SH2-B promotes insulin receptor substrate 1 (IRS1)- and IRS2-mediated activation of the phosphatidylinositol 3-kinase pathway in response to leptin

Abstract

Leptin regulates energy homeostasis primarily by binding and activating its long form receptor (LRb). Deficiency of either leptin or LRb causes morbid obesity. Leptin stimulates LRb-associated JAK2, thus initiating multiple pathways including the Stat3 and phosphatidylinositol (PI) 3-kinase pathways that mediate leptin biological actions. Here we report that SH2-B, a JAK2-interacting protein, promotes activation of the PI 3-kinase pathway by recruiting insulin receptor substrate 1 (IRS1) and IRS2 in response to leptin. SH2-B directly bound, via its PH and SH2 domain, to both IRS1 and IRS2 both in vitro and in intact cells and mediated formation of a JAK2/SH2-B/IRS1 or IRS2 tertiary complex. Consequently, SH2-B dramatically enhanced leptin-stimulated tyrosine phosphorylation of IRS1 and IRS2 in HEK293 cells stably expressing LRb, thus promoting association of IRS1 and IRS2 with the p85 regulatory subunit of PI 3-kinase and phosphorylation and activation of Akt. SH2-B mutants with lower affinity for IRS1 and IRS2 exhibited reduced ability to promote association of JAK2 with IRS1, tyrosine phosphorylation of IRS1, and association of IRS1 with p85 in response to leptin. Moreover, deletion of the SH2-B gene impaired leptin-stimulated tyrosine phosphorylation of endogenous IRS1 in mouse embryonic fibroblasts (MEF), which was reversed by reintroduction of SH2-B. Similarly, SH2-B promoted growth hormone-stimulated tyrosine phosphorylation of IRS1 in both HEK293 and MEF cells. Our data suggest that SH2-B is a novel mediator of the PI 3-kinase pathway in response to leptin or other hormones and cytokines that activate JAK2.

Fig. 1. SH2-B directly binds to IRS1 and IRS2

A, HEK293 cells were transiently cotransfected with expression plasmids encoding IRS1 (0.8 μg) and SH2-Bβ (1 μg). The cell extracts were prepared 48 h after transfection, immunoprecipitated (IP) with anti-SH2-B (αSH2-B), and immunoblotted (IB) with αIRS1. The same blot was reprobed with αSH2-B. The cell extracts were also immunoblotted with αIRS1. In a parallel experiment, the cell extracts were immunoprecipitated with αIRS1 and immunoblotted with αSH2-B. The same blot was reprobed with αIRS1. B, HEK293 cells were transiently cotransfected with expression plasmids encoding IRS2 (0.8 μg) and SH2-Bβ (1 μg). The cell extracts were prepared 48 h after transfection, immunoprecipitated αSH2-B, and immunoblotted with αIRS2. The same blot was reprobed with αSH2-B. The cell extracts were also immunoblotted with αIRS2. In a parallel experiment, the cell extracts were immunoprecipitated with αIRS2 and immunoblotted with αSH2-B. The same blot was reprobed with αIRS2. C, HEK293 cells were transiently transfected with expression plasmids encoding IRS1 (1 μg) or IRS2 (1 μg). The cell extracts were prepared 48 h after transfection and immunoprecipitated with αIRS1 or αIRS2, respectively. Immunopurified IRS1 and IRS2 were resolved by SDS-PAGE and transferred to nitrocellulose membrane. The blot was incubated with GST-SH2-B, and subsequently immunoblotted with αSH2-B. D, FAO cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoprecipitated with αIRS1, αIRS2, or αSH2-B, respectively, and immunoblotted with αSH2-B. The same blots were reprobed with αIRS1 or αIRS2 as indicated.

Fig. 2. SH2-B binds to IRS1 and IRS2 via multiple sites

A, HEK293 cells were transiently cotransfected with expression plasmids encoding IRS1 (1 μg) and SH2-Bβ (0.8 μg) or SH2-B(R555E) (0.8 μg). The cell extracts were prepared 48 h after transfection, immunoprecipitated (IP) with αIRS1, and immunoblotted (IB) with αSH2-B. The same blot was reprobed with αIRS1. B, HEK293 cells were transiently cotransfected with expression plasmids encoding IRS2 (0.7 μg) and Myc-tagged ΔN504 (1.2 μg). The cell extracts were prepared 48 h after transfection, immunoprecipitated with αIRS2, and immunoblotted with αMyc. The same blot was reprobed with αIRS2. C, schematic representation of full-length and N-terminal truncated SH2-Bβ. D, HEK293 cells were transiently cotransfected with expression plasmids encoding IRS1(Y18F) (1 μg) and Myc-tagged full-length or truncated SH2-Bβ (1 μg). The cell extracts were prepared 48 h after transfection, immunoprecipitated with αSH2-B, and immunoblotted with αIRS1. The cell extracts were immunoblotted with αMyc to estimate the expression of full-length or various N-terminally truncated SH2-Bβ.

Fig. 3. SH2-B mediates a JAK2/SH2-B/IRS1 or IRS2 tertiary complex

A, HEK293 cells were transiently cotransfected with expression plasmids encoding IRS1 (1 μg), JAK2 (0.8 μg), and SH2-Bβ (0.6 μg) as indicated. The cell extracts were prepared 48 h after transfection, immunoprecipitated (IP) with αJAK2, and immunoblotted (IB) sequentially with αIRS1, αSH2-B, and αJAK2. B, HEK293^LRb cells were transiently cotransfected with expression plasmids encoding IRS1 (1.2 μg), JAK2 (0.6 μg), and SH2-Bβ or ΔN504 (0.6 μg) as indicated. The cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoprecipitated with αJAK2 and immunoblotted with αIRS1. The same blot was reprobed with anti-phosphotyrosine (αPY). C, HEK293^LRb cells were transiently cotransfected with expression plasmids encoding IRS2 (1.2 μg), JAK2 (0.6 μg), and SH2-Bβ or ΔN504 (0.6 μg) as indicated. The cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoprecipitated with αJAK2 and immunoblotted with αIRS2. The same blot was reprobed with anti-phosphotyrosine.

Fig. 4. SH2-B mediates leptin-stimulated tyrosine phosphorylation of IRS1 and IRS2

A, HEK293^LRb cells were transiently cotransfected with expression plasmids encoding IRS1 (1 μg) and SH2-Bβ, SH2-B(R555E), or ΔN504 (0.8 μg) as indicated. The cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoblotted (IB) with anti-phosphotyrosine. The positions of IRS1, SH2-Bβ, SH2-B(R555E), and ΔN504 were marked. CON, control. B, HEK293^LRb cells were transiently cotransfected with expression plasmids encoding IRS2 (1 μg) and SH2-Bβ (0.8 μg). The cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoblotted with αPY. The positions of IRS2 and SH2-Bβ were marked. C, HEK293^LRb cells were transiently cotransfected with expression plasmids encoding IRS2 (0.8 μg), Stat3 (0.4 μg), and Myc-tagged SH2-Bβ (0.3 μg) as indicated. The cells were treated with 200 ng/ml leptin for 10 min. The cell extracts were immunoblotted with anti-phosphotyrosine (αPY), anti-IRS2, anti-phospho-Stat3, anti-Stat3, and anti-Myc as indicated. The positions of IRS2, Stat3, and SH2-Bβ were marked. The phosphorylation of IRS2 and Stat3 was quantitated and normalized to total IRS2 or Stat3, respectively. D, SH2-B^−/−/LRb and SH2-B^+/+/LRb MEFs stably expressing LRb were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoprecipitated (IP) with αIRS1 and immunoblotted with anti-phosphotyrosine. The same blot was reprobed with αIRS1. E, SH2-B^−/−/LRb MEFs were infected with control (Con) or SH2-Bβ retroviruses, and stable clones were selected. The cell extracts were prepared from SH2-B^+/+/LRb and SH2-B^−/−/LRb MEFs infected with control or SH2-Bβ retroviruses, immunoprecipitated with αSH2-B, and immunoblotted with αSH2-B. F, control or SH2-Bβ retroviruses-infected SH2-B^−/−/LRb MEFs were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoprecipitated with αIRS1 and immunoblotted with αPY. The same blot was reprobed with αIRS1.

Fig. 5. SH2-B enhances leptin-induced activation of the PI 3-kinase pathway

A, HEK293^LRb cells were transiently cotransfected with expression plasmids encoding IRS1 (1 μg) and SH2-Bβ, SH2-B(R555E), or ΔN504 (0.8 μg) as indicated. The cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoprecipitated (IP) with αp85 and immunoblotted (IB) with αIRS1. The same blot was reprobed with αp85. B, HEK293^LRb cells were transiently cotransfected with expression plasmids encoding IRS1 (1 μg), Akt1 (0.8 μg), and SH2-Bβ or SH2-B(R555E) (0.8 μg) as indicated. The cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoblotted with αphospho-Akt (Thr³⁰⁸), αAkt, and αSH2-B as indicated. Con, control.

Fig. 6. SH2-B mediates GH-stimulated tyrosine phosphorylation of IRS1

A, HEK293 cells were transiently cotransfected with expression plasmids encoding GH receptor (0.7 μg) IRS1 (1.2 μg), Stat5b (0.5 μg), and SH2-Bβ (0.6 μg) as indicated. The cells were treated with 8 × 10⁻³ IU/ml GH for 10 min. The cell extracts were immunoblotted (IB) with anti-phosphotyrosine (αPY), anti-IRS1, anti-phospho-Stat5b, and anti-Stat5b as indicated. The positions of IRS1 and Stat5b are marked. CON, control. B, control (Con) or SH2-Bβ retrovirus-infected SH2-B^−/−/LRb MEFs were treated with 8 × 10⁻³ IU/ml GH for 10 min. The cell extracts were immunoprecipitated (IP) with αIRS1 and immunoblotted with αPY. The same blot was reprobed with αIRS1.

Fig. 7. A Model of SH2-B action

SH2-B binds to and potentiates activation of JAK2, globally enhancing the activation of pathways downstream of JAK2 (mechanism 1). SH2-B binds simultaneously to both JAK2 and IRS1 or IRS2, mediating a JAK2/SH2-B/IRS1 or IRS2 complexes. Consequently, SH2-B mediates specifically tyrosine phosphorylation of IRS1 and IRS2, resulting in activation of the PI 3-kinase pathway (mechanism 2).