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

2003; Elsevier BV; Volume: 278; Issue: 8 Linguagem: Inglês

10.1074/jbc.m207433200

1083-351X

Makiko Otsuki, Toshiki Itoh, Tadaomi Takenawa,

Force Microscopy Techniques and Applications

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 activationin 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. 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 activationin 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. Four principal steps in vesicular membrane traffic can be distinguished. First, the coated membrane is invaginated by the assembly of coat proteins and formation of the coated pits. This is followed by a pinching off of the coated vesicles, transport of the vesicles to the appropriate acceptor membrane, and fusion of the vesicles with this membrane. Numerous proteins and lipids are involved in and regulated properly in a molecular cascade (1Sorkin A. J. Cell Sci. 2000; 113: 4375-4376Google Scholar). In mammalian cells, endocytosis of vesicles can occur via clathrin-coated pits, a clathrin-independent pathway, or caveolae. It is thought that endocytosis occurs at rafts, which are plasma membrane domains enriched in cholesterol and sphingolipids (2Simons K. Ikonen E. Nature. 1997; 387: 569-572Google Scholar, 3Ikonen E. Curr. Opin. Cell Biol. 2001; 13: 470-477Google Scholar). Rafts also contain glycosylphosphatidylinositol-anchored proteins and various transmembrane proteins (4Skibbens J.E. Roth M.G. Matlin K.S. J. Cell Biol. 1989; 108: 821-832Google Scholar, 5Sargiacomo M. Sudol M. Tang Z. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Google Scholar, 6Danielsen E.M. Biochemistry. 1995; 34: 1596-1605Google Scholar). Activation of receptors by ligands results in internalization of receptor complexes, and these complexes are rapidly recycled from endosomes back to the cell surface (7Sorkin A. Carpenter G. J. Biol. Chem. 1991; 266: 23453-23460Google Scholar, 8Lamaze C. Baba T. Redelmeier T.E. Schmid S.L. Mol. Biol. Cell. 1993; 4: 715-727Google Scholar). Endocytosis of epidermal growth factor (EGF) 1The abbreviations used are: EGF, epidermal growth factor; N-WASP, neural Wiskott-Aldrich syndrome protein; EGFR, epidermal growth factor receptor; EA, endophilin A; SH3, Src homology 3; PIP2, phosphatidylinositol 4,5-bisphosphate; LPA-AT, lysophosphatidic acid acyltransferase; LPA, lysophosphatidic acid; PA, phosphatidic acid; Arp2/3, actin-related protein 2/3; GST, glutathioneS-transferase; GFP, green fluorescence protein; YFP, yellow fluorescence protein; EEA1, early endosome antigen 1; CDX, methyl-β-cyclodextrin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; DTT, dithiothreitol; VCA, verprolin-homology-cofilin-homology-acidic; MES, 4-morpholineethanesulfonic acid; PC, phosphatidylcholine; PI, phosphatidylinositol 1The abbreviations used are: EGF, epidermal growth factor; N-WASP, neural Wiskott-Aldrich syndrome protein; EGFR, epidermal growth factor receptor; EA, endophilin A; SH3, Src homology 3; PIP2, phosphatidylinositol 4,5-bisphosphate; LPA-AT, lysophosphatidic acid acyltransferase; LPA, lysophosphatidic acid; PA, phosphatidic acid; Arp2/3, actin-related protein 2/3; GST, glutathioneS-transferase; GFP, green fluorescence protein; YFP, yellow fluorescence protein; EEA1, early endosome antigen 1; CDX, methyl-β-cyclodextrin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; DTT, dithiothreitol; VCA, verprolin-homology-cofilin-homology-acidic; MES, 4-morpholineethanesulfonic acid; PC, phosphatidylcholine; PI, phosphatidylinositol receptor served as a model system for studies of ligand-dependent receptor trafficking for many years (9Sorkin A. Waters C.M. Bioessays. 1993; 15: 375-382Google Scholar). EGF-activated receptors are internalized via the clathrin-coated pit pathway (10Sorkin A. Mazzotti M. Sorkina T. Scotto L. Beguinot L. J. Biol. Chem. 1996; 271: 13377-13384Google Scholar) through interactions with the clathrin adaptor complex AP2 that recruits the clathrin triskelion (11Sorkin A. Carpenter G. Science. 1993; 261: 612-615Google Scholar). Various accessory proteins, such as Eps15 and Epsin, are recruited to the AP2-clathrin complex and subsequently form an endocytic clathrin coat (12Brodin L. Low P. Shupliakov O. Curr. Opin. Neurobiol. 2000; 10: 312-320Google Scholar). The clathrin-adaptor coats undergo rearrangement, resulting in invagination of the coated membrane, i.e. the clathrin-coated pit (13Springer S. Spang A. Schekman R. Cell. 1999; 97: 145-148Google Scholar). For constriction from shallow to deeply invaginated coated pits and fission, endophilin A (EA) and dynamin are recruited to the rim of the polymerizing clathrin coat (14Hannah M.J. Schmidt A.A. Huttner W.B. Annu. Rev. Cell Dev. Biol. 1999; 15: 733-798Google Scholar). EA has lysophosphatidic acid acyltransferase (LPA-AT) activity that catalyzes transfer of fatty acids from co-enzyme A to LPA, thereby generating phosphatidic acid (PA) (15Schmidt A. Wolde M. Thiele C. Fest W. Kratzin H. Podtelejnikov A.V. Witke W. Huttner W.B. Soling H.D. Nature. 1999; 401: 133-141Google Scholar). This shift in the biophysical properties of phospholipids in the cytoplasmic leaflet of the membrane bilayer would cause inward distortion of the luminal leaflets and subsequent membrane fission (16Scales S.J. Scheller R.H. Nature. 1999; 401: 123-124Google Scholar). This finding was confirmed by a recent study (17Ringstad N. Gad H. Low P. Di Paolo G. Brodin L. Shupliakov O. De Camilli P. Neuron. 1999; 24: 143-154Google Scholar) showing that microinjection of an antibody against EA into lamprey reticulospinal synapses interferes with synaptic vesicle recycling and clathrin-coated vesicle formation. The 100-kDa GTPase dynamin acts as an essential factor in the fission stage of clathrin-mediated endocytosis (18Sever S. Damke H. Schmid S.L. Traffic. 2000; 1: 385-392Google Scholar). Dynamin assembles at the site of fission and garrotes the membrane in a process driven by GTP hydrolysis (19Takei K. McPherson P.S. Schmid S.L. De Camilli P. Nature. 1995; 374: 186-190Google Scholar). It was reported that purified dynamin causes vesiculation of liposomes in vitro in a GTP-dependent fashion (20Sever S. Muhlberg A.B. Schmid S.L. Nature. 1999; 398: 481-486Google Scholar). The newly formed detached clathrin-coated vesicles were internalized and moved through the cytoplasm to early endosomes. Vesicular trafficking at the plasma membrane would require rearrangement of the cortical actin filaments to remove the barrier to vesicular fusion or budding events (21Qualmann B. Kessels M.M. Kelly R.B. J. Cell Biol. 2000; 150: 111-116Google Scholar). Actin filaments and actin-based motor proteins play an essential role in endocytosis in yeast (22Kubler E. Riezman H. EMBO J. 1993; 12: 2855-2862Google Scholar, 23Benedetti H. Raths S. Crausaz F. Riezman H. Mol. Biol. Cell. 1994; 5: 1023-1037Google Scholar, 24Munn A.L. Stevenson B.J. Geli M.I. Riezman H. Mol. Biol. Cell. 1995; 6: 1721-1742Google Scholar, 25Geli M.I. Riezman H. Science. 1996; 272: 533-535Google Scholar). It was also reported that treatment of several actin-disrupting agents inhibit receptor-mediated endocytosis in mammalian cells (26Gottlieb T.A. Ivanov I.E. Adesnik M. Sabatini D.D. J. Cell Biol. 1993; 120: 695-710Google Scholar, 27Lamaze C. Fujimoto L.M. Yin H.L. Schmid S.L. J. Biol. Chem. 1997; 272: 20332-20335Google Scholar). These data suggest that the actin cytoskeleton plays an essential role in endocytosis. The actin-related protein 2/3 (Arp2/3) complex enhances the nucleation and polymerization of actin filaments to promote filament assembly in vivo (28Machesky L.M. Atkinson S.J. Ampe C. Vandekerckhove J. Pollard T.D. J. Cell Biol. 1994; 127: 107-115Google Scholar,29Mullins R.D. Heuser J.A. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6181-6186Google Scholar). Neural Wiskott-Aldrich syndrome protein (N-WASP) plays an essential role in Arp2/3-dependent actin dynamics by enhancing Arp2/3 complex-induced nucleation of actin filaments (30Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Google Scholar). N-WASP contains a WASP homology domain, a Cdc42 binding domain, a proline-rich domain, two G-actin-binding verprolin-homology domains, a cofilin-homology domain, and a carboxyl-terminal acidic segment (31Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Google Scholar). The verprolin-homology-cofilin-homology-acidic (VCA) domain of N-WASP is an essential minimal region for activation of Arp2/3 complex. At rest, N-WASP is thought to be an auto-inhibited through an intramolecular interaction between its Cdc42 interaction and COOH-terminal domains (32Prehoda K.E. Scott J.A. Mullins R.D. Lim W.A. Science. 2000; 290: 801-806Google Scholar). Binding of GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate (PIP2) to N-WASP causes a conformational change in N-WASP that allows the VCA domain to interact with the Arp2/3 complex and initiate actin polymerization (33Rohatgi R. Ho H.Y. Kirschner M.W. J. Cell Biol. 2000; 150: 1299-1310Google Scholar). The yeast homologue of WASPs, Las17, was implicated in endocytosis (34Naqvi S.N. Zahn R. Mitchell D.A. Stevenson B.J. Munn A.L. Curr. Biol. 1998; 8: 959-962Google Scholar), and lymphocytes from WASP knockout mice exhibited reduced actin polymerization and defective T cell receptor endocytosis (35Zhang J. Shehabeldin A. da Cruz L.A. Butler J. Somani A.K. McGavin M. Kozieradzki I. dos Santos A.O. Nagy A. Grinstein S. Penninger J.M. Siminovitch K.A. J. Exp. Med. 1999; 190: 1329-1342Google Scholar). These data suggest that N-WASP plays various roles at many steps of endocytosis. Here we show that EGF induces recruitment of N-WASP from the cytoplasm to rafts. We also show that N-WASP interacts with EA through its proline-rich domain, playing an essential role in the fission step of clathrin-mediated endocytosis. For expression in mammalian cells, several N-WASP constructs, inducing full-length N-WASP and proline-rich domain (amino acids 271–385)-deleted N-WASP (N-WASPΔP), were constructed in pcDL-SRα and pEYFP (Clontech). EA3 cDNA (gift from Dr. T. Endo, University of Chiba, Japan) was amplified by PCR with primers that introduced 5′ BamHI and 3′ HindIII sites. PCR products were cloned into theBamHI-HindIII sites of pCMV-tag3B (Clontech) and into the BglII andHindIII sites of pEGFP (Clontech) to produce proteins tagged with Myc and green fluorescence protein (GFP). To obtain the glutathione S-transferase (GST) fusion protein of EA (GST-EA), full-length EA3 cDNA was subcloned into pCMV-tag3B, cut with BamHI and XhoI, and inserted into pGEX-2T (Amersham Biosciences). GST fusion proteins of EA3, GST-EAΔSH3 (amino acids 1–254) and GST-SH3 (amino acids 278–348), were produced by in-frame insertion of the PCR-amplified fragment corresponding to each sequence into theBamHI-EcoRI sites of pGEX-2T. Anti-Myc antibody was purchased from Santa Cruz Biotechnology, and the anti-clathrin heavy chain antibody (Ab-1) was from Oncogene. The anti-dynamin antibody (mouse clone 41), the anti-caveolin 1, the anti-Rab5, and the anti-phosphotyrosine antibody (PY20) were from Transduction Laboratories. The anti-N-WASP antibody was prepared as described (36Miki H. Takenawa T. Biochem. Biophys. Res. Commun. 1998; 243: 73-78Google Scholar). The secondary antibodies linked to peroxidase were from Cappel. The secondary antibodies linked to fluorescein, Texas Red, and Cy5 were from Molecular Probes. HeLa cells plated on 150-mm dishes were serum-starved for 24 h and then stimulated with 100 ng/ml EGF (Invitrogen). After two washes with ice-cold phosphate-buffered saline (PBS), HeLa cells were scraped into 800 μl of 500 mmsodium carbonate (pH 11.0). The cell lysates were extruded through a 23-gauge needle 10 times and then sonicated for 5 min in a sonicator bath. One milliliter of the homogenate was then adjusted to 45% sucrose by the addition of 1 ml of 90% sucrose prepared in MES buffered saline (MBS) (25 mm MES, pH 6.5, 90% sucrose, 150 mm NaCl) and placed at the bottom of an ultracentrifuge tube (14 × 89 mm, Beckman Instruments). A discontinuous sucrose gradient was formed (2 ml of 5% sucrose/3 ml of 25% sucrose/3 ml of 35% sucrose; all in MBS containing 250 mm sodium carbonate) and centrifuged at 100,000 × g for 3 h in an SW41 rotor (Beckman Instruments). Proteins at the 5/25, 25/35, and 35/45% interfaces were collected and separated by SDS-PAGE (10% acrylamide) followed by Western blot analysis with the ECL detection system (Pierce). HeLa cells, A431 cells, and COS7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. For immunofluorescence microscopy, 1 × 105 cells were plated on 2% gelatin-coated coverslips in 35-mm dishes. Cells were serum-starved for 24 h and then stimulated with 100 ng/ml EGF and fixed with 3.7% formaldehyde in PBS. Fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min and then incubated with primary antibodies for 60 min. After washing, cells were incubated with secondary antibodies. To stain EGF receptors, biotin-conjugated anti-EGF receptor (EGFR1, Biogenesis) was used as primary antibody and ultra avidin-rhodamine (Leinca Technologies, Inc.) as secondary antibody. To visualize actin filaments, rhodamine-conjugated phalloidin (Molecular Probes) was also added during the incubation with secondary antibodies. After a 30-min incubation, coverslips were washed and mounted on glass slides. Cells were observed with a confocal laser scanning microscope (MRC 1024; Bio-Rad). HeLa cells plated on coverslips were incubated with 10 mmmethyl-β-cyclodextrin (CDX, Sigma) in the serum-free DMEM, 50 mm HEPES (pH 7.6) at 37 °C for 1 h. After incubation in serum-free DMEM without CDX for 15 min, they cells were stimulated with 100 ng/ml EGF and fixed. COS7 cells were transfected in Opti-MEM (Invitrogen) using 6 μl of LipofectAMINE (Invitrogen) and 3 μg of plasmid DNA per 35-mm dish according to the manufacturer's instructions. The DNA/LipofectAMINE was maintained on the cells for 4 h, and the medium was then exchanged with maintenance medium. Twenty-four hours after transfection, cells were subjected to EGF internalization assay. For EGF stimulation, transfected cells were cultured for 2 h in maintenance medium and then for 24 h in serum-free DMEM. To obtain cell lysates, 20 μg of recombinant plasmid was mixed with 107 cells, and the mixtures were subjected to electroporation with a Gene Pulser (Bio-Rad). EGF-treated or transfected cells in 100-mm dishes were washed twice with ice-cold PBS and lysed in 200 μl of TGH buffer (50 mm HEPES (pH 7.6), 50 mmNaCl, 5 mm EDTA, 1 mm orthovanadate, 10% glycerol, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride). After addition of 800 μl of IP buffer (50 mmHEPES (pH 7.6), 50 mm NaCl, 5 mm EDTA, 1 mm orthovanadate, 10% glycerol, 1 mmphenylmethylsulfonyl fluoride), lysates were extruded 10 times through a 23-gauge needle and centrifuged at 100,000 × g for 5 min. Supernatants were mixed with 10 μg of anti-N-WASP antibody or anti-Myc antibody (Santa Cruz Biotechnology) for 2 h. As a negative control, normal mouse IgG (Santa Cruz Biotechnology) or pre-immune rabbit serum was used. Then protein A- and G-agarose beads (Pierce) were added, and the mixtures were incubated for 1 h. Immunoprecipitates were washed three times with IP buffer and then analyzed by Western blotting. GST fusion proteins were expressed inEscherichia coli JM109 and purified from E. colilysates with glutathione-Sepharose beads (Amersham Biosciences) according to standard methods. GST fusion proteins were eluted with 50 mm glutathione in 10 mm HEPES (pH 7.6). Glutathione in the samples was removed by dialysis with IP buffer. Protein concentrations were measured by Bradford assays with bovine serum albumin as a standard. Twenty micrograms of the GST fusion proteins were immobilized on glutathione-Sepharose beads and mixed with HeLa cell lysates or COS7 cell lysates. After the beads were washed with IP buffer, they were suspended in SDS sample buffer and subjected to SDS-PAGE and Western blot analysis. Actin was purified from rabbit skeletal muscle, and monomeric actin was isolated by gel filtration on Superdex 200 (Amersham Biosciences) in G buffer (2 mm Tris-HCl (pH 8.0), 0.2 mm ATP, 0.2 mm CaCl2, 0.5 mm DTT). Arp2/3 complex was purified from bovine brain extracts as described previously (37Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Google Scholar). N-WASP was prepared with a baculovirus system as described previously (30Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Google Scholar). A GST fusion protein containing the verplorin homology, cofilin homology, acidic region (VCA domains) and the GST fusion protein of the Ash/Grb2 NH2-terminal SH3 domain was used as a positive control (36Miki H. Takenawa T. Biochem. Biophys. Res. Commun. 1998; 243: 73-78Google Scholar). All GST fusion proteins used in the actin polymerization assay were eluted with 50 mm glutathione in 10 mmHEPES (pH 7.6). For the actin polymerization assay, phospholipid vesicles were prepared from phosphatidylcholine, egg (PC) (Avanti), phosphatidylinositol (PI) (Doosan Sedary Research Laboratories), PIP2 (Cell Signals, Inc), phosphatidic acid, dioleoyl (PA) (Sigma), and lysophosphatidic acid, oleoyl (LPA) (Sigma). Chloroform-dissolved phospholipids were mixed in the appropriate ratios (PC/PI (50:50), PC/PI/PIP2(48:48:4), PC/PI/PA (48:48:4), or PC/PI/LPA (48:48:4)) and dried under nitrogen. The dried lipid mixture was resuspended in lipid buffer (10 mm HEPES (pH 7.7), 100 mm NaCl, 5 mm EGTA, 50 mm sucrose) to a final concentration of 4 mm and then extruded through a 100-nm pore polycarbonate filter (Avanti) with the Mini-Extruder. The lipid vesicles were used at a final concentration of 2 or 5 μmin the assay. Actin polymerization was measured as the change in the fluorescence intensity of pyrene-labeled actin as described previously (36Miki H. Takenawa T. Biochem. Biophys. Res. Commun. 1998; 243: 73-78Google Scholar). To follow actin polymerization with purified components, pyrene-labeled G-actin or unlabeled G-actin was isolated by incubation with freshly thawed proteins in G buffer (5 mm Tris-HCl (pH 8.0), 0.2 mm CaCl2, 0.2 mm ATP, 0.2 mm DTT) for 12 h at 4 °C followed by removal of residual F-actin by centrifugation at 40,000 × g for 1 h. Polymerization reaction mixtures contained 60 nm Arp2/3 complex and various proteins and lipids in 95 μl of the assay buffer (10 mmHEPES (pH 7.6), 100 mm KCl, 1 mmMgCl2, 0.1 mm EDTA, 1 mm DTT) and were preincubated for 5 min. The reaction was initiated by adding a 5-μl mixture of 40 μm unlabeled actin, 4 μm labeled actin, and 4 mm ATP to the preincubated reaction mixtures. The change in fluorescence was measured at 407 nm with excitation at 365 nm in a fluorescence spectrometer (Jasco). All kinetic analyses were performed with the software provided by the manufacturer. To study the function of N-WASP, we examined localization of N-WASP during EGF-induced endocytosis. It is believed that endocytosis occurs at particular sites in the plasma membrane called rafts (2Simons K. Ikonen E. Nature. 1997; 387: 569-572Google Scholar, 3Ikonen E. Curr. Opin. Cell Biol. 2001; 13: 470-477Google Scholar). EGF-induced clathrin-coated vesicles are sorted primarily to early endosomes. Both rafts and early endosomes are rich in cholesterol and sphingolipid and can be separated from the cytosol by discontinuous sucrose gradient fractionation (38Song K.S. Scherer P.E. Tang Z. Okamoto T. Li S. Chafel M. Chu C. Kohtz D.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 15160-15165Google Scholar). The components of rafts and early endosomes were fractionated from lysates of EGF-stimulated HeLa cells to determine the EGF-dependent distribution of N-WASP. Raft components are enriched at the 5/25% interface of the gradient, whereas the early endosome marker early endosome antigen-1 (EEA1) is found at the 25/35% interface. The rest of the cell lysates, mainly cytosolic proteins, are localized at the 35/45% interface (38Song K.S. Scherer P.E. Tang Z. Okamoto T. Li S. Chafel M. Chu C. Kohtz D.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 15160-15165Google Scholar, 39Gagescu R. Demaurex N. Parton R.G. Hunziker W. Huber L.A. Gruenberg J. Mol. Biol. Cell. 2000; 11: 2775-2791Google Scholar). In the present study, caveolins, marker proteins of rafts, were detected in all fractions including the 5/25% interface (Fig. 1 A). This distribution did not change after EGF stimulation (data not shown). Rab5, a small GTPase associated with EEA1, was localized at the 25/35% interface (Fig. 1 A). These data indicate that raft components were localized at the 5/25% interface, and those of early endosomes were at the 25/35% interface. Hereafter, the collections of proteins at the 5/25, 25/35, and 35/45% interfaces will be referred to as the rafts fraction, EEA1 fraction, and cytosol fraction, respectively. Activated EGF-receptors were detected with an anti-phosphotyrosine antibody and used as a marker of internalized clathrin-coated vesicles. Phosphorylated receptors were detected in the rafts fraction at 1.5 min after EGF treatment and were then detected in the EEA1 fraction after 15 and 30 min (Fig. 1 B). This change in distribution supports the idea that EGF receptors are internalized from rafts to early endosomes via a clathrin-dependent pathway. Clathrin, localized at early endosomes and cytosol in unstimulated cells, was recruited to rafts and then subsequently moved to early endosomes similar to EGF receptors. In contrast to clathrin, dynamin was not detected in early endosomes prior to EGF stimulation. Dynamin was also recruited to rafts after EGF stimulation, but this recruitment was later than that of clathrin. This time difference supports the hypothesis that dynamin is recruited to the edge of the neck of the clathrin-coated pit (14Hannah M.J. Schmidt A.A. Huttner W.B. Annu. Rev. Cell Dev. Biol. 1999; 15: 733-798Google Scholar). Like dynamin and many other proteins, N-WASP was detected in the cytosol fraction. After EGF treatment, N-WASP was recruited to rafts and then internalized into early endosomes (Fig. 1 B). The results of sucrose fractionation indicate that after EGF treatment, N-WASP was recruited approximately at the same time as clathrin. To visualize the recruitment of N-WASP induced by EGF, HeLa cells were stained with anti-N-WASP antibody (Fig. 1 C) before and after EGF stimulation. In serum-starved cells, N-WASP was localized in a dot-like pattern around the perinuclear region. At 1.5–5 min after EGF stimulation, N-WASP was located throughout the cytoplasm, and a portion was present at the plasma membrane (Fig. 1 C,left). The translocation of N-WASP was well observed at the cell periphery (Fig. 1 C), where membrane ruffles were induced upon EGF stimulation. After 15 and 30 min, N-WASP had returned to its resting localization pattern. CDX is a useful tool to extract cholesterol from biological membranes with high preference over other lipid species (40Rodal S.K. Skretting G. Garred O. Vilhardt F. van Deurs B. Sandvig K. Mol. Biol. Cell. 1999; 10: 961-974Google Scholar, 41Subtil A. Gaidarov I. Kobylarz K. Lampson M.A. Keen J.H. McGraw T.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6775-6780Google Scholar). The major component of rafts is cholesterol, and therefore, CDX treatment should disrupt rafts in living cells. In the present study, CDX treatment inhibited uptake of the Texas Red-labeled EGF into HeLa cells (data not shown), suggesting that clathrin-mediated endocytosis is dependent upon rafts. In CDX-treated cells, N-WASP remained at the perinuclear region after EGF stimulation (Fig. 1 C,right), and no translocation to the plasma membrane was observed. These data suggest that EGF induces the recruitment of N-WASP to rafts in the plasma membrane and confirm the sucrose fractionation data. To examine whether N-WASP co-localizes with EGF receptor (EGFR) in rafts, EGF-treated A431 cells were double-stained with anti-EGFR and anti-N-WASP antibodies. At 1.5–5 min after EGF stimulation, EGFRs were accumulated to the plasma membranes together with N-WASP (Fig.1 D) and subsequently internalized with the clathrin-coated vesicles. The internalized EGFRs were co-localized clathrin and dynamin (data not shown). In CDX-treated cells, neither EGFR nor N-WASP showed recruitment to the plasma membranes (data not shown). These data suggest that both EGFR and N-WASP were recruited to raft fractions upon EGF stimulation. Some components of the endocytic machinery, such as Pacsin and Intersectin (42Merrifield C.J. Moss S.E. Ballestrem C. Imhof B.A. Giese G. Wunderlich I. Almers W. Nat. Cell Biol. 1999; 1: 72-74Google Scholar, 43Roos J. Kelly R.B. J. Biol. Chem. 1998; 273: 19108-19119Google Scholar), have been reported to bind to both dynamin and N-WASP. These proteins have SH3 domains and associate with dynamin and N-WASP through the proline-rich domains. EA also has an SH3 domain in its COOH terminus and binds to dynamin (44Simpson F. Hussain N.K. Qualmann B. Kelly R.B. Kay B.K. McPherson P.S. Schmid S.L. Nat. Cell Biol. 1999; 1: 119-124Google Scholar). EA has LPA-AT activity and plays an essential role in the fission step of clathrin-mediated endocytosis (45Huttner W.B. Schmidt A. Curr. Opin. Neurobiol. 2000; 10: 543-551Google Scholar). We hypothesized that EA associates with N-WASP through its SH3 domain. To evaluate this hypothesis, we performed co-immunoprecipitation experiments. Myc-tagged full-length EA expression plasmids (Myc-EA) and full-length N-WASP expression plasmids were co-transfected transiently into COS7 cells, and the EA/N-WASP interaction was detected by Western blot analysis after precipitation of cell lysates with anti-N-WASP antibody or anti-Myc antibody (Fig.2 A). Both immunoprecipitates contained Myc-EA and N-WASP. Positive signals were specific, as preimmune serum and control IgG immunoprecipitates did not yield positive signals. These findings indicate that EA associates with N-WASP in vivo. We then examined whether the SH3 domain of EA can associate with the proline-rich domain of N-WASP. As shown in Fig. 2 B, we constructed a variety of GST fusion proteins of EA, including full-length (GST-EA), SH3 domain-deleted (GST-EAΔSH3), and domain only (GST-SH3). These fusion proteins were mixed with COS7 cell lysates for pull-down assays. Western blotting revealed that the precipitates collected with GST-EA and GST-SH3 contained dynamin and N-WASP (Fig.2 C). When the same assay was performed with lysates of cells in which proline-rich domain-deleted N-WASP (N-WASPΔP) was overexpressed, endogenous N-WASP but not N-WASPΔP was detected in precipitates (Fig. 2 D). These results show that the SH3 domain of EA interacts with the proline-rich domain. As shown in Fig. 2, when both Myc-EA and N-WASP were expressed transiently, association of EA and N-WASP was revealed by co-immunoprecipitation. But surprisingly, when only Myc-EA was transfected into COS7 cells and anti-N-WASP antibody immunoprecipitates were blotted with anti-Myc antibody, however, no signal was detected (Fig. 3 A). Because EA is a cytosolic enzyme, it may be recruited to rafts in an EGF-dependent manner together with N-WASP. Therefore, we tested the possibility that EGF might induce the interaction between N-WASP and EA. COS7 cells transfected with Myc-EA were stimulated with EGF, and immunoprecipitation with anti-N-WASP antibody or with anti-Myc an