The beta -Amyloid Precursor Protein APP Is Tyrosine-phosphorylated in Cells Expressing a Constitutively Active Form of the Abl Protoncogene*

Nicola ZambranoDagger , Paola BruniDagger §, Giuseppina MinopoliDagger §, Rosario MoscaDagger , Diana MolinoDagger , Claudio Russo, Gennaro Schettini, Marius Sudol||, and Tommaso RussoDagger **

From the Dagger  Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, I-80131 Napoli, Italy, the  Section of Pharmacology, Department of Oncology, Biology and Genetics, University of Genova and Service of Pharmacology and Neuroscience/IST, I-16132 Genova, Italy, and the || Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029-6574

Received for publication, January 29, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

The cytosolic domain of the beta -amyloid precursor protein APP interacts with three PTB (phosphotyrosine binding domain)-containing adaptor proteins, Fe65, X11, and mDab1. Through these adaptors, other molecules can be recruited at the cytodomain of APP; one of them is Mena, that binds to the WW domain (a protein module with two conserved tryptophans) of Fe65. The enabled and disabled genes of Drosophila, homologues of the mammalian Mena and mDab1 genes, respectively, are genetic modulators of the phenotype observed in flies null for the Abl tyrosine kinase gene. The involvement of Mena and mDab1 in the APP-centered protein-protein interaction network suggests the possibility that Abl plays a role in APP biology. We show that Fe65, through its WW domain, binds in vitro and in vivo the active form of Abl. Furthermore, in cells expressing the active form of Abl, APP is tyrosine-phosphorylated. Phosphopeptide analysis and site-directed mutagenesis support the hypothesis that Tyr682 of APP695 is the target of this phosphorylation. Co-immunoprecipitation experiments demonstrate that active Abl and tyrosine-phosphorylated APP also form a stable complex, which could result from the interaction of the pYENP motif of the APP cytodomain with the SH2 domain of Abl. These results suggest that Abl, Mena, and mDab1 are involved in a common molecular machinery and that APP can play a role in tyrosine kinase-mediated signaling.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The amyloid plaques, the major pathological hallmark of Alzheimer's disease (AD),1 contain the beta -amyloid peptide, which results from the proteolytic cleavage of a membrane protein known as beta -amyloid precursor protein (APP) (for review see Ref. 1). The functions of APP are still poorly understood,despite a significant amount of information concerning its involvement in a complex protein-protein interaction network. APP is a type I membrane protein with a small cytosolic domain that has been shown to interact with several soluble proteins. Three of these proteins, Fe65, X11, and mDab1, interact with APP through a PTB domain (2-8). They have the characteristics of adaptor proteins, and thus they could connect APP to different intracellular molecular pathways. In addition to the PTB domain, X11 has two PDZ (PSD-95/DlgA/ZO-1) domains and forms oligomeric complexes with two other proteins, Munc-18 and CASK (9, 10). Fe65 possesses two PTB domains (PTB1 and PTB2), one of which interacts with APP (5) and the other of which interacts with LRP (low density lipoprotein receptor-related protein), a scavenger receptor structurally related to the low density lipoprotein receptor (11), and with the transcription factor CP2/LSF/LBP1 (12). At the N terminus, Fe65 possesses a WW domain, through which it forms complexes with several proteins including Mena (13), the mammalian homologue of the product of the Drosophila enabled gene. The third PTB-containing protein that interacts with APP is mDab1 (7, 8), the mammalian homologue of the product of the Drosophila disabled gene. Genetic studies have demonstrated that insects bearing the mutation of the gene homologous to the mammalian c-abl tyrosine kinase gene survive past morphogenesis with some defects in eye structure, but when this mutation is associated with heterozygous or homozygous mutations of the disabled gene, defects in the formation of central nervous system are observed. Insects that are homozygous for mutations in both Abl and disabled genes show an almost complete loss of proper axonal connections (14-16). These defects are significantly ameliorated by mutations of the enabled gene (17, 18). The functions of the corresponding mammalian proteins, Mena and mDab1, are probably similar. In fact, mice lacking mDab1 show severe abnormalities in the development of the central nervous system, including defects in migration of neurons (19), whereas the mutation of Mena causes defects in neurulation and commissure formation (20).

The c-abl proto-oncogene is the cellular counterpart of the viral oncogene of the Abelson murine leukemia virus, and its gene is involved in the Philadelphia chromosome translocation t (9;22) that generates the BCR-Abl fusion protein in human leukemia (for review see Ref. 21). Abl is a nonreceptor tyrosine kinase similar to c-Src. At the N-terminal half, Abl contains an SH3 and an SH2 domain, and in the middle it contains a kinase domain. In contrast to Src and other kinases, its C terminus is long and contains nuclear localization, nuclear export signals, and domains that are able to interact with DNA and F-actin (for recent reviews, see Refs. 22 and 23). The intracellular distribution of Abl is very peculiar; among the various isoforms of Abl, one (type IV) is membrane-associated and another is soluble (type I). The latter can be found in either the cytosol or the nucleus. The cytosolic Abl interacts with the actin cytoskeleton (24), whereas in the nucleus it can bind DNA and other proteins (25).

Despite the large amount of experimental data on Abl, its functions are still not fully understood. In particular the molecular mechanisms connecting Drosophila Abl to disabled and enabled and mammalian Abl to mDab1 and Mena are not known. However, the opposite effects of the mutations of the enabled and disabled genes in Drosophila suggest that the products of these genes may be involved in the same molecular pathway. This conjecture is in agreement with the observations that both mDab1 and Mena participate in the protein-protein interaction network centered at the cytodomain of APP and supports the hypothesis that c-Abl also could be involved in the APP-centered molecular machinery. In this report we demonstrate that APP is tyrosine-phosphorylated in cells expressing a constitutively active form of Abl. Active Abl is tightly connected to the oligomeric complexes formed by APP, because Abl binds to the WW domain of Fe65 and is co-immunoprecipitated with APP.

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Recombinant Constructs-- The Fe65 and APP695 expression constructs have been described (5). Mouse c-Abl type IV cDNA, cloned into the pCDNA3 vector (Invitrogen), was a kind gift of A. Costanzo. Abl-PP expression vector and pGEX-Abl-SH2 (26) were kindly provided by D. Barilà and G. Superti-Furga. To generate the APPY653F, Y682F, and Y687A constructs, missense mutations in the APP695 cDNA were introduced by the QuikChangeTM site-directed mutagenesis kit (Stratagene) according to manufacturer's instructions, with the following six primer pairs (Ceinge): 5'-GGTGATGCTGAAGAAGAAACAGTTCACATCCATTCATCATGGTGTGG-3'; 5'-CCACACCATGATGAATGGATGTGAACTGTTTCTTCTTCAGCATCACC-3'; 5'-CGGCTACGAAAATCCAACCGCCAAGTTCTTTGAGCAGATGC-3'; 5'-GCATCTGCTCAAAGAACTTGGCGGTTGGATTTTCGTAGCCG-3'; 5'-CCAAGATGCAGCAGAACGGCTTCGAAAATCCAACCTACAAGTTCTTTG-3'; 5'-CAAAGAACTTGTAGGTTGGATTTTCGAAGCCGTTCTGCTGCATCTTGG-3'.

The recombinant constructs were sequenced by the dideoxy terminator method with the Sequenase kit (Amersham Pharmacia Biotech) to confirm the inserted mutations. All plasmids were propagated following standard procedures (27) and purified on Qiagen Maxi-columns (Qiagen).

Cell Culture, Transfections, and Extract Preparation-- COS7 african green monkey kidney cells were cultured in Dulbecco's modified minimal medium supplemented with 10% fetal calf serum at 37 °C in a 5% CO2 atmosphere. For transfection, 3 × 106 cells were electroporated at 250 microfarad and 220 V with 5 µg each of the indicated recombinant constructs. When multiple DNAs were co-transfected, the total amount of DNA was maintained constant by the addition of pRc-CMV empty vector DNA (Invitrogen). 36 h after transfection, cells were harvested in ice-cold phosphate-buffered saline and gently lysed in lysis buffer (50 mM Tris/HCl, pH 7.5, 150 mM sodium chloride, 0.5% Triton X-100, 10% glycerol, 50 mM sodium fluoride, 1 mM sodium vanadate) in the presence of a protease inhibitor mixture (Complete, Roche Molecular Biochemicals). The extracts were clarified by centrifugation at 16,000 × g at 4 °C, and the protein concentration was determined by the Bio-Rad protein assay according to manufacturer's instructions.

Pull-down, Co-immunoprecipitation, and Kinase Assays-- The GST fusion proteins containing the SH2 domain of Abl or the WW, PTB1, and PTB2 domains of Fe65 were obtained by isopropyl-thiogalactoside induction of Escherichia coli cells harboring the corresponding constructs and purified from the bacterial lysates with glutathione-Sepharose resin (Amersham Pharmacia Biotech) according to the instructions of the manufacturer. For the pull-down experiments, purified fusion proteins were used to saturate glutathione-Sepharose beads (10 µl) and challenged to transfected COS7 cell extracts (500 µg/sample). Unbound proteins were removed by washing three times the beads with lysis buffer, while retained proteins were resolved by SDS-PAGE, electroblotted to polyvinylidene difluoride Immobilon-P membrane (Millipore) and analyzed by Western blot with the indicated antibodies. Loading of the GST proteins was checked by staining the filters with Ponceau S solution (Sigma).

Immunoprecipitations were carried out on COS7 lysates from transfected cells with the following antibodies: anti-Fe65 (5); anti-APP 369 (28) or anti-APP monoclonal antibodies 4G8 and 6E10 (Senetek PLC); anti-Abl Ab-3 monoclonal antibody; anti-phosphotyrosine PY99 (Santa Cruz Biotechnology); and rabbit preimmune sera and purified mouse IgGs (Sigma), which were used as control antibodies. Immune complexes were collected with 20 µl of protein A/G plus Sepharose (Santa Cruz), washed three times for 5 min on a rotatory wheel at 4 °C with lysis buffer, and separated on SDS-PAGE gels. The detection of proteins from immunoprecipitation experiments was achieved in Western blot experiments with the indicated above mentioned antibodies or with the anti-APP monoclonal antibody 22C11 (Chemicon), which was followed by enhanced chemiluminescence with the ECL kit (Amersham Pharmacia Biotech).

For the kinase assay (29), washed immune complexes, obtained with anti-Abl sc-23 antibody (Santa Cruz) from Abl-PP-transfected COS7 cell extracts (100 µg), were equilibrated in assay buffer (50 mM Tris/HCl, pH 7.5, 5 mM manganese chloride, 1 mM dithiothreitrol) and washed. The kinase reaction was carried out at room temperature for 15 min by incubating the beads with 20 µl of assay buffer in the presence of 1 µg of synthetic 38-mer APP peptide (Primm) and 5 µCi of [gamma -32P]ATP (3000 Ci/mmol, Amersham Pharmacia Biotech). The sequence of the synthetic 38-mer peptide is the following: K6DAAVTPEERHLSKMQQNGYENPTYKFFEQMQN. Control reactions were carried out in the absence of any protein source and in the presence of nonspecific immune complex. The reaction products were fractionated onto 12.5% SDS-PAGE, stained with Coomassie R-250 (Bio-Rad), and exposed to autoradiographic films. For phosphopeptide analysis (30, 31), the reaction products were resolved by SDS-PAGE and transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore). The labeled peptide band, visualized by autoradiography, was excised and subjected to hydrolysis in the presence of 6 N HCl for 90 min at 110 °C. Lyophilized phospho-amino acids were resuspended in water and resolved by thin layer chromatography on cellulose-coated glass plates (Sigma) in propionic acid, 1 M ammonium hydroxide, 2-propanol solvent (45/17.5/17.5, v/v/v). Detection of the labeled sample was by autoradiography. Phospho-amino acid standards L-phosphoserine, L-phosphothreonine, and L-phosphotyrosine (Sigma) were run on adjacent lanes and detected by ninhydrin spraying.

    RESULTS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Abl Interacts with the WW Domain of the Fe65 Adaptor Protein-- It was recently hypothesized that Abl could interact with APP through the adaptor proteins, which are known to bind to the APP cytodomain (32). We explored the possibility that Fe65, one of the multimodular proteins that forms complexes with APP, could be one of the adaptors that tethers Abl to the cytodomain of APP. To address this point we performed pull-down experiments in which GST-Fe65 fusion proteins were used as baits to entrap Abl from extracts of COS7 cells transfected with plasmid vectors driving the expression of type IV mouse c-Abl or of Abl-PP, in which the mutation of two prolines of c-Abl (Pro242 and Pro249), located between the SH2 domain and the TK domain, results in a constitutive TK activity (33). As shown in Fig. 1A, both PTB domains of Fe65 do not interact with Abl-PP. On the contrary, Abl-PP is affinity-purified by the GST-Fe65 fusion protein containing the WW domain. A similar result was observed by using the extracts from cells transfected with c-Abl, which similarly interacts only with the GST-Fe65-WW protein.


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Fig. 1.   Fe65 and active Abl interact through the WW domain of Fe65. A, glutathione-Sepharose beads were saturated with wild type GST (lanes 2 and 7) or with recombinant GST-Fe65 fusion proteins containing the PTB1 (lane 3), PTB2 (lane 4), or WW (lanes 5 and 8) domains of Fe65 and were incubated with extracts from COS7 cells transfected with Abl-PP (lanes 1-5) or c-Abl (lanes 6-8) expression vectors. Bound proteins were eluted from the resin and resolved by 8% SDS-PAGE. Western blot (WB) was with Abl antibody. The bottom panels (lanes 2'-5', 7', and 8') show the Ponceau S staining of the filters that indicates the amount of each GST protein used. Lanes 1 and 6 contain 10 µg of the lysates used in the pull-down experiments. B, protein extracts from COS7 cells transfected with either Abl-PP (lanes 1-6) or mouse type IV c-Abl (lanes 7-9) expression vectors were immunoprecipitated (IP) with Fe65 antibody or, as a control, with preimmune serum (PI) and resolved by 8% SDS-PAGE. Western blot (lanes 1, 2, and 7-9) was with Abl antibody. The same filter of lanes 1 and 2 was reprobed with anti-pTyr antibody (lanes 3 and 4) and then with Fe65 antibody (lanes 5 and 6). The arrowhead indicates the Abl band and the asterisk the Fe65 bands.

This interaction between Fe65 and Abl, observed in vitro, was analyzed in extracts from COS7 cells transfected with c-Abl or Abl-PP and immunoprecipitated with the Fe65 antibody. As shown in Fig. 1B, this experiment demonstrated that the constitutively active Abl-PP mutant is present in the proteins immunoprecipitated by Fe65 antibody (see lane 2), whereas c-Abl is not (see lane 9). Looking at the amino acid sequence of Abl, there is at least one motif that can be recognized by the WW of Fe65 (PPPPPA) located between amino acids 899 and 904 (13, 34).

The Cytosolic Domain of APP Is Tyr-phosphorylated in Cells Expressing a Constitutively Active Form of Abl-- The Fe65-Abl-PP interaction supports the hypothesis that Fe65 functions as a docking site to bring Abl close to phosphorylation targets. To evaluate whether Fe65 itself is a target of Abl TK, the same blot of Fig. 1B was challenged with an anti-pTyr antibody. This experiment demonstrated that Fe65 is not recognized by this antibody (see Fig. 1B, lane 4), and therefore its tyrosine phosphorylation is unlikely. It was documented that APP is phosphorylated on Ser and Thr in vitro and in vivo (35, 36). However, APP was never observed to be Tyr-phosphorylated. We analyzed the possibility that Tyr residues present in the cytodomain of APP are substrates of Abl TK activity. To this aim, COS7 cells were transfected with an APP695 expression vector and/or with the vector driving the expression of Abl-PP. Fig. 2A shows that proteins immunoprecipitated using the APP antibody contained a band of the same size as that of APP and clearly stained with an anti-phosphotyrosine antibody only in cells that express the constitutively active mutant form of Abl (see lane 2). Conversely, APP is present in the proteins immunoprecipitated with the anti-pTyr antibody, again only in cells expressing the active form of Abl (see Fig. 2A, lanes 6 and 7).


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Fig. 2.   APP is tyrosine-phosphorylated in cells expressing the active form of Abl. A, extracts from COS7 cells transfected with Abl-PP and/or APP695 expression vectors (as indicated) were immunoprecipitated (IP) with anti-APP 369 (lanes 1-4) or anti-pTyr (lanes 6 and 7) antibodies and analyzed by Western blot (WB) with anti-pTyr (lanes 1 and 2) or anti-APP 369 (lanes 3-7) antibodies. Lanes 3 and 4 refer to the same filter as in lanes 1 and 2 stripped and reblotted with APP antibody. Lane 5 contains 10 µg of extract from cells trasfected with Abl-PP and APP expression vectors. B, a 38-amino acid-long peptide designed on the basis of the extreme C-terminal sequence common to all of the APP isoforms was incubated in the presence of [gamma -32P]ATP with immunoprecipitated proteins from COS7 cells transfected with Abl-PP expression vector. Abl antibody was used for immunoprecipitation, and mouse IgG were used as a control. Reaction mixtures were separated by 12.5% SDS-PAGE. Lanes 1-4 show the Coomassie staining of the gel, and lanes 2'-4' show the autoradiography of the same gel. Lanes 2 and 2', peptide incubated with [gamma -32P]ATP; lanes 3 and 3', peptide incubated with [gamma -32P]ATP and mouse IgG-immunoprecipitated proteins; lanes 4 and 4', peptide incubated with [gamma -32P]ATP and anti-Abl-immunoprecipitated proteins. The arrowhead indicates the migration of peptide band. C, the 32P-labeled peptide was hydrolyzed, and the resulting amino acids were separated by thin layer chromatography as described under "Experimental Procedures." The migration of the phospho-amino acid standard is reported: pTyr; pThr, phosphothreonine; and pSer, phosphoserine. D, extracts from COS7 cells transfected with Abl-PP and with wild type APP695 (lanes 1 and 5) or mutants APP Y653F (lanes 2 and 6), APP Y682F (lanes 3 and 7), or APP Y687A (lanes 4 and 8) expression vectors were immunoprecipitated with anti-APP antibodies 4G8 and 6E10 and analyzed by Western blot with anti-pTyr antibody (lanes 1-4). The same filter was reprobed with anti-APP 369 antibody (lanes 5-8).

To evaluate whether the band immunoprecipitated by the APP antibody and stained by the anti-pTyr antibody is the consequence of a Tyr phosphorylation of the APP cytosolic domain, a peptide was synthesized on the basis of the C-terminal sequence of APP, common to all the APP isoforms. This peptide was incubated in the presence of [gamma -32P]ATP with an Abl-immunoprecipitated extract of COS7 cells expressing Abl-PP. Fig. 2B shows the autoradiography of the SDS-PAGE gel and the peptide results to be 32P-labeled only when incubated with the proteins immunoprecipitated with the Abl antibody and not with the proteins immunoprecipitated with unrelated antibodies. The labeled band was transferred onto a polyvinylidene difluoride filter, and the peptide was hydrolyzed as described under "Experimental Procedures." The resulting amino acids were separated by thin layer chromatography, and 32P-labeled amino acids were detected by autoradiography. Fig. 2C shows that the only 32P-labeled residue found in the hydrolyzed peptide was phosphotyrosine.

There are three tyrosines located in the cytodomain of APP (residues 653, 682, and 687 of APP695). To analyze their involvement in the above described phenomena, we generated three mutant APP695 expression vectors in which Tyr653 or Tyr682 has been changed into phenylalanine or Tyr687 has been changed into alanine. These vectors were transfected in COS7 cells with the Abl-PP-expressing vector. As shown in Fig. 2D, the APP-Y653F and APP-Y687A are recognized by the anti-pTyr antibody, whereas the APP-Y682F is not; this suggests the hypothesis that Tyr682 is the actual target of the phosphorylation of the citodomain of APP. This result is in agreement with the data concerning the substrate preference of protein tyrosine kinases, indicating that c-Abl preferentially phosphorylates proteins at the level of the YXXP motif (37), where X are hydrophilic residues, which fits with the YENP sequence we found to be phosphorylated in APP.

APP Co-immunoprecipitates with the Active Form of Abl-- The Western blot experiment of Fig. 2A (see lane 2), in addition to the APP bands also shows a slower migrating band recognized by the anti-pTyr antibody and co-immunoprecipitated by the APP antibody. This band is present only in the samples from COS7 cells co-transfected with Abl-PP, and its size is compatible with that of Abl itself. This possibility was analyzed in the experiments reported in Fig. 3; panel A shows the Western blot analysis with Abl antibody of proteins immunoprecipitated by APP antibody that demonstrated the presence of Abl in the immunoprecipitates. The co-immunoprecipitation of Abl with APP has been observed also in cells not transfected with APP (see Fig. 3A, lane 3), thus suggesting that the transfected, active Abl could also be co-immunoprecipitated with endogenous APP751, present in significant amounts in COS7 cells. The co-immunoprecipitation of APP and Abl was confirmed in the experiment reported in Fig. 3B, in which protein extracts from COS7 cells were immunoprecipitated with Abl antibody and blotted with APP. Also in this case APP is present in the extract immunoprecipitated by the Abl antibody.


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Fig. 3.   APP695 co-immunoprecipitates with active Abl-PP. A, extracts from COS7 cells transfected with Abl-PP and/or with wild type APP695 expression vectors, as indicated, were immunoprecipitated (IP) with APP 4G8 and 6E10 monoclonal antibodies (lanes 2-5) or, as a control, with mouse IgG (lanes 6-9) and analyzed by Western blot with Abl antibody. Lane 1 shows the Western blot analysis with Abl antibody of 10 µg of Abl-PP expressing COS7 cell extract. B, extracts from COS7 cells transfected with Abl-PP and/or with wild type APP695 expression vectors, as indicated, were immunoprecipitated with Abl antibody and analyzed by Western blot with APP 22C11 monoclonal antibody. Lane 1 shows the Western blot analysis with APP antibody of 10 µg of APP expressing COS7 cell extract. C, glutathione-Sepharose beads were saturated with wild type GST (lanes 3 and 5) or with recombinant GST-Abl-SH2 fusion protein (lanes 4 and 6) and were incubated with extracts from COS7 cells transfected with APP695 alone (lanes 3 and 4) or with APP695 plus Abl-PP (lanes 5 and 6). Bound proteins were eluted from the resin and resolved by 8% SDS-PAGE. Western blot (WB) was with APP antibody 369. Lanes 1 and 2 contain 10 µg of the lysates used in the pull-down experiments. The bottom panels (lanes 3'-6') show the Ponceau S staining of the filters that indicates the amount of each GST protein used.

It was demonstrated that the various known protein tyrosine kinases phosphorylate Tyr-containing sites that, upon phosphorylation, are recognized by their own or closely related SH2 domains (38). This finding suggests that the phosphorylation of the APP cytodomain at the level of the YENP motif generates a new motif, pYXXP, that could interact with Abl SH2 domain. To examine this point, COS7 cells were transfected with APP695 or with APP695 and Abl-PP expression vectors. Total extracts from these cells were analyzed in pull-down experiments using a GST fusion protein containing the SH2 domain of Abl. As shown in Fig. 3C, GST-Abl-SH2 protein is able to bind to APP only in cells overexpressing active Abl, thus supporting the hypothesis that tyrosine-phosphorylated APP can form complexes with Abl through the Abl SH2 domain. In any case, it is worth noting that the APP Y682F mutant, which is not tyrosine-phosphorylated, still co-immunoprecipitates with Abl, thus suggesting that in addition to an APP-Abl interaction mediated by the SH2 domain, other complexes could contain both APP and Abl.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We addressed the question of the possible involvement of Abl in the protein-protein interaction network centered at the cytosolic domain of APP and found that APP is tyrosine-phosphorylated in cells expressing a constitutively active Abl. In addition we showed that this protein forms complexes with Fe65 and with APP itself. Our results suggest that Fe65 could bind at the same time APP, through its PTB2 domain, and Abl, through its WW domain, thus allowing the formation of a heterotrimeric complex. As a consequence of this interaction, active Abl is docked close to the APP cytodomain, and this could favor the phosphorylation of its Tyr682 (see a hypothetical model in Fig. 4). The observed co-immunoprecipitation of Tyr-phosphorylated APP and Abl could be the consequence of APP-Abl interaction through the SH2 domain of Abl, which has a high affinity for the pYXXP motif (38). Furthermore, the phosphorylation of Tyr682 is expected to be deleterious for the binding of the PTB domains of Fe65, X11, and mDab1 because it was demonstrated that, at least for Fe65 and X11, this residue is crucial for the formation of the complexes with APP (3), and it is a hydrophobic residue in most of the known PTB binding sites (39).


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Fig. 4.   Proposed model for the recruitment of active Abl to the APP cytodomain. A, Fe65 could bind transiently both APP, through its PTB2 domain, and active Abl, through its WW domain, thus allowing the formation of a heterotrimeric complex. As a consequence of this interaction, active Abl could phosphorylate the Tyr682 residue of APP. mDab1 and Mena are expected to compete with Abl for the binding to APP. B, once phosphorylated, APP could directly interact with the SH2 domains of various proteins including Abl itself. The phosphorylation of Tyr682 is expected to be deleterious for the binding of the PTB domains of Fe65 to APP because this requires a hydrophobic amino acid in that position.

As reported in Fig. 4, there are several lines of evidence suggesting that Abl, mDab1, and Mena are involved in common molecular machineries. It was, in fact, demonstrated that mDab1 binds through its PTB domain with the cytosolic domain of APP (7, 8) and that Mena interacts with the WW domain of Fe65 (13), which, in turn, interacts with APP. Considering the results reported in this paper, it can be hypothesized that Abl, mDab1, and Mena compete for the anchoring to the same intracellular site: mDab1 by competing with Fe65 for the binding to the cytodomain of APP; and Abl and Mena by competing for the binding to the WW domain of Fe65 (Fig. 4).

It is worth noting that the Tyr682 of human APP695 and the YENP motif are both conserved among all the known APPs in primates, rodents, Drosophila, and Caenorhabditis and are present also in the APP-related proteins APLP1 and APLP2. Considering that the overall sequence identity between Drosophila APP (Appl) and the mammalian APPs is less than 30%, the 100% conservation of the cytosolic motif containing the phosphorylated tyrosine suggests that it plays a key functional role. This means that the understanding of the molecular basis of the different phenotypes observed in insects bearing mutations of Drosophila Abl (DAbl) and/or disabled and/or enabled should also take into account the involvement of APP. Appl null flies show behavioral defects that are rescued by human APP, and the possible correlation with the defects caused by DAbl, disabled, and enabled gene mutations is not apparent. However, one could gain better insight by the analysis of the phenotypes of insects bearing combined mutations of Appl with the other three genes. For example, the effects of disabled gene mutation on the Abl -/- flies also could be the consequence of the direct interaction of these two proteins with APP, whereas the amelioration observed in Drosophila Abl -/- disabled -/- following the mutation of the enabled gene could be also based on the competition between the enabled and DAbl gene products for the binding to Appl through Drosophila Fe65.

Although the WW domain of Fe65 interacts in vitro with both c-Abl and Abl-PP, only the complexes between Fe65 and the active form of Abl, and not those with the wild type c-Abl, were found in cell extracts. This effect could be due to a lower amount of c-Abl than Abl-PP available for the formation of the in vivo complexes; or it could be due to a low affinity of c-Abl for the WW domain of Fe65 so that, in vivo, it cannot form a significant number of complexes with Fe65 because of the competition of the other ligands of the WW domain of this protein. Furthermore, active Abl probably has a different conformation than c-Abl, thus acquiring a higher affinity for the WW domain. On the contrary, the APP-Abl direct interaction probably requires an active Abl, because the binding is based on a pTyr-SH2 interaction.

It has been hypothesized often that APP could have some role in signaling, and in a recent review article, Bothwell and Giniger (32) suggested the possibility that intracellular signaling could be involved in the development of AD. Their hypothesis takes into account the numerous reports on various proteins that could be involved in the pathogenesis of AD and suggests a role for c-Abl as a modulator of APP biology. Our results support their hypothesis. A point that deserves attention, besides those discussed in the mentioned review article, concerns the possible involvement of p73 in the molecular machinery under examination. In fact, this protein is a key regulator of apoptosis that binds to and is activated by Abl as a response to DNA damage (40-42). An isoform of p73 functions as an anti-apoptotic protein in developing neurons (43), and the role of its phosphorylation by Abl has not been addressed. The finding that active Abl binds to APP suggested an examination of the possible regulatory effects of this binding on the p73 phosphorylation by Abl and the consequences on this regulation of the enhanced APP proteolytic processing characteristic of AD.

Our results support the hypothesis that Fe65 connects Abl to the APP-centered molecular machinery. However, other possible roles of the Fe65·Abl complex should be examined, as for example those suggested by the observation that both Fe65 and Abl, further than in the cytoplasm, are also localized in the nucleus (24, 44).

    ACKNOWLEDGEMENTS

We thank A. Costanzo, D. Barilà, and G. Superti-Furga for expression vectors and M. Santoro for helpful discussion.

    FOOTNOTES

* This work was supported by grants from the V Framework program (Contract QLK6-1999-02238), European Union, the Consiglio Nazionale delle Ricerche-Italy "Programma Biotecnologie MURST L.95/95," and the "Progetto strategico Basi biologiche delle malattie degenerative del Sistema Nervoso Centrale" (to T. R.), the Alzheimer Association (RG 99-1670), and the Human Frontier Science Program Organization (Grant RG 234 to M. S.).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.

§ Supported by Biogem fellowships.

** To whom correspondence should be addressed: Dipartimento di Biochimica e Biotecnologie Mediche, Via S. Pansini 5, I-80131 Napoli, Italy. Tel.: +390817463131; Fax: +390817464359; E-mail: russot@dbbm.unina.it.

Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M100792200

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; APP, beta -amyloid precursor protein; GST, glutathione S-transferase; PTB, phosphotyrosine binding domain; pTyr, phosphotyrosine; PAGE, polyacrylamide gel electrophoresis; SH2/3, Src homology domains 2 and 3; TK, tyrosine kinase.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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