Abstract
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 Tyr(1077) (which we demonstrate to be phosphorylated during receptor activation) and a secondary role for LepRb Tyr(1138) in the acute phosphorylation of STAT5a and STAT5b. Tyr(1138) and STAT3 attenuate STAT5-dependent transcription over the long-term, however. In contrast, Tyr(985) (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 Tyr(1077) 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.
Highlights
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)
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 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
Summary
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; ␣PY, anti-phosphotyrosine antibody; ARC, arcuate nucleus of the hypothalamus. LepRb has 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 controls the activation of phosphatidylinositol (PI) 3Ј-kinase and pathways that regulate the phosphorylation of ribosomal protein S6 in the hypothalamus, and mediates the tissuespecific regulation of the AMP-dependent protein kinase (32–36). 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). 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
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