Inhibitory roles for SHP-1 and SOCS-3 following pituitary proopiomelanocortin induction by leukemia inhibitory factor - PubMed (original) (raw)
Inhibitory roles for SHP-1 and SOCS-3 following pituitary proopiomelanocortin induction by leukemia inhibitory factor
C Bousquet et al. J Clin Invest. 1999 Nov.
Abstract
Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that stimulates the hypothalamo-pituitary-adrenal (HPA) axis through JAK-STAT activation. We show here that LIF-induced JAK2 and STAT3 tyrosine phosphorylation is transient, disappearing within 20 and 40 minutes, respectively. LIF activates the SH2 domain-containing tyrosine phosphatase, SHP-1, with maximal stimulation observed at 30 minutes. SHP-1 is constitutively associated with JAK2, and LIF induces recruitment of phosphorylated STAT3 to this complex. Overexpression of wild-type or dominant negative forms of SHP-1 shows decreased or increased LIF-induced proopiomelanocortin (POMC) promoter activity, respectively. LIF-induced JAK2 and STAT3 dephosphorylation is delayed until after 60 minutes in cells that overexpress the mutant SHP-1. In addition, SOCS-3, a negative regulator of LIF signaling, binds to JAK2 after 60 minutes of LIF stimulation, after which the complex is degraded by the proteasome. SOCS-3 overexpression blocks LIF-induced JAK2 tyrosine phosphorylation, confirming a role for SOCS-3 in deactivating JAK2 by direct association. Using SOCS-3 fusion proteins, we also define regions of the SOCS-3 protein that are critical for inhibition of LIF-induced POMC promoter activity. Corticotrophic signaling by LIF is thus subject to 2 forms of negative autoregulation: dephosphorylation of JAK2 and STAT3 by the SHP-1 tyrosine phosphatase, and SOCS-3-dependent inactivation of JAK2.
Figures
Figure 1
Time course of LIF-induced JAK2 and STAT3 phosphorylation. AtT20 cells were treated for the indicated times with 1 nM LIF. After cell lysis and immunoprecipitation with polyclonal antibody recognizing either JAK2 (a) or STAT3 (b), immunoprecipitates were separated on a 7.5% polyacrylamide-SDS gel. Immunoblotting was then performed with a monoclonal antiphosphotyrosine antibody. To verify equal protein loading in each lane, membranes were stripped and immunoblotted with antibodies recognizing either JAK2 (a) or STAT3 (b).
Figure 2
SHP-1 forms a constitutive complex with JAK2, and the recruitment of STAT3 is LIF-inducible. AtT20 cells were treated for the indicated times with 1 nM LIF. After cell lysis, 500 μg of protein extracts was immunoprecipitated with an mAb recognizing SHP-1 (a, lanes 1–8 ; b, lanes 1–5) or with a mouse preimmune serum (PI). To verify the identity of JAK2 and STAT3, 50 μg of protein extracts was immunoprecipitated with polyclonal antibodies recognizing JAK2 (a, lane 9) and STAT3 (b, lane 6). Immunoprecipitates were separated on a 7.5% polyacrylamide-SDS gel, and immunoblotting was performed with a polyclonal anti-JAK2 antibody (a) or a polyclonal anti-STAT3 antibody (b).
Figure 3
SHP-1 catalytic activity is LIF-inducible. AtT20 cells were stimulated for the indicated times with 1 nM LIF. After cell lysis and immunoprecipitation with mAb recognizing SHP-1, immunoprecipitates were subjected to a phosphatase assay using [32P]poly (Glu, Tyr). The results are representative of 3 separate experiments and are expressed as percentage of control (100% = no treatment).
Figure 4
LIF-induced dephosphorylations of JAK2 and STAT3 are delayed in the mutant SHP-1–overexpressing transformants. Mutant SHP-1–overexpressing cells were treated for the indicated times with 1 nM LIF. After cell lysis, immunoprecipitation and Western blots were performed as described in Figure 1, a and b (a and b, respectively). Quantification of kinetics of LIF-induced JAK2 (c) and STAT3 (d) tyrosine phosphorylation, observed in 3 different experiments from 3 independent clones of mock or mutant SHP-1–transfected cells, was performed. *P < 0.05%, LIF-treated versus control untreated cells. †P < 0.05%, SHP-1 mutant-transfected versus mock-transfected cells.
Figure 5
SHP-1 catalytic activity influences LIF-induced POMC promoter activity. Mock-, wild-type (WT) SHP-1–, or mutant (mut) SHP-1–transfected AtT20 cells were transiently transfected with rat POMC promoter fused to the luciferase reporter. Cells were then treated for 6 hours with 1 nM LIF (a) or 5 mM dibutyryl cAMP (b). Luciferase activity was measured as described in Methods. The results are representative of 3 separate experiments using 3 independent clones and are expressed as fold-induction above control.
Figure 6
SOCS-3 protein is LIF–inducible, binds in vivo to JAK2, and blocks LIF-induced JAK2 tyrosine phosphorylation. (a) AtT20 cells were treated for the indicated times with 1 nM LIF. After cell lysis, half the lysates were used in immunoprecipitations using a polyclonal antibody recognizing COOH-terminal of SOCS-3; the immunoprecipitates were separated on a 15% SDS-polyacrylamide gel and then immunoblotted with a polyclonal antibody recognizing NH2-terminal of SOCS-3. To verify equal loading of proteins, the other half of the lysates were used in immunoprecipitations using a polyclonal anti-JAK2 antibody, run on a 7.5% SDS-polyacrylamide gel and blotted with an anti-JAK2 antibody. (b) AtT20 cells were pretreated with 1% DMSO (vehicle) (lanes 2–4) or with 10 μM LLnL (dissolved in DMSO) for 15 minutes (lanes 1, 5–8), and then treated with 1 nM LIF for the indicated time (lanes 2–8). The experiment was also performed as described in a. (c) AtT20 cells were treated for the indicated times with 1 nM LIF. After cell lysis, half of the lysates were used in immunoprecipitations using a polyclonal antibody recognizing COOH-terminal of SOCS-3; the immunoprecipitates were separated on a 7.5% SDS-polyacrylamide gel and then immunoblotted with a polyclonal anti-JAK2 antibody. To verify equal loading of proteins, the other half of the lysates were used in immunoprecipitations using a polyclonal anti-JAK2 antibody, run on a 7.5% SDS-polyacrylamide gel, and blotted with an anti-JAK2 antibody. (d) Treatment of mock-transfected AtT20 cells (lanes 4–6) or SOCS-3–overexpressing cells (lanes 1–3) were performed as indicated. Immunoprecipitation was then performed using an anti-JAK2 antibody. The immune complexes were run on a 7.5% SDS-polyacrylamide gel, and a Western blot was performed using an antiphosphotyrosine antibody. To verify equal loading of proteins, the membrane was stripped and subjected to a Western blot using an anti-JAK2 antibody.
Figure 7
Definition of the functional domains of SOCS-3. (a) Description of the different domains of SOCS-3. (b) Fusion protein corresponding to a NH2-terminal tag from the pcDNA3.1/His vector (Invitrogen) fused to different portions of SOCS-3. These constructs were transiently transfected with rat POMC promoter fused to the luciferase reporter in AtT20 cells. Cells were then treated for 6 hours with 1 nM LIF. Luciferase activity was measured as described in Methods. The results are representative of 3 separate experiments and are expressed as percentage of control (100% = full-length SOCS-3).
Figure 8
Model for SHP-1 and SOCS-3 regulation of LIF transduction signal. (a) SHP-1 is constitutively associated with JAK2 in a tyrosine phosphorylation–independent manner. (b) After LIF stimulation, heterodimerization occurs between LIFRα and gp130 subunits, which activates JAK2 activity. JAK2 tyrosine phosphorylates itself as well as gp130, which allows recruitment of STAT3 through its SH2 domain onto gp130 docking sites. STAT3 is also tyrosine-phosphorylated by JAK2 and therefore activated. STAT3 then homodimerizes or heterodimerizes with STAT1, which accounts for its nuclear translocation. (c) SHP-1 might be activated because its partners (JAK2 and STAT3) become tyrosine-phosphorylated. Increased tyrosine phosphatase activity of SHP-1 directly dephosphorylates JAK2, therefore inhibiting its kinase activity and resulting in dephosphorylation of its substrates. Once gp130, JAK2, and STAT3 are dephosphorylated, STAT3 leaves the receptor complex, which also returns to the basal state. Activation of SHP-1 is maximal at 30 minutes of LIF stimulation. (d) Sustained deactivation of LIF signaling also occurs through LIF-induced SOCS-3 protein synthesis and binding to JAK2, which appears maximally between 40 and 60 minutes of LIF stimulation. SOCS-3 inhibits JAK2 kinase activity and is then degraded after 90 minutes.
References
- Hibi M, Nakajima K, Hirano T. IL-6 cytokine family and signal transduction: a model of the cytokine system. J Mol Med. 1996;74:1–12. -PubMed
- Chesnokova V, Auernhammer CJ, Melmed S. Murine LIF gene disruption attenuates the hypothalamo-pituitary-adrenal axis stress response. Endocrinology. 1998;139:2209–2216. -PubMed
- Wang Z, Ren SG, Melmed S. Hypothalamic and pituitary LIF gene expression in-vivo: a novel endotoxin-inducible neuroendocrine interface. Endocrinology. 1996;137:2947–2953. -PubMed
- Bousquet C, Ray DW, Melmed S. A common proopiomelanocortin element mediates LIF and CRH transcriptional synergy. J Biol Chem. 1997;272:10551–10557. -PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Miscellaneous