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First published online 9 May 2006
doi: 10.1242/jcs.02947

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A complex interaction pattern of CIS and SOCS2 with the leptin receptor

¹ Department of Medical Protein Research, Faculty of Medicine and Health Sciences, Flanders Interuniversity Institute for Biotechnology, VIB09, Ghent University, A. Baertsoenkaai 3, 9000 Ghent, Belgium
² Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University, 52074 Aachen, Germany

View larger version (36K): [in a new window]   Fig. 1. (A) Overview of LR signalling and its interaction partners. The murine LR carries three conserved tyrosines in its cytoplasmic tail at positions Y985, Y1077 and Y1138. JAK2 is constitutively associated with the LR at the conserved Box 1 and 2 motifs. Upon leptin stimulation, the JAKs become fully activated through cross-phosphorylation and phosphorylate the tyrosine residues in the receptor. STAT3 is recruited to the phosphorylated Y1138 docking site. Upon phosphorylation, STAT3 translocates as dimers to the nucleus, and induces specific gene expression. SHP2 is recruited to the Y985 docking site and couples to the Ras/Raf signalling cascade. The PI-3K pathway is also involved in LR signalling. Tyrosines Y985 and Y1077 take part in negative regulation of the leptin signal by binding SOCS3. PTP-1B is involved in negative regulation by dephosphorylation of JAK2 after internalisation of the LR complex. (B) MAPPIT principle. A particular bait protein is linked C-terminally to the chimeric receptor consisting of the extracellular part of the EpoR and the intracellular part of the LR with all three tyrosines mutated to phenylalanine, whereas the prey protein is fused to the STAT3 recruitment sites of the gp130 chain. The bait-receptor is incapable of recruiting STAT3 upon stimulation. However, when bait and prey proteins interact, the C-terminal part of the gp130 chain is brought in close proximity to the JAK kinases allowing its tyrosine phosphorylation and subsequent STAT3 activation. Read-out is based on a STAT3-responsive reporter construct. (C) GGS-MAPPIT. For GGS-MAPPIT the bait protein is attached C-terminally to a variant of the chimeric EpoR-LR receptor. The cytosolic domain of the LR following the JAK2 association domain is replaced by a GGS-array, preventing any background activation resulting from prey association with the LR-F3. (D) LR-MAPPIT. Here, the LR itself functions as bait protein. Owing to the Y1138F mutation, no STAT3 recruitment or activation can occur. Upon stimulation, the two membrane proximal tyrosines can nevertheless be phosphorylated by JAK2. Interaction of the prey protein with the LR, which may depend on phosphorylation, allows STAT3 activation and subsequent reporter induction.

View larger version (29K): [in a new window]   Fig. 2. Differential association of CIS and SOCS2 with the LR. (A) HEK293T cells were transiently co-transfected with plasmids encoding different pMET7-LR variants and the pMG2-CIS and pMG2-SOCS2 prey constructs, or with mock vector, combined with the pXP2d2-rPAP1-luci. The transfected cells were either stimulated for 24 hours with leptin or were left untreated (NS, not stimulated). Luciferase measurements were performed in triplicate. Data are expressed as mean fold induction (leptin stimulated/NS) + s.d. (B) LR expression levels were measured on the same transfected cells by incubation for 2 hours with leptin-SEAP fusion protein with or without a 100-fold excess of unlabelled leptin. Mean bound SEAP activity + s.d. of triplicate measurements is plotted. (C) Western blot analysis of CISprey and SOCS2prey expression. Expression of the FLAG-tagged fusion prey proteins in the same transfected cells was verified on lysates using anti-FLAG antibody.

View larger version (30K): [in a new window]   Fig. 3. SOCS2 interaction with the peptide matching the Y1077 motif of the LR is phosphorylation dependent. FLAG-tagged SOCS2 was expressed in HEK293T cells and lysates were incubated with phosphorylated or non-phosphorylated peptides corresponding to the Y1077 or Y985 motif. Immunoblotting with anti-FLAG antibody revealed specific interaction of SOCS2 with the tyrosine-phosphorylated Y1077 motif.

View larger version (44K): [in a new window]   Fig. 4. Stability of the CIS and SOCS2 interactions with the LR. (A) HEK293T cells were transiently co-transfected with plasmids encoding different pMET7-LR variants, the pMG2-CIS or pMG2-SOCS2 prey construct, pEF-FLAG-I/mCIS or pEF-FLAG-I/mSOCS2, or the appropriate amount of mock vector together with the pXP2d2-rPAP1-luci. The transfected cells were either stimulated for 24 hours with leptin or were left untreated (NS, not stimulated). Luciferase measurements were performed in triplicate. Data are expressed as mean fold induction (leptin stimulated/NS) + s.d. (B) Western blot analysis of CISprey, SOCS2prey, CIS and SOCS2 expression. Expression of the FLAG-tagged fusion proteins, CIS and SOCS2 was verified on lysates of transfected cells using anti-FLAG antibody.

View larger version (19K): [in a new window]   Fig. 5. GGS-MAPPIT analysis of CIS and SOCS2 interactions with the LR. (A,B) HEK293T cells (A) or TF-1 cells (B) were transiently co-transfected with plasmids encoding the chimeric bait constructs with the different LR motifs or with the FKBP12 control bait, and the pMG2-CIS, pMG2-SOCS2 prey constructs, combined with the pXP2d2-rPAP1-luci. The transfected cells were either stimulated for 24 hours with Epo or were left untreated (NS, not stimulated). Luciferase measurements were performed in triplicate. Data are expressed as mean fold induction (Epo stimulated/NS) + s.e.m. (C) FACS analysis shows the expression of the different chimeric GGS bait receptors in TF-1 cells. The grey filled curves represent the parental TF-1 cells; open lines, the transiently transfected TF-1 cells.

View larger version (12K): [in a new window]   Fig. 6. SOCS2 interferes with the association of a STAT5a prey at Y1077. (A,B) HEK293T cells (A) or TF-1 cells (B) were transiently co-transfected with the plasmid encoding the GGS bait construct with the Y1077 LR motif, the pMG2-STAT5aSH2 prey construct, the pMET7-FLAG-SOCS2 or pMET7-FLAG-SOCS2 box, or the appropriate amount of mock vector together with the pXP2d2-rPAP1-luci. The transfected cells were either stimulated for 24 hours with leptin or were left untreated (NS, not stimulated). Luciferase measurements were performed in triplicate. Data are expressed as mean fold induction (Epo stimulated/NS) + s.d.

View larger version (13K): [in a new window]   Fig. 7. SOCS2 interacts with CIS. (A) MAPPIT analysis. HEK293T cells were transiently co-transfected with plasmids encoding the chimeric EpoR-LR(F3) construct as a negative control or with the full-length (FL) CIS bait, and the pMG2-SOCS2 prey constructs, combined with the pXP2d2-rPAP1-luci reporter. The transfected cells were either stimulated for 24 hours with Epo or were left untreated (NS, not stimulated). Luciferase measurements were performed in triplicate. Data are expressed as mean fold induction (Epo stimulated/NS) + s.d. (B) Co-immunoprecipitation. HEK293T cells were transiently co-transfected with pMET7-Flag-SOCS2 and pMET7-Etag-CIS. Cell lysates were immunoprecipitated (IP) with anti-FLAG and subsequently immunoblotted (IB) with anti-E. (C) SOCS2 interacts with the SOCS box of CIS. HEK293T cells were transiently co-transfected with plasmids encoding the chimeric EpoR-LR(F3) construct as a negative control or with the CIS SOCS box bait, and the pMG2-SOCS2 prey construct or the appropriate amount of mock vector, combined with the pXP2d2-rPAP1-luci. The transfected cells were either stimulated for 24 hours with Epo or were left untreated (NS, not stimulated). Luciferase measurements were performed in triplicate. Data are expressed as mean fold induction (Epo stimulated/NS) + s.d.

View larger version (27K): [in a new window]   Fig. 8. (A) Generation of a SOCS2 mutant deficient in elongin B/C binding. HEK293T cells were transiently transfected with the pMET7TAP2-SOCS2 and pMET7TAP2-SOCS2(LC-QQ) constructs. Cell lysates were purified using the TAP2 tag and loaded on a polyacrylamide gel and silverstained. From a parallel experiment, the indicated bands were identified as cullin 5, elongin B and elongin C by mass spectrometry. (B) The SOCS2(LC-QQ) mutant still binds CIS. HEK293T cells were transiently co-transfected with plasmids encoding the chimeric EpoR-LR(F3) construct as a negative control or with the CIS SOCS box bait, and the pMG2-SOCS2 or pMG2-SOCS2 (LC-QQ) prey constructs, combined with the pXP2d2-rPAP1-luci. The transfected cells were either stimulated for 24 hours with Epo or were left untreated (NS, not stimulated). Luciferase measurements were performed in triplicate. Data are expressed as mean fold induction (Epo stimulated/NS) + s.d. (C) Differential effects of the SOCS2(LC-QQ) mutant on CIS interaction with the LR recruitment motifs. HEK293T cells were transiently co-transfected with plasmids encoding different pMet7-LR variants, the pMG2-CIS prey construct, pMet7-FLAG-SOCS2 or pMet7-FLAG-SOCS2(LC-QQ), or the appropriate amount of mock vector together with the pXP2d2-rPAP1-luci. The transfected cells were either stimulated for 24 hours with leptin or were left untreated (NS, not stimulated). Luciferase measurements were performed in triplicate. Data are expressed as mean fold induction (leptin stimulated/NS) + s.d.