Neural Wiskott-Aldrich Syndrome Protein Is Recruited to Rafts and Associates with Endophilin A in Response to Epidermal Growth Factor

• Published in: The Journal of biological chemistry Vol. 278; no. 8; pp. 6461 - 6469
• Main Authors: Otsuki, Makiko, Itoh, Toshiki, Takenawa, Tadaomi
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 21.02.2003
• Subjects: Acyltransferases - metabolism; Adaptor Proteins, Signal Transducing; Animals; Carrier Proteins - drug effects; Carrier Proteins - physiology; Cell Fractionation; Centrifugation, Density Gradient; Chlorocebus aethiops; Clathrin - physiology; COS Cells; DNA Primers; Dynamins - physiology; Epidermal Growth Factor - pharmacology; Glutathione Transferase - genetics; HeLa Cells; Humans; Kinetics; Mammals; Membrane Microdomains - drug effects; Membrane Microdomains - physiology; Membrane Microdomains - ultrastructure; Nerve Tissue Proteins - drug effects; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - physiology; Recombinant Fusion Proteins - metabolism; Transfection; Wiskott-Aldrich Syndrome - physiopathology; Wiskott-Aldrich Syndrome Protein, Neuronal
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M207433200

Neural Wiskott-Aldrich syndrome protein (N-WASP) has been implicated in endocytosis; however, little is known about how it interacts functionally with the endocytic machinery. Sucrose gradient fractionation experiments and immunofluorescence studies with anti-N-WASP antibody revealed that N-WASP is recruited together with clathrin and dynamin, which play essential roles in clathrin-mediated endocytosis, to lipid rafts in an epidermal growth factor (EGF)-dependent manner. Endophilin A (EA) binds to dynamin and plays an essential role in the fission step of clathrin-mediated endocytosis. In the present study, we show that the Src homology 3 (SH3) domain of EA associates with the proline-rich domain of N-WASP and dynamin in vitro . Co-immunoprecipitation assays with anti-N-WASP antibody revealed that EGF induces association of N-WASP with EA. In addition, EA enhances N-WASP-induced actin-related protein 2/3 (Arp2/3) complex activation in vitro . Immunofluorescence studies revealed that actin accumulates at sites where N-WASP and EA are co-localized after EGF stimulation. Furthermore, studies of overexpression of the SH3 domain of EA indicate that EA may regulate EGF-induced recruitment of N-WASP to lipid rafts. These results suggest that, upon EGF stimulation, N-WASP interacts with EA through its proline-rich domain to induce the fission step of clathrin-mediated endocytosis.