Heteromeric MAPPIT: a Novel Strategy to Study Modification-dependent Protein-protein Interactions in Mammalian Cells

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2003 Jul 11

PMID 12853652

Citations 10

Authors

Irma Lemmens

Sven Eyckerman

Lennart Zabeau

Dominiek Catteeuw

Els Vertenten

Kristin Verschueren

Danny Huylebroeck

Joel Vandekerckhove

Jan Tavernier

Affiliations

Soon will be listed here.

Abstract

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 (beta)c chain and its modifying enzyme to the GM-CSFRalpha chain (or vice versa). We demonstrate several serine phosphorylation-dependent interactions in the TGFbeta/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.

Citing Articles

Split Intein-Mediated Protein Ligation for detecting protein-protein interactions and their inhibition.

Yao Z, Aboualizadeh F, Kroll J, Akula I, Snider J, Lyakisheva A Nat Commun. 2020; 11(1):2440.

PMID: 32415080 PMC: 7229206. DOI: 10.1038/s41467-020-16299-1.

Straightforward Protein-Protein Interaction Interface Mapping via Random Mutagenesis and Mammalian Protein Protein Interaction Trap (MAPPIT).

Vyncke L, Masschaele D, Tavernier J, Peelman F Int J Mol Sci. 2019; 20(9).

PMID: 31027327 PMC: 6539206. DOI: 10.3390/ijms20092058.

Predictive and Experimental Approaches for Elucidating Protein-Protein Interactions and Quaternary Structures.

Nealon J, Philomina L, McGuffin L Int J Mol Sci. 2017; 18(12).

PMID: 29206185 PMC: 5751226. DOI: 10.3390/ijms18122623.

Application guide for omics approaches to cell signaling.

Yao Z, Petschnigg J, Ketteler R, Stagljar I Nat Chem Biol. 2015; 11(6):387-97.

PMID: 25978996 DOI: 10.1038/nchembio.1809.

Ryanodine receptors are targeted by anti-apoptotic Bcl-XL involving its BH4 domain and Lys87 from its BH3 domain.

Vervliet T, Lemmens I, Vandermarliere E, Decrock E, Ivanova H, Monaco G Sci Rep. 2015; 5:9641.

PMID: 25872771 PMC: 4397538. DOI: 10.1038/srep09641.

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