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.
Tcherkasowa
Anna Ewgenjewna
AE
Institut für Immunologie and I Medizinische Klinik, Christian-Albrechts-Universität Kiel, Germany.
Adam-Klages
Sabine
S
Kruse
Marie-Luise
ML
Wiegmann
Katja
K
Mathieu
Sabine
S
Kolanus
Waldemar
W
Krönke
Martin
M
Adam
Dieter
D
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D019556:COS Cells; D002460:Cell Line; D004789:Enzyme Activation / Q000235:genetics / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D025941:Protein Interaction Mapping / Q000379:methods; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D047888:Receptors, Tumor Necrosis Factor, Type I; D020449:Repetitive Sequences, Amino Acid; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D015703:Antigens, CD; D047908:Intracellular Signaling Peptides and Proteins; C102036:NSMAF protein, human; D010446:Peptide Fragments; D011506:Proteins; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D011493:Protein Kinase C; D013108:Sphingomyelin Phosphodiesterase