Heteromeric MAPPIT: a novel strategy to study modification-dependent protein-protein interactions in mammalian cells.
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.
Lemmens
Irma
I
Department of Medical Protein Research VIB09, Faculty of Medicine and Health Sciences, Ghent University, Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
Eyckerman
Sven
S
Zabeau
Lennart
L
Catteeuw
Dominiek
D
Vertenten
Els
E
Verschueren
Kristin
K
Huylebroeck
Danny
D
Vandekerckhove
Joël
J
Tavernier
Jan
J
D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D014408:Biomarkers, Tumor / Q000235:genetics; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D053614:Janus Kinase 2; D037181:Lectins, C-Type / Q000235:genetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D000073718:Pancreatitis-Associated Proteins; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D051381:Rats; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D017467:Receptors, Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D054411:Receptors, Leptin; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; D037181:Lectins, C-Type; D000073718:Pancreatitis-Associated Proteins; D011518:Proto-Oncogene Proteins; C000612141:REG3A protein, human; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D054411:Receptors, Leptin; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D051901:Smad4 Protein; C490811:Smad4 protein, rat; C494088:Stat3 protein, rat; D015534:Trans-Activators; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507927:Jak2 protein, rat; D053614:Janus Kinase 2