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The WW Domain of Neural Protein FE65 Interacts with Proline-rich Motifs in Mena, the Mammalian Homolog of DrosophilaEnabled*

Open AccessPublished:December 26, 1997DOI:https://doi.org/10.1074/jbc.272.52.32869
      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 the Drosophila 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.
      FE65 is a brain-enriched protein with the modular structure of a typical adapter containing two types of protein-protein interaction domains: the WW domain and two phosphotyrosine binding (PTB)
      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.
      1The 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.
      domains (Refs.
      • Fiore F.
      • Zambrano N.
      • Minopoli G.
      • Donini V.
      • Duilio A.
      • Russo T.
      and
      • Ermekova K.S.
      • Chang A.
      • Zambrano N.
      • de Candia P.
      • Russo T.
      • Sudol M.
      ; see Fig. 1). WW domains bind proline-rich proteins containing the PPXY or PPLP core motifs (where X signifies any amino acid) (
      • Chen H.I.
      • Sudol M.
      ,
      • Chan D.C.
      • Bedford M.T.
      • Leder P.
      ,
      • Sudol M.
      ). PTB domains bind proteins containing the consensus sequence NPXY; in some instances, the tyrosine must be phosphorylated to mediate high-affinity binding (
      • Bork P.
      • Margolis B.
      ,
      • Kavanaugh W.M.
      • Turck C.W.
      • Williams L.T.
      ,
      • van der Geer P.
      • Pawson T.
      ). Importantly, the carboxyl-terminal PTB domain of FE65 binds in vitro and in vivo to the cytoplasmic portion of the β-amyloid precursor protein (βAPP), a large transmembrane protein implicated in Alzheimer disease (
      • Fiore F.
      • Zambrano N.
      • Minopoli G.
      • Donini V.
      • Duilio A.
      • Russo T.
      ,
      • Guenette S.Y.
      • Chen J.
      • Jondro P.D.
      • Tanzi R.E.
      ,
      • Zambrano N.
      • Buxbaum J.D.
      • Minopoli G.
      • Fiore F.
      • De Candia P.
      • De Renzis S.
      • Faraonio R.
      • Sabo S.
      • Cheetham J.
      • Sudol M.
      • Russo T.
      ,
      • McLoughlin D.M.
      • Miller C.C.J.
      ). βAPP is a precursor protein of the β-amyloid peptide, the major constituent of the extracellular senile plaques in the Alzheimer brain. In pathological conditions, the level of β-amyloid peptide production and/or accumulation is increased dramatically compared with the normal physiological state. The formation of amyloid plaques correlates well with the onset of Alzheimer disease (
      • Selkoe D.J.
      , ,
      • Kang J.
      • Lemaire H.
      • Unterbeck A.
      • Salbaum J.M.
      • Masters C.L.
      • Grzeschik K.
      • Multhaup G.
      • Beyreuther K.
      • Muller-Hill B.
      ). The biogenesis of βAPP is a complex process that involves specific proteolytic activities, as well as other steps, which include additional posttranslational modifications, trafficking, and secretion (
      • Oltersdorf T.
      • Ward P.J.
      • Henriksson T.
      • Beattie E.C.
      • Neve R.
      • Lieberburg I.
      • Fritz L.C.
      ,
      • Esch F.S.
      • Keim P.S.
      • Beattie E.C.
      • Blacher R.W.
      • Culwell A.R.
      • Oltersdorf T.
      • McClure D.
      • Ward P.J.
      ,
      • Shoji M.
      • Golde T.E.
      • Ghiso J.
      • Cheung T.T.
      • Estus S.
      • Shaffer L.M.
      • Cai X.
      • McKay D.M.
      • Tintner R.
      • Frangione B.
      • Younkin S.G.
      ,
      • Koo E.H.
      • Squazzo S.L.
      ,
      • Cai X.-D.
      • Golde T.E.
      • Younkin S.G.
      ). Little is known about the partner molecules that interact with βAPP and control its processing. In addition to FE65, three other proteins have been shown recently to interact with the cytoplasmic domain of βAPP: the heterotrimeric G protein Go (
      • Nishimoto I.
      • Okamoto T.
      • Matsuura Y.
      • Takahashi S.
      • Okamoto T.
      • Murayama Y.
      • Ogata E.
      ,
      • Okamoto T.
      • Takeda S.
      • Murayama Y.
      • Ogata E.
      • Nishimoto I.
      ,
      • Yamatsuji T.
      • Matsui T.
      • Okamoto T.
      • Komatsuzaki K.
      • Takeda S.
      • Fukumoto H.
      • Iwatsubo T.
      • Suzuki N.
      • Asami-Okada A.
      • Ireland S.
      • Kinane B.
      • Giambarella U.
      • Nishimoto I.
      ), the neuron-specific X11 protein (
      • Borg J.-P.
      • Ooi J.
      • Levy E.
      • Margolis B.
      ), and a 59-kDa βAPP-binding protein 1 (
      • Chow N.
      • Korenberg J.R.
      • Chen X.-N.
      • Neve R.L.
      ).
      Figure thumbnail gr1
      Figure 1Schematic diagram of the domain structure of FE65. The FE65 protein contains two distinct types of protein binding domains. The WW domain is located toward the amino-terminal sequence. PTB1 and PTB2 are located toward the carboxyl-terminal part of the FE65 protein, followed by the nuclear localization signal (NLS). The sequences of wild-type and mutant WW domains of FE65 are indicated at the bottom, distinguished by the mutations W69F and P72A. These two mutations render the WW of FE65 inactive, in terms of ligand binding, with minimal apparent effect on the structure of the domain. Signature tryptophans, the conserved carboxyl-terminal proline, and substituted amino acids in the mutant are shown in boldface.H 2 N and COOH indicate the amino and carboxyl termini, respectively. Numbers indicate positions of amino acids.
      The modular, adapter-like structures of FE65 and X11 proteins suggest that they may link the cytoplasmic portion of βAPP to cytosolic proteins in a manner similar to signal transduction cascades involving growth factor or hormone receptors and integrins (reviewed in Ref.
      • Ermekova K.S.
      • Chang A.
      • Zambrano N.
      • de Candia P.
      • Russo T.
      • Sudol M.
      ). The molecular components and the significance of the putative βAPP signaling remain to be defined, but it is possible that defects in this pathway could be involved in the pathogenesis of Alzheimer disease (
      • Ermekova K.S.
      • Chang A.
      • Zambrano N.
      • de Candia P.
      • Russo T.
      • Sudol M.
      ,
      • LaFerla F.M.
      • Tinkle B.T.
      • Bieberich C.J.
      • Haudenschild C.C.
      • Jay G.
      ,
      • Oster-Granite M.L.
      • McPhie D.L.
      • Greenan J.
      • Neve R.L.
      ,
      • Yamatsuji T.
      • Okamoto T.
      • Takeda S.
      • Murayama Y.
      • Tanaka N.
      • Nishimoto I.
      ).
      Biochemical and genetic analyses of proto-oncogenes have elucidated many facets of signaling processes that underlie neuronal function and development (reviewed in Ref.
      • Sudol M.
      • Grant S.G.N.
      • Maisonpierre P.C.
      ). The study of the Drosophila homolog of the Abl proto-oncogene implicated this gene in the processes of axonal outgrowth and fasciculation (reviewed in Ref.
      • Hoffmann M.F.
      ). Using genetic modifier screens, mutations in several new genes have been identified that modify Abl-dependent phenotypes. These genes include Disabled, Prospero, Failed Axon Connection, and Enabled (Ena) (
      • Gertler F.B.
      • Bennet R.L.
      • Clark M.J.
      • Hoffman F.M.
      ,
      • Gertler F.B.
      • Hill K.K.
      • Clark M.J.
      • Hoffmann F.M.
      ).Ena was identified in a specific screen for dominant mutations that alleviate the Abl phenotype (
      • Gertler F.B.
      • Doctor J.S.
      • Hoffman F.M.
      ). The Ena protein is axonally localized and is a substrate for the Drosophila Abl kinase and a ligand for the SH3 domains of Abl and Src (
      • Gertler F.B.
      • Comer A.R.
      • Juang J.L.
      • Ahern S.M.
      • Clark M.J.
      • Liebl E.C.
      • Hoffmann F.M.
      ). Recently, two vertebrate proteins closely related to Ena were identified. These two proteins, mammalian Enabled (Mena) and Ena-VASP-like are closely related to Ena (
      • Gertler F.B.
      • Niebuhr K.
      • Reinhard M.
      • Wehland J.
      • Soriano P.
      ) as well as to vasodilator-stimulated phosphoprotein (VASP) (
      • Haffner C.
      • Jarchau T.
      • Reinhard M.
      • Hoppe J.
      • Lohmann S.M.
      • Walter U.
      ). Together, these molecules make up the Ena/VASP family of proteins and share three distinct regions of similarity: the amino-terminal 115 amino acids (the Ena-VASP homology 1 (EVH1) domain), a proline-rich core, and the carboxyl-terminal 226 amino acids (the EVH2 domain). The EVH1 domain mediates subcellular targeting of Ena/VASP family proteins by engaging in protein-protein interactions with a distinct proline-rich motif (
      • Gertler F.B.
      • Niebuhr K.
      • Reinhard M.
      • Wehland J.
      • Soriano P.
      ,
      • Niebuhr K.
      • Ebel F.
      • Frank R.
      • Reinhard M.
      • Domann E.
      • Carl U.D.
      • Walter U.
      • Gertler F.B.
      • Wehland J.
      • Chakraborty T.
      ). Ena/VASP family proteins are concentrated in focal adhesions and actin stress fibers and are found in areas of dynamic actin remodeling, such as lammellipodia and axonal growth cones. The subcellular distribution, interactions with profilin, and a small actin monomer-binding protein implicated in the regulation of actin dynamics suggest a role for Ena/VASP family proteins in the regulation of cell motility and morphology by modulating the actin-based cytoskeleton (Ref.
      • Gertler F.B.
      • Niebuhr K.
      • Reinhard M.
      • Wehland J.
      • Soriano P.
      ; reviewed in Ref.
      • Machesky L.
      ). A specialized role for Mena in the nervous system is suggested by a 140-kDa neuron-specific isoform of Mena that is present in the developing and adult brain along with the broadly expressed 80-kDa form of Mena (
      • Gertler F.B.
      • Niebuhr K.
      • Reinhard M.
      • Wehland J.
      • Soriano P.
      ).
      Here we report the identification of a cognate ligand for the WW domain of FE65. Using a combination of functional screens of mouse embryo cDNA expression libraries, the SPOTs technique of peptide synthesis, pull-down experiments from mouse brain lysates, and coimmunoprecipitation from mammalian cell extracts, we were able to characterize the potential ligands of the FE65 WW domain. We identified one of these ligands as Mena, and we demonstrate herein the interaction of the FE65 WW domain with Mena through specific proline-rich motifs that contain the PPLP core.

      DISCUSSION

      We identified the Mena protein as one of the cognate ligands for the WW domain of FE65 adapter protein and mapped the sites of interaction on Mena to polyproline-rich regions containing the signature PPLP motif. More importantly, we documented the binding between Mena and the FE65 WW domain in vitro and in vivo by pull-down experiments from mouse brain lysates and by coimmunoprecipitations of the complex from COS cells overexpressing the FE65 gene product.
      The following aspects of the work deserve brief comment: (a) specificity of the interaction between FE65 and Mena; (b) biological role of the FE65-Mena complex; and (c) functional implications of the FE65-Mena complex for the biogenesis of the βAPP.
      Given the numerous examples of specificity and degeneracy in the protein-protein interactions mediated by SH2, SH3, and known WW domains (
      • Chen H.I.
      • Sudol M.
      ,
      • Chan D.C.
      • Bedford M.T.
      • Leder P.
      ,
      • Staub O.
      • Dho S.
      • Henry P.
      • Correa J.
      • Ishikawa T.
      • McGlade J.
      • Rotin D.
      ,
      • Pawson T.
      ), we consider the FE65-Mena complex a cognate pair. The major criterion for this conclusion is the expression of these two proteins in the same cellular compartment (
      • Gertler F.B.
      • Niebuhr K.
      • Reinhard M.
      • Wehland J.
      • Soriano P.
      ,
      • Simeone A.
      • Duilio A.
      • Fiore F.
      • Acampora R.
      • De Felice P.
      • Faraonio R.
      • Paolocci S.
      • Cimino F.
      • Russo T.
      ) and tissues (i.e. cytoplasm and neural tissues) and formation of the complex in vivo as revealed by its coimmunoprecipitation. Two other observations support the specificity of binding. First, the WW domain of FE65 belongs to the subset of WW domains that contain three aromatic positions in the middle of the linear sequence of the domain. This feature seems to correlate with the preference of the domain for polyproline ligands containing PPLP cores. In contrast, the WW domains with two consecutive aromatic amino acids in the middle bind ligands with PPXY cores (
      • Ermekova K.S.
      • Chang A.
      • Zambrano N.
      • de Candia P.
      • Russo T.
      • Sudol M.
      ,
      • Chen H.I.
      • Sudol M.
      ,
      • Chan D.C.
      • Bedford M.T.
      • Leder P.
      ). Our results are consistent with this observation in that the strongest relative binding of the FE65 WW is to Mena peptides containing PPLP cores. Interestingly, the PPSY motif that is present in the neural isoform of Mena exhibited binding in vitro only to the WW domains of YAP and Nedd4. The unique stretches of homoprolines interrupted by leucines form binding sites to other modules, including the SH3 domains, EVH1 domains, and profilin. Although seemingly degenerate, these interactions may require specific core sequences within the polyproline regions as proposed in the “binary switch” hypothesis for overlapping core motifs for SH3 and WW domains (
      • Sudol M.
      ,
      • Bedford M.T.
      • Chan D.C.
      • Leder P.
      ,
      • Sudol M.
      ). Because the proline-rich core of Mena binds to the profilin, WW, and SH3 domains, it will be important to determine which of these interactions are compatible and which are competitive. Second, the competition experiment, in which PPLP-containing polypeptides were used to dissociate interactions between Mena and FE65, supports our conclusion on the specificity of binding, although the control, “scrambled” peptide was not used due to an intrinsic difficulty in permuting homoprolines and the significant affinity of the FE65 WW domain for hexamer or longer polyprolines.
      Three other proteins present in mouse brain lysate bind to the WW domain of FE65, one of which, the 75-kDa protein, either is a strong binder or is present in relatively higher quantity in the lysates. The identification of these proteins and insight into their biological properties will further our understanding of FE65 function. Perhaps different ligands of the FE65 WW domain compete with each other and thus modulate the transduction of the signal from βAPP to the intracellular pathway (
      • Ermekova K.S.
      • Chang A.
      • Zambrano N.
      • de Candia P.
      • Russo T.
      • Sudol M.
      ).
      The FE65 PTB2 domain interacts with carboxyl-terminal tail of βAPPin vivo (
      • Fiore F.
      • Zambrano N.
      • Minopoli G.
      • Donini V.
      • Duilio A.
      • Russo T.
      ,
      • van der Geer P.
      • Pawson T.
      ,
      • Guenette S.Y.
      • Chen J.
      • Jondro P.D.
      • Tanzi R.E.
      ,
      • Zambrano N.
      • Buxbaum J.D.
      • Minopoli G.
      • Fiore F.
      • De Candia P.
      • De Renzis S.
      • Faraonio R.
      • Sabo S.
      • Cheetham J.
      • Sudol M.
      • Russo T.
      ). Proteins that bind to PTB1 and WW domains of FE65 could be involved in biogenesis of βAPP. Specifically, cellular trafficking of βAPP and its derivatives is a complex process during which βAPP, as well as products of βAPP secretory cleavage, are transported to the endosomes/lysosomes in the clathrin-coated vesicles (
      • Nordstedt C.
      • Caporaso G.L.
      • Thyberg J.
      • Gandy S.E.
      • Greengard P.
      ). The sequence NPTY, which is present in the cytodomain of βAPP and shown to be responsible for interaction with the FE65, was also found to be a specific targeting signal for internalization into the clathrin-coated vesicles of low-density lipoprotein receptor and other proteins (
      • Trowbridge I.S.
      • Collawn J.F.
      • Hopkins C.R.
      ). Therefore, FE65 may serve as an adapter protein that brings other partner molecules into the complex with βAPP, which would effect βAPP secretion, internalization, and/or trafficking. In light of this, Mena, being a cytoskeletal protein involved in microfilament assembly, is a good candidate to participate in cellular network of proteins interacting with βAPP in the cytoplasm. Interestingly, expression of the neuron-enriched isoform of Mena in fibroblasts induces the formation of actin-rich cellular outgrowths implicating Mena in the establishment of cytoskeletal microfilament connections (
      • Gertler F.B.
      • Niebuhr K.
      • Reinhard M.
      • Wehland J.
      • Soriano P.
      ). Similar to its relative, VASP, Mena has been shown to interact with the G-actin binding protein profilin. Furthermore, Mena, profilin, and vinculin (shown to bind to Mena in vitro) are concentrated in axonal growth cones (
      • Gertler F.B.
      • Niebuhr K.
      • Reinhard M.
      • Wehland J.
      • Soriano P.
      ). It is expected that the study of the Mena-FE65 complex may elucidate molecular events that affect or regulate βAPP biogenesis and may provide clues to the molecular changes that underlie Alzheimer disease.

      Acknowledgments

      We thank Joseph Buxbaum, Xavier Espanel, and Paul Greengard for valuable discussions; Alex Chang for the pGEX-2TKMsb1WW construct; and George Pendergast and Edward Ziff for the anti-mbh1 antibodies and constructive advice. We are grateful to Jurgen Wehland and Ronald Frank for their generous gift of the Mena SPOTs filter.

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