Design and application of a cytokine-receptor-based interaction trap.
Ligand-induced clustering of type I cytokine receptor subunits leads to trans-phosphorylation and activation of associated cytosolic janus kinases (JAKs). In turn, JAKs phosphorylate tyrosine residues in the receptor tails, leading to recruitment and activation of signalling molecules. Among these, signal transducers and activators of transcription (STATs) are important in the direct transmission of signals to the nucleus. Here, we show that incorporation of an interaction trap in a signalling-deficient receptor allows the identification of protein-protein interactions, using a STAT-dependent complementation assay. Mammalian protein-protein interaction trap (MAPPIT) adds to existing yeast two-hybrid procedures, as originally explored by Fields and Song, and permits the detection of both modification-independent and of phosphorylation-dependent interactions in intact human cells. We also demonstrate that MAPPIT can be used to screen complex complementary DNA libraries, and using this approach, we identify cytokine-inducible SH2-containing protein (CIS) and suppressor of cytokine signalling-2 (SOCS-2) as interaction partners of the phosphotyrosine 402 (Tyr 402)-binding motif in the erythropoietin receptor (EpoR). Importantly, this approach places protein-protein interactions in their normal physiological context, and is especially applicable to the in situ analysis of signal transduction pathways.
Eyckerman
S
S
Flanders Interuniversity Institute for Biotechnology, VIB09, Department of Medical Protein Research, Faculty of Medicine and Health Sciences, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
Verhee
A
A
der Heyden
J V
JV
Lemmens
I
I
Ostade
X V
XV
Vandekerckhove
J
J
Tavernier
J
J
D000818:Animals; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D015723:Gene Library; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D018121:Receptors, Cytokine / Q000235:genetics*; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D050794:STAT1 Transcription Factor; D015398:Signal Transduction; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains / Q000502:physiology
D004268:DNA-Binding Proteins; D011506:Proteins; D018121:Receptors, Cytokine; D017467:Receptors, Erythropoietin; D012097:Repressor Proteins; C106643:SOCS2 protein, human; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; C491409:Socs2 protein, mouse; C494080:Stat1 protein, mouse; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators; D019000:Phosphotyrosine