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Nucleic Acids Research, 2003, Vol. 31, No. 14 e75
© 2003 Oxford University Press

Heteromeric MAPPIT: a novel strategy to study modification-dependent protein–protein interactions in mammalian cells

Irma Lemmens, Sven Eyckerman, Lennart Zabeau, Dominiek Catteeuw, Els Vertenten, Kristin Verschueren1, Danny Huylebroeck1, Joël Vandekerckhove and Jan Tavernier*

Department of Medical Protein Research VIB09, Faculty of Medicine and Health Sciences, Ghent University, Albert Baertsoenkaai 3, B-9000 Ghent, Belgium and 1 Department of Developmental Biology VIB07 and Laboratory of Molecular Biology (Celgen), University of Leuven, Herestraat 49, B-3000 Leuven, Belgium

*To whom correspondence should be addressed. Tel: +32 9 331 33 01; Fax: +32 9 331 35 99; Email: jan.tavernier{at}rug.ac.be

We recently reported a two-hybrid trap for detecting protein–protein interactions in intact mammalian cells (MAPPIT). The bait protein was fused to a STAT recruitment-deficient, homodimeric cytokine receptor and the prey protein to functional STAT recruitment sites. In such a configuration, STAT-dependent responses can be used to monitor a given bait–prey interaction. Using this system, we were able to demonstrate both modification-independent and tyrosine phosphorylation- dependent interactions. Protein modification in this approach is, however, strictly dependent on the receptor-associated JAK tyrosine kinases. We have now extended this concept by using extracellular domains of the heteromeric granulocyte/macrophage colony-stimulating factor receptor (GM-CSFR). Herein, the bait was fused to the ßc chain and its modifying enzyme to the GM-CSFR{alpha} chain (or vice versa). We demonstrate several serine phosphorylation-dependent interactions in the TGFß/Smad pathway using the catalytic domains of the ALK4 or ALK6 serine/threonine kinase receptors. In all cases tested, STAT-dependent signaling was completely abolished when mutant baits were used wherein critical serine residues were replaced by alanines. This approach operates both in transient and stable expression systems and may not be limited to serine phosphorylation but has the potential for studying various different types of protein modification-dependent interactions in intact cells.


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