Neural Wiskott-Aldrich syndrome protein is recruited to rafts and associates with endophilin A in response to epidermal growth factor.
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.
Otsuki
Makiko
M
Department of Biochemistry, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.
Itoh
Toshiki
T
Takenawa
Tadaomi
T
D000217:Acyltransferases / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000187:drug effects / Q000502:physiology*; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000502:physiology; D017931:DNA Primers; D034281:Dynamins / Q000502:physiology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008322:Mammals; D021962:Membrane Microdomains / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014923:Wiskott-Aldrich Syndrome / Q000503:physiopathology*; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D002966:Clathrin; D017931:DNA Primers; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C107640:SH3GL2 protein, human; C495771:WASL protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D004815:Epidermal Growth Factor; D000217:Acyltransferases; C018764:2-acylglycerophosphate acyltransferase; D005982:Glutathione Transferase; D034281:Dynamins