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.
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.
Mesh Terms:
Amino Acid Motifs, Amino Acid Sequence, Animals, Antigens, CD, COS Cells, Cell Line, Enzyme Activation, Hela Cells, Humans, Intracellular Fluid, Intracellular Signaling Peptides and Proteins, Jurkat Cells, Molecular Sequence Data, Peptide Fragments, Precipitin Tests, Protein Binding, Protein Interaction Mapping, Protein Kinase C, Proteins, Receptors, Cell Surface, Receptors, Tumor Necrosis Factor, Receptors, Tumor Necrosis Factor, Type I, Repetitive Sequences, Amino Acid, Signal Transduction, Sphingomyelin Phosphodiesterase
Amino Acid Motifs, Amino Acid Sequence, Animals, Antigens, CD, COS Cells, Cell Line, Enzyme Activation, Hela Cells, Humans, Intracellular Fluid, Intracellular Signaling Peptides and Proteins, Jurkat Cells, Molecular Sequence Data, Peptide Fragments, Precipitin Tests, Protein Binding, Protein Interaction Mapping, Protein Kinase C, Proteins, Receptors, Cell Surface, Receptors, Tumor Necrosis Factor, Receptors, Tumor Necrosis Factor, Type I, Repetitive Sequences, Amino Acid, Signal Transduction, Sphingomyelin Phosphodiesterase
J. Immunol.
Date: Nov. 01, 2002
PubMed ID: 12391233
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