Leptin Stimulates Both JAK2-dependent and JAK2-independent Signaling Pathways

Zhiqin Li,Liangyou Rui,Lin Jiang

doi:10.1074/jbc.m805545200

Zhiqin Li, Liangyou Rui + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m805545200

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Abstract

Leptin controls body weight by activating the long form of the leptin receptor (LEPRb). Janus kinase 2 (JAK2) is associated with LEPRb and autophosphorylates in response to leptin. JAK2 also phosphorylates LEPRb, STAT3, and multiple other downstream molecules. Surprisingly, here we show that JAK2 is not required for leptin stimulation of STAT3 phosphorylation. Leptin time- and dose-dependently stimulated tyrosine phosphorylation of STAT3 in both human and mouse JAK2-null cells. Leptin also increased the viability of JAK2-null cells. Overexpression of c-Src or Fyn, two Src family members, promoted STAT3 phosphorylation, whereas inhibition of the endogenous Src family members by either pharmacological inhibitors or dominant negative Src(K298M) decreased the ability of leptin to stimulate the phosphorylation of STAT3 and ERK1/2. Leptin also stimulated tyrosine phosphorylation of kinase-inactive JAK2(K882E) in JAK2-null cells. Overexpression of JAK2(K882E) enhanced the ability of leptin to stimulate STAT3 phosphorylation in JAK2-null cells. Tyr1138 in LEPRb was required for leptin-stimulated phosphorylation of STAT3 but not JAK2(K882E). These data suggest that leptin stimulates non-JAK2 tyrosine kinase(s), including the Src family members, which phosphorylate JAK2, STAT3, and other molecules downstream of LEPRb. JAK2 mediates leptin signaling by both phosphorylating its substrates and forming a signaling complex as a scaffolding/adaptor protein. The non-JAK2 kinase(s) and JAK2 may act coordinately and synergistically to mediate leptin response.

Highlights

Humans [1,2,3,4,5,6,7]
Leptin Stimulates Janus kinase 2 (JAK2)-independent Tyrosine Phosphorylation of STAT3 in ␥2ALEPRb Cells—To determine whether JAK2 is required for leptin signaling, LEPRb was stably expressed in JAK2-deficient ␥2A human fibroblast cells (␥2ALEPRb) [33]
SHP2 mediates leptin stimulation of the MAPK pathway, which is involved in the regulation of body weight [38, 39]

Summary

EXPERIMENTAL PROCEDURES

Material—␣pSTAT3 (pY705), ␣STAT3, and ␣ERK2 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). PC12LRb cells were grown on collagen-coated dishes in DMEM supplemented with 25 mM glucose, 100 units/ml penicillin, 100 ␮g/ml streptomycin, 10% heat-inactivated horse serum, and 5% FBS. PC12LRb cells were cultured for 6 days in DMEM supplemented with 25 mM glucose, 2% horse serum, 1% FBS, 100 ng/ml NGF, 100 units/ml penicillin, and 100 ␮g/ml streptomycin. Protein A-agarose bead-bound JAK2 was washed extensively with a kinase reaction buffer (50 mM HEPES, pH 7.6, 10 mM MnCl2, 100 mM NaCl, 0.5 mM dithiothreitol, 1 mM Na3VO4) and incubated at room temperature for 30 min in the kinase reaction buffer supplemented with [␥-32P]ATP (10 ␮Ci), 20 ␮M cold ATP, 10 ␮g/ml aprotinin, and 10 ␮g/ml leupeptin. Protein A-agarose bead-bound JAK2 was washed with lysis buffer, boiled for 5 min in the SDS-PAGE sample buffer, resolved by SDS-PAGE, and transferred onto nitrocellulose membranes. The absorbance at 570 nm (A570) of the cells prior to H2O2 treatment was converted to 100% viability, and all other A570 was normalized to these values as a percentage of viability

RESULTS

LEPRb was stably introduced into

Blot Blot αFlag

DISCUSSION