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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Motifs
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Amino Acid Sequence
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Animals
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Antigens, CD / chemistry
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Antigens, CD / genetics
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Antigens, CD / physiology*
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COS Cells
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Cell Line
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Enzyme Activation / genetics
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Enzyme Activation / immunology
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HeLa Cells
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Humans
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Intracellular Fluid / metabolism
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Intracellular Signaling Peptides and Proteins
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Jurkat Cells
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Molecular Sequence Data
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Peptide Fragments / chemistry
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Peptide Fragments / genetics
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Peptide Fragments / metabolism
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Precipitin Tests
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Protein Binding / genetics
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Protein Binding / immunology
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Protein Interaction Mapping / methods
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Protein Kinase C / chemistry
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Protein Kinase C / genetics
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Protein Kinase C / metabolism*
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Proteins / chemistry
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Proteins / genetics
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Proteins / metabolism*
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Receptors for Activated C Kinase
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Receptors, Cell Surface / chemistry
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Receptors, Cell Surface / genetics
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Receptors, Cell Surface / metabolism*
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Receptors, Tumor Necrosis Factor / chemistry
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Receptors, Tumor Necrosis Factor / genetics
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Receptors, Tumor Necrosis Factor / physiology*
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Receptors, Tumor Necrosis Factor, Type I
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Repetitive Sequences, Amino Acid
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Signal Transduction / genetics
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Signal Transduction / immunology*
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Sphingomyelin Phosphodiesterase / chemistry
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Sphingomyelin Phosphodiesterase / genetics
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Sphingomyelin Phosphodiesterase / metabolism*
Substances
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Antigens, CD
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Intracellular Signaling Peptides and Proteins
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NSMAF protein, human
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Peptide Fragments
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Proteins
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Receptors for Activated C Kinase
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Receptors, Cell Surface
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Receptors, Tumor Necrosis Factor
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Receptors, Tumor Necrosis Factor, Type I
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Protein Kinase C
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Sphingomyelin Phosphodiesterase