Growth Factor Receptor-bound Protein 2 Interaction with the Tyrosine-phosphorylated Tail of Amyloid β Precursor Protein Is Mediated by Its Src Homology 2 Domain
2004; Elsevier BV; Volume: 279; Issue: 24 Linguagem: Inglês
10.1074/jbc.m400488200
ISSN1083-351X
AutoresDawang Zhou, Cristiana Noviello, Chiara D’Ambrosio, Andrea Scaloni, Luciano D'adamio,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoThe sequential processing of the familial disease gene product amyloid β precursor protein (AβPP) by β- and γ-secretases generates amyloid β, which is considered to be the pathogenic factor of Alzheimer's disease, and the AID peptide (AβPP intracellular domain). The AID peptide acts as a positive regulator of apoptosis and modulates transcription and calcium release. To gain clues about the molecular mechanisms regulating the function of AβPP and AID, proteins interacting with the AID region of AβPP have been isolated using the yeast two-hybrid system. Recent evidence indicates that AβPP undergoes post-translational modification events in the AID region and that phosphorylation might regulate its affinity for interacting proteins. To test this possibility and to uncover AβPP-binding partners whose interaction depends on AβPP phosphorylation, we used a proteomic approach. Here we describe a protein, growth factor receptor-bound protein 2 (Grb2), that specifically binds AβPP, phosphorylated in Tyr682. Furthermore, we show that this interaction is direct and that Grb2 binds to phospho-AβPP via its Src homology 2 region. Together with the evidence that Grb2 is in complex with AβPP in human brains and that these complexes are augmented in brains from Alzheimer's cases, our data indicate that Grb2 may mediate some biological and possibly pathological AβPP-AID function. The sequential processing of the familial disease gene product amyloid β precursor protein (AβPP) by β- and γ-secretases generates amyloid β, which is considered to be the pathogenic factor of Alzheimer's disease, and the AID peptide (AβPP intracellular domain). The AID peptide acts as a positive regulator of apoptosis and modulates transcription and calcium release. To gain clues about the molecular mechanisms regulating the function of AβPP and AID, proteins interacting with the AID region of AβPP have been isolated using the yeast two-hybrid system. Recent evidence indicates that AβPP undergoes post-translational modification events in the AID region and that phosphorylation might regulate its affinity for interacting proteins. To test this possibility and to uncover AβPP-binding partners whose interaction depends on AβPP phosphorylation, we used a proteomic approach. Here we describe a protein, growth factor receptor-bound protein 2 (Grb2), that specifically binds AβPP, phosphorylated in Tyr682. Furthermore, we show that this interaction is direct and that Grb2 binds to phospho-AβPP via its Src homology 2 region. Together with the evidence that Grb2 is in complex with AβPP in human brains and that these complexes are augmented in brains from Alzheimer's cases, our data indicate that Grb2 may mediate some biological and possibly pathological AβPP-AID function. IntroductionAlzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid β; AβPP, amyloid β precursor protein; AID, AβPP intracellular domain; PTB, phosphotyrosine binding; Grb2, growth factor receptor-bound protein 2; SH, Src homology; GST, glutathione S-transferase; fl, full-length; MALDI-TOF-MS, matrix-assisted laser desorption ionization-time-of-light-mass spectrometry; strep, streptactin; YFP, yellow fluorescent protein; RTK, tyrosine kinase receptor; WT, wild type; WB, Western blot; MEF, mouse embryonic fibroblast; HEK, human embryonic kidney; MAPK, mitogen-activated protein kinase. constitutes approximately two-thirds of all cases of dementia (1Nussbaum R.L. Ellis C.E. N. Engl. J. 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In differentiated PC12 cells and in SH-SY5Y cells AβPP Thr668 phosphorylation is mediated by Cdk5 (34Iijima K. Ando K. Takeda S. Satoh Y. Seki T. Itohara S. Greengard P. Kirino Y. Nairn A.C. Suzuki T. J. Neurochem. 2000; 75: 1085-1091Crossref PubMed Scopus (204) Google Scholar). Glycogen synthase-3β and more efficiently c-Jun N-terminal kinase-3 also phosphorylate Thr668 in vitro (35Aplin A.E. Jacobsen J.S. Anderton B.H. Gallo J.M. Neuroreport. 1997; 8: 639-643Crossref PubMed Scopus (41) Google Scholar, 36Inomata H. Nakamura Y. Hayakawa A. Takata H. Suzuki T. Miyazawa K. Kitamura N. J. Biol. Chem. 2003; 278: 22946-22955Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). More recently, it has been shown that c-Jun N-terminal kinase-1 and -2 phosphorylate Thr668of AβPP in a JIP1-dependent (36Inomata H. Nakamura Y. Hayakawa A. Takata H. Suzuki T. Miyazawa K. Kitamura N. J. Biol. Chem. 2003; 278: 22946-22955Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 37Scheinfeld M.H. Ghersi E. Davies P. D'Adamio L. J. Biol. Chem. 2003; 278: 42058-42063Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and -independent manner (37Scheinfeld M.H. Ghersi E. Davies P. D'Adamio L. J. Biol. Chem. 2003; 278: 42058-42063Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) in vivo and in vitro. Phosphorylation of Tyr682 (YpENPTY, following the AβPP695 isoform numbering) (where Yp is phosphotyrosine) can be mediated by a constitutively active form of the tyrosine kinase Abl (2Sisodia S.S. St George-Hyslop P.H. Nat. Rev. Neurosci. 2002; 3: 281-290Crossref PubMed Scopus (484) Google Scholar, 38Zambrano N. Bruni P. Minopoli G. Mosca R. Molino D. Russo C. Schettini G. Sudol M. Russo T. J. Biol. Chem. 2001; 276: 19787-19792Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 39Lau L.F. Ahlijanian M.K. Neurosignals. 2003; 12: 209-214Crossref PubMed Scopus (55) Google Scholar, 40Scheinfeld M.H. Ghersi E. Laky K. Fowlkes B.J. D'Adamio L. J. Biol. Chem. 2002; 277: 44195-44201Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) or by overexpression of the nerve growth factor receptor TrkA (41Tarr P.E. Contursi C. Roncarati R. Noviello C. Ghersi E. Scheinfeld M.H. Zambrano N. Russo T. D'Adamio L. Biochem. Biophys. Res. Commun. 2002; 295: 324-329Crossref PubMed Scopus (46) Google Scholar). These phosphorylation events are detected in the brain of normal subjects as well as in patients with Alzheimer's disease. We have found that phosphorylation of Tyr682 affects the interaction of AβPP with some binding partners. ShcA and -C, members of a family of cytoplasmic adaptor proteins that also includes ShcB, contain a PTB region that binds to the YENPTY AβPP motif (42Tarr P.E. Roncarati R. Pelicci G. Pelicci P.G. D'Adamio L. J. Biol. Chem. 2002; 277: 16798-16804Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). However, unlike the other PTB-containing proteins that interact with AβPP, ShcA and -C associate with AβPP only when Tyr682 is phosphorylated. Interestingly, the expression level of ShcA protein is augmented in AD brains as compared with normal brain samples (43Russo C. Dolcini V. Salis S. Venezia V. Violani E. Carlo P. Zambrano N. Russo T. Schettini G. Ann. N. Y. Acad. Sci. 2002; 973: 323-333Crossref PubMed Scopus (31) Google Scholar, 44Russo C. Dolcini V. Salis S. Venezia V. Zambrano N. Russo T. Schettini G. J. Biol. Chem. 2002; 277: 35282-35288Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Moreover, Shc-AβPP complexes are found in human brains and are increased in AD samples.These data underscore the biological relevance of AβPP phosphorylation. Moreover, they suggest that these phosphorylation events may control AβPP functions by regulating the affinity of AβPP for distinct binding partners. Because the yeast two-hybrid system is not ideal to identify interactions that are regulated by post-translational modification events, we used a biochemical approach to isolate brain proteins in which the binding to AβPP is modulated by AβPP phosphorylation. In this study, we describe one of the interactors isolated using this strategy, growth factor receptor-bound protein 2 (Grb2). We present data showing that Grb2 directly interacts with AβPP and that this interaction requires phosphorylation of Tyr682 in AβPP. Unlike the other interactors that bind the YENPTY AβPP motif via their PTB domain, Grb2 binds to YpENPTY via its Src homology (SH) 2 region. These data, together with the finding that a complex of Grb2-AβPP is formed in human brains and that these complexes are increased in AD brains, suggest that Grb2 may mediate some biological and perhaps pathological AβPP-AID function.MATERIALS AND METHODSStrep-tag Peptides Synthesis—Strep-tag peptides including the strep-tag alone (control), strep-tag AID, strep-tag AID phosphothreonine (AID-Thr(P)668), strep-tag AID phosphotyrosine (AID-Tyr(P)682), and strep-tag AID phosphothreonine and phosphotyrosine (AID-Thr(P)668/Tyr(P)682) were synthesized and purified by Tufts University Core Facility (Boston, MA). The sequences of the peptides used are indicated in Fig. 1A.cDNA Constructs—The GST-Grb2 full-length (fl) and GST-Grb2 N- and C-terminal SH3 constructs were generously provided by Dr. Robert A. Weinberg. The GST-Grb2-SH2 construct was prepared by PCR amplification of the SH2 domain. The PCR primers used for the cloning (restriction sites are indicated in boldface) are the following: SH2 sense, 5′-AAAAGGATCCGCCATGTGGTTT-3′; SH2 antisense, 5′-AAA-ACTCGAGTTATTCTATGTC-3′. The amplified product was cloned into pGEX vector (Amersham Biosciences) and sequenced. The GST-AID constructs have been described previously (42Tarr P.E. Roncarati R. Pelicci G. Pelicci P.G. D'Adamio L. J. Biol. Chem. 2002; 277: 16798-16804Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). All of the YFP-Grb2 constructs were prepared by PCR amplification, cloned into YFP-N1 vector (Clontech), and sequenced.Cell Culture—Stock cultures of wild type and ShcA knock-out mouse embryonic fibroblast (MEF) cells (a gift from Dr. G. Pelicci) and human embryonic kidney 293 cells stably transfected with AβPP695 were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (Biofluids, Rockville, MD) and penicillin/streptomycin in a humidified atmosphere containing 7% (v/v) CO2 at 37 °C.GST Fusion Proteins—Recombinant GST fusion proteins were constructed as described above and expressed in Escherichia coli strain BL21 (Invitrogen) to make non-phosphorylated proteins and were expressed in strain TKB1 (Stratagene) to make tyrosine-phosphorylated proteins (42Tarr P.E. Roncarati R. Pelicci G. Pelicci P.G. D'Adamio L. J. Biol. Chem. 2002; 277: 16798-16804Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Proteins were purified using glutathione-Sepharose beads (Amersham Biosciences). Phosphorylation of fusion proteins was confirmed by immunoblotting.Antibodies—Rabbit polyclonal Grb2 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and mouse monoclonal ShcA antibody was from Transduction Laboratories (Lexington, KY). Phosphotyrosine-100 mouse monoclonal antibody was obtained from Cell Signaling Technology (Beverly, MA), and mouse monoclonal P2-1 antibody was a generous gift of W. E. Van Nostrand (45Van Nostrand W.E. Wagner S.L. Shankle W.R. Farrow J.S. Dick M. Rozemuller J.M. Kuiper M.A. Wolters E.C. Zimmerman J. Cotman C.W. Cunningham D.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2551-2555Crossref PubMed Scopus (181) Google Scholar). 22C11 monoclonal antibody was purchased from Chemicon International (Temecula, CA), and horse-radish peroxidase-conjugated secondary antibodies were purchased from Southern Biotechnology Associates (Birmingham, AL).GST Pulldowns, Immunoprecipitation, and Western Blot Analysis— For GST pull-down experiments, cells were collected, washed with phosphate-buffered saline, and lysed with lysis buffer (50 mm Tris/HCl, pH 7.4, 70 mm NaCl, 1% (v/v) Triton X-100, 50 mm sodium fluoride, 1 mm sodium vanadate, pH 7.5) to which was added 2 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride (42Tarr P.E. Roncarati R. Pelicci G. Pelicci P.G. D'Adamio L. J. Biol. Chem. 2002; 277: 16798-16804Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). 6 μg of each GST fusion protein was incubated with cell lysates for 2 h. Samples were washed four times with lysis buffer and then boiled with SDS-PAGE sample buffer. For immunoprecipitation experiments cells were collected 48 h after transfection, washed with phosphate-buffered saline, and lysed with lysis buffer. After 30 min of precleaning with ImmunoPure Plus immobilized protein AG (Pierce), cell lysates were incubated overnight with the antibodies and then processed as described previously. The eluted proteins were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and probed with the primary antibodies for 1 h or overnight. Immunoblots were revealed using horse-radish peroxidase-conjugated secondary antibodies followed by chemiluminescence (Supersignal West Pico, Pierce).In Vitro Protein Interaction Assays—Equivalent molar amounts (3 nmol) of strep-tag AID peptides were incubated with 30 μl of 50% Strep-Tactin matrix (IBA) in a total volume of 400 μl of NET-N buffer (150 mm NaCl, 1 mm EDTA, 50 mm Tris/HCl, 1% (v/v) Nonidet P-40, pH 8.0) for 1 h at 4 °C. The beads were washed two times with 400 μl of NET-N buffer and then incubated with 6 μg of each GST fusion protein in 400 μl of NET-N buffer for 2–4 h at 4 °C. The beads were then washed with 1 ml of NET-N four times. The bound proteins were eluted from the beads by boiling the samples at 95 °C in SDS-PAGE loading buffer for 4 min. Proteins were analyzed by NuPAGE® Novex Bis-Tris 4–12% gel (Invitrogen) electrophoresis, and then each gel was stained with Coomassie Blue.Strep Pulldown from Mice Brain and MEF Cells—The strep pull-down experiment from MEFs was performed as the GST pull-down procedure described above, except that 3 nmol of strep-tag peptide were used instead of 6 μg of GST proteins. To prepare the mice brain lysates, adult BALB/c mice (age 3 months) were euthanized, and brains were removed and homogenized in buffer containing 100 mm Tris/HCl, 150 mm NaCl, 1% (v/v) Triton X-100, 1 mm EDTA, pH 8.0, protease and phosphatase inhibitors. To preclean the lysate, it was passed through the column containing strep-tag beads, and then equal amounts of precleaned lysate were applied onto each column containing Strep-Tactin matrix incubated previously with the different strep-tag peptides. The beads were washed four times with lysis buffer and then eluted with 10 mm desthiobiotin. The pulled samples were analyzed in parallel by SDS-PAGE followed by ammoniacal silver staining or Western blotting.Mass Spectrometry Analysis—Protein bands from SDS-PAGE were excised from the gel, triturated, and washed with water. Samples were reduced in gel, S-alkylated, and digested with trypsin as reported previously (46Talamo F. D'Ambrosio C. Arena S. Del Vecchio P. Ledda L. Zehender G. Ferrara L. Scaloni A. Proteomics. 2003; 3: 440-460Crossref PubMed Scopus (141) Google Scholar). Digests were subjected to a desalting/concentration step on μZipTipC18 (Millipore Corp., Bedford, MA) before mass analysis. Peptide mixtures were loaded on the MALDI target together with α-cyano-4-hydroxycinnamic acid as matrix using the dried droplet technique. Samples were analyzed with a Voyager-DE PRO mass spectrometer (Applied Biosystems, Framingham, MA) (47Gianni D. Zambrano N. Bimonte M. Minopoli G. Mercken L. Talamo F. Scaloni A. Russo T. J. Biol. Chem. 2003; 278: 9290-9297Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar); spectra were acquired in reflectron mode and elaborated using the Data Explorer 4.1 software provided by the manufacturer. Internal mass calibration was performed with peptides derived from enzyme autoproteolysis. The PROWL software package was used to identify bands unambiguously from independent Swiss Protein and NCBI non-redundant sequence data bases.Enzyme-linked Immunosorbent Assay—The human amyloid β-(1–40) enzyme-linked immunosorbent assay kit was purchased from Immuno-Biological Laboratories (Fujioka, Japan). 48 h after transient transfection of YFP and YFP-Grb2 fusion proteins, Aβ-(1–40) was assayed in triplicates from conditioned media of HEK 293 stably transfected with AβPP695. The assay was performed according to the manufacturer's instructions.RESULTSIdentification of Growth Factor Receptor-bound Protein 2 as Binding Protein of Phosphorylated AβPP Tyrosine—With the goal of identifying new AβPP interacting proteins in which binding is regulated by phosphorylation events, a direct AID-proteomic approach was used. Five synthetic peptides, i.e. control peptide, AID, AID phosphothreonine (AID-Thr(P)668), AID phosphotyrosine (AID-Tyr(P)682), and AID phosphothreonine and tyrosine (AID-Thr(P)668/Tyr(P)682) were immobilized on different samples of Strep-Tactin resin (Fig. 1A). A mouse brain cell lysate was applied in parallel on separate columns packed with these differently coated solid supports, was washed, and then was eluted by SDS-PAGE loading buffer. Before binding experiments, lysate was passed twice on resin to remove aspecific ligands. The pulled samples were analyzed in parallel by SDS-PAGE and stained by ammoniacal silver (Fig. 1B). Several bands were detected that did not occur in control, thus demonstrating the suitability of this methodology to isolate AβPP AID interacting proteins. A species migrating with an apparent mass of ∼28 kDa seemed to occur depending on the nature of coated support. In fact, this protein specifically interacted with AID-Tyr(P)682 and AID-Thr(P)668/Tyr(P)682 but not with control, AID, and AID-Thr(P)668 peptides, indicating that this binding to AID requires specific AID phosphorylation on Tyr682. Different bands were excised from the gel, treated, and digested as described under "Materials and Methods" and analyzed by MALDI-TOF-MS. Peptide mass fingerprint experiments and non-redundant sequence data base searching identified the 28-kDa protein as Grb2 (Fig. 1C). These findings were confirmed by analyzing the same samples by SDS-PAGE and Western blot using an anti-Grb2 antibody (WB: αGrb2) (Fig. 1D). The αGrb2 antibody detected a component migrating at 28 kDa only in the samples eluted from the columns coated with AID-Tyr(P)682 and AID-Thr(P)668/Tyr(P)682 peptide and not with control, AID, and AID-Thr(P)668 peptides.The Grb2-AβPP Interaction Is Not Mediated by ShcA—Grb2 is an adaptor protein containing an SH2 domain flanked on both sides by an Src homology domain 3 (SH3) called SH3 N and C termini. The SH2 region of Grb2 interacts with phosphorylated tyrosine residues present in several proteins. Well described Grb2 interactors are tyrosine kinase receptors (RTKs) (48Lowenstein E.J. Daly R.J. Batzer A.G. Li W. Margolis B. Lammers R. Ullrich A. Skolnik E.Y. Bar-Sagi D. Schlessinger J. Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1331) Google Scholar, 49Zhu X. Raina A.K. Rottkamp C.A. Aliev G. Perry G. Boux H. Smith M.A. J. Neurochem. 2001; 76: 435-441Crossref PubMed Scopus (367) Google Scholar, 50Wang Y. Pennock S.D. Chen X. Kazlauskas A. Wang Z. J. Biol. Chem. 2004; 279: 8038-8046Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Upon activation by specific ligands, RTKs undergo tyrosine autophosphorylation. These phosphotyrosines function as docking sites for the SH2 domain of Grb2 and other cytoplasmic adaptors, which include Shc. Receptor-bound Shc proteins can also be phosphorylated by active RTKs and function as docking sites for an indirect recruitment of Grb2 to RTKs. Therefore, Grb2 can bind RTKs either directly or indirectly through Shc. In view of the finding that Shc family members directly bind to tyrosine-phosphorylated AβPP, we first tested whether ShcA proteins bridge Grb2 to AβPP-Tyr(P)682. To this end, we used ShcA knock-out mouse embryonic fibroblasts (kindly provided by G. Pelicci). Protein lysates from ShcA knock-out cells, as well as from wild type MEFs used as a control, were purified by affinity chromatography with control, AID, and AID-Tyr(P)682synthetic peptides, as described under "Materials and Methods." Pulled samples were analyzed by SDS-PAGE and Western blot using either an anti-ShcA (αShcA) or an anti-Grb2 antibody. The lower part of the gel was stained with Coomassie Blue to verify that the same amount of peptides was used for each pulldown (Fig. 2A, lower panel). The three ShcA isoforms of 46, 52, and 66 kDa were identified in the total lysate of wild type but not ShcA knock-out MEFs (lane I). As shown in the top panel of Fig. 2A, the 52- and 46-kDa ShcA isoforms interacted with AID-Tyr(P)682 but not with control and AID peptides. Grb2 protein, which is highly expressed in both wild type and ShcA knock-out MEFs, also interacted specifically with AID-Tyr(P)682 peptide (Fig. 2A, WB αGrb2). Interestingly, AID-Tyr(P)682 was still able to recruit Grb2 from ShcA knock-out MEFs, suggesting that ShcA is not required for the formation of the Grb2-AID-Tyr(P)682 complex.Fig. 2Pull-down experiments from ShcA WT and knock-out MEFs. A, the same amount of total protein was precipitated from ShcA WT and knock-out (KO) MEFs using control, AID, and AID-Tyr(P)682 synthetic peptides. The samples were then analyzed by WB and revealed by αShcA (top panel) and αGrb2 (middle panel) antibodies. All ShcA isoforms, 46-, 52-, and 66-kDa, are detected in the ShcA WT total lysates only (I.). The p52 and p46 ShcA isoforms interact specifically with AID-Tyr(P)682 and not with control or AID peptides. The αGrb2 WB reveals that AID-Tyr(P)682 peptide precipitates Grb2 protein in both ShcA WT and knock-out cells suggesting that ShcA is not necessary for the formation of Grb2-AID-Tyr(P)682 complex. The Coomassie Blue staining in the lower panel shows the amount of peptides used for each pulldown. B, total lysate from ShcA WT and knock-out cells was pulled down using GST-AID and GST-AID-Tyr(P)682 proteins. The WB with the αTyr(P) antibody verifies the phosphorylation state of the GST-AID-Tyr(P)682 proteins (bottom panel). The αShcA WB reveals that all ShcA isoforms are precipitated specifically by GST-AID-Tyr(P)682 and not by GST-AID proteins (top panel). In accord with the strep-tag pull-down experiment Grb2 interaction with GST-AID-Tyr(P)682 proteins does not require ShcA as shown by the αGrb2 WB (second panel). The apparent size differences in the bands detected by the αShcA and αGrb2 antibodies are caused by the gel distortion during the electrophoresis. In t
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