mediated by a non-SH2 domain region of SH2B1 and a non-Tyr813 region of JAK2, this non-SH2 domain region of SH2B1- and the non-Tyr813 region in JAK2-mediated constitutive binding of SH2B1 to JAK2 may inhibit basal JAK2 activity.

Phosphorylated Tyr813 in JAK2 Mediates SH2B1-Promoted JAK2 Activation
To determine whether SH2B1 promotes JAK2 activation by directly binding to phosphorylated Tyr813, SH2B1ß was transiently cooverexpressed with JAK2 or JAK2(Y813F) in human embryonic kidney (HEK)293 cells. Cell extract was immunoblotted with {alpha}phospho-JAK2 ({alpha}pY1007/1008). {alpha}pY1007/1008 detects Tyr1007/1008-phosphorylated and active JAK2. SH2B1 robustly stimulated Tyr1007/1008 phosphorylation in a dose-dependent manner (Fig. 3AGo). Similarly, SH2B1 markedly increased the total levels of JAK2 autophosphorylation as measured by immunoblotting JAK2 with {alpha}PY (Fig. 3BGo). In contrast, SH2B1ß was unable to increase either Tyr1007/1008 phosphorylation or total autophosphorylation of JAK2(Y813F) (Fig. 3Go, A and B). These results suggest that Tyr813 phosphorylation is required for SH2B1 to enhance the activity of overexpressed and constitutively active JAK2.


Figure 3
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  		Fig. 3. Tyr813 Is Required for SH2B1-Stimulated JAK2 Activation

JAK2 plasmids (0.2 µg) were transiently cotransfected with the indicated amounts of SH2B1 plasmids in HEK293 cells. Cell extract was prepared 22 h after transfection and immunoblotted with the indicated antibodies. IB, Immunoblotting.

 
To determine whether SH2B1 enhances leptin stimulation of JAK2, {gamma}2ALEPRb/JAK2 and {gamma}2ALEPRb/JAK2/SH2B1 cells were treated with 100 ng/ml leptin for 10 min, and cell extract was immunoblotted with {alpha}pY1007/1008. Leptin stimulated Tyr1007/1008 phosphorylation to a higher extent in {gamma}2ALEPRb/JAK2/SH2B1 cells than in {gamma}2ALEPRb/JAK2 cells (Fig. 4AGo). Phosphorylated JAK2 was quantified and normalized to total JAK2 protein levels. SH2B1 significantly increased leptin-stimulated Tyr1007/1008 phosphorylation by 75 ± 21% (mean ± SEM, n = 5, P < 0.05).


Figure 4
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  		Fig. 4. Leptin-Stimulated Phosphorylation of Tyr813 in JAK2 Mediates SH2B1-Promoted JAK2 Activation

A–C, {gamma}2ALEPRb/JAK2, {gamma}2ALEPRb/Y813F, {gamma}2ALEPRb/JAK2/SH2B1, and {gamma}2ALEPRb/Y813F/SH2B1 cells were stimulated with leptin (100 ng/ml in both A and B and 50 ng/ml in C) for 10 min, and cell extract was prepared. Cell extract was immunoblotted with {alpha}pY1007/1008 or {alpha}JAK2 (A). JAK2 in cell extract was immunoprecipitated with {alpha}JAK2 and immunoblotted with {alpha}PY (B) or {alpha}pY813 (C). The same blots were reprobed with {alpha}JAK2 (B and C). D, {gamma}2ALEPRb/JAK2 or {gamma}2ALEPRb/Y813F cells were infected with SH2B1 or ß-galactosidase control adenoviruses. Infected cells were treated with 100 ng/ml leptin for 10 min, and cell extract was immunoblotted with {alpha}pY1007/1008, {alpha}JAK2, or {alpha}SH2B1. IB, Immunoblotting; IP, immunoprecipitation.

 
To estimate the effect of SH2B1 on the total level of JAK2 autophosphorylation, JAK2 was immunoprecipitated with polyclonal {alpha}JAK2 and immunoblotted with {alpha}PY. Leptin-stimulated JAK2 autophosphorylation was higher in {gamma}2ALEPRb/JAK2/SH2B1 cells than in {gamma}2ALEPRb/JAK2 cells (Fig. 4BGo). Phosphorylated JAK2 was quantified and normalized to total JAK2 protein levels. SH2B1 significantly increased leptin-stimulated JAK2 autophosphorylation by 58 ± 11% (mean ± SEM, n = 4, P < 0.05). SH2B1 also enhanced leptin-stimulated phosphorylation of Tyr813 in JAK2 (Fig. 4CGo).

To determine whether Tyr813 phosphorylation is required for SH2B1 to enhance leptin-stimulated JAK2 activation, {gamma}2ALEPRb/Y813F and {gamma}2ALEPRb/Y813F/SH2B1 cells were treated with 100 ng/ml leptin for 10 min. JAK2(Y813F) activation was examined by immunoblotting cell extract with {alpha}pY1007/1008, and the total level of JAK2(Y813F) autophosphorylation was examined by immunoblotting {alpha}JAK2 precipitates with {alpha}PY. Overexpression of SH2B1 did not increase either Tyr1007/1008 phosphorylation ({gamma}2ALEPRb/Y813F: 1.10 ± 0.07; {gamma}2ALEPRb/Y813F/SH2B1: 1.14 ± 0.05. Arbitrary units, n = 8, P = 0.18) or total autophosphorylation of JAK2(Y813F) ({gamma}2ALEPRb/Y813F: 0.45 ± 0.09; {gamma}2ALEPRb/Y813F/SH2B1: 0.42 ± 0.08. Arbitrary units, n = 4, P = 0.67) (Fig. 4Go, A and B). In addition, SH2B1 was transiently overexpressed in {gamma}2ALEPRb/JAK2 or {gamma}2ALEPRb/Y813F cells via adenoviral-mediated gene transfer. Transient overexpression of SH2B1 enhanced both basal and leptin-stimulated Tyr1007/1008 phosphorylation in JAK2, but not in JAK2(Y813F) (Fig. 4DGo). Phosphorylated JAK2 was quantified and normalized to total JAK2 protein levels. SH2B1 significantly increased leptin-stimulated Tyr1007/1008 phosphorylation in JAK2 by 214 ± 65% (mean ± SEM, n = 4, P < 0.05) in {gamma}2ALEPRb/JAK2 cells but not in JAK2(Y813F) in {gamma}2ALEPRb/Y813F cells. Together, these observations suggest that leptin stimulates the phosphorylation of Tyr813. Phospho-Tyr813 directly binds to the SH2 domain of SH2B1, thereby enhancing leptin-stimulated JAK2 activation and autophosphorylation.

Phosphorylation of Tyr813 in JAK2 Is Not Required for Leptin-Stimulated STAT3 Activation
To determine whether SH2B1 and Tyr813 phosphorylation increases leptin-stimulated phosphorylation and activation of STAT3, {gamma}2ALEPRb/JAK2, {gamma}2ALEPRb/JAK2/SH2B1, {gamma}2ALEPRb/Y813F, and {gamma}2ALEPRb/Y813F/SH2B1 cells were treated with 100 ng/ml leptin for 10 min. Cell extract was immunoblotted with {alpha}phospho-STAT3 ({alpha}pSTAT3) or {alpha}STAT3. {alpha}pSTAT3 specifically recognizes active STAT3 that is phosphorylated on Tyr705. Surprisingly, leptin stimulated STAT3 phosphorylation to a similar extent in {gamma}2ALEPRb/JAK2, {gamma}2ALEPRb/JAK2/SH2B1, {gamma}2ALEPRb/Y813F, and {gamma}2ALEPRb/Y813F/SH2B1 cells (Fig. 5AGo). To determine whether Tyr813 phosphorylation affects the ability of leptin to stimulate STAT3 activation, {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells were treated with leptin at various concentrations, and STAT3 activation was estimated by immunoblotting with {alpha}pSTAT3. The degree of leptin-stimulated STAT3 phosphorylation was similar between {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells at all concentrations of leptin (Fig. 5BGo). To determine whether Tyr813 phosphorylation prolongs STAT3 activation, {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells were treated with leptin for 0, 5, 15, 30, 60, and 120 min. Leptin-stimulated STAT3 phosphorylation in {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells was similar at each time point (Fig. 5CGo). To determine whether SH2B1 enhances STAT3 activation at a lower concentration of leptin, {gamma}2ALEPRb/JAK2, {gamma}2ALEPRb/JAK2/SH2B1, {gamma}2ALEPRb/Y813F, and {gamma}2ALEPRb/Y813F/SH2B1 cells were treated with 50 ng/ml leptin for 10 min. Leptin-stimulated STAT3 phosphorylation was similar both between {gamma}2ALEPRb/JAK2 and {gamma}2ALEPRb/JAK2/SH2B1 and between {gamma}2ALEPRb/Y813F and {gamma}2ALEPRb/Y813F/SH2B1 cells (Fig. 5DGo). Together, these observations suggest that leptin stimulates STAT3 activation independent of Tyr813 phosphorylation and subsequent binding of phosphorylated Tyr813 to the SH2 domain of SH2B1.


Figure 5
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  		Fig. 5. Phosphorylation of Tyr813 in JAK2 Is Not Required for Leptin-Stimulated Activation of STAT3

A, {gamma}2ALEPRb/JAK2, {gamma}2ALEPRb/Y813F, {gamma}2ALEPRb/JAK2/SH2B1, and {gamma}2ALEPRb/Y813F/SH2B1 cells were treated with 100 ng/ml leptin for 10 min, and cell extract was immunoblotted with {alpha}pSTAT3 or {alpha}STAT3. B, {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells were stimulated for 10 min with leptin at various concentrations, and cell extract was immunoblotted with {alpha}pSTAT3 or {alpha}STAT3. C, {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells were stimulated with 100 ng/ml leptin for various times, and cell extract was immunoblotted with {alpha}pSTAT3 or {alpha}STAT3. D, {gamma}2ALEPRb/JAK2, {gamma}2ALEPRb/Y813F, {gamma}2ALEPRb/JAK2/SH2B1, and {gamma}2ALEPRb/Y813F/SH2B1 cells were stimulated with 50 ng/ml leptin for 10 min, and cell extract was immunoblotted with {alpha}pSTAT3 or {alpha}STAT3. IB, Immunoblotting.

 
SH2B1 Promotes Leptin-Stimulated IRS1 Phosphorylation Independent of the Phosphorylation of Tyr813 in JAK2
IRS protein-mediated activation of the PI 3-kinase pathway is required for leptin to regulate energy balance, body weight, and glucose metabolism (16, 17, 43, 44). We reported previously that genetic deletion of the SH2B1 gene abrogates leptin-stimulated tyrosine phosphorylation of hypothalamic IRS2 in SH2B1 knockout mice (38). Genetic deletion of SH2B1 also markedly decreases leptin-stimulated tyrosine phosphorylation of endogenous IRS1 in cultured mouse embryonic fibroblasts (13). These findings suggest that endogenous SH2B1 is required for leptin stimulation of the IRS proteins/PI 3-kinase pathway. To determine whether SH2B1 promotes leptin-stimulated tyrosine phosphorylation of IRS1 via binding to phosphorylated Tyr813 in JAK2, various {gamma}2A cells were stimulated with 50 ng/ml leptin for 10 min, and endogenous IRS1 was immunoprecipitated with {alpha}IRS1 and immunoblotted with {alpha}PY. Leptin stimulated IRS1 phosphorylation in {gamma}2ALEPRb/JAK2, {gamma}2ALEPRb/Y813F, {gamma}2ALEPRb/JAK2/SH2B1, and {gamma}2ALEPRb/Y813F/SH2B1 but not {gamma}2ALEPRb cells (Fig. 6Go). SH2B1 markedly enhanced both JAK2- and JAK2(Y813F)-mediated tyrosine phosphorylation of IRS1 in response to leptin (Fig. 6Go). Phosphorylated IRS1 was quantified and normalized to total IRS1 protein levels. SH2B1 enhanced JAK2-mediated IRS1 phosphorylation by 197 ± 86% (mean ± SEM, n = 7; P < 0.05) and JAK2(Y813F)-mediated IRS1 phosphorylation by 294 ± 99% (mean ± SEM, n = 7, P < 0.05). These data indicate that JAK2 is required for leptin to stimulate tyrosine phosphorylation of IRS1, but Tyr813 phosphorylation is not required for IRS1 phosphorylation. SH2B1 enhances leptin-stimulated IRS1 phosphorylation independent of Tyr813 phosphorylation in JAK2.


Figure 6
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  		Fig. 6. Tyr813 in JAK2 Is Not Required for SH2B1 to Enhance IRS1 Phosphorylation in Response to Leptin

{gamma}2ALEPRb/JAK2, {gamma}2ALEPRb/Y813F, {gamma}2ALEPRb/JAK2/SH2B1, {gamma}2ALEPRb/Y813F/SH2B1, and {gamma}2ALEPRb cells were treated with 50 ng/ml leptin or vehicle for 5 min. Cell extract was immunoprecipitated with {alpha}IRS1 and immunoblotted with {alpha}PY. The same blots were reprobed with {alpha}IRS1. IB, Immunoblotting; IP, immunoprecipitation.

 
Leptin Promotes the Association of SH2B1 with IRS1
SH2B1 could be enhancing leptin-stimulated IRS1 phosphorylation by increasing the accessibility of IRS1 to JAK2. To determine whether SH2B1 forms a multiprotein complex with IRS1 and JAK2, JAK2 and IRS1 were transiently coexpressed with or without SH2B1 in HEK293 cells, and cell extract was immunoprecipitated with {alpha}SH2B1 and immunoblotted with {alpha}IRS1, {alpha}JAK2, or {alpha}PY. SH2B1 coimmunoprecipitated with both IRS1 and JAK2 (Fig. 7AGo, lane 3). Both SH2B1-bound IRS1 and JAK2 were tyrosyl phosphorylated (Fig. 7AGo, panel 4). Interestingly, SH2B1 also bound to IRS1 in the absence of JAK2 overexpression (Fig. 7AGo, lane 1); however, JAK2 overexpression slightly increased the coimmunoprecipitation of SH2B1 with IRS1 (Fig. 7AGo, lanes 1 vs. 3). In parallel experiments, IRS1 in cell extract was immunoprecipitated with {alpha}IRS1 and immunoblotted with {alpha}PY (to detect both phosphorylated IRS1 and JAK2) or {alpha}Myc (to detect Myc-tagged SH2B1). IRS1 coimmunoprecipitated with SH2B1 in the absence of overexpressed JAK2; IRS1 was associated with both SH2B1 and JAK2 in the presence of overexpressed JAK2 (Fig. 7BGo). Consistently, SH2B1 promoted tyrosine phosphorylation of IRS1 (Fig. 7BGo, upper panel).


Figure 7
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  		Fig. 7. Leptin Stimulates the Interaction of IRS1 with SH2B1 Independent of Tyr813 Phosphorylation

A and B, IRS1 plasmids were transiently cotransfected with SH2B1 plasmids in the presence or absence of JAK2 plasmids in HEK293 cells. Myc-tagged SH2B1 in cell extract was immunoprecipitated with {alpha}SH2B1 and immunoblotted with {alpha}IRS1, {alpha}Myc, {alpha}JAK2, or {alpha}PY as indicated. Cell extract was also immunoblotted with {alpha}IRS1 or {alpha}JAK2 (A). In parallel experiments, IRS1 was immunoprecipitated with {alpha}IRS1 and immunoblotted with {alpha}PY, {alpha}Myc, or {alpha}IRS1 as indicated (B). C, {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells were treated with 100 ng/ml leptin for 10 min. IRS1 in cell extract was immunoprecipitated with {alpha}IRS1 and immunoblotted with {alpha}SH2B1 or {alpha}IRS1. D, {gamma}2ALEPRb/JAK2 and {gamma}2ALEPRb/Y813F cells were treated with 100 ng/ml leptin for 10 min. IRS1 in cell extract was immunoprecipitated with {alpha}SH2B1 and immunoblotted with {alpha}IRS1 or {alpha}SH2B1. IB, Immunoblotting; IP, immunoprecipitation.

 
To determine whether leptin promotes the interaction between SH2B1 and IRS1, {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells were stimulated with 100 ng/ml leptin for 10 min, and endogenous IRS1 in cell extract was immunoprecipitated with {alpha}IRS1 and immunoblotted with {alpha}SH2B1. Leptin increased the binding of IRS1 to SH2B1 in both {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells, indicating that leptin promotes the association of SH2B1 with IRS1 independent of Tyr813 phosphorylation (Fig. 7CGo). To determine whether leptin promotes association of endogenous SH2B1 with endogenous IRS1, {gamma}2ALEPRb/JAK2 and {gamma}2ALEPRb/Y813F cells were stimulated with 100 ng/ml leptin for 10 min, and endogenous IRS1 in cell extract was immunoprecipitated with {alpha}IRS1 and immunoblotted with {alpha}SH2B1. Endogenous IRS1 bound to endogenous SH2B1, and the binding was increased in response to leptin stimulation in both {gamma}2ALEPRb/JAK2 and {gamma}2ALEPRb/Y813F cells (Fig. 7DGo). Together, these observations suggest that binding of SH2B1 to IRS1 contributes to leptin-stimulated tyrosine phosphorylation of IRS1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We previously provided multiple lines of genetic evidence demonstrating that SH2B1 is a key endogenous positive regulator of leptin sensitivity in animals. Genetic deletion of the SH2B1 gene results in severe leptin resistance and morbid obesity in SH2B1 null mice (30, 38). Tissue-specific restoration of SH2B1 in neurons, the main targets of leptin, rescues leptin resistance and obesity in SH2B1 knockout mice (40). Moreover, neuron-specific overexpression of SH2B1 protects against high-fat diet-induced leptin resistance and obesity in SH2B1 transgenic mice (40). In this study, we provide biochemical evidence demonstrating that SH2B1 regulates leptin signaling by multiple mechanisms. In the absence of leptin, SH2B1 constitutively interacted with JAK2 that was not tyrosyl phosphorylated and thus inactive; the SH2 domain of SH2B1 was not required for this constitutive association; however, leptin significantly increased the interaction of SH2B1 with JAK2. Leptin rapidly and robustly stimulated the phosphorylation of Tyr813 in JAK2, and phosphorylated Tyr813 subsequently bound to the SH2 domain of SH2B1. Consistent with these observations, the PH domain of SH2B1 is both required and sufficient for SH2B1 to bind to a kinase-inactive JAK2 mutant (45). Crystal structural analysis reveals that a phospho-Tyr813 peptide of JAK2 physically binds to the SH2 domain of SH2B1 (24). Therefore, SH2B1 appears to bind to JAK2 by multiple sites. A non-SH2 domain region in SH2B1 constitutively binds to a non-Tyr813 site in JAK2, whereas the SH2 domain of SH2B1 binds to phosphorylated Tyr813 in JAK2.

Surprisingly, leptin was unable to stimulate both SH2B1(R555E)-bound JAK2 and SH2B1-bound JAK2(Y813F). Both SH2B1(R555E)-JAK2 interaction and SH2B1-JAK2(Y813F) interaction are mediated by a non-SH2 domain region of SH2B1 and a non-Tyr813 site in JAK2. A C-terminal truncated SH2B1 lacking a functional SH2 domain, which constitutively binds to JAK2 via the interaction of the non-SH2 domain region of SH2B1 and the non-Tyr813 site in JAK2, inhibits JAK2 activity (45). Therefore, the interaction of the non-SH2 domain region of SH2B1 with the non-Tyr813 site in JAK2 may inhibit basal JAK2 activity and autophosphorylation.

In contrast, SH2B1 significantly enhanced leptin-stimulation of JAK2 that was phosphorylated on Tyr813. The mechanism of SH2B1 stimulation of JAK2 remains unknown. SH2B1 forms homodimers via its N-terminal DD domain (25, 46). SH2B1 dimerization is predicted to induce the dimerization of SH2B1-bound JAK2, thereby promoting JAK2 activation (25). However, overexpression of either N-terminal truncated SH2B1, which lacks the DD domain, or full-length SH2B1, activates JAK2 to a similar extent, and the SH2 domain of SH2B1 is both required and sufficient to activate JAK2 (45, 47). These results suggest that the interaction of the SH2 domain of SH2B1 with phospho-Tyr813 stabilizes JAK2 in an active conformation.

We show that leptin stimulates tyrosine phosphorylation of IRS1 in {gamma}2ALEPRb/JAK2 but not in {gamma}2ALEPRb cells, providing strong evidence that JAK2 mediates leptin-stimulated tyrosine phosphorylation of IRS1. Because SH2B1 promotes JAK2 activation, SH2B1 significantly increased leptin-stimulated tyrosine phosphorylation of IRS1, a JAK2 substrate, as expected. In agreement with these findings, genetic deletion of SH2B1 markedly reduces leptin-stimulated tyrosine phosphorylation of IRS1 in mouse embryonic fibroblasts, and this reduction is reversed by the expression of recombinant SH2B1 (13). Surprisingly, SH2B1 also enhanced JAK2(Y813F)-mediated tyrosine phosphorylation of IRS1 in response to leptin, even though SH2B1 did not enhance JAK2(Y813F) activation. SH2B1 directly binds to both IRS1 and IRS2, and the SH2 domain of SH2B1 is sufficient to bind to both tyrosyl-phosphorylated IRS1 and IRS2 (13). Leptin promoted the association of SH2B1 with IRS1 similarly in both {gamma}2ALEPRb/JAK2 and {gamma}2ALEPRb/Y813F cells. These observations suggest that SH2B1 enhances leptin-stimulated tyrosine phosphorylation of IRS proteins by an additional mechanism independent of SH2B1 enhancement of JAK2 activation.

We propose a model of SH2B1 regulation of leptin signaling (Fig. 8Go). In the absence of leptin, the constitutive binding of SH2B1 (either as monomers or dimers) to JAK2, which is mediated by the non-SH2 domain region(s) of SH2B1 and the non-Tyr813 region(s) of JAK2, not only increases the local concentration of SH2B1 close to JAK2 but also inhibits basal JAK2 activity (Fig. 8AGo). Leptin stimulates JAK2 autophosphorylation on Tyr813, and phosphorylated Tyr813 binds to the SH2 domain of SH2B1 (either as monomers or dimers) (Fig. 8BGo). The physical interaction of the SH2 domain of SH2B1 with phospho-Tyr813 in JAK2 robustly and quickly promotes JAK2 activation, thus globally enhancing leptin signaling (Fig. 8BGo, left side of LEPRb). Leptin also stimulates the binding of IRS1 to SH2B1 (either as monomers or dimers). SH2B1-homodimers binds simultaneously to both JAK2 and IRS1, thereby increasing the concentration of IRS1 proximal to JAK2 and facilitating JAK2-mediated phosphorylation of IRS1 (Fig. 8BGo, right side of LEPRb). In addition, SH2B1 binds, via its SH2 domain, directly to phosphotyrosine(s) in IRS1, thereby protecting IRS1 from dephosphorylation by protein tyrosine phosphatase(s) (Fig. 8BGo, low side). Tyrosyl-phosphorylated IRS1 and/or IRS2 mediate leptin stimulation of the PI 3-kinase pathway.


Figure 8
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  		Fig. 8. A Model of SH2B1 Action

A, In the absence of leptin, SH2B1 (either as monomers or dimers) binds via its non-SH2 domain region(s) to inactive and non-tyrosyl-phosphorylated JAK2, thereby both increasing the local concentration of SH2B1 close to JAK2 and inhibiting basal JAK2 activity. B, Leptin stimulates phosphorylation of JAK2 on Tyr813. SH2B1 binds via its SH2 domain to phosphorylated Tyr813, thus enhancing leptin stimulation of JAK2 (left side of LEPRb). SH2B1 (either as monomers or dimers) also directly binds to IRS1 or IRS2, thereby recruiting IRS proteins to JAK2 and/or stabilizing JAK2/SH2B1/IRS protein complexes, which facilitates tyrosine phosphorylation of IRS proteins by JAK2 (right side of LEPRb). In addition, the binding of SH2B1 to IRS proteins may protect IRS proteins from protein tyrosine phosphatase-mediated dephosphorylation (low side). Tyrosyl-phosphorylated IRS proteins activate the PI 3-kinse pathway. PTP, Protein tyrosine phosphatase.

 
JAK2 Tyr813 phosphorylation is not required for leptin-stimulated phosphorylation of STAT3. The time course and dose response of leptin-stimulated STAT3 phosphorylation were indistinguishable between {gamma}2ALEPRb/JAK2/SH2B1 and {gamma}2ALEPRb/Y813F/SH2B1 cells. Even though leptin-induced JAK2 activation was enhanced by overexpression of SH2B1 in {gamma}2ALEPRb/JAK2/SH2B1 cells, SH2B1 did not enhance leptin-stimulated STAT3 phosphorylation. One explanation for these interesting data is that STAT3 is already maximally phosphorylated by JAK2Y813F or by JAK2 at endogenous levels of SH2B1.

In summary, we demonstrated that in the absence of leptin, SH2B1 constitutively interacted with non-tyrosyl-phosphorylated JAK2, inhibiting basal JAK2 activity. Leptin stimulated JAK2 phosphorylation on Tyr813, which subsequently bound to the SH2 domain of SH2B1, resulting in an enhancement of JAK2 activation. JAK2 is required for leptin stimulation of tyrosine phosphorylation of IRS1. SH2B1 bound to IRS1 and enhanced leptin-stimulated tyrosine phosphorylation of IRS1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of {gamma}2A Cell Lines Stably Expressing LEPRb, JAK2, JAK2(Y813F), and SH2B1
Murine LEPRb cDNA was inserted into a retroviral vector (pQCXIH) to generate recombinant LEPRb retroviruses. {gamma}2A cells, which are derived from human fibroblasts and do not express endogenous JAK2 (42), were infected with the LEPRb retroviruses and selected by hygromycin. Hygromycin-resistant cells were pooled and designated as {gamma}2ALEPRb cells. {gamma}2ALEPRb cells stably express LEPRb as confirmed by immunoblotting with {alpha}LEPRb (data not shown). {gamma}2ALEPRb cells were subsequently infected with recombinant JAK2, JAK2(Y813F), or JAK2(K882E) retroviruses and selected by puromycin to generate {gamma}2ALEPRb/JAK2, {gamma}2ALEPRb/Y813F, and {gamma}2ALEPRb/K882E cell lines stably expressing JAK2, JAK2(Y813F), or JAK2(K882E), respectively. JAK2(Y813F) and JAK2(K882E) were generated by replacing Tyr813 with Phe and Lys882 (the ATP-binding site) with Glu using a site-directed mutagenesis kit (Stratagene, La Jolla, CA), respectively. {gamma}2ALEPRb/JAK2, {gamma}2ALEPRb/Y813F, or {gamma}2ALEPRb/K882E cells were infected with recombinant SH2B1ß retroviruses that coexpress green fluorescent protein. Green fluorescent protein-positive cells (top 40%) were sorted by fluorescence-activated cell sorting to generate {gamma}2ALEPRb/JAK2/SH2B1, {gamma}2ALEPRb/Y813F/SH2B1, and {gamma}2ALEPRb/K882E/SH2B1 cell lines stably expressing rat SH2B1ß.

Parental {gamma}2A and all derived cells were cultured at 37 C in 5% CO2 in DMEM supplemented with 25 mM glucose, 100 U/ml penicillin, 100 µg/ml streptomycin, and 6% fetal calf serum. HEK293 cells were grown at 37 C in 5% CO2 in DMEM supplemented with 25 mM glucose, 100 U/ml penicillin, 100 µg/ml streptomycin, and 8% calf serum.

Adenoviral Infection
{gamma}2ALEPRb/JAK2 or {gamma}2ALEPRb/Y813F cells were grown to confluence in 100-mm culture dishes. Cells were infected with SH2B1ß or ß-gal control recombinant adenoviruses (3.8 x 1010 viral particles in 4 ml growth medium per plate) for 4 h, and grown in growth medium. Cells were deprived, 48 h after infection, of serum overnight and then treated with leptin. Cell extract was prepared and used for immunoprecipitation and immunoblotting analysis.

Transfection
HEK293 cells were split at 2 x 105 cells per well in a 12-well culture dish. The next day, JAK2 or JAK2(Y813F) expression plasmids (0.2 µg) were cotransfected with 0, 0.05, 0.1, 0.2, or 0.4 µg SH2B1 expression plasmids using Lipofectamine 2000 reagents (Invitrogen Corp., Carlsbad, CA). The total amount of plasmid DNA was maintained constant for each individual condition by adding empty prk5 vector. Cells were lysed 22 h after transfection and subjected to immunoprecipitation and immunoblotting assays.

Immunoprecipitation and Immunoblotting
Confluent cells were deprived of serum overnight (~16 h) in DMEM containing 0.6% BSA and treated with leptin as indicated in the figure legends. The cells were rinsed two times with ice-cold PBS, solubilized in lysis buffer (50 mM Tris, pH 7.5; 1% Nonidet P-40; 150 mM NaCl; 2 mM EGTA; 1 mM Na3VO4; 100 mM NaF; 10 mM Na4P2O7; 1 mM phenylmethylsulfonyl fluoride; 10 µg/ml aprotinin; 10 µg/ml leupeptin), and centrifuged at 14,000 x g for 15 min at 4 C. Protein concentration in the supernatant (cell extract) was determined using a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). Cell extract (same amount of protein for each individual condition) was incubated with the indicated antibody on ice for 2 h. The immune complexes were collected on protein A-agarose during a 1-h incubation at 4 C. The beads were washed three times with washing buffer (50 mM Tris, pH 7.5; 1% Nonidet P-40; 150 mM NaCl; 2 mM EGTA; 100 mM NaF; and 10 mM Na4P2O7) and boiled for 5 min in SDS-PAGE sample buffer (50 mM Tris-HCl, pH 6.8; 2% SDS; 2% ß-mercaptoethanol; 10% glycerol; 0.005% bromophenol blue). The solubilized proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane (Amersham International Plc, Little Chalfont, Buckinghamshire, UK), and detected by immunoblotting with the indicated antibody using enhanced chemiluminescence or Odyssey detection system. Some membranes were subsequently incubated at 55 C for 30 min in stripping buffer (100 mM ß-mercaptoethanol; 2% SDS; 62.5 mM Tris-HCl, pH 6.7) to prepare them for a second round of immunoblotting. Phosphorylated JAK2 was quantified using the Odyssey software and normalized to total JAK2 protein levels. Similarly, phosphorylated IRS1 was quantified and normalized to total IRS1 protein levels. {alpha}IRS1, {alpha}JAK2, and {alpha}phospho-JAK2 were from Biosource (Camarillo, CA) or Upstate Biotechnology, Inc. (Charlottesville, VA). {alpha}STAT3 and {alpha}phospho-STAT3 were from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) or Cell Signaling Technology, Inc. (Beverly, MA).

Statistical Analysis
The data are presented as the mean ± SEM. Student’s t tests were used for comparisons between two groups. P < 0.05 was considered statistically significant.


   ACKNOWLEDGMENTS
 
We thank David Morris and Drs. Lawrence Argetsinger and Jason Kurzer for helpful discussion. We thank Dr. George Stark (Cleveland Clinic Foundation, Cleveland, OH) for providing {gamma}2A cells.


   FOOTNOTES
 
This work was supported by Career Development Award (7-03-CD-11) from the American Diabetes Association and RO1 DK 065122 and RO1 DK073601 from National Institutes of Health (NIH) (all to L.R.). This work utilized the cores supported by the Michigan Diabetes Research and Training Center (funded by NIH Grant 5P60 DK20572) and University of Michigan’s Cancer Center (funded by NIH Grant 5 P30 CA46592). C.C.S. was supported by NIH Grants DK34171 and DK54222; and M.G.M.J. was supported by NIH Grant DK56731.

Disclosure Statement: All authors have nothing to declare.

First Published Online June 12, 2007

Abbreviations: DD, Dimerization domain; HEK, human embryonic kidney; IRS1, insulin receptor substrate 1; JAK2, Janus kinase 2; LEPRb: long-isoform of leptin receptor; PH, pleckstrin homology domain; PI 3, phosphatidylinositol 3; {alpha}PY, antiphosphotyrosine antibody; SH2: Src homology 2; STAT, signal transducer and activator of transcription.

Received for publication February 27, 2007. Accepted for publication June 4, 2007.


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 MATERIALS AND METHODS
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