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Phosphorylation of Jak2 on Ser(523) inhibits Jak2-dependent leptin receptor signaling.

ABSTRACT: The leptin receptor, LRb, and other cytokine receptors are devoid of intrinsic enzymatic activity and rely upon the activity of constitutively associated Jak family tyrosine kinases to mediate intracellular signaling. In order to clarify mechanisms by which Jak2, the cognate LRb-associated Jak kinase, is regulated and mediates downstream signaling, we employed tandem mass spectroscopic analysis to identify phosphorylation sites on Jak2. We identified Ser523 as the first-described site of Jak2 serine phosphorylation and demonstrated that this site is phosphorylated on Jak2 from intact cells and mouse spleen. Ser523 was highly phosphorylated in HEK293 cells independently of LRb-Jak2 activation, suggesting a potential role for the phosphorylation of Ser523 in the regulation of LRb by other pathways. Indeed, mutation of Ser523 sensitized and prolonged signaling by Jak2 following activation by the intracellular domain of LRb. The effect of Ser523 on Jak2 function was independent of Tyr570-mediated inhibition. Thus, the phosphorylation of Jak2 on Ser523 inhibits Jak2 activity and represents a novel mechanism for the regulation of Jak2-dependent cytokine signaling.

SUBMITTER: Ishida-Takahashi R

PROVIDER: S-EPMC1489076 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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Phosphorylation of Jak2 on Ser(523) inhibits Jak2-dependent leptin receptor signaling.

Ishida-Takahashi Ryoko R Rosario Felicia F Gong Yusong Y Kopp Keely K Stancheva Zlatina Z Chen Xiaohong X Feener Edward P EP Myers Martin G MG

Molecular and cellular biology 20060601 11

The leptin receptor, LRb, and other cytokine receptors are devoid of intrinsic enzymatic activity and rely upon the activity of constitutively associated Jak family tyrosine kinases to mediate intracellular signaling. In order to clarify mechanisms by which Jak2, the cognate LRb-associated Jak kinase, is regulated and mediates downstream signaling, we employed tandem mass spectroscopic analysis to identify phosphorylation sites on Jak2. We identified Ser523 as the first-described site of Jak2 se ...[more]

PMID: 16705160

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Project description:ObjectiveDifferentiated articular chondrocytes express a functional bisoform of the leptin receptor (LRb); however, leptin-LRb signaling in these cells is poorly understood. We hypothesized that leptin-LRb signaling in articular chondrocytes functions to modulate canonical Wnt signaling events by altering the expression of Frizzled (FZD) receptors.MethodsHuman chondrocyte cell lines and primary articular chondrocytes were grown in serum containing growth media for 24h, followed by a media change to Dulbecco's modified Eagle's medium (DMEM) containing 1% Nutridoma-SP to obtain a serum-deficient environment for 24h before treatment. Treatments included recombinant human leptin (10-100nM), recombinant human IL-6 (0.3-3nM), or recombinant human erythropoietin (Epo) (10mU/ml). Cells were harvested 30min-48h after treatment and whole cell lysates were analyzed using immunoblots or luciferase assays.ResultsTreatment of cells with leptin resulted in activation of Janus kinase 2 (JAK2) and subsequent phosphorylation of specific tyrosine residues on LRb, followed by dose- and time-dependent increases in the expression of Frizzled-1 (FZD1) and Frizzled-7 (FZD7). Leptin-mediated increases in the expression of FZD1 were blocked by pre-treatment with the protein synthesis inhibitor cycloheximide or the JAK2 inhibitor AG490. Experiments using a series of hybrid Epo extracellular domain-leptin intracellular domain receptors (ELR) harboring mutations of specific tyrosine residues in the cytoplasmic tail showed that increases in the expression of FZD1 were dependent on LRb-mediated phosphorylation of STAT3, but not ERK1/2 or STAT5. Leptin pre-treatment of chondrocytes prior to Wnt3a stimulation resulted in an increased magnitude of canonical Wnt signaling.ConclusionThese experiments show that leptin-LRb signaling in articular chondrocytes modulates expression of canonical Wnt signaling receptors and suggests that direct cross-talk between these pathways is important in determining chondrocyte homeostasis.

Project description:BackgroundNutrition intake during growth strongly influences ovarian follicle development and egg laying in chicken hens, yet the underlying endocrine regulatory mechanism is still poorly understood. The relevant research progress is hindered by difficulties in detection of leptin gene and its expression in the chicken. However, a functional leptin receptor (LEPR) is present in the chicken which has been implicated to play a regulatory role in ovarian follicle development and egg laying. The present study targeted LEPR by immunizing against its extracellular domain (ECD), and examined the resultant ovarian follicle development and egg-laying rate in chicken hens.MethodsHens that have been immunized four times with chicken LEPR ECD were assessed for their egg laying rate and feed intake, numbers of ovarian follicles, gene expression profiles, serum lipid parameters, as well as STAT3 signaling pathway.ResultsAdministrations of cLEPR ECD antigen resulted in marked reductions in laying rate that over time eventually recovered to the levels exhibited by the Control hens. Together with the decrease in egg laying rate, cLEPR-immunized hens also exhibited significant reductions in feed intake, plasma concentrations of glucose, triglyceride, high-density lipoprotein, and low-density lipoprotein. Parallelled by reductions in feed intake, mRNA gene expression levels of AgRP, orexin, and NPY were down regulated, but of POMC, MC4R and lepR up-regulated in Immunized hen hypothalamus. cLEPR-immunization also promoted expressions of apoptotic genes such as caspase3 in theca and fas in granulosa layer, but severely depressed IGF-I expression in both theca and granulosa layers.ConclusionsImmunization against cLEPR ECD in egg-laying hens generated antibodies that mimic leptin bioactivity by enhancing leptin receptor transduction. This up-regulated apoptotic gene expression in ovarian follicles, negatively regulated the expression of genes that promote follicular development and hormone secretion, leading to follicle atresia and interruption of egg laying. The inhibition of progesterone secretion due to failure of follicle development also lowered feed intake. These results also demonstrate that immunization against cLEPR ECD may be utilized as a tool for studying bio-functions of cLEPR.

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Phosphorylation of Jak2 on Ser(523) inhibits Jak2-dependent leptin receptor signaling.

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Phosphorylation of Jak2 on Ser(523) inhibits Jak2-dependent leptin receptor signaling.

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