FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2031565865> ?p ?o ?g. }

• W2031565865 endingPage "20078" @default.
• W2031565865 startingPage "20070" @default.
• W2031565865 abstract "We have isolated a novel protein based on its association with Drosophila APP-like protein (APPL), a homolog of the β-amyloid precursor protein (APP) that is implicated in Alzheimer's disease. This novelAPPL-interactingprotein 1 (APLIP1) contains a Src homology 3 domain and a phosphotyrosine interaction domain and is expressed abundantly in neural tissues. The phosphotyrosine interaction domain of APLIP1 interacts with a sequence containing GYENPTY in the cytoplasmic domain of APPL. APLIP1 is highly homologous to the carboxyl-terminal halves of mammalian c-Jun NH2-terminal kinase (JNK)-interacting protein 1b (JIP1b) and 2 (JIP2), which also contain Src homology 3 and phosphotyrosine interaction domains. The similarity of APLIP1 to JIP1b and JIP2 includes interaction with component(s) of the JNK signaling pathway and with the motor protein kinesin and the formation of homo-oligomers. JIP1b interacts strongly with the cytoplasmic domain of APP (APPcyt), as APLIP1 does with APPL, but the interaction of JIP2 with APPcyt is weak. Overexpression of JIP1b slightly enhances the JNK-dependent threonine phosphorylation of APP in cultured cells, but that of JIP2 suppresses it. These observations suggest that the interactions of APP family proteins with APLIP1, JIP1b, and JIP2 are conserved and play important roles in the metabolism and/or the function of APPs including the regulation of APP phosphorylation by JNK. Analysis of APP family proteins and their associated proteins is expected to contribute to understanding the molecular process of neural degeneration in Alzheimer's disease. We have isolated a novel protein based on its association with Drosophila APP-like protein (APPL), a homolog of the β-amyloid precursor protein (APP) that is implicated in Alzheimer's disease. This novelAPPL-interactingprotein 1 (APLIP1) contains a Src homology 3 domain and a phosphotyrosine interaction domain and is expressed abundantly in neural tissues. The phosphotyrosine interaction domain of APLIP1 interacts with a sequence containing GYENPTY in the cytoplasmic domain of APPL. APLIP1 is highly homologous to the carboxyl-terminal halves of mammalian c-Jun NH2-terminal kinase (JNK)-interacting protein 1b (JIP1b) and 2 (JIP2), which also contain Src homology 3 and phosphotyrosine interaction domains. The similarity of APLIP1 to JIP1b and JIP2 includes interaction with component(s) of the JNK signaling pathway and with the motor protein kinesin and the formation of homo-oligomers. JIP1b interacts strongly with the cytoplasmic domain of APP (APPcyt), as APLIP1 does with APPL, but the interaction of JIP2 with APPcyt is weak. Overexpression of JIP1b slightly enhances the JNK-dependent threonine phosphorylation of APP in cultured cells, but that of JIP2 suppresses it. These observations suggest that the interactions of APP family proteins with APLIP1, JIP1b, and JIP2 are conserved and play important roles in the metabolism and/or the function of APPs including the regulation of APP phosphorylation by JNK. Analysis of APP family proteins and their associated proteins is expected to contribute to understanding the molecular process of neural degeneration in Alzheimer's disease. β-Amyloid precursor protein (APP), 1The abbreviations used are: APPAlzheimer's β-amyloid precursor proteinAPPcytthe cytoplasmic domain of APPAPPsAPP family proteinsAPLP1amyloid precursor-like protein 1APLP2amyloid precursor-like protein 2APPLDrosophilaAPP-like proteinAPPLcytthe cytoplasmic domain of APPLAPLIP1APPL-interacting protein 1CHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidDJNKDrosophila JNKDKLCDrosophila kinesin light chainDLKdual leucine zipper-bearing kinaseGSTglutathione S-transferaseHAhemagglutininHepHemipterousJNKc-Jun NH2-terminal kinaseJIPJNK-interacting proteinMAPmitogen-activated proteinPI domainphosphotyrosine interaction domainrp49ribosomal protein 49RT-PCRreverse transcription PCRSH3 domainSrc homology domain 31The abbreviations used are: APPAlzheimer's β-amyloid precursor proteinAPPcytthe cytoplasmic domain of APPAPPsAPP family proteinsAPLP1amyloid precursor-like protein 1APLP2amyloid precursor-like protein 2APPLDrosophilaAPP-like proteinAPPLcytthe cytoplasmic domain of APPLAPLIP1APPL-interacting protein 1CHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidDJNKDrosophila JNKDKLCDrosophila kinesin light chainDLKdual leucine zipper-bearing kinaseGSTglutathione S-transferaseHAhemagglutininHepHemipterousJNKc-Jun NH2-terminal kinaseJIPJNK-interacting proteinMAPmitogen-activated proteinPI domainphosphotyrosine interaction domainrp49ribosomal protein 49RT-PCRreverse transcription PCRSH3 domainSrc homology domain 3 implicated in the cause and progression of Alzheimer's disease, is a receptor-like transmembrane protein consisting of an extracellular domain, a transmembrane domain, and a short cytoplasmic domain (for review, see Ref. 1Selkoe D.J. Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1515) Google Scholar). APP belongs to the conserved APP family (APPs), which includes amyloid precursor-like protein 1 and 2 (APLP1 and APLP2) in mammals, APP747 in Xenopus, elAPP in electric ray, APL-1 in Caenorhabditis elegans, and APP-like (APPL) in Drosophila (for review, see Ref. 2Coulson E.J. Paliga K. Beyreuther K. Masters C.L. Neurochem. Int. 2000; 36: 175-184Crossref PubMed Scopus (156) Google Scholar). Besides the involvement of APP in the pathogenic process of Alzheimer's disease, APPs are expected to be functionally important because mice lacking all of the APP family genes die in the early postnatal period (3Heber S. Herms J. Gajic V. Hainfellner J. Aguzzi A. Rulicke T. Kretzschmar H. von Koch C. Sisodia S. Tremml P. Lipp H.P. Wolfer D.P. Müller U. J. Neurosci. 2000; 20: 7951-7963Crossref PubMed Google Scholar), but the physiological role of APPs has not been analyzed sufficiently in mammal. Except for mammalian APPs, Drosophila APPL is the most characterized (4Rosen D.R. Martin-Morris L. Luo L.Q. White K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2478-2482Crossref PubMed Scopus (170) Google Scholar). APPL is expressed abundantly in neural tissue (5Martin-Morris L.E. White K. Development. 1990; 110: 185-195PubMed Google Scholar) and has been associated with synaptic differentiation at the neuromuscular junction (6Torroja L. Packard M. Gorczyca M. White K. Budnik V. J. Neurosci. 1999; 19: 7793-7803Crossref PubMed Google Scholar). APPL is metabolized in the same manner as mammalian APPs (7Luo L.Q. Martin-Morris L.E. White K. J. Neurosci. 1990; 10: 3849-3861Crossref PubMed Google Scholar), and human APP expressed in fly is transported normally in neurons (8Yagi Y. Tomita S. Nakamura M. Suzuki T. Mol. Cell. Biol. Res. Commun. 2000; 4: 43-49Crossref PubMed Scopus (23) Google Scholar). Furthermore, the behavioral defect of APPL-null flies is partially rescued by the expression of human APP (9Luo L. Tully T. White K. Neuron. 1992; 9: 595-605Abstract Full Text PDF PubMed Scopus (226) Google Scholar). These facts suggest that the molecular mechanism of metabolism and physiological roles of APPs may be basically conserved among species. Revealing the characteristics of APPs which have been conserved among species may be helpful in understanding the character of APP and the molecular mechanism of the pathogenic process of Alzheimer's disease. Alzheimer's β-amyloid precursor protein the cytoplasmic domain of APP APP family proteins amyloid precursor-like protein 1 amyloid precursor-like protein 2 DrosophilaAPP-like protein the cytoplasmic domain of APPL APPL-interacting protein 1 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Drosophila JNK Drosophila kinesin light chain dual leucine zipper-bearing kinase glutathione S-transferase hemagglutinin Hemipterous c-Jun NH2-terminal kinase JNK-interacting protein mitogen-activated protein phosphotyrosine interaction domain ribosomal protein 49 reverse transcription PCR Src homology domain 3 Alzheimer's β-amyloid precursor protein the cytoplasmic domain of APP APP family proteins amyloid precursor-like protein 1 amyloid precursor-like protein 2 DrosophilaAPP-like protein the cytoplasmic domain of APPL APPL-interacting protein 1 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Drosophila JNK Drosophila kinesin light chain dual leucine zipper-bearing kinase glutathione S-transferase hemagglutinin Hemipterous c-Jun NH2-terminal kinase JNK-interacting protein mitogen-activated protein phosphotyrosine interaction domain ribosomal protein 49 reverse transcription PCR Src homology domain 3 The cytoplasmic domains of APPs are more highly conserved than the other domains among a wide variety of species and are important for intracellular metabolism (10Perez R.G. Soriano S. Hayes J.D. Ostaszewski B. Xia W. Selkoe D.J. Chen X. Stokin G.B. Koo E.H. J. Biol. Chem. 1999; 274: 18851-18856Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 11Tomita S. Kirino Y. Suzuki T. J. Biol. Chem. 1998; 273: 19304-19310Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and for physiological function (12Ando K. Oishi M. Takeda S. Iijima K. Isohara T. Nairn A.C. Kirino Y. Greengard P. Suzuki T. J. Neurosci. 1999; 19: 4421-4427Crossref PubMed Google Scholar, 13Allinquant B. Hantraye P. Mailleux P. Moya K. Bouillot C. Prochiantz A. J. Cell Biol. 1995; 128: 919-927Crossref PubMed Scopus (232) Google Scholar, 14Perez R.G. Zheng H. Van der Ploeg L.H. Koo E.H. J. Neurosci. 1997; 17: 9407-9414Crossref PubMed Google Scholar, 15Xu X. Yang D. Wyss-Coray T. Yan J. Gan L. Sun Y. Mucke L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7547-7552Crossref PubMed Scopus (78) Google Scholar). Several proteins have been reported to interact with the cytoplasmic domain of APP (APPcyt) in mammals (16Borg J.P. Ooi J. Levy E. Margolis B. Mol. Cell. Biol. 1996; 16: 6229-6241Crossref PubMed Scopus (429) Google Scholar, 17Guenette S.Y. Chen J. Jondro P.D. Tanzi R.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10832-10837Crossref PubMed Scopus (149) Google Scholar, 18Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 19Zheng P. Eastman J. Vande Pol S. Pimplikar S.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14745-14750Crossref PubMed Scopus (106) Google Scholar, 20Trommsdorff M. Borg J.P. Margolis B. Herz J. J. Biol. Chem. 1998; 273: 33556-33560Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 21Watanabe T. Sukegawa J. Sukegawa I. Tomita S. Iijima K. Oguchi S. Suzuki T. Nairn A.C. Greengard P. J. Neurochem. 1999; 72: 549-556Crossref PubMed Scopus (43) Google Scholar), although the importance and physiological function(s) of these interactions are not well understood. In invertebrates the protein interaction of APPs has been scarcely identified. In this study we identified a novel gene in Drosophila, named APLIP1, which encodes a protein that interacts with the cytoplasmic domain of APPL (APPLcyt). APLIP1 was homologous to mammalian scaffold proteins JIP1b and JIP2, which were implicated in the JNK signaling cascade and/or intracellular transport (22Dickens M. Rogers J.S. Cavanagh J. Raitano A. Xia Z. Halpern J.R. Greenberg M.E. Sawyers C.L. Davis R.J. Science. 1997; 277: 693-696Crossref PubMed Scopus (624) Google Scholar, 23Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (581) Google Scholar, 24Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (403) Google Scholar, 25Verhey K.J. Meyer D. Deehan R. Blenis J. Schnapp B.J. Rapoport T.A. Margolis B. J. Cell Biol. 2001; 152: 959-970Crossref PubMed Scopus (489) Google Scholar). They could interact with APPs and in addition shared abundant expression in neural tissue and the properties to bind JNK kinase and kinesin. Moreover, we propose a possible function of mammalian JIP to modulate the phosphorylation of APP. Analysis of this evolutionarily conserved interaction of APPs with APLIP1 and JIP contributes to our knowledge of the mechanism of Alzheimer's disease progression. The yeast two-hybrid screening was executed with the MATCH MAKER two-hybrid system (Clontech) as described (18Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The pGBT9APPLcyt “bait” plasmid encoding the cytoplasmic domain (amino acids 834–886) of APPL (4Rosen D.R. Martin-Morris L. Luo L.Q. White K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2478-2482Crossref PubMed Scopus (170) Google Scholar) and a Drosophila whole adult cDNA library cloned into the vector pACT2 (Clontech) were cotransfected into the yeast HF7c strain. Cotransfectants were grown on a selective medium lacking Trp, Leu, and His and were assayed for their activation of lacZ reporter genes. Positive clones were recloned into Escherichia coli HB101, and their nucleotide sequences were determined. Quantification of the β-galactosidase activity of cotransfectants was executed with a liquid assay using o-nitrophenyl galactopyranoside according to the manufacturer's protocol (Clontech) and was described in Miller units. The cDNA encoding full-length APLIP1 was isolated from a Drosophila embryonic cDNA library (a kind gift from Dr. Ueda) using a radiolabeled probe prepared from a partial fragment of APLIP1 (nucleotides 841–1788). Hybridizations were carried out with a standard procedure (26Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual, 2nd Ed., Chap. 12, Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar). Positive clones were subjected to excision of the pBluescript phagemid from the λZAPII vector using the ExAssist/SOLR system (Stratagene). Mouse clones were isolated by cross-hybridization with a mouse brain cDNA library cloned into the UNIZAP XR phage vector (Stratagene) using an APLIP1 fragment as a probe. The 5′-upstream sequence of mouse JIP2 cDNA was obtained by 5′-rapid amplification of cDNA ends (version 2; Invitrogen) from mouse whole brain total RNA using a reverse primer (5′-CCTTTTCACAGTGGTCCGAG-3′) and a nested PCR reverse primer (5′-TAAGGCCCAGGCCACAGT-3′). The cDNA encoding APPLcyt was produced by RT-PCR using adult total RNA, and the resulting cDNA fragment encoding amino acid positions 834–886 of APPL (4Rosen D.R. Martin-Morris L. Luo L.Q. White K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2478-2482Crossref PubMed Scopus (170) Google Scholar) was cloned into pGBT9 (Clontech) at EcoRI/BamHI sites for yeast two-hybrid screening. Various deletions of APPLcyt were produced by PCR and cloned into pGBT9 for two-hybrid analysis: ΔN12 (846–886), ΔN21 (855–886), ΔC25 (834–861), and Δ873–882 (internal 873–882 deletion in APPLcyt 834–886). cDNAs encoding APPLcyt(834–886) and APPLcytΔ873–882 were inserted into a pGEX-4T-1 vector (Amersham Biosciences) at EcoRI/SalI sites to produce GST fusion proteins. The cDNA encoding the full-length APLIP1 protein (APLIP1) and the PCR-produced carboxyl-terminal deletion construct CΔ137 (amino acids 1–353) were cloned into pGAD424 (Clontech) at SmaI/PstI sites. Other various amino-terminal truncated proteins of APLIP1 (NΔ84 (amino acids 85–490), NΔ207 (208–490), NΔ280 (281–490), NΔ330 (331–490), and NΔ387 (388–490)) were also produced by PCR and cloned into pGAD424 (Clontech) at EcoRI/PstI. cDNAs of APLIP1, NΔ207, and CΔ137 were cloned into the mammalian expression vector pcDNA3.1Myc/HisA (Invitrogen) to produce pcDNA3.1APLIP1Myc/HisA, pcDNA3.1APLIP1-NΔ207Myc/HisA, and pcDNA3.1APLIP1-CΔ137Myc/HisA, respectively; these constructs express proteins tagged with a c-Myc epitope at the carboxyl terminus in mammalian cells. Full-length cDNAs of Drosophila APPL (GenBank J04516),Drosophila JNK (DJNK; GenBank U49249), and Drosophila hemipterous (Hep; GenBank U93032), and a partial cDNA encoding amino acids 162–508 of the Drosophila kinesin light chain (DKLC; GenBank L11013) were obtained by RT-PCR from poly(A)+ RNA of 12–18-h embryos. APPL cDNA was inserted into pcDNA3 at EcoRI/XbaI. Hep and DKLC cDNA were inserted into pcDNA3 at EcoRI/XhoI with a FLAG tag at the amino terminus. DJNK and Hep were cloned into pGEX-4T-1 (AmershamBiosciences) at SmaI/XhoI and EcoRI/XhoI sites, respectively, to produce GST fusion proteins. The constructs pcDNA3APP695 (18Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) and pcDNA3-FLAG-APP695 (12Ando K. Oishi M. Takeda S. Iijima K. Isohara T. Nairn A.C. Kirino Y. Greengard P. Suzuki T. J. Neurosci. 1999; 19: 4421-4427Crossref PubMed Google Scholar) have been described. Full-length cDNAs of JIP1 (GenBank AF054611) and JIP2 (GenBank AF220195) were obtained from mouse brain total RNA by RT-PCR and cloned into pcDNA3 at EcoRI/XbaI and EcoRI/XhoI sites, respectively, with FLAG or hemagglutinin (HA) tags at the their amino termini. The cDNA encoding JIP1a (GenBank AF003115), which is also known as a splicing variant of JIP1 with an internal deletion of amino acids 557–603 in JIP1b (22Dickens M. Rogers J.S. Cavanagh J. Raitano A. Xia Z. Halpern J.R. Greenberg M.E. Sawyers C.L. Davis R.J. Science. 1997; 277: 693-696Crossref PubMed Scopus (624) Google Scholar, 23Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (581) Google Scholar), was produced by PCR and cloned into the EcoRI and XbaI sites of pcDNA3 (Invitrogen), with the HA sequence at the amino terminus. pcDNA3-HA-X11L were produced by inserted HA sequence into amino terminus of full-length cDNA of X11L cloned into pcDNA3 (18Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The cDNA encoding dual leucine zipper-bearing kinase (DLK; GenBank NM006301) was obtained from human brain total RNA (Clontech) by RT-PCR and cloned into pcDNA3.1Myc/HisA to express proteins tagged with a c-Myc epitope at the carboxyl terminus. Poly(A)+ RNA was isolated from various developmental stages of embryos, larvae, pupae, and adult flies (Canton-S) using a μMACS RNA isolation kit (PerkinElmer Life Sciences). For Northern blotting, 1 μg of poly(A)+ RNA was loaded onto a 1% (w/v) agarose gel under denatured conditions, electrophoresed, and transferred to a membrane. Radiolabeled probes (>108 cpm/μg DNA) of APLIP1 and ribosomal protein 49 (rp49) were prepared with [α-32P]dCTP (3,000 Ci/mmol, PerkinElmer Life Sciences) and a random primer labeling kit (Roche Diagnostics). Hybridization was carried out using ExpresHyb hybridization solution (Clontech) according to the user's manual, and signals were detected by autoradiography. A cDNA of rp49 (GenBank U92431) was obtained by RT-PCR with adult head poly(A)+RNA. For RT-PCR analysis, poly(A)+ RNA, prepared from adult heads and bodies separated using glass beads and a sieve, were reverse transcribed with SuperScript II (Invitrogen) using an oligo(dT) primer for 2 h at 37 °C and treated with RNase H (Takara). A PCR was performed with Ex-Taq (Takara) using [α-32P]dCTP and primer sets specific for APLIP1 and rp49 as follows: APLIP1, 5′-AATGCGGCTACTTGATG-3′ and 5′-GTGGTGCCGCGGCACAAA-3′; rp49, 5′-AGTCGGATCGATATGCTAAG-3′ and 5′-AGTAAACGCGGTTCTGCATG-3′. The resulting products were electrophoresed on 5% (w/v) acrylamide gels and quantified with a Fuji BAS image analyzer. A digoxigenin-labeled RNA probe was prepared with T7 RNA polymerase (Roche Diagnostics), digoxigenin-11-UTP, and APLIP1 cDNA in pBluescript SKII digested with EcoRI as a template for the antisense probe. Embryos were dechorionated and fixed as described previously (27Yagi Y. Hayashi S. Genes Cells. 1997; 2: 41-53Crossref PubMed Scopus (27) Google Scholar). Hybridization was performed with antisense probes and visualized using an anti-digoxigenin antibody conjugated to alkaline phosphatase and an nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate substrate (Roche Diagnostics). The rabbit polyclonal anti-APP antibody G369 has been described previously (28Oishi M. Nairn A.C. Czernik A.J. Lim G.S. Isohara T. Gandy S.E. Greengard P. Suzuki T. Mol. Med. 1997; 3: 111-123Crossref PubMed Google Scholar). A rabbit polyclonal anti-APPL antibody raised against the extracellular domain of APPL (Ab952) was a kind gift from Dr. K. White (29Torroja L. Luo L. White K. J. Neurosci. 1996; 16: 4638-4650Crossref PubMed Google Scholar). Anti-FLAG (M2; Sigma), anti-HA (12CA5; Roche Diagnostics), and anti-c-Myc (Invitrogen) monoclonal antibodies were purchased. Polyclonal APP phosphorylation state-specific antibody (pAbThr-668) UT-33 was raised against a phosphopeptide APP665–673[Cys][PiThr-668] of APP695 and described previously (12Ando K. Oishi M. Takeda S. Iijima K. Isohara T. Nairn A.C. Kirino Y. Greengard P. Suzuki T. J. Neurosci. 1999; 19: 4421-4427Crossref PubMed Google Scholar, 30Iijima 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 (201) Google Scholar, 31Ando K. Iijima K.I. Elliott J.I. Kirino Y. Suzuki T. J. Biol. Chem. 2001; 276: 40353-40361Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). African green monkey kidney COS-7 and mouse neuroblastoma Neuro-2a cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum. To express proteins, 5 × 105 ∼1 × 106 COS-7 or ∼1 × 106 Neuro-2a cells were transiently transfected with 0.5–2 μg of each plasmid in LipofectAMINE 2000 (Invitrogen) or LipofectAMINE (Invitrogen) according to the manufacturer's protocol and cultured for 24–48 h in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum. The cells were harvested and lysed in CHAPS lysis buffer (phosphate-buffered saline containing 10 mm CHAPS, 5 μg/ml chymostatin, 5 μg/ml leupeptin, 5 μg/ml pepstatin A, 1 mm Na3VO4, and 1 mm NaF) for 0.5 h at 4 °C. Microcystine-LR (1 μm) was added to the buffer to analyze the phosphorylation. The lysed cells were centrifuged at 12,000 ×g for 10 min at 4 °C, and the supernatant was used for the following analysis. A pGEX-4T-1 cDNA construct was introduced into E. coli BL21, and a GST fusion protein was prepared. The protein was purified with glutathione-Sepharose 4B (Amersham Biosciences) as described (18Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), and the purity of the protein was examined by staining with Coomassie Brilliant Blue R-250 after SDS-PAGE. The purified GST fusion proteins (∼1 nmol) coupled to glutathione-Sepharose 4B were incubated for 2 h at 4 °C with cell lysates derived from COS-7 cells transiently expressing the recombinant protein. The beads were washed with phosphate-buffered saline (10 mm sodium phosphate, pH 7.4, 140 mm NaCl) three times, and the protein bound to the GST fusion protein was eluted with SDS sample buffer and analyzed by Western blot with ECL (Amersham Biosciences) or125I-protein A (Amersham Biosciences). The supernatant prepared from double transfected COS-7 cells was incubated with 5–10 μg of antibody at 4 °C for 1 h. The resulting immunocomplex bound to mouse antibodies was recovered using protein G-Sepharose beads (Amersham Biosciences) and analyzed by Western blot with the antibody indicated. APPs are thought to function through interaction of their cytoplasmic domains with cytoplasmic proteins. Therefore, we screened a Drosophila adult cDNA library with the yeast two-hybrid system using APPLcyt as bait and obtained clones encoding part of a novel protein. The full-length cDNA was isolated from an embryonic cDNA library by plaque hybridization using partial cDNA as a probe. The full-length cDNA obtained encoded a protein consisting of 490 amino acids and containing putative SH3 and PI domains in its carboxyl-terminal half (Fig. 1, A and B). We called this novel protein APLIP1, for APP-like proteininteracting protein 1. From an analysis of the Drosophila genomic data base (32Celera Genomics, Inc. Berkeley Drosophila Genome Project teamScience. 2000; 287: 2185-2195Crossref PubMed Scopus (4744) Google Scholar), the APLIP1 gene was revealed to be composed of five exons and to be located on region 61F1–61F4 of chromosome 3L. No other gene resembling APLIP1 was found in the data base. In this two-hybrid screening, a cDNA encoding the carboxyl-terminal fragment of the Drosophila homolog of X11L (dX11L) (GenBank AF208839) 2M. Hase, Y. Yagi, H. Taru, S. Tomita, A. Sumioka, K. Hori, Y. Miyamoto, T. Sasamura, M. Nakamura, K. Matsuno, and T. Suzuki, J. Neurochem. (2002) in press. and a putative Ser/Thr protein kinase, Bin4 (GenBank AF096866), were also selected as positive clones. The binding activities of these clones with APPL examined in yeast are comparable with that of APLIP1. 3H. Taru, K. Iijima, M. Hase, Y. Kirino, Y. Yagi, and T. Suzuki, unpublished observation. APPL is expressed specifically in neural tissues during development (4Rosen D.R. Martin-Morris L. Luo L.Q. White K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2478-2482Crossref PubMed Scopus (170) Google Scholar, 7Luo L.Q. Martin-Morris L.E. White K. J. Neurosci. 1990; 10: 3849-3861Crossref PubMed Google Scholar). If APLIP1 interacts with the cytoplasmic domain of APPL in vivo, we would expect APLIP1 to be expressed in neural tissues as well. Therefore, we examined the expression profile of APLIP1. Northern blot analysis was performed against poly(A)+ RNA prepared from various developmental stages (Fig. 2 A). A 2.4-kb transcript showed strong expression during late embryonic (12–18 h after egg deposition (E12–18)) to adult stages. No signal could be detected in the lane containing RNA from an E0–6 embryo, and only a weak signal was observed in E6–12 embryos. This expression profile is similar to that of APPL, although expression of APPL is weak in larval stages (4Rosen D.R. Martin-Morris L. Luo L.Q. White K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2478-2482Crossref PubMed Scopus (170) Google Scholar). Next we examined whether the expression of APLIP1 is enriched in the nervous system. We prepared poly(A)+ RNA from the heads and bodies of adult flies and quantified the amount of transcript of the APLIP1 and rp49 by RT-PCR using specific primer sets (Fig. 2 B). A larger amount of the APLIP1 PCR product was obtained from the heads than from the bodies, whereas comparable amounts of the PCR product derived from the rp49 RNA were observed in the heads and bodies. This result suggests that APLIP1 is expressed largely in neural tissues, as is APPL. The abundant expression of the APLIP1 in the neural tissues was confirmed by in situ hybridization (Fig. 2 C).APLIP1 expression was detected in the brain and central nervous system of late embryos (Fig. 2 C), whereas no signal was observed in early embryos (data not shown). These observations suggest the possibility that APLIP1 interacts with APPL in neural tissues. The APLIP1 protein interacted with APPL in yeast cells. To investigate whether they interact in other systems, we examined protein-protein interactions between APLIP1 and APPL in vitro using a GST fusion protein. A GST-APLIP1 fusion protein was prepared in bacteria, purified, immobilized on glutathione-Sepharose, and incubated with a lysate of COS-7 cells expressing APPL. APPL expressed in COS-7 cells exhibits several bands probably differing in glycosylation. APPL attached to the beads was washed, eluted together with GST-APLIP1, and detected by Western blot analysis using an anti-APPL antibody (Fig. 3 A). APPL bound to the beads coupled with the GST-APLIP1 fusion protein but not to GST alone. We examined the interaction of APLIP1 with APPL in cells (Fig. 3 B). APLIP1 tagged with FLAG epitope (FLAG-APLIP1) was expressed in COS-7 cells together with or without APPL (left panel of Fig. 3 B). The proteins were immunoprecipitated from the cell lysate with an anti-FLAG antibody and analyzed by immunoblot with anti-APPL and anti-FLAG antibodies. We observed that APPL was coimmunoprecipitated from the cell lysate with the FLAG-APLIP1 (middle panel of Fig. 3 B). Conversely, when we immunoprecipitated APPL from the cell lysates with anti-APPL antibody, FLAG-APLIP1 was recovered with APPL (right panel of Fig. 3 B). These results indicate that APLIP1 binds to APPL both in vitro and in the cell. The region required for the interaction between APLIP1 and APPL was determined. The ability of various truncated protein constructs derived from APLIP1 to bind to the APPLcyt was examined using the yeast two-hybrid system. Growth in a selective medium and β-galactosidase activity were analyzed (Fig. 4 A). Proteins that were truncated but still contained the putative SH3 and PI domains in the carboxyl-terminal half (NΔ84, NΔ207, and NΔ280) were able to bind APPLcyt. A transformant harboring a protein lacking the SH3 region (NΔ330) showed no significant difference in β-galactosidase activity compared with the control (mock) but still could grow in the selective medium. However, NΔ387, which lacked the amino-terminal side of the PI domain, and CΔ137, which lacks the carboxyl-terminal region including the PI domain, exhibited neither β-galactosidase activity nor growth in the selective medium. This result suggests that the carboxyl-terminal region containing the PI domain of APLIP1 is essential for binding to APPLcyt and that the SH3 domain may be involved in the preservation of the struct" @default.
• W2031565865 created "2016-06-24" @default.
• W2031565865 creator A5008474125 @default.
• W2031565865 creator A5013078816 @default.
• W2031565865 creator A5017043028 @default.
• W2031565865 creator A5040622924 @default.
• W2031565865 creator A5053314310 @default.
• W2031565865 creator A5065167441 @default.
• W2031565865 date "2002-05-01" @default.
• W2031565865 modified "2023-10-14" @default.
• W2031565865 title "Interaction of Alzheimer's β-Amyloid Precursor Family Proteins with Scaffold Proteins of the JNK Signaling Cascade" @default.
• W2031565865 cites W1527604508 @default.
• W2031565865 cites W1587773919 @default.
• W2031565865 cites W1757544724 @default.
• W2031565865 cites W1882078023 @default.
• W2031565865 cites W1902275498 @default.
• W2031565865 cites W1967218150 @default.
• W2031565865 cites W1972188808 @default.
• W2031565865 cites W1977052613 @default.
• W2031565865 cites W1983795337 @default.
• W2031565865 cites W1994825168 @default.
• W2031565865 cites W2006300948 @default.
• W2031565865 cites W2007133768 @default.
• W2031565865 cites W2014873177 @default.
• W2031565865 cites W2017957082 @default.
• W2031565865 cites W2022762164 @default.
• W2031565865 cites W2022840078 @default.
• W2031565865 cites W2023531011 @default.
• W2031565865 cites W2034201162 @default.
• W2031565865 cites W2039750814 @default.
• W2031565865 cites W2040732053 @default.
• W2031565865 cites W2041076714 @default.
• W2031565865 cites W2041581676 @default.
• W2031565865 cites W2042057922 @default.
• W2031565865 cites W2045395146 @default.
• W2031565865 cites W2045929031 @default.
• W2031565865 cites W2046402345 @default.
• W2031565865 cites W2047604959 @default.
• W2031565865 cites W2057840062 @default.
• W2031565865 cites W2065384834 @default.
• W2031565865 cites W2075385027 @default.
• W2031565865 cites W2088795208 @default.
• W2031565865 cites W2090560595 @default.
• W2031565865 cites W2094597369 @default.
• W2031565865 cites W2096910934 @default.
• W2031565865 cites W2106882534 @default.
• W2031565865 cites W2129645512 @default.
• W2031565865 cites W2136653849 @default.
• W2031565865 cites W2140851951 @default.
• W2031565865 cites W2141652419 @default.
• W2031565865 cites W2147103035 @default.
• W2031565865 cites W2150596288 @default.
• W2031565865 cites W2155483393 @default.
• W2031565865 cites W2157303458 @default.
• W2031565865 cites W2162051767 @default.
• W2031565865 cites W2164063328 @default.
• W2031565865 cites W39375236 @default.
• W2031565865 cites W4299940357 @default.
• W2031565865 cites W4302977162 @default.
• W2031565865 doi "https://doi.org/10.1074/jbc.m108372200" @default.
• W2031565865 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11912189" @default.
• W2031565865 hasPublicationYear "2002" @default.
• W2031565865 type Work @default.
• W2031565865 sameAs 2031565865 @default.
• W2031565865 citedByCount "128" @default.
• W2031565865 countsByYear W20315658652012 @default.
• W2031565865 countsByYear W20315658652013 @default.
• W2031565865 countsByYear W20315658652014 @default.
• W2031565865 countsByYear W20315658652015 @default.
• W2031565865 countsByYear W20315658652016 @default.
• W2031565865 countsByYear W20315658652017 @default.
• W2031565865 countsByYear W20315658652018 @default.
• W2031565865 countsByYear W20315658652019 @default.
• W2031565865 countsByYear W20315658652020 @default.
• W2031565865 countsByYear W20315658652021 @default.
• W2031565865 countsByYear W20315658652022 @default.
• W2031565865 countsByYear W20315658652023 @default.
• W2031565865 crossrefType "journal-article" @default.
• W2031565865 hasAuthorship W2031565865A5008474125 @default.
• W2031565865 hasAuthorship W2031565865A5013078816 @default.
• W2031565865 hasAuthorship W2031565865A5017043028 @default.
• W2031565865 hasAuthorship W2031565865A5040622924 @default.
• W2031565865 hasAuthorship W2031565865A5053314310 @default.
• W2031565865 hasAuthorship W2031565865A5065167441 @default.
• W2031565865 hasBestOaLocation W20315658651 @default.
• W2031565865 hasConcept C126322002 @default.
• W2031565865 hasConcept C136229726 @default.
• W2031565865 hasConcept C179104552 @default.
• W2031565865 hasConcept C185592680 @default.
• W2031565865 hasConcept C2777633098 @default.
• W2031565865 hasConcept C2779134260 @default.
• W2031565865 hasConcept C31705614 @default.
• W2031565865 hasConcept C502032728 @default.
• W2031565865 hasConcept C53645450 @default.
• W2031565865 hasConcept C55493867 @default.
• W2031565865 hasConcept C62478195 @default.
• W2031565865 hasConcept C71924100 @default.
• W2031565865 hasConcept C86803240 @default.

• next