The Long Form of the Leptin Receptor Regulates STAT5 and Ribosomal Protein S6 via Alternate Mechanisms

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 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. 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. 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 (1Ogden C.L. Carroll M.D. Curtin L.R. McDowell M.A. Tabak C.J. Flegal K.M. J. Am. Med. Assoc. 2006; 295: 1549-1555Crossref PubMed Scopus (7402) Google Scholar, 2Tierney E.F. Gregg E.W. Narayan K.M. J. Am. Med. Assoc. 2006; 295: 383Crossref Scopus (9) Google Scholar, 3Narayan K.M. Boyle J.P. Thompson T.J. Sorensen S.W. Williamson D.F. J. Am. Med. Assoc. 2003; 290: 1884-1890Crossref PubMed Scopus (1236) Google Scholar). 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 (4Marx J. Science. 2003; 299: 846-849Crossref PubMed Scopus (99) Google Scholar, 5Elmquist J.K. Maratos-Flier E. Saper C.B. Flier J.S. Nat. 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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 (15Mantzoros C.S. Flier J.S. J. Clin. Endocrinol. Metab. 2000; 85: 4000-4002Crossref PubMed Scopus (107) Google Scholar, 16El Haschimi K. Pierroz D.D. Hileman S.M. Bjorbaek C. Flier J.S. J. Clin. Investig. 2000; 105: 1827-1832Crossref PubMed Scopus (714) Google Scholar, 17Munzberg H. Flier J.S. Bjorbaek C. Endocrinology. 2004; 145: 11797-11802Crossref Scopus (578) Google Scholar). Potential mechanisms to explain this leptin resistance include alterations in leptin signaling, among others (18Banks W.A. Peptides. 2004; 25: 331-338Crossref PubMed Scopus (124) Google Scholar, 19Munzberg H. Bjornholm M. Bates S.H. Myers Jr., M.G. Cell Mol. Life Sci. 2005; 62: 642-652Crossref PubMed Scopus (203) Google Scholar). Leptin binding activates the constitutively LepRb-associated Janus kinase (Jak) 3The 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; αPY, anti-phosphotyrosine antibody; ARC, arcuate nucleus of the hypothalamus. 2 tyrosine kinase to mediate tyrosine phosphorylation-dependent leptin signaling via several pathways (8Myers Jr., M.G. Recent Prog. Horm. Res. 2004; 59: 287-304Crossref PubMed Scopus (337) Google Scholar, 20Baumann H. Morella K.K. White D.W. Dembski M. Bailon P.S. Kim H. Lai C.F. Tartaglia L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8374-8378Crossref PubMed Scopus (763) Google Scholar, 21Tartaglia L.A. Dembski M. Weng X. Deng N. Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. 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A. 1996; 93: PubMed Scopus Google STAT3 and STAT5 have and the of leptin to STAT5 in D. J. Endocrinology. 2001; PubMed Google Scholar). the regulation of by leptin in D. K. S.C. S.C. Science. 2006; PubMed Scopus Google the mechanisms by which leptin mediates and the effects of this regulation Here we demonstrate the regulation of STAT5 by leptin in and the phosphorylation by which leptin regulates STAT5 and ribosomal protein S6 phosphorylation and and and have S.M. Bates S.H. Myers Jr., M.G. J. 2000; Full Full PubMed Scopus Google Scholar, Myers Jr., M.G. Mol. Cell. 2004; PubMed Scopus Google Scholar). for phosphorylated and Tyr1138 of LepRb in by of a phosphorylated on the phosphorylated in and on the to a of and by over to and to of phosphorylation and to the form of the used for and RSK, and Cell human and and secondary for of and have S.M. Bates S.H. Myers Jr., M.G. J. 2000; Full Full PubMed Scopus Google and used as for the to and The of the mutations and the of mutations by of Cell for in with and in a and at in as Myers Jr., M.G. Mol. Cell. 2004; PubMed Scopus Google Scholar). to each by in or with for the at in 2 2 and 2 by at at for of the the protein and and of protein to the for or in for by with at by with protein for by and in in and by as Myers Jr., M.G. Mol. Cell. 2004; PubMed Scopus Google Scholar). the with to the in and as of in with the the for STAT3 C. Bates S.H. S.M. Flier J.S. Myers Jr., M.G. J. 2000; Full Full PubMed Scopus Google or (the the of and of (the of S. A. J. 1995; Full Full PubMed Scopus Google and control with or of for the with a (the of which cap-dependent of the and of the D. H. J. N. J. J. Full Full Scopus Google Scholar). and for and the system on a Life of our at the of or with in to and and by the on the and of and arcuate nucleus and and as H. Flier J.S. Bjorbaek C. Endocrinology. 2004; 145: 11797-11802Crossref Scopus (578) Google the with leptin or for and with of the and as In in and with or with and for or with a for a that and H. Flier J.S. Bjorbaek C. Endocrinology. 2004; 145: 11797-11802Crossref Scopus (578) Google Scholar). of STAT5 and S6 during LepRb in and in the phosphorylation of STAT5 and S6 and S6(P), in cultured a of the receptor extracellular domain with the intracellular domain the long form of the leptin receptor (LepRb) In this the of signals is the control of S.M. Bates S.H. Myers Jr., M.G. J. 2000; Full Full PubMed Scopus Google Scholar). and others have to signal by the intracellular domain of the intracellular signaling by LepRb S.M. Bates S.H. Myers Jr., M.G. J. 2000; Full Full PubMed Scopus Google Scholar, C. S.M. Bates S.H. Pierroz D.D. H. Myers Jr., M.G. Flier J.S. J. 2001; Full Full PubMed Scopus Google Scholar, Bjornholm M. Bates S.H. M. Myers Jr., M.G. Mol. Endocrinol. 2005; PubMed Scopus Google Scholar, J. M. L. A. 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J. 2005; PubMed Scopus (99) Google this analysis that Tyr1138 is required and for In contrast, loss of to the phosphorylation of of Tyr1077 Tyr985 to the of to mediate the of Tyr1077 and Tyr1138 for the of for LepRb tyrosine phosphorylation and we the phosphorylation of by Tyr1138 mediates the phosphorylation of as well as STAT3 the of STAT5 and produced by and K. Y. P. J. L. K. L. 2001; PubMed Scopus Google we the phosphorylation of STAT5a and by for Tyr1077 Tyr1138 to the for these in the regulation of STAT5 STAT5a and phosphorylated during in cells, and the phosphorylation of each of these required the of Tyr1077 and The phosphorylation of STAT3 by STAT5 The analysis suggests in to Tyr985 and which LepRb phosphorylation Tyr1077 to be a site of as phosphorylated to a tyrosine phosphorylation site C.A. C.A. Science. PubMed Scopus Google Scholar). Our analysis of phosphorylation with a S.M. Bates S.H. Myers Jr., M.G. J. 2000; Full Full PubMed Scopus Google that Tyr1077 is phosphorylated or our that the Tyr1077 is with the phosphorylation of and Tyr1138 we a of with the phosphorylated form of each of these the on the and each of these on an with for individual tyrosine in the or of Epo, and the phosphorylation of and each site on the receptor by with the analysis the tyrosine phosphorylation of protein by each as with that the intracellular domain of LepRb the of each receptor each at the phosphorylated of each tyrosine on the intracellular domain of LepRb with in a and to only to receptor a at its phosphorylation data that each the phosphorylated form of the LepRb tyrosine and that each of the intracellular tyrosine on LepRb is phosphorylated during receptor have that of Tyr1077 the phosphorylation of is this of LepRb phosphorylation or results a in with for Tyr1077 and Tyr1138 in STAT5-dependent for Tyr1077 and Tyr1138 in STAT5-dependent we the of tyrosine in the J. 1995; Full Full PubMed Scopus Google for this is but for individual of Tyr985 in the increased with the of Tyr985 to mediate the of protein and with the of this phosphorylation site to the LepRb signaling SOCS3, to mediate of LepRb signaling C. Bates S.H. S.M. Flier J.S. Myers Jr., M.G. J. 2000; Full Full PubMed Scopus Google Scholar). of Tyr1138 of the of the and the of Tyr1077 decreased that STAT5 signaling activates the the of STAT3 the response of this in (which to a of STAT3 to the regulation of and STAT5-dependent transcription by Tyr1077 and Tyr1138 during signaling, we by for these during of STAT3 STAT5a and of of these increased with that in the of STAT, that this the regulation by the as the of the increased by STAT3 required the of Tyr1138 as for STAT3 a of with a but of high STAT3 The of during STAT5 a role for Tyr1077 in the regulation of and as the high of each STAT5 a of STAT5 analysis an role for Tyr1138 in STAT5-dependent as increased transcription with and increased transcription with results that Tyr1077 and Tyr1138 to the phosphorylation of STAT5 during acute Tyr1077 in the regulation of and transcription during and that Tyr1138 STAT3 mediate of transcription these a role for attenuation of STAT5-dependent transcription we the effect of STAT3 and on the by this of In to the results of the for Tyr1138 in the transcription and for Tyr1077 in this analysis that STAT3 STAT3 at high levels such that a of STAT3 STAT3 only in this and this STAT3 the of in or the by which STAT3 STAT5-dependent transcription is for with the that STAT3 mediates to the effects of in with our that Tyr1138 and STAT3 promote to mediate the of phosphorylation during LepRb signaling over a S.M. Bates S.H. Myers Jr., M.G. J. 2000; Full Full PubMed Scopus Google Scholar, Bjornholm M. Bates S.H. M. Myers Jr., M.G. Mol. Endocrinol. 2005; PubMed Scopus Google Scholar). data that the contributions of and to in only the in each but the of each in a the in STAT5 in of the arcuate nucleus by the of STAT5 with in (which in R.L. Bjornholm M. H. Myers Jr., M.G. 2006; PubMed Scopus Google analysis a of in STAT5 in of the arcuate in for Tyr985 and ERK in during LepRb the mechanisms by which leptin regulates S6(P), we during of and for individual tyrosine phosphorylation or for to mediate the phosphorylation of or In this to mediate as by with the phosphorylated form of of on Whereas and each S6(P), to mediate S6(P), and at as well as Whereas the of decreased in with this a of decreased of the in this by decreased and decreased phosphorylation of Tyr985 on as in and with ERK by this Thus, Tyr985 is required for the of by the intracellular domain of LepRb in cells. Tyr985 to the ERK during signaling, we that this be in the regulation of an of the of ERK, to the role of the ERK in the regulation of during signaling tyrosine phosphorylation of but phosphorylation of the ERK and the of to increase the phosphorylation of S6 in response to data that the regulation of by leptin in cultured is via the ERK ERK signaling the of RSK, an mediator of D. H. J. N. J. J. Full Full Scopus Google Scholar). Thus, the that Tyr985 and ERK mediate a role for in the regulation of by we to the phosphorylation of S6 kinase by in the intracellular domain of LepRb the phosphorylation of via Tyr985 we a which cap-dependent and mediates D. H. J. N. J. J. Full Full Scopus Google Scholar, J. Cell. 2005; Full Full PubMed Scopus Google to the regulation of cap-dependent by LepRb analysis that of the intracellular domain of LepRb in increased cap-dependent and that this effect required Thus, these data that the regulation of and cap-dependent by LepRb via a in cells. In to the phosphorylation of STAT5 and S6 by leptin action in our results the phosphorylation of LepRb on intracellular tyrosine and demonstrate for in the regulation of STAT5 and S6 phosphorylation by LepRb phosphorylation in cultured cells. In these data for Tyr985 in the regulation of transcription by LepRb and for STAT3 in the attenuation of STAT5 action Whereas the physiologic of STAT3 by leptin in the C. Halaas J.L. Jr., M. Friedman J.M. Nat. 1996; PubMed Scopus Google and data an important role for STAT3 in the regulation of body energy homeostasis S.H. M. Y. Maratos-Flier E. Myers Jr., M.G. Nature. 2003; PubMed Scopus Google the physiologic role for STAT5 in leptin action D. J. Endocrinology. 2001; PubMed Google Scholar, C. Halaas J.L. Jr., M. Friedman J.M. Nat. 1996; PubMed Scopus Google Scholar). demonstrate the tyrosine phosphorylation of STAT5 in the of the role for STAT5 in leptin action in vivo. Whereas body of STAT5 a of due to the role by STAT5 in signaling by growth and M. S. C. Cell. Full Full PubMed Scopus Google Scholar, S. PubMed Scopus Google with central nervous system of STAT5 normal with the of obesity in the of elevated leptin Thus, STAT5 an important role in the regulation of energy homeostasis by leptin, and the of a role for STAT5 in the regulation of by leptin will be have the phosphorylation of Tyr1077 on the intracellular domain of LepRb and have that this to signaling by the phosphorylation and of others have that Tyr1077 a phosphorylation site on or Tyr1077 have to with and of the and at in or in P. J. S. H. D. J. J. 2005; PubMed Scopus (99) Google Scholar, D. J. L. J. K. S. J. J. Cell Sci. 2006; PubMed Scopus Google Scholar, S. D. A. J. J. 2000; PubMed Scopus Google Scholar, J. D. J. D. J. J. 2006; Scholar). Our data role for Tyr1077 in the attenuation of LepRb signaling for the of Tyr1077 increase the of LepRb signal that we have but only and STAT5-dependent The of decreased Tyr985 phosphorylation by suggests the that Tyr1077 a role in the phosphorylation of Whereas Tyr1077 and Tyr1138 of LepRb to acute the role of in results in a effect of Tyr1138 on and Tyr1077 mediates the important of of STAT5 is with on phosphorylation during receptor activation, and with the that leptin (e.g. of and in of LepRb STAT3 signaling Bjornholm M. Bates S.H. M. Myers Jr., M.G. Mol. Endocrinol. 2005; PubMed Scopus Google Scholar, S.H. M. Y. Maratos-Flier E. Myers Jr., M.G. Nature. 2003; PubMed Scopus Google Scholar, S.H. M. M. Maratos-Flier E. Myers Jr., M.G. Diabetes. 2004; PubMed Scopus Google Scholar). The of the STAT5 signal among as the and levels of these and STAT5 levels the and LepRb have the phosphorylation of S6 in the of Whereas S. D. A. J. J. 2000; PubMed Scopus Google the phosphorylation of and S6 kinase as well as S6 in response to leptin and in the of we to these phosphorylation in response to LepRb signaling that the intracellular domain of LepRb stimulates via Tyr985 and of the ERK in cultured cells. Tyr985 cap-dependent and the phosphorylation of RSK, the mediator of and cap-dependent translation. Thus, these data that Tyr985 of LepRb acts via the to control and cap-dependent in cells. the of hypothalamic the that the regulation of these signals in be that in cells. the regulation of protein kinase by leptin by of the Y. N. Kim A. J. P. B.B. Nature. 2004; PubMed Scopus Google Scholar, Y. Kim C. D. B.B. Nature. PubMed Scopus Google and demonstrate alternate regulation of by leptin C.B. K. S. J. Clin. Investig. 2005; PubMed Scopus Google Scholar). Thus, will be important to the mechanisms by which LepRb regulates these signals in our results the phosphorylation of STAT5 and S6 in the demonstrate the phosphorylation of Tyr1077 on and define for Tyr1077 and Tyr985 in the regulation of STAT5 and in cultured cells. the role for Tyr985 in the of LepRb to its role in ERK activation) and the of signaling for the of leptin, will be to understand the physiologic among these as well as the of Tyr1077 and STAT5 signaling to leptin for results to and for STAT5 and S. A. for