Design and application of a cytokine-receptor-based interaction trap.

Nature cell biology (2002-01-10)
S Eyckerman, A Verhee, J V der Heyden, I Lemmens, X V Ostade, J Vandekerckhove, J Tavernier
RESUMO

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.

MATERIAIS
Número do produto
Marca
Descrição do produto

Sigma-Aldrich
Puromycin dihydrochloride, Ready Made Solution, from Streptomyces alboniger, 10 mg/mL in H2O, 0.2 μm filtered