The WW Domain of Neural Protein FE65 Interacts with Proline-rich Motifs in Mena, the Mammalian Homolog of DrosophilaEnabled*
- Kira S. Ermekova‡,
- Nicola Zambrano§,
- Hillary Linn‡,
- Giuseppina Minopoli§,
- Frank Gertler¶,
- Tommaso Russo§ and
- Marius Sudol‡‖
- From the ‡Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, the§Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi di Napoli “Federico II,” Via S. Pansini 5, I-80131 Napoli, Italy, and the ¶Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02138-4307
Abstract
The neural protein FE65 contains two types of protein-protein interaction modules: one WW binding domain and two phosphotyrosine binding domains. The carboxyl-terminal phosphotyrosine binding domain of FE65 interacts in vivo with the β-amyloid precursor protein, which is implicated in Alzheimer disease. To understand the function of this adapter protein, we identified binding partners for the FE65 WW domain. Proline-rich sequences sharing a proline-proline-leucine-proline core motif were recovered by screening expression libraries for ligands of the FE65 WW domain. Five proteins of molecular masses 60, 75, 80, 140, and 200 kDa could be purified from mouse brain lysates by affinity to the FE65 WW domain. We identified two of these five proteins as the 80- and 140-kDa isoforms encoded by Mena, the mammalian homolog of theDrosophila Enabled gene. Using the SPOTs technique of peptide synthesis, we identified the sequences in Mena that interact with the FE65 WW domain and found that they contain the signature proline-proline-leucine-proline motif. Finally, we demonstrated that Mena binds to FE65 in vivo by coimmunoprecipitation assay from COS cell extracts. The specificity of the Mena-FE65 WW domain association was confirmed by competition assays. Further characterization of the FE65-Mena complex may identify a physiological role for these proteins in β-amyloid precursor protein biogenesis and may help in understanding the mechanism of molecular changes that underlie Alzheimer disease.
Footnotes
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↵* This work was supported by Grants CA45757 and CAO1605 from NCI, National Institutes of Health, by a Human Frontier Science Program grant, by a Muscular Dystrophy Association grant (to M. S.), by Leukemia Society of America Special Fellowship 3337-95 (to F. G.), and by Telethon Grant E522 (to T. R.). Special thanks go to Philippe Soriano for generous support of F. G.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF020191 (clone 13), AF020311 (clone 7), AF020312 (clone 9-1), and AF020313 (clone 48).
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↵‖ To whom correspondence should be addressed: Department of Biochemistry, Box 1020, The Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029-6574. Tel.: 212-241-9431; Fax: 212-426-1483; E-mail: M_Sudol{at}smtplink.mssm.edu.
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↵1 The abbreviations used are: PTB, phosphotyrosine binding domain; GST, glutathione S-transferase; βAPP, β-amyloid precursor protein; Ena, Drosophila Enabled; Mena, mammalian Enabled; VASP, vasodilator-stimulated phosphoprotein; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; MAP, microtubule-associated protein; EVH, Ena-VASP homology; mbh1,myc basic motif homolog-1; RIPA buffer, 10 mmTris HCl, pH 7.4, 5 mm EDTA, 300 mm NaCl, 0.1% SDS; Tris/Tween buffer, 50 mm Tris HCl, pH 7.5,100 mm NaCl, 1 mm EDTA, 0.1% Tween 20, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride; TBS-T buffer, 20 mm Tris HCl, pH 7.5, 150 mm NaCl, 0.05% Tween 20; HMK buffer, 20 mmTris HCl, pH 7.5, 100 mm NaCl, 12 mmMgCl2; CMV, cytomegalovirus.
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↵2 T. Russo, unpublished data.
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↵3 F. Gertler, unpublished data.
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- Received August 8, 1997.
- Revision received September 23, 1997.
