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• W2135606345 abstract "The Wiskott-Aldrich syndrome is an inherited X-linked immunodeficiency characterized by thrombocytopenia, eczema, and a tendency toward lymphoid malignancy. Lymphocytes from affected individuals have cytoskeletal abnormalities, and monocytes show impaired motility. The Wiskott-Aldrich syndrome protein (WASP) is a multi-domain protein involved in cytoskeletal organization. In a two-hybrid screen, we identified the protein Cdc42-interacting protein 4 (CIP4) as a WASP interactor. CIP4, like WASP, is a Cdc42 effector protein involved in cytoskeletal organization. We found that the WASP-CIP4 interaction is mediated by the binding of the Src homology 3 domain of CIP4 to the proline-rich segment of WASP. Cdc42 was not required for this interaction. Co-expression of CIP4 and green fluorescent protein-WASP in COS-7 cells led to the association of WASP with microtubules. In vitro experiments showed that CIP4 binds to microtubules via its NH2 terminus. The region of CIP4 responsible for binding to active Cdc42 was localized to amino acids 383–417, and the mutation I398S abrogated binding. Deletion of the Cdc42-binding domain of CIP4 did not affect the colocalization of WASP with microtubules in vivo. We conclude that CIP4 can mediate the association of WASP with microtubules. This may facilitate transport of WASP to sites of substrate adhesion in hematopoietic cells. The Wiskott-Aldrich syndrome is an inherited X-linked immunodeficiency characterized by thrombocytopenia, eczema, and a tendency toward lymphoid malignancy. Lymphocytes from affected individuals have cytoskeletal abnormalities, and monocytes show impaired motility. The Wiskott-Aldrich syndrome protein (WASP) is a multi-domain protein involved in cytoskeletal organization. In a two-hybrid screen, we identified the protein Cdc42-interacting protein 4 (CIP4) as a WASP interactor. CIP4, like WASP, is a Cdc42 effector protein involved in cytoskeletal organization. We found that the WASP-CIP4 interaction is mediated by the binding of the Src homology 3 domain of CIP4 to the proline-rich segment of WASP. Cdc42 was not required for this interaction. Co-expression of CIP4 and green fluorescent protein-WASP in COS-7 cells led to the association of WASP with microtubules. In vitro experiments showed that CIP4 binds to microtubules via its NH2 terminus. The region of CIP4 responsible for binding to active Cdc42 was localized to amino acids 383–417, and the mutation I398S abrogated binding. Deletion of the Cdc42-binding domain of CIP4 did not affect the colocalization of WASP with microtubules in vivo. We conclude that CIP4 can mediate the association of WASP with microtubules. This may facilitate transport of WASP to sites of substrate adhesion in hematopoietic cells. Wiskott-Aldrich syndrome protein Cdc42-interacting protein 4 glutathione S-transferase polymerase chain reaction phosphate-buffered saline Src homology pleckstrin homology green fluorescent protein WASP-interacting protein 1,4-piperazinediethanesulfonic acid polyacrylamide gel electrophoresis tetramethylrhodamine B isothiocyanate GTPase binding domain Cdc42/Rac interacting and binding guanyl-5′-yl thiophosphate guanosine 5′-O-(thiotriphosphate) protein kinase C and casein kinase 2 substrate in neurons proline, serine, threonine phosphatase-interacting protein insulin-like growth factor 1-receptor β The Wiskott-Aldrich syndrome is an inherited X-linked immunodeficiency with associated thrombocytopenia, eczema, and a tendency toward development of malignancy of the lymphoreticular system (1.Aldrich R.A. Steinberg A.G. Campbell D.C. Pediatrics. 1954; 13: 133-138PubMed Google Scholar, 2.Rosen F.S. Cooper M.D. Wedgwood M.D. N. Engl. J. Med. 1995; 333: 431-440Crossref PubMed Scopus (342) Google Scholar). Affected individuals have lymphocytes that respond poorly to certain stimulants in vitro (3.Molina I.J. Sancho J. Terhorst C. Rosen F.S. Remold-O'Donnell E. J. Immunol. 1993; 151: 4383-4390PubMed Google Scholar, 4.Simon H.U. Mills G.B. Hashimoto S. Siminovitch K.A. J. Clin. Invest. 1992; 90: 1396-1405Crossref PubMed Scopus (75) Google Scholar) and show abnormalities of cytoskeletal structure (5.Gallego M.D. Santamaria M. Pena J. Molina I.J. Blood. 1997; 90: 3089-3097Crossref PubMed Google Scholar, 6.Kenney D. Cairns L. Remold-O'Donnel E. Peterson J. Rosen F.S. Parkman R. Blood. 1986; 68: 1329-1332Crossref PubMed Google Scholar, 7.Molina I.J. Kenney D.M. Rosen F.S. Remold-O'Donnell E. J. Exp. Med. 1992; 176: 867-874Crossref PubMed Scopus (118) Google Scholar, 8.Facchetti F. Blanzuoli L. Vermi W. Notarangelo L.D. Giliani S. Fiorini M. Fasth A. Stewart D.M. Nelson D.L. J. Pathol. 1998; 185: 99-107Crossref PubMed Scopus (50) Google Scholar). Impaired monocyte motility is also observed (9.Altman L.C. Snyderman R. Blaese R.M. J. Clin. Invest. 1974; 54: 486-493Crossref PubMed Scopus (63) Google Scholar, 10.Badolato R. Sozzani S. Malacarne F. Bresciani S. Fiorini M. Borsatti A. Albertini A. Mantovani A. Ugazio A.G. Notarangelo L.D. J. Immunol. 1998; 161: 1026-1033PubMed Google Scholar, 11.Zicha D. Allen W.E. Brickell P.M. Kinnon C. Dunn G.A. Jones G.E. Thrasher A.J. Br. J. Hematol. 1998; 101: 659-665Crossref PubMed Scopus (201) Google Scholar). The gene mutated in Wiskott-Aldrich syndrome was cloned in 1994, and was shown to encode a proline-rich protein of 502 amino acids (12.Derry J.M.J. Ochs H.D. Francke U. Cell. 1994; 78: 635-644Abstract Full Text PDF PubMed Scopus (816) Google Scholar, 13.Derry J.M.J. Ochs H.D. Francke U. Cell. 1994; 79: 923PubMed Google Scholar, 14.Kwan S. Hagemann T.L. Radtke B.E. Blaese R.M. Rosen F.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4706-4710Crossref PubMed Scopus (96) Google Scholar). Although the function of WASP1 was not immediately apparent from knowledge of its primary structure, a variety of interactions have been discovered showing that WASP plays a role in cytoskeletal organization. The NH2 terminus of WASP binds to WASP-interacting protein (WIP), a protein with profilin binding and actin binding motifs, overexpression of which causes changes in cytoskeletal structure (15.Ramesh N. Anton I.M. Hartwig J.H. Geha R.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14671-14676Crossref PubMed Scopus (302) Google Scholar). Missense mutations known to cause Wiskott-Aldrich syndrome impair the WASP-WIP interaction (16.Stewart D.M. Tian L. Nelson D.L. J. Immunol. 1999; 162: 5019-5024PubMed Google Scholar). A GBD/CRIB motif for binding the active forms of the small GTPases Rac and Cdc42, proteins that are involved in control of cytoskeletal organization, is present in WASP (18.Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar, 19.Kolluri R. Tolias K.F. Carpenter C.L. Rosen F.S. Kirchhausen T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5615-5618Crossref PubMed Scopus (187) Google Scholar, 20.Rivero-Lezcano O.M. Marcilla A. Sameshima J.H. Robbins K.C. Mol. Cell. Biol. 1995; 15: 5725-5731Crossref PubMed Scopus (279) Google Scholar). The proline-rich segment of WASP, between amino acids 310 and 420, interacts with a number of proteins containing SH3 domains, many of which are involved in the regulation of cytoskeletal structure. Among the SH3 proteins known to interact with WASP are the adaptor proteins Nck (21.Quilliam L.A. Lambert Q.T. Mickelson-Young L.A. Westwick J.K. Sparks A.B. Kay B.K. Jenkins N.A. Gilbert D.J. Copeland N.G. Der C.J. J. Biol. Chem. 1996; 271: 28772-28776Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 22.She H.Y. Rockow S. Tang J. Nishimura R. Skolnik E.Y. Chen M. Margolis B. Li W. Mol. Biol. Cell. 1997; 8: 1709-1721Crossref PubMed Scopus (104) Google Scholar) and Grb2 (23.Zhu Q. Watanabe C. Liu T. Hollenbaugh D. Blaese R.M. Kanner S.B. Aruffo A. Ochs H.D. Blood. 1997; 90: 2680-2689Crossref PubMed Google Scholar,24.Banin S. Truong O. Katz D.R. Waterfield M.D. Brickell P.M. Gout I. Curr. Biol. 1996; 6: 981-988Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), Src family kinases (23.Zhu Q. Watanabe C. Liu T. Hollenbaugh D. Blaese R.M. Kanner S.B. Aruffo A. Ochs H.D. Blood. 1997; 90: 2680-2689Crossref PubMed Google Scholar, 25.Finan P.M. Soames C.J. Wilson L. Nelson D.L. Stewart D.M. Truong O. Hsuan J.J. Kellie S. J. Biol. Chem. 1996; 271: 26291-26295Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 26.Cory G.O. MacCarthy-Morrogh L. Banin S. Gout I. Brickell P.M. Levinsky R.J. Kinnon C. Lovering R.C. J. Immunol. 1996; 157: 3791-3795PubMed Google Scholar, 27.Kinnon C. Cory G.O. MacCarthy-Morrogh L. Banin S. Gout I. Lovering R.C. Brickell P.M. Biochem. Soc. Trans. 1997; 25: 648-650Crossref PubMed Scopus (10) Google Scholar), phospholipase Cγ (23.Zhu Q. Watanabe C. Liu T. Hollenbaugh D. Blaese R.M. Kanner S.B. Aruffo A. Ochs H.D. Blood. 1997; 90: 2680-2689Crossref PubMed Google Scholar, 25.Finan P.M. Soames C.J. Wilson L. Nelson D.L. Stewart D.M. Truong O. Hsuan J.J. Kellie S. J. Biol. Chem. 1996; 271: 26291-26295Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), and Tec family kinases (26.Cory G.O. MacCarthy-Morrogh L. Banin S. Gout I. Brickell P.M. Levinsky R.J. Kinnon C. Lovering R.C. J. Immunol. 1996; 157: 3791-3795PubMed Google Scholar, 28.Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (544) Google Scholar). Motifs in the COOH terminus of WASP show homology to the actin-binding proteins verprolin (verprolin homology domain, amino acids 430–446) and cofilin (cofilin homology domain, amino acids 469–487) (18.Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar, 29.Miki H. Tanekawa T. Biochem. Biophys. Res. Commun. 1998; 243: 73-78Crossref PubMed Scopus (113) Google Scholar). WASP has been shown to interact directly with actin through the verprolin homology domain (30.Machesky L.M Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar). A WASP homolog expressed in neural and other tissues, N-WASP, has the ability to depolymerize actin filaments in vitro (28.Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (544) Google Scholar, 29.Miki H. Tanekawa T. Biochem. Biophys. Res. Commun. 1998; 243: 73-78Crossref PubMed Scopus (113) Google Scholar). WASP, N-WASP, and the related proteins Scar1 in human and Bee1/Las17 in yeast interact with the Arp2/3 complex, a key regulator of actin polymerization (31.Winter D. Lechler T. Li R. Curr. Biol. 1999; 9: 501-504Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 32.Yarar D. To W. Abo A Welch M.D. Curr. Biol. 1999; 9: 555-558Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). WASP-coated microspheres exhibit cytoplasmic actin-based motility, dependent on the presence of the Arp2/3 complex (33.Aspenström P. Curr. Biol. 1997; 7: 479-487Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Taken together, these studies suggest that WASP is a mediator of Cdc42/Rac signaling, and has direct effects on the actin cytoskeleton via actin-binding domains and activation of the Arp2/3 complex. SH3 protein interactions may play a role in localization of WASP and/or transmission of extracellular signals to and through WASP. We performed a yeast two-hybrid screen to look for novel WASP-interacting proteins. One WASP-interacting clone encoded a portion of the protein CIP4. This protein was identified previously as interacting with the active form of Cdc42 (34.Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1316) Google Scholar). CIP4 is a 545-amino acid protein, widely expressed, that has an NH2-terminal domain of unknown function with homology to the NH2 termini of the non-receptor tyrosine kinase Fer and the Fes/Fps proto-oncogene, which was termed the FCH domain. The central region of the protein (amino acids 293–481) was shown to interact with Cdc42, although it does not contain a GBD/CRIB motif. An SH3 domain is found in the COOH terminus of CIP4. We performed a series of studies exploring the mechanism of WASP/CIP4 interaction, and the effects of co-expression of CIP4 on WASP distribution and actin cytoskeletal structure in COS-7 cells. We used the Interaction Trap two-hybrid screen (35.Golemis E.A. Gyuris J. Brent R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York1994: 13.14.1-13.14.17Google Scholar). Yeast and vectors were obtained from Roger Brent (University of Massachusetts, Boston, MA). An human T-cell lymphotrophic virus type I-transformed T-cell line cDNA library for this system was purchased from CLONTECH (Palo Alto, CA). The screen was performed as described (16.Stewart D.M. Tian L. Nelson D.L. J. Immunol. 1999; 162: 5019-5024PubMed Google Scholar, 36.Stewart D. Treiber-Held S. Kurman C.C. Facchette F. Notarangelo L.D. Nelson D.L. J. Clin. Invest. 1996; 97: 2627-2634Crossref PubMed Scopus (105) Google Scholar) using full-length WASP as bait in the LexA system. Additional methods were obtained from material supplied with the library. The specificity of interaction of clones expressing the interaction phenotype was tested with a bait plasmid containing an open reading frame of Drosophilabicoid protein that was included with the materials obtained from Dr. R. Brent, and with a bait plasmid containing an open reading frame of human IGF1-Rβ cytoplasmic domain that was a gift from Bhakta Dey (NCI Metabolism Branch, Bethesda, MD). A cDNA encoding full-length WASP was cloned into pQBI-25 (Quantum Biotechnologies Inc., Laval, Quebec, Canada) in order to produce GFP-WASP, and into pCR2 (Invitrogen, Carlsbad, CA) for in vitro translation. GST-Cdc42 expression constructs were generously provided by Dr. Anne Ridley (Ludwig Institute for Cancer Research, University College, London, United Kingdom). A full-length CIP4 cDNA PCR product from a B-cell cDNA library (gift from Dr. Colin Duckett, National Institutes of Health, Bethesda, MD) was cloned into pCR2.1 vector (Invitrogen). This CIP4 cDNA was used as a template to clone CIP4 and CIP4 fragments into pCR2.1 for in vitro translation and into pGEX4T-2 for expression of GST-tagged proteins. CIP4 cDNA was cloned into a derivative of the mammalian expression vector pRK5 (PharMingen, San Diego, CA) that was modified to include an NH2-terminal myc epitope tag (pRK5-myc) in order to express myc-tagged CIP4 in mammalian cells. Point mutations were produced by whole vector PCR with primers containing the desired base change using the QuikChangeTMsite-directed mutagenesis kit (Stratagene, La Jolla, CA). Internal deletion mutants of CIP4 and WASP were generated by whole vector PCR using the QuikChangeTM kit, with primers flanking the sites of the intended deletion. T4 DNA ligase was then used for blunt end ligation of the PCR products. All constructs were sequenced to confirm the presence of the intended mutations. In vitro binding assays of WASP with CIP4, and WASP or CIP4 with Cdc42 were performed using the GST pull-down technique. In these assays, an in vitrotranslated, [35S]methionine-radiolabeled protein was incubated with a potential binding partner in the form of an unlabeled GST fusion protein bound to beads of glutathione-agarose. Specifically, GST fusion protein (10 μg) bound to glutathione-agarose was incubated at 4 °C for 2 h with 10 μl of the in vitrotranslated protein in PBS, 1% Triton X-100 or, in the case of loaded Cdc42, with 50 mm Tris-HCl, pH 7.5, 150 mmNaCl, 1 mm dithiothreitol, 1% Triton X-100, 5 mm MgCl2, 0.2% bovine serum albumin in a total volume of 100 μl, then washed three times with the same buffer. Proteins adhering to the beads (labeled protein and unlabeled GST protein) were released from the beads, and any interaction disrupted by boiling in SDS gel loading buffer containing 2% 2-mercaptoethanol. This protein mixture was analyzed on an SDS-polyacrylamide gel and the gel exposed to x-ray film. Any labeled protein that had bound to the GST fusion protein will appear as a band on the film. Radiolabeled WASP or CIP4 was prepared by in vitrotranslation using the TnT® coupled transcription-translation system (Promega, Madison WI) in the presence of [35S]methionine. GST fusion proteins were expressed using the protease-deficient Escherichia coli bacterial strain BL21. Cells from induced cultures were collected, resuspended in 50 μl of ice-cold PBS/ml of culture and disrupted by sonication. Triton X-100 was added to a final concentration of 1% and the lysate was mixed for 30 min. The lysate was centrifuged at 12,000 ×g for 10 min, diluted 5-fold with 1% Triton X-100/PBS, and reduced glutathione-agarose (Sigma) added. After binding of GST fusion proteins, the glutathione-agarose was washed and stored at 4 °C. The quantity and purity of the GST fusion proteins was assessed by SDS-PAGE, followed by Coomassie Blue staining. GST-Cdc42 proteins were loaded with the non-hydrolyzable GTP analog GTPγS immediately before use by adding equal volumes of the purified fusion protein bound to glutathione-agarose in PBS and 100 mm Tris-HCl, pH 7.5, 15 mm EDTA, 1 mg/ml bovine serum albumin, 2 mmdithiothreitol, 1 mm GTPγS or GDPβS at 37 °C for 30 min and fixed with 12.5 mm MgCl2. Affinity-purified anti-CIP4 antibody was prepared as follows. A PCR product encoding CIP4 residues 118–481 was cloned into pFLAG-ATS vector (Kodak-IBI) and recombinant FLAG-CIP4 expressed in E. coli BL-21. The FLAG-CIP4 was purified by affinity chromatography on anti-FLAG M2 agarose according to manufacturer's directions. Rabbits were immunized with 100 μg of purified FLAG-CIP4 initially, followed by 10 μg/week for 10 weeks. Preimmune and immune sera were collected and assayed by enzyme-linked immunosorbent assay using GST or GST-CIP4 as antigens. Anti-CIP4 antibody was purified from the immune sera by affinity chromatography on GST-CIP4 bound to cyanogen bromide-activated Sepharose (Amersham Pharmacia Biotech). Rabbit anti-tubulin antibody, mouse anti-tubulin monoclonal antibody DM1A, and phalloidin-TRITC were purchased from Sigma. Goat anti-rabbit IgG AlexaTM594 and goat anti-mouse IgG AlexaTM488 were purchased from Molecular Probes. Rabbit anti-myc antibody was purchased from Upstate Biotechnology, and mouse monoclonal anti-myc was purchased from Invitrogen. Monoclonal anti-WASP was obtained as described (37.Masson D. Kreis T.E. J. Cell Biol. 1993; 123: 357-371Crossref PubMed Scopus (87) Google Scholar). Plasmid DNA was prepared using the Qiagen Maxi Prep kit. COS-7 cells were maintained in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and 2 mml-glutamine. Cells were transfected by electroporation. COS-7 cells transfected with GFP-WASP and myc-CIP4 were lysed at 1.2 × 107cells/ml in 1% digitonin, 50 mm Tris-Cl, pH 7.4, 150 mm NaCl supplemented with Complete® proteinase inhibitor mixture (Sigma) at 4 °C for 20 min and centrifuged at 16,000 × g for 10 min. The clarified lysate was diluted 1:3 with washing buffer (50 mm Tris-Cl, pH 7.4, 150 mm NaCl with proteinase inhibitor), and affinity-purified rabbit anti-CIP4 or preimmune serum was added to a final concentration of 10 μg/ml or 5 μl/ml, respectively. After incubation at 4 °C for 2 h, protein A-Sepharose (50 μl of a 50% slurry in wash buffer) was added, and the suspension rocked at 4 °C for 1 h. The pellet was washed three times with washing buffer and bound proteins analyzed by SDS-PAGE, followed by Western blot using either monoclonal anti-myc or monoclonal anti-WASP. Microtubules were obtained by incubating 5 mg/ml tubulin (Molecular Probes, Eugene, OR) in G-PEM buffer (100 mm K-PIPES, 1 mm EGTA, 1 mm MgSO4, 1 mm GTP, pH 6.8) with 30% glycerol at 35 °C for 10 min. Microtubules were stabilized by 10 μm paclitaxel (Molecular Probes) as instructed by the manufacturer. The microtubule binding experiments were performed as described (38.Linder S. Nelson D. Weiss M. Aepfelbacher M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9648-9653Crossref PubMed Scopus (351) Google Scholar). Briefly, 50 μl of in vitro translated, [35S]methionine-labeled CIP4 or CIP4 mutants was diluted in 200 μl of PB (80 mm K-PIPES, pH 6.9, 1 mmEGTA, 1 mm CaCl2) containing Complete® proteinase inhibitor mixture (Sigma). The mixture was spun at 50,000 × g for 1 h at 4 °C in a 70.1Ti rotor (Beckman). 100 μl of supernatant was incubated with or without 30 μg of microtubules for 30 min at 37 °C in the presence of 10 μm paclitaxel. The microtubule and CIP4 mixtures were layered over 1 ml of 15% sucrose in PB, and spun for 30 min at 30,000 × g in a SW55 rotor (Beckman). Both supernatants (above the sucrose) and pellets were analyzed for the presence of CIP4 and its mutants by 4–20% SDS-PAGE, followed by autoradiography. The gels were then stained with Coomassie Blue to confirm that an equal mass of microtubules was loaded on the gel in each sample. COS-7 cells were transfected with pQBI25-GFP-WASP in the presence or absence of pRK5-myc-CIP4 by electroporation. Cells were plated on coverslips in 24-well culture plates. 24–48 h after transfection, cells were fixed and permeabilized by Cytoperm/Fix kit (PharMingen). Primary human macrophages were prepared and cultured for 7 days as described (39.Avila J. Brandt R. Kosik K.S. Brain Microtubule Associated Proteins. Harwood Academic Publishers, Boston1998Google Scholar). To visualize microtubules, cells were fixed and permeabilized by 4% formaldehyde, 0.1% Triton X-100, 80 mm K-PIPES, 1 mm EGTA, 1 mm MgSO4, 30% glycerol at room temperature for 30 min (40.Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (556) Google Scholar) or by 4% paraformaldehyde, 0.3% Triton X-100, 0.05% glutaraldehyde in cytoskeleton buffer (10 mm PIPES, pH 7.0, 150 mm NaCl, 5 mm EGTA, 5 mmMgCl2, 100 μg/ml streptomycin) at room temperature for 15 min. Both methods gave similar results. After staining, coverslips were mounted on glass slides by FluorSave® reagent (Calbiochem). Confocal laser scanning microscopy was performed using an Olympus confocal laser scanning microscope equipped with argon and krypton ion lasers. Series of optical sections through the cells were collected. Images were assembled using Adobe PhotoShop software. Several unique WASP-interacting clones were identified in the two-hybrid screen. One clone, our number 4–2, encoded a portion of the protein CIP4, a 545 amino acid protein that previously had been identified as an SH3-domain containing protein with partial homology to the non-receptor kinase Fer that interacts with the active form of Cdc42 (33.Aspenström P. Curr. Biol. 1997; 7: 479-487Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The nucleic acid sequence of clone 4–2 was identical to bases 390–1949 of the published CIP4 sequence (GenBank accession number AJ000414) with the exception of a single C insertion at 1911 in the 3′-untranslated region. The cDNA encoded CIP4 amino acids 118–545. The interaction was specific to the WASP-LexA bait protein; no interaction of CIP4 with eitherDrosophila bicoid protein or the cytoplasmic domain of IGF1-Rβ was detected in the two-hybrid system (data not shown). The region of WASP responsible for interaction with CIP4 was mapped by deletion mutation analysis in the two-hybrid system. This showed that CIP4 interacted with a WASP construct retaining amino acids 1–442, but failed to interact with a WASP construct retaining amino acids 1–201 (data not shown). Since the WASP 1–442 has both the GBD (230–260) and the proline-rich region (310–420), two mechanisms of interaction were thought possible. First, the CIP4 SH3 domain might bind directly to WASP in the proline-rich region. Alternatively, since the yeast homolog of Cdc42 is similar to the human protein, the binding of endogenous yeast Cdc42 to WASP and CIP4 simultaneously could contribute to the interaction. We therefore performed an in vitro binding assay to explore the mechanisms of CIP4/WASP interaction. The interaction between radiolabeled, in vitro translated WASP of different lengths and GST-CIP4 full-length and deletion mutants is shown in Fig. 1. Full-length CIP4 (GST-CIP4 wild type (WT)) bound strongly to WASP mutants retaining amino acids 1–442 and 1–502, which contain both the polyproline region and the GBD. WASP mutants lacking the polyproline region (1–302 and 1–201) bound CIP4 weakly, whether or not the WASP GBD was present (1–302) or absent (1–201). This indicates that the proline-rich region of WASP is necessary for effective CIP4 binding. However, full-length WASP bound weakly to GST-CIP4 SH3, which contains the isolated CIP4 SH3 domain. WASP mutant 1–442, which retains the polyproline region but lacks the COOH-terminal actin binding region, bound GST-CIP4 SH3 strongly. This difference may be due to a WASP intramolecular interaction between COOH-terminal acidic residues and basic residues just NH2-terminal of the GBD, which may render the proline-rich region less available for binding to the CIP4 SH3 domain (41.Kato M. Miki H. Imai K. Nonoyama S. Suzuki T. Sasakawa C. Takenawa T. J. Biol. Chem. 1999; 274: 27225-27230Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 42.Glaven J.A. Whitehead I. Bagrodia S. Kay R. Cerione R.A. J. Biol. Chem. 1999; 274: 2279-2285Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). WASP mutants lacking the polyproline region (1–302 and 1–201) failed to bind the CIP4 SH3 domain. A CIP4 mutant lacking the SH3 domain (GST-CIP4 118–481) bound all WASP mutants weakly, if at all. These results demonstrate that the WASP-CIP4 interaction is mediated chiefly by the binding of the CIP4 SH3 domain to the polyproline region of WASP, and that the presence or absence of the WASP GBD has no influence on this binding. To further establish that the CIP4-WASP interaction can occur independently of Cdc42 binding, the binding of in vitrotranslated radiolabeled WASP to both CIP4 and the constitutively active Cdc42 mutant V12 was examined. As shown in Fig.2, a deletion mutant of WASP specifically lacking the GBD retains the ability to bind CIP4, but Cdc42 binding is lost. This shows that CIP4 can bind to WASP in the absence of Cdc42 binding. To further investigate the CIP4-WASP interaction, COS-7 cells were co-transfected with GFP-WASP and myc-tagged CIP4. GFP-WASP co-immunoprecipitated with CIP4 as shown in Fig.3. The CIP4 118–481 mutant did not show binding to WASP, again showing the necessity of the CIP4 SH3 domain. When expressed alone, the CIP4 SH3 domain localized entirely in the cell nucleus as detected by immunofluorescence (data not shown), and was not tested for interaction with WASP by co-immunoprecipitation. The primary sequence of CIP4 does not contain the GBD/CRIB motif characteristic of the binding site of Cdc42. We therefore tested a series of CIP4 deletion mutants for the ability to interact with the active form of Cdc42. Full-length in vitrotranslated CIP4 interacted specifically with GST-Cdc42-GTPγS or with the constitutively active mutant Cdc42-V12-GTPγS but not to the GDPβS-loaded forms (not shown). The dominant negative Cdc42 mutant N17 failed to bind CIP4 regardless of GTP or GDP loading (not shown). As shown in Fig. 4, full-length CIP4 (1–545) or CIP4 constructs retaining amino acids 1–417 and 1–423 reacted with Cdc42, but CIP4 1–383 or 1–407 did not. A series of substitution mutations in this region of the CIP4 construct 1–417 revealed that the mutation I398S abrogated binding of CIP4 to active Cdc42 (Fig. 4 B). We conclude that the CIP4 amino acids 383–417 and the amino acid Ile-398 are critical for Cdc42 binding to CIP4. Wiskott-Aldrich syndrome patients show structural abnormalities of platelets and peripheral lymphocytes, and decreased density of surface microvilli (3.Molina I.J. Sancho J. Terhorst C. Rosen F.S. Remold-O'Donnell E. J. 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Immunol. 1998; 161: 1026-1033PubMed Google Scholar, 11.Zicha D. Allen W.E. Brickell P.M. Kinnon C. Dunn G.A. Jones G.E. Thrasher A.J. Br. J. Hematol. 1998; 101: 659-665Crossref PubMed Scopus (201) Google Scholar). Recent studies suggest a role for WASP in actin polymerization since overexpression of WASP induces the formation of actin-containing clusters (18.Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar). In order to test the role of CIP4 on WASP-induced cytoskeleton changes, WASP with GFP at the COOH terminus was transfected into COS-7 cells in the absence or presence of myc-tagged CIP4. In the absence of CIP4, the colocalization o" @default.
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• W2135606345 title "Cdc42-interacting Protein 4 Mediates Binding of the Wiskott-Aldrich Syndrome Protein to Microtubules" @default.
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