Leptin stimulates both JAK2-dependent and JAK2-independent signaling pathways

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.

FIGURE 1.

JAK2 is not required for leptin stimulation of tyrosine phosphorylation of STAT3 in γ2A cells. A, γ2A, γ2A^LEPRb, and γ2A^LEPRb/JAK2 cells were treated without or with 100 ng/ml leptin for 10 min. The cell extracts were immunoblotted with anti-phospho-STAT3 (Tyr(P)⁷⁰⁵)(αpSTAT3), αSTAT3, or αJAK2 as indicated. B, γ2A^LEPRb cells were treated with leptin for 10 min at the concentrations of 0, 1, 5, 10, 50, or 100 ng/ml. The cell extracts were immunoblotted with αpSTAT3 or αSTAT3, respectively. C, γ2A^LEPRb cells were treated with 100 ng/ml leptin for 0, 5, 10, 30, 60, and 120 min. The cell extracts were immunoblotted with αpSTAT3 or αSTAT3, respectively. D, γ2A^LEPRb and γ2A^{LEPRb(Y1138F)} cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoblotted with αpSTAT3 or αSTAT3.

FIGURE 2.

Leptin stimulates tyrosine phosphorylation of kinase-inactive JAK2(K882E) in γ2A cells. A, γ2A^LEPRb, γ2A^LEPRb/K882E, or γ2A^LEPRb/JAK2 cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoprecipitated (IP) with anti-JAK2 antibody (αJAK2) and immunoblotted with anti-phosphotyrosine antibody (αPY). The same blots were reprobed with anti-phospho-JAK2 (pY813) (αpY813) or αJAK2. In parallel experiments, cell extracts were immunoblotted with anti-phospho-JAK2 (pY1007/1008) (αpY1007/8) or αJAK2. B, γ2A^LEPRb/K882E or γ2A^LEPRb/JAK2 cells were treated with 100 ng/ml leptin for 0, 5, 10, 30, and 60 min. The cell extracts were immunoblotted with αpY813, αpY1007/8, or αJAK2 as indicated. C, γ2A^LEPRb, γ2A^LEPRb/K882E, γ2A^Y1138F, and γ2A^Y1138F/K882E cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoblotted with αpY1007/8 or αJAK2. In parallel experiments, the cell extracts were immunoprecipitated with αJAK2, immunoblotted with αPY, and reprobed with αJAK2. D, γ2A^LEPRb, γ2A^LEPRb/K882E, and γ2A^LEPRb/JAK2 cells were treated with leptin for 10 min at the indicated concentrations. The cell extracts were immunoblotted with αpSTAT3 or αSTAT3, respectively.

FIGURE 3.

JAK2 is not required for leptin stimulated phosphorylation of STAT3 and ERK1/2 in MEF^KO cells. A, MEF^KO-LEPRb, MEF^{KO-LEPRb/K882E}, and MEF ^{KO-LEPRb/JAK2} cells were treated with 200 ng/ml leptin for 10 min. The cell extracts were immunoblotted with the indicated antibodies. B, MEF^{KO-LEPRb/K882E} and MEF ^{KO-LEPRb/JAK2} cells were treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoprecipitated with αJAK2. Immunopurified JAK2 was subjected to in vitro kinase assays in the presence of [γ-³²P]ATP, resolved by SDS-PAGE, and transferred onto nitrocellulose membranes. JAK2 was visualized by autoradiography and subsequently immunoblotted with αJAK2.

FIGURE 4.

Src inhibitors inhibit leptin signaling in multiple cell types. A, γ2A^LEPRb cells were pretreated with Me₂SO or SU6656 (10 μm) for 4 h and treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoblotted with αpSTAT3 or αSTAT3. B, γ2A^LRb/K882E cells were pretreated with Me₂SO or SU6656 (10 μm) for 4 h, and treated with 100 ng/ml leptin for 10 min. The cell extracts were immunoblotted with αpSTAT3 or αSTAT3. In parallel, the cell extracts were immunoprecipitated (IP) with αJAK2 and immunoblotted with αPY. The same blots were reprobed with αJAK2. C, MEF^{KO-LEPRb/K882E} cells were pretreated with Me₂SO or SU6656 (10 μm) for 4 h and treated with 100 ng/ml leptin for an additional 10 min. The cell extracts were immunoblotted with the indicated antibodies. D, representative images of differentiated PC12^LEPRb cells. PC12^LEPRb cells were incubated with 100 ng/ml NGF for 6 days to induce neuronal differentiation. E, PC12^LRb cells were differentiated into neurons with 100 ng/ml NGF treatment for 6 days. Differentiated cells were pretreated with dimethyl sulfoxide, (DMSO), SU6656 (10 μm), or PP2 (10 μm) for 4 h, and treated with 100 ng/ml leptin for an additional 10 min. The cell extracts were immunoblotted with the indicated antibodies.

FIGURE 5.

The Src family members phosphorylate both STAT3 and JAK2. A, c-Src was transiently coexpressed with STAT3 in HEK293^LEPRb cells. The cell extracts were immunoblotted with αpSTAT3, αSTAT3, anti-phospho-Src (pTyr416) (αpSrc), or αSrc as indicated. B, FLAG-tagged Fyn was transiently coexpressed with STAT3 in HEK293^LEPRb cells. The cell extracts were immunoblotted with αpSTAT3, αSTAT3, or αFLAG, respectively. C, c-Src was transiently coexpressed with kinase inactive-JAK2(K882E) in HEK293^LEPRb cells. The cell extracts were immunoprecipitated with αJAK2 and immunoblotted with αPY. The same blots were reprobed with αJAK2. The cell extracts were immunoblotted with αpSrc or αSrc. D, STAT3 expression vectors (0.4 μg) were transiently cotransfected with either c-Src (2 ng) or Fyn expression plasmids (2 ng) into HEK293^LEPRb cells. 48 h after transfection, the cells were deprived of serum overnight and treated with leptin for 10 min. The cell extracts were immunoblotted with either αpSTAT3 or αSTAT3, respectively. E, STAT3 expression vectors (0.4 μg) were transiently cotransfected with kinase-inactive Src(K298M) expression plasmids (from 0.4 to 1.6 μg) into HEK293^LEPRb cells. The cells were treated with leptin for 10 min, and the cell extracts were immunoblotted with either αpSTAT3 or αSTAT3 as described for D. Con, control.

FIGURE 6.

The non-JAK2 pathway(s) promote cell survival. A, γ2A^LEPRb cells were pretreated with or without 100 ng/ml leptin for 8 h and then treated with 600 mm H₂O₂ for 0, 24, 48, 72, 96, or 120 h in the absence or presence of 100 ng/ml leptin. Cell viability was measured by MTT assays. B, MEF^KO-LEPRb cells were pretreated with or without 100 ng/ml leptin for 8 h and then treated with 100 mm H₂O₂ for 0 or 144 h in the absence or presence of 100 ng/ml leptin. Cell viability was measured by MTT assays. *, p < 0.05. Con, control.

FIGURE 7.

JAK2 is required for growth hormone-stimulated phosphorylation of STAT3. A, γ2A^GHR and γ2A^GHR/JAK2 cells were treated with GH (8 × 10^–3 unit/ml) for 10 min. The cell extracts were immunoblotted with αpSTAT3 or αSTAT3. In parallel experiments, cell extracts were immunoprecipitated (IP) with αJAK2 and immunoblotted with αPY. The same blots were reprobed with αJAK2. B, γ2A^GHR/K882E and γ2A^GHR/JAK2 cells were treated with GH (8 × 10^–3 unit/ml) for 10 min. The cell extracts were immunoprecipitated with αJAK2 and immunoblotted with αPY. The same blots were immunoblotted with αJAK2.