Identification of SAP97 as an intracellular binding partner of TACE.
Tumor necrosis factor alpha converting enzyme (TACE) is the metalloprotease-disintegrin responsible for the ectodomain shedding of several proteins, including tumor necrosis factor alpha. Using the yeast two-hybrid system, we identified the scaffolding protein synapse associated protein 97 (SAP97) as a binding partner of the cytoplasmic domain of TACE. By deletions and site-directed mutagenesis, we demonstrated that this interaction involved the PDZ3 domain of SAP97 and the extreme C-terminal amino-acid sequence of TACE. This interaction as well as the identification of the specific domains involved was confirmed in vitro by affinity purification and in mammalian cells by co-immunoprecipitation and alteration of localization analyzed by immunofluorescence microscopy. In addition, confocal microscopy showed that endogenous TACE and SAP97 colocalized in some intracellular areas of COS-7 cells and CACO-2 cells. Furthermore, overexpression of SAP97, unlike that of a mutant form of SAP97 deleted for its PDZ3 domain, altered the ability of TACE to release its substrates. Altogether, these results demonstrate an interaction between TACE and SAP97, which may have a functional implication for the regulation of TACE shedding activity.
Peiretti
Franck
F
INSERM EPI 99-36, Laboratoire d'Hématologie, Faculté de Médecine, 27 Bd Jean Moulin, Marseilles 13385 Cedex 5, France. Franck.Peiretti@medecine.univ-mrs.fr
Deprez-Beauclair
Paule
P
Bonardo
Bernadette
B
Aubert
Helene
H
Juhan-Vague
Irene
I
Nalbone
Gilles
G
D051722:ADAM Proteins; D000072198:ADAM17 Protein; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D018938:Caco-2 Cells; D018076:DNA, Complementary / Q000378:metabolism; D000074288:Discs Large Homolog 1 Protein; D005434:Flow Cytometry; D017353:Gene Deletion; D015723:Gene Library; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins; D008666:Metalloendopeptidases / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000502:physiology*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; C513314:DLG1 protein, human; D018076:DNA, Complementary; D000074288:Discs Large Homolog 1 Protein; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D005982:Glutathione Transferase; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human