Title
Design and application of a cytokine-receptor-based interaction trap
Author
Faculty/Department
Faculty of Pharmaceutical, Biomedical and Veterinary Sciences . Biomedical Sciences
Publication type
article
Publication
London ,
Subject
Human medicine
Source (journal)
Nature cell biology. - London
Volume/pages
3(2001) :12 , p. 1114-1119
ISSN
1465-7392
ISI
000172603700017
Carrier
E
Target language
English (eng)
Full text (Publishers DOI)
Abstract
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 molecules1. Among these, signal transducers and activators of transcription (STATs) are important in the direct transmission of signals to the nucleus2. 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 Song3, 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.
E-info
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