1. Interaction with factor associated with neutral sphingomyelinase activation, a WD motif-containing protein, identifies receptor for activated C-kinase 1 as a novel component of the signaling pathways of the p55 TNF receptor
- Author
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Sabine Mathieu, Marie-Luise Kruse, Waldemar Kolanus, Dieter Adam, Anna Ewgenjewna Tcherkasowa, Sabine Adam-Klages, Martin Krönke, and Katja Wiegmann
- Subjects
Scaffold protein ,Intracellular Fluid ,Repetitive Sequences, Amino Acid ,Immunoprecipitation ,Immunology ,Amino Acid Motifs ,Molecular Sequence Data ,Receptors, Cell Surface ,Biology ,Receptors for Activated C Kinase ,Receptors, Tumor Necrosis Factor ,Cell Line ,Jurkat Cells ,Antigens, CD ,Protein Interaction Mapping ,Immunology and Allergy ,Animals ,Humans ,Short linear motif ,Amino Acid Sequence ,Receptor ,Protein Kinase C ,Receptor for activated C kinase 1 ,Intracellular Signaling Peptides and Proteins ,Colocalization ,Proteins ,Transfection ,Precipitin Tests ,Peptide Fragments ,Cell biology ,Enzyme Activation ,Sphingomyelin Phosphodiesterase ,Biochemistry ,Receptors, Tumor Necrosis Factor, Type I ,COS Cells ,Signal transduction ,human activities ,HeLa Cells ,Protein Binding ,Signal Transduction - Abstract
Factor associated with neutral sphingomyelinase activation (FAN) represents a p55 TNFR (TNF-R55)-associated protein essential for the activation of neutral sphingomyelinase. By means of the yeast interaction trap system, we have identified the scaffolding protein receptor for activated C-kinase (RACK)1 as an interaction partner of FAN. Mapping studies in yeast revealed that RACK1 is recruited to the C-terminal WD-repeat region of FAN and binds to FAN through a domain located within WD repeats V to VII of RACK1. Our data indicate that binding of both proteins is not mediated by linear motifs but requires folding into a secondary structure, such as the multibladed propeller characteristic of WD-repeat proteins. The interaction of FAN and RACK1 was verified in vitro by glutathione S-transferase-based coprecipitation assays as well as in eukaryotic cells by coimmunoprecipitation experiments. Colocalization studies in transfected cells suggest that TNF-R55 forms a complex with FAN and that this complex recruits RACK1 to the plasma membrane. Furthermore, activation of N-SMase by TNF was strongly enhanced when RACK1, FAN, and a noncytotoxic TNF-R55 mutant were expressed concurrently, suggesting RACK1 as a modulator of N-SMase activation. Together, these findings implicate RACK1 as a novel component of the signaling pathways of TNF-R55.
- Published
- 2002