A systematic scan of interactions with tyrosine motifs in the erythropoietin receptor using a mammalian 2-hybrid approach

Montoye, Tony; Lemmens, Irma; Catteeuw, Dominiek; Eyckerman, Sven; Tavernier, Jan

doi:10.1182/blood-2004-07-2733

Cited by 20 publications

(23 citation statements)

References 36 publications

(47 reference statements)

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“…Expression plasmid vectors pEF-FLAG-I/mSOCS2 and pEF-FLAG-I/mCIS were a gift from R. Starr (The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia). Generation of the prey constructs pMG2-CIS and pMG2-SOCS2 both containing part of the gp130 chain (aa 905-918) in duplicate was described elsewhere (Montoye et al, 2005).…”

Section: Constructsmentioning

confidence: 99%

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

Lavens

Montoye

Piessevaux

et al. 2006

Journal of Cell Science

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Hypothalamic leptin receptor signalling plays a central role in weight regulation by controlling fat storage and energy expenditure. In addition, leptin also has direct effects on peripheral cell types involved in regulation of diverse body functions including immune response, bone formation and reproduction. Previous studies have demonstrated the important role of SOCS3 (suppressor of cytokine signalling 3) in leptin physiology. Here, we show that CIS (cytokine-inducible SH2 protein) and SOCS2 can also interact with the leptin receptor. Using MAPPIT (mammalian protein-protein interaction trap), a cytokine receptor-based two-hybrid method operating in intact cells, we show specific binding of CIS with the conserved Y985 and Y1077 motifs in the cytosolic domain of the leptin receptor. SOCS2 only interacts with the Y1077 motif, but with higher binding affinity and can interfere with CIS and STAT5a prey recruitment at this site. Furthermore, although SOCS2 does not associate with Y985 of the leptin receptor, we find that SOCS2 can block interaction of CIS with this position. This unexpected interference can be explained by the direct binding of SOCS2 on the CIS SOCS box, whereby elongin B/C recruitment is crucial to suppress CIS activity.

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Section: Constructsmentioning

confidence: 99%

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

Lavens

Montoye

Piessevaux

et al. 2006

Journal of Cell Science

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“…Cells were simultaneously starved (removal of serum and hIL5 or mIL3) for 24 h and stimulated with 5 ng/ml hEpo or 100 ng/ ml leptin. Activation of the pGL2-SPI2.1-luciferase reporter (gift by Dr. Walter Becker [17]) was measured as described before [14]. All MAPPIT data in this manuscript are expressed as fold induction (stimulated/non-stimulated or NS) and are representative of at least three independent experiments.…”

Section: Cell Lines Electroporation Conditions Reporter Assays Expmentioning

confidence: 99%

“…All other members of the SOCS family were cloned as pMbc-prey fusion proteins. Murine CIS and SOCS2 were already cloned as pMG2-preys [14], and were transferred as EcoRI-XbaI fragments. Murine SOCS1 was amplified by PCR using primers 7 and 8 and was ligated into EcoRI-NotI opened pMbc vector.…”

Section: Constructsmentioning

confidence: 99%

“…A splice variant lacking exon 4 of murine SOCS7 (Dr. Martens N, personal communication) was amplified by PCR with primers 17 and 18 on cDNA of mouse hypothalamic N-38 cells (gift from Dr. Belsham) and inserted in the EcoRI-XbaI opened pMbc vector. pMbc-prey vectors carrying hPLCc(2· SH2) and hSTAT5a(F694) were obtained by EcoRI-XbaI insert swaps from the parental pMG2-based vectors [14]. The hPLCc(2· SH2) insert was also cloned in the pMET7-expression vector [13] by exchanging EcoRI-XbaI digested inserts.…”

Section: Constructsmentioning

confidence: 99%

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Analysis of leptin signalling in hematopoietic cells using an adapted MAPPIT strategy

et al. 2006

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The adipocyte-secreted hormone leptin participates in the regulation of hematopoiesis and enhances proliferation of hematopoietic cells. We used an adaptation of the MAPPIT mammalian two-hybrid method to study leptin signalling in a hematopoietic setting. We confirmed the known interactions of suppressor of cytokine signalling 3 (SOCS3) and STAT5 with the Y985 and Y1077 motifs of the leptin receptor, respectively. We also provide evidence for novel interactions at the Y1077 motif, including phospholipase C gamma and several members of the SOCS protein family, further underscoring the important role of the Y1077 motif in leptin signalling.

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“…This way, protein interactions can be studied with the LR in its normal oligomeric configuration [6]. In another MAPPIT setup, we can use the wild-type EpoR ( Figure 1D) [7]. Since the EpoR signals via STAT5 and not STAT3 and since the levels of STAT5 are low in the commonly used HEK-293T cells [human embryonic kidney cells expressing the…”

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confidence: 99%

MAPPIT: a versatile tool to study cytokine receptor signalling

Lemmens

Lievens

Tavernier

2008

Biochemical Society Transactions

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MAPPIT (mammalian protein-protein interaction trap) is a cytokine receptor-based two-hybrid method that operates in intact mammalian cells. A bait is fused C-terminally to a STAT (signal transducer and activator of transcription) recruitment-deficient receptor, whereas the prey is linked to functional STATbinding sites. When bait and prey interact a ligand-dependent complementation of the STAT recruitment deficiency occurs, leading to activation of a STAT-responsive reporter. MAPPIT is very well suited to study protein interactions involving activated cytokine receptors as the technique allows modification of the bait protein in a physiologically optimal environment. Cytokines and their receptorsCytokines are small messenger molecules that are involved in cell-cell communication and are critical to many vital processes such as embryonic development, growth, haemopoiesis and immune responses. They cannot cross the cellular membrane but make use of transmembrane receptors that capture cytokines and transmit their signal inside the cell. In this mini-review, we will mainly focus on homomeric type I cytokine receptors such as the EpoR (erythropoietin receptor), GHR (growth hormone receptor), LR (leptin receptor) and GCSFR (granulocyte colony-stimulating factor receptor). These cytokine receptors have no intrinsic kinase activity but make use of associated JAKs (Janus kinases). Upon ligand binding the JAKs are brought into close proximity enabling them to cross-phosphorylate each other. Subsequently the JAKs phosphorylate tyrosine residues in the cytoplasmic receptor tail, thereby creating docking sites for signalling molecules such as STAT (signal transducer and activator of transcription) proteins. After binding of these STATs they are phosphorylated in turn by the JAKs and translocate as dimers to the nucleus where they initiate transcription of target genes ( Figure 1A). Several mechanisms exist to modulate and terminate cytokine signalling. Among these, the SOCS (suppressor of cytokine signalling) protein Key words: cytokine receptor, mammalian protein-protein interaction trap (MAPPIT), signal transducer and activator of transcription (STAT), signal transduction, suppressor of cytokine signalling (SOCS), Toll-like receptor (TLR). Abbreviations used: βc, beta common; Epo, erythropoietin; EpoR, Epo receptor; GCSFR, granulocyte colony-stimulating factor receptor; GHR, growth hormone receptor; GM-CSF, granulocyte/macrophage colony-stimulating factor; gp130, glycoprotein 130; HEK-293T cells, human embryonic kidney cells expressing the large T-antigen of SV40 (simian virus 40); IRAK, interleukin-1-receptor-associated kinase; IRS4, insulin receptor substrate 4; JAK, Janus kinase; LR, leptin receptor; MAPPIT, mammalian protein-protein interaction trap; MyD88, myeloid differentiation primary response protein 88; Mal, MyD88 adaptor-like protein; PLCγ , phospholipase Cγ ; SH2, Src homology 2; CIS, cytokine-inducible SH2 domain-containing protein; SOCS, suppressor of cytokine signalling; STAT, signal transducer and ...

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A systematic scan of interactions with tyrosine motifs in the erythropoietin receptor using a mammalian 2-hybrid approach

Cited by 20 publications

References 36 publications

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

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

Analysis of leptin signalling in hematopoietic cells using an adapted MAPPIT strategy

MAPPIT: a versatile tool to study cytokine receptor signalling

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