Title: A Scaffold Protein JIP-1b Enhances Amyloid Precursor Protein Phosphorylation by JNK and Its Association with Kinesin Light Chain 1
Abstract: Amyloid precursor protein (APP) is the precursor molecule of β-amyloid peptides, the major components of amyloid plaque in patients with Alzheimer's disease. In this study, we isolated JIP-1b, a JNK signaling scaffold protein, as a binding protein of APP, and analyzed the roles of JIP-1b in APP phosphorylation by JNK and the association of kinesin light chain 1 with APP. APP phosphorylation at threonine 668 by JNK was enhanced on the JIP-1b scaffold in vitro and in cultured cells exogenously expressing APP. APP phosphorylation in nerve growth factor-differentiated PC12 cells was mediated by activation of JNK signaling. JIP-1b also enhanced the association of kinesin light chain 1 with APP. Our results suggest that JIP-1b may function as a protein linking the kinesin-I motor protein to the cargo receptor, APP, and that the JNK signaling pathway may regulate the phosphorylation of this cargo protein through the JIP-1b scaffold. Amyloid precursor protein (APP) is the precursor molecule of β-amyloid peptides, the major components of amyloid plaque in patients with Alzheimer's disease. In this study, we isolated JIP-1b, a JNK signaling scaffold protein, as a binding protein of APP, and analyzed the roles of JIP-1b in APP phosphorylation by JNK and the association of kinesin light chain 1 with APP. APP phosphorylation at threonine 668 by JNK was enhanced on the JIP-1b scaffold in vitro and in cultured cells exogenously expressing APP. APP phosphorylation in nerve growth factor-differentiated PC12 cells was mediated by activation of JNK signaling. JIP-1b also enhanced the association of kinesin light chain 1 with APP. Our results suggest that JIP-1b may function as a protein linking the kinesin-I motor protein to the cargo receptor, APP, and that the JNK signaling pathway may regulate the phosphorylation of this cargo protein through the JIP-1b scaffold. The deposition of amyloid plaque is a principal pathological feature in the brain parenchyma and blood vessel walls of patients with Alzheimer's disease. The major components of amyloid plaque are β-amyloid (Aβ) 1The abbreviations used are: Aβ, β-amyloid; APP, amyloid precursor protein; TPR, tetratricopeptide repeat; KLC, kinesin light chain; JNK, c-Jun NH2-terminal kinase; JIP, JNK interacting protein; MKK7, mitogen-activated protein kinase kinase 7; MLK3, mixed lineage kinase 3; GST, glutathione S-transferase; JBD, JNK-binding domain; SH3, Src homology 3; PID, phosphotyrosine interacting domain; KB, kinesin binding; AICD, APP intracellular domain; HA, hemagglutinin; NGF, nerve growth factor; APPC, cytoplasmic region of APP. peptides, including Aβ40 and Aβ42, which are derived by proteolytic cleavage of amyloid precursor protein (APP) (for a review see Ref. 1Selkoe D.J. Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1534) Google Scholar). APP is a type I membrane spanning protein. Three major isoforms of APP comprising 695, 751, and 770 amino acids are generated by alternative splicing. Enzymes termed β- and γ-secretase cleave APP to form the amino and carboxyl termini of the Aβ peptides. Besides its pathological role in Alzheimer's disease, APP is thought to be functionally important because mice lacking all APP family genes die in the early postnatal period (2Heber S. Herms J. Gajic V. Hainfellner J. Aguzzi A. Rulicke T. von Kretzschmar H. von Koch C. Sisodia S. Tremml P. Lipp H.P. Wolfer D.P. Muller U. J. Neurosci. 2000; 20: 7951-7963Crossref PubMed Google Scholar). Although numerous studies have shown various roles of APP, the physiological function of APP is not yet clear. In neurons, APP is transported within axons by the fast anterograde axonal transport system from cell bodies to nerve terminals (3Sisodia S.S. Koo E.H. Hoffman P.N. Perry G. Price D.L. J. Neurosci. 1993; 13: 3136-3142Crossref PubMed Google Scholar). When expression of the kinesin heavy chain is inhibited with antisense oligonucleotides, axonal transport of APP is disturbed in cultured hippocampal neurons, suggesting that axonal transport of APP requires the motor protein kinesin-I (4Ferreira A. Caceres A. Kosik K.S. J. Neurosci. 1993; 13: 3112-3123Crossref PubMed Google Scholar). Recently, it was shown that APP may function as a cargo receptor for kinesin-I. APP forms a complex with kinesin-I by binding directly to the tetratricopeptide repeat (TPR) domain of the kinesin light chain (KLC) subunit (5Kamal A. Stokin G.B. Yang Z. Xia C.-H. Goldstein L.S.B. Neuron. 2000; 28: 449-459Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). Neuronal overexpression or deletion of APPL, the Drosophila functional homolog of APP, disrupt axonal transport in Drosophila (6Torroja L. Chu H. Kotovsky I. White K. Curr. Biol. 1999; 9: 489-492Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 7Gunawardena S. Goldstein L.S.B. Neuron. 2001; 32: 389-401Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). Furthermore, the fast anterograde axonal transport of β-secretase and presenilin-1 is mediated by APP and kinesin-I, and processing of APP to Aβ by secretases can occur in an axonal membrane compartment transported by kinesin-I (8Kamal A. Almenar-Queralt A. LeBlanc J.F. Roberts E.A. Goldstein L.S.B. Nature. 2001; 414: 643-648Crossref PubMed Scopus (501) Google Scholar). APP is a phosphorylated protein and one known phosphorylation site is threonine 668 (Thr668) (numbering for the APP695 isoform) within the cytoplasmic region of APP (9Suzuki T. Oishi M. Marshak D.M. Czernik A.J. Nairn A.C. Greengard P. EMBO J. 1994; 13: 1114-1122Crossref PubMed Scopus (212) Google Scholar). Constitutive phosphorylation of APP is observed at Thr668 specifically in neurons. Phosphorylation of APP appears to regulate its function and localization (10Ando 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). Thus, it is important to elucidate the mechanism of APP phosphorylation. Several kinases were reported to phosphorylate Thr668 of APP. Cdc2 kinase phosphorylates the Thr668 during the G2/M phase of the cell cycle (9Suzuki T. Oishi M. Marshak D.M. Czernik A.J. Nairn A.C. Greengard P. EMBO J. 1994; 13: 1114-1122Crossref PubMed Scopus (212) Google Scholar). Thr668 of APP is phosphorylated in adult rat brain and in differentiated PC12 cells (10Ando 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), and this phosphorylation is mediated by Cdk5, a neuronal homolog of Cdc2 kinase (11Iijima 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 (208) Google Scholar). Glycogen synthase kinase-3β also phosphorylates Thr668 of APP in vitro (12Aplin A.E. Gibb G.M. Jacobsen J.S. Gallo J.-M. Anderton B.H. J. Neurochem. 1996; 67: 699-707Crossref PubMed Scopus (164) Google Scholar). Recently, it was shown that APP is more efficiently phosphorylated at Thr668 by c-Jun NH2-terminal kinase-3 (JNK-3) than by Cdk5 or glycogen synthase kinase-3β in vitro (13Standen C.L. Brownlees J. Grierson A.J. Kesavapany S. Lau K-F. McLoughlin D.M. Miller C.C.J. J. Neurochem. 2001; 76: 316-320Crossref PubMed Scopus (112) Google Scholar). However, it is unknown whether JNK-3 phosphorylates APP in neurons. In this study, we isolated human JNK interacting protein-1b (JIP-1b) as a novel molecule interacting with the cytoplasmic region of APP. Although JIP-1b was originally isolated as a cytoplasmic inhibitor of the JNK signal transduction pathway (14Dickens 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 (629) Google Scholar), a subsequent study revealed that JIP-1b is a scaffold protein that interacts with specific multiple components of the JNK pathway namely JNK, mitogen-activated protein kinase kinase 7 (MKK7), and mixed lineage kinase 3 (MLK3) (15Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (589) Google Scholar). JNK is activated in cells treated with inflammatory cytokines or in cells exposed to environmental stress (for a review see Ref. 16Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar). Although the main targets of the JNK pathway include the transcription factor c-Jun, ATF2, and Elk-1, JNK-3 also induces robust phosphorylation of Thr668 in the cytoplasmic region of APP (13Standen C.L. Brownlees J. Grierson A.J. Kesavapany S. Lau K-F. McLoughlin D.M. Miller C.C.J. J. Neurochem. 2001; 76: 316-320Crossref PubMed Scopus (112) Google Scholar). Thus, JIP-1b could recruit JNK to APP and in turn enhance the phosphorylation of APP by JNK. Moreover, JIP-1b was identified as a binding partner of the TPR domain of KLC, and was suggested to be a scaffold protein linking the kinesin-I motor protein to its membrane cargo (17Verhey 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 (499) Google Scholar). Thus, APP could associate with kinesin-I via JIP-1b. In this study, we further examined whether JIP-1b functions as a scaffold protein for the association of APP with JNK and KLC1. Our results demonstrate that APP associates with JNK and KLC1 on the JIP-1b scaffold, and that APP phosphorylation by JNK is enhanced by the association of APP with JIP-1b. These results suggest that JIP-1b may function as a linker protein of the kinesin-I motor protein to the cargo receptor, APP, and that the JNK signaling pathway may regulate the phosphorylation of the cargo protein through the JIP-1b scaffold. Expression Plasmids—To construct expression vectors (pcDNA/HA and pcDNA/FLAG) with the HA tag or FLAG tag sequence, oligonucleotide fragments encoding HA or FLAG were inserted into the EcoRI and XhoI sites of the expression vector pcDNA3 (Invitrogen). Expression vectors encoding the HA-tagged or FLAG-tagged proteins were constructed by inserting cDNAs into pcDNA/HA or pcDNA/FLAG. Expression vectors encoding the HA-tagged JNK1 and GST-c-Jun were described previously (18Nakano K. Yamauchi J. Nakagawa K. Itoh H. Kitamura N. J. Biol. Chem. 2000; 275: 20533-20539Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). APP cDNA and MLK3 cDNA were obtained by PCR using oligonucleotide primers and human brain Marathon-Ready™ cDNA (Clontech). MKK7 cDNA and KLC1 cDNA were kindly provided by Dr. J. Yamauchi (Nara Institute of Science and Technology) and Dr. A. Armin (McGill University), respectively. Deletion and point mutants were constructed with a QuikChange site-directed mutagenesis kit (Stratagene). Antibodies—To prepare a polyclonal anti-JIP-1b antibody, the human JIP-1b cDNA (amino acid residues 162–433) was inserted into the pGEX-4T-2 vector (Amersham Biosciences). The fusion protein with glutathione S-transferase (GST) was then expressed in Escherichia coli, and purified. Rabbits were immunized with the purified protein, and the antiserum (anti-JIP-1b) was raised. Anti-APP antibodies, UT18 and G369, were previously described (19Ando K. Iijima K. Elliott J.I. Kirino Y. Suzuki T. J. Biol. Chem. 2001; 276: 40353-40361Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 20Taru H. Iijima K. Hase M. Kirino Y. Yagi Y. Suzuki T. J. Biol. Chem. 2002; 277: 20070-20078Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The anti-FLAG (M2) and anti-β-tubulin antibodies were purchased from Sigma, the anti-HA antibody (3F10) from Roche Diagnostics, the anti-APP antibody (6E10) from Signet, the anti-JNK1 antibody from Pharmingen, and the anti-phospho-c-Jun and anti-phospho-APP antibodies from Cell Signaling Technology. Yeast Two-hybrid Screening—To isolate cDNA clones encoding APP-interacting proteins, yeast two-hybrid screening was performed as previously described (21Takata H. Kato M. Denda K. Kitamura N. Genes Cells. 2000; 5: 57-69Crossref PubMed Scopus (70) Google Scholar). A human brain cDNA library inserted down-stream of the Ga14 DNA activation domain in the pACT2 vector was obtained from Clontech. The bait plasmid and library were co-transformed into yeast (L40 strain). Cell Culture—HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. PC12 cells were cultured on collagen-coated dishes in RPMI 1640 medium supplemented with 10% horse serum, 5% fetal bovine serum, and 1% nonessential amino acids. Neuronal differentiation of PC12 cells was induced by NGF (50 ng/ml) in RPMI 1640 medium supplemented with 1% horse serum and 1% non-essential amino acids (differentiation medium). Transfection—HEK293 cells were transfected with plasmid expression vectors using FuGENE 6 transfection reagent (Roche Diagnostics). For transfection of the HA-tagged JNK binding domain (JBD) expression vector into PC12 cells, the cells were plated in a 12-well dish at 5 × 105 cells per well. At 24 h after plating, the cells were transfected with 1.6 μg of plasmid per well by the LipofectAMINE 2000 (Invitrogen). At 6 h after transfection, the cells were replated on collagen-coated cover-slips and cultured in differentiation medium. At 72 h later, the cells were fixed and subjected to immunofluorescence staining. Transfection of the HA-tagged MLK3 expression vector into PC12 cells was performed as described previously (22Xu Z. Maroney A.C. Dobrzanski P. Kukekov N.V. Greene L.A. Mol. Cell. Biol. 2001; 21: 4713-4724Crossref PubMed Scopus (223) Google Scholar). At 4 to 5 days after NGF treatment, PC12 cells were transfected with 1.6 μg of plasmid per well in a 12-well dish by LipofectAMINE 2000. At 6 h after transfection, the medium was replaced with fresh medium containing NGF, and 24 h later, the cells were subjected to immunofluorescence staining. Assay for in Vitro Binding to Immobilized GST Fusion Proteins (GST Pull-down)—Cells were lysed in lysis buffer (50 mm Tris-Cl, pH 7.4, 1% Nonidet P-40, 20 mm EDTA, 1 mm sodium orthovanadate, 10 mm NaF, 20 mm glycerophosphate, 1 mm phenylmethylsulfonyl fluoride, and 1 μg/ml each of aprotinin, leupeptin, and pepstatin). The cell lysate was incubated with purified GST fusion proteins bound to glutathione-Sepharose 4B beads at 4 °C. After the beads were washed three times with washing buffer (10 mm Tris-Cl, pH 7.4, 150 mm NaCl, 1 mm sodium orthovanadate, and 0.1% Nonidet P-40), the binding proteins were released by boiling in 2× sample buffer (25 mm Tris-Cl, pH 6.5, 1% SDS, 1% mercaptoethanol, and 5% glycerol), and then separated by SDS-PAGE under reducing conditions, before being analyzed by immunoblotting. Immunoprecipitation and Immunoblotting—Cells were lysed in lysis buffer. Immunoprecipitation and immunoblotting were performed as described previously (21Takata H. Kato M. Denda K. Kitamura N. Genes Cells. 2000; 5: 57-69Crossref PubMed Scopus (70) Google Scholar). In Vitro Phosphorylation of APP—To prepare purified JIP-1b proteins, HEK293 cells were transfected with expression vectors encoding FLAG-tagged JIP-1b proteins. At 48 h after transfection, the cells were lysed in kinase lysis buffer (20 mm HEPES, pH 7.4, 0.5% Nonidet P-40, 3 mm MgCl2, 100 mm NaCl, 1 mm dithiothreitol, 1 mm sodium orthovanadate, 10 mm NaF, 20 mm glycerophosphate, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 mm EGTA). The cell lysate was incubated with an anti-FLAG M2-agarose affinity gel (Sigma) at 4 °C. The binding JIP-1b proteins were eluted with FLAG peptide (Sigma), and concentrated with CENTRICON (Millipore) in reaction buffer (20 mm HEPES, pH 7.4, 1 mm dithiothreitol, 0.01 mm sodium orthovanadate, 2 mm glycerophosphate, 0.1 mm phenylmethylsulfonyl fluoride, 0.1 μg/ml leupeptin, and 0.1 mm EGTA). The amount of each JIP-1b protein was measured by Coomassie Brilliant Blue staining. The GST-fused cytoplasmic region of APP (APPC) proteins was expressed in E. coli using the pGEX vector and purified by glutathione 4B beads (Amersham Biosciences). To prepare activated JNK, HEK293 cells were transfected with an expression vector encoding HA-tagged JNK. At 48 h after transfection, the cells were treated with anisomycin (30 μg/ml) for 30 min. The cells were then lysed in kinase lysis buffer. The cell lysate was incubated with an anti-HA antibody at 4 °C, and after the addition of protein G beads the mixture was incubated at 4 °C. The immune complexes were precipitated and then washed twice with kinase lysis buffer and twice with reaction buffer. The precipitates were incubated in 30 μl of reaction buffer containing 18 μm ATP, 5 μCi of [γ-32P]ATP (Amersham Biosciences), 5 μg of GST-APPC, or its mutants with purified JIP-1b or its mutants for 25 min at 30 °C. The reaction was stopped by adding sample buffer, and the mixture was heated at 95 °C for 5 min. The proteins were then separated by SDS-PAGE. The radioactivity incorporated into APPC and JIP-1b was detected by autoradiography. Immunofluorescence Staining—PC12 cells were cultured on collagen-coated glass coverslips and treated with NGF (50 ng/ml). The cells were fixed in 4% paraformaldehyde in phosphate-buffered saline containing 4% sucrose for 10 min at room temperature and then permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 5 min. The cells were then rinsed with phosphate-buffered saline, and incubated with the primary antibody for 1 h at room temperature, followed by an incubation with Alexa-488- or Alexa-594-conjugated secondary antibody (Molecular Probes). Cells were examined by confocal immunofluorescence microscopy. Binding of the COOH-terminal Cytoplasmic Region in APP to JIP-1b—To isolate cDNA clones encoding APP-interacting proteins, we screened a human brain cDNA library using a yeast two-hybrid system. We inserted a cDNA fragment encoding the COOH-terminal cytoplasmic region (amino acid 649–695) of human APP into a pBTM116 plasmid vector, and used it as bait. We isolated 27 clones that reacted positively for β-galactosidase with the bait vector, but not by themselves. Among them, 24 clones encoded proteins that have previously been shown to bind to the cytoplasmic region of APP (22 clones of FE65, a clone of FE65-L2, and a clone of X11). Besides them, three clones, which encoded human JIP-1b, were isolated. Although JIP-1b has recently been shown to bind to APP (20Taru H. Iijima K. Hase M. Kirino Y. Yagi Y. Suzuki T. J. Biol. Chem. 2002; 277: 20070-20078Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 23Matsuda S. Yasukawa T. Homma Y. Ito Y. Niikura T. Hiraki T. Hirai S. Ohno S. Kita Y. Kawasumi M. Kouyama K. Yamamoto T. Kyriakis J.M. Nishimoto I. J. Neurosci. 2001; 21: 6597-6607Crossref PubMed Google Scholar), the significance of the binding has not been characterized. Because the 5′-portion of cDNA was missing, this portion was obtained by PCR amplification of human genomic DNA (Clontech) and then connected to the cDNA clone. The full-length human JIP-1b that we obtained consisted of 711 amino acid residues, and contained a JBD in the NH2-terminal region, a Src homology (SH) 3 domain in the middle region, and a phosphotyrosine interacting domain (PID) and kinesin binding (KB) domain in the COOH-terminal region. Binding sites between APP and JIP-1b were first determined by the yeast two-hybrid system. A series of deletion mutants of JIP-1b were inserted into a pACT2 vector, and assayed for their binding to the cytoplasmic region of APP. The NH2-terminal truncated mutant including the PID (Del 4) bound to the cytoplasmic region of APP, whereas the mutant with a further 47-amino acid deletion (Del 6) did not (Fig. 1A). The PID deletion mutant lacking the COOH-terminal 25 amino acids (Del 8) did not bind to the cytoplasmic region of APP. These results suggest that the PID of JIP-1b is the domain that binds to the cytoplasmic region of APP, but did not exclude the possibility that the KB domain is also required for the binding. It is known that the PID of FE65 and X11 recognizes the NPTY motif within the cytoplasmic region of APP and binds to it (24Borg J.P. Ooi J. Levy E. Margolis B. Mol. Cell. Biol. 1996; 16: 6229-6241Crossref PubMed Scopus (436) Google Scholar). To examine whether this motif is also required for JIP-1b to bind to the cytoplasmic region of APP, the cytoplasmic region of APP and its deletion mutant lacking the NPTY motif were inserted into a pBTM116 vector, and assayed for their binding to JIP-1b. The mutant did not bind to JIP-1b (Fig. 1B), indicating that JIP-1b recognizes the NPTY motif within the cytoplasmic region of APP. Next, the binding of APP to JIP-1b was examined by the GST pull-down method. Recently, it was shown that the COOH-terminal tail region (11 amino acids) of JIP-1b interacts with the TPR domain of kinesin light chain (17Verhey 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 (499) Google Scholar). Thus, we also examined whether this KB domain affects the binding of APP to JIP-1b. The cytoplasmic region of APP (APPC) and its mutant lacking the NPTY motif (ΔNPTY) were expressed as GST fusion proteins in E. coli. The FLAG-tagged full-length JIP-1b and its truncated mutants lacking the KB domain (ΔKB) or lacking the PID and KB domain (ΔPID) (Fig. 1C) were expressed in HEK293 cells, and assayed for binding to the GST-fused cytoplasmic region of APP (GST-APPC) and its deletion mutant (GST-ΔNPTY). The full-length JIP-1b bound to the GST-APPC, but not to the GST-ΔNPTY (Fig. 1D). ΔKB that retained the PID was still able to bind to the GST-APPC, suggesting that the KB domain is not required for the binding of APP to JIP-1b. The truncated mutant lacking the whole PID (ΔPID) did not bind to the GST-APPC. These results confirmed that the NPTY motif of APP and the PID of JIP-1b are responsible for the binding between APP and JIP-1b. The binding of APP and JIP-1b in mammalian cells was examined by coimmunoprecipitation. The FLAG-tagged JIP-1b with or without APP was expressed in HEK293 cells. Lysates of the cells were immunoprecipitated with an anti-FLAG or anti-APP (6E10) antibody, and the immunoprecipitates were immunoblotted with an anti-APP (UT18) or anti-FLAG antibody. APP was detected in the immunoprecipitates with the anti-FLAG antibody from cells expressing JIP-1b and APP (Fig. 1E). The coimmunoprecipitated APP corresponded to the immature form of APP. Thus, the immature form appeared to be more efficiently coimmunoprecipitated with JIP-1b than the mature forms, although a long exposure of the immunoblot showed that the mature forms of APP were also coimmunoprecipitated with JIP-1b (data not shown). Although a small amount of JIP-1b was immunoprecipitated by the anti-APP antibody (6E10), the increased amount of JIP-1b was detected in the immunoprecipitates with the anti-APP antibody from cells expressing JIP-1b and APP. Because JIP-1b was shown to selectively bind JNK, it is possible that JNK may affect the binding of APP to JIP-1b. To examine the effect of JNK on the binding, the FLAG-tagged JIP-1b was expressed together with APP and the HA-tagged JNK in HEK293 cells, and a similar coimmunoprecipitation experiment was performed. The coimmunoprecipitation of APP and JIP-1b was not affected by the presence of JNK. These results suggest that APP associates with JIP-1b in mammalian cells, and that the binding of JIP-1b to JNK does not affect the association. APP Associates with JNK and Is Phosphorylated on the JIP-1b Scaffold—It was shown that JNK is able to phosphorylate APP on Thr668 (13Standen C.L. Brownlees J. Grierson A.J. Kesavapany S. Lau K-F. McLoughlin D.M. Miller C.C.J. J. Neurochem. 2001; 76: 316-320Crossref PubMed Scopus (112) Google Scholar). Thus, it is possible that JIP-1b functions as a scaffold protein linking JNK to APP. To examine this possibility, we first tested whether APP associates with JNK through JIP-1b. The FLAG-tagged JIP-1b and/or HA-tagged JNK were expressed in HEK293 cells, and assayed for binding to the GST-APPC. JNK alone did not bind to the GST-APPC. Coexpression of JIP-1b led to the binding of JNK to the GST-APPC, whereas coexpression of JIP-1b lacking the JNK binding domain (ΔJBD) did not although the ΔJBD did bind to the GST-APPC (Fig. 2). These results suggest that APP associates with JNK on the JIP-1b scaffold. Next we examined the effect of the presence of JIP-1b on the in vitro phosphorylation of APP by JNK. It was shown that overexpression of JIP-1b inhibits the JNK activity (14Dickens 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 (629) Google Scholar). Thus, to examine APP phosphorylation quantitatively, JNK and JIP-1b were separately expressed and purified, before being used for the in vitro kinase assay with GST-APPC and its mutants as substrates. The HA-tagged JNK was expressed in HEK293 cells, and the cells were treated with anisomycin to activate the JNK. The activated JNK was purified by immunoprecipitation with an anti-HA antibody, and an equal amount of the activated JNK was used in each reaction of the in vitro kinase assay. The FLAG-tagged JIP-1b and its mutants were expressed in HEK293 cells, and purified with an anti-FLAG antibody bound to agarose gel. The purified JIP-1b and its mutants were detected by immunoblotting (Fig. 3A), and their amounts were measured by Coomassie Brilliant Blue staining. In addition the vector (pcDNA3) used for the expression of JIP-1b was also transfected into HEK293 cells, and the cell lysate was treated as was done for the purification of the FLAG-tagged JIP-1b, for use as the control (pcDNA3). The presence of the purified JIP-1b (1.25 μg) enhanced the phosphorylation of APPC by the activated JNK, whereas the level of phosphorylation detected was much less in the presence of the control (pcDNA3) (Fig. 3B). JIP-1b was also phosphorylated by JNK. When the phosphorylation site (Thr668) of APPC was mutated to an alanine (T668A), no phosphorylation was observed (Fig. 3C), confirming that the site phosphorylated by JNK is Thr668. The deletion mutant of APP (ΔNPTY) was phosphorylated to a lower extent. The presence of more JIP-1b (3.75 μg) further enhanced the phosphorylation of APPC by JNK. A mutant JIP-1b with a ΔJBD did not enhance the phosphorylation of either APPC or JIP-1b, whereas a deletion of the kinesin-binding domain (ΔKB) did not affect the enhancement of phosphorylation (Fig. 3D). In addition mutant JIP-1b with a deletion in a part of the PID (Δdel) or a deletion of the PID and KB domain (ΔPID) did not enhance APPC phosphorylation, although their own phosphorylation was not affected (Fig. 3D). Note that APPC phosphorylation by JNK occurred in the absence of JIP-1b (compare lanes a and d in Fig. 3D). Thus, these results suggest that JIP-1b linking APP to JNK enhances the in vitro phosphorylation of APP by JNK. The phosphorylation of APP in mammalian cells was examined in HEK293 cells exogenously expressing APP, JNK, and JIP-1b. The cells were treated with anisomycin to activate JNK, and the JNK activity was measured by in vitro kinase assay using GST-c-Jun as a substrate. The phosphorylation of APP was detected by immunoblotting using an anti-phospho-APP antibody. JNK activation induced the phosphorylation of APP (Fig. 4A). As previously reported (14Dickens 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 (629) Google Scholar), overexpression of JIP-1b suppressed the activity of JNK. In proportion to the suppression, APP phosphorylation was decreased, suggesting that APP phosphorylation depends on the JNK activity. Over-expression of the ΔPID mutant of JIP-1b, which lacked the APP binding site, further decreased APP phosphorylation, although the level of JNK activity was similar to that in the cells over-expressing the wild-type JIP-1b. These results suggest that JIP-1b functions as a scaffold protein linking JNK to APP in mammalian cells. Overexpression of the ΔJBD mutant of JIP-1b that lacked the JNK binding domain did not affect the activity of JNK, but reduced APP phosphorylation, suggesting that association of ΔJBD with APP inhibited the phosphorylation of APP by JNK. When the Thr668 phosphorylation site of APP was mutated to an alanine, no phosphorylation by activated JNK was observed, indicating that the site phosphorylated by JNK in mammalian cells is Thr668. JIP-1b was shown to be a scaffold protein that interacts with specific multiple components of the JNK pathway namely JNK, MKK7, and MLK3. It was shown that overexpression of JIP-1b enhances the activity of JNK induced by MKK7 or MLK3 (15Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (589) Google Scholar). Thus, the upstream components of JNK may affect the APP phosphorylation by JNK through JIP-1b. To examine the effect of upstream components, MKK7 or MLK3 were expressed together with APP, JNK, and JIP-1b in HEK293 cells, and the JNK activity as well as APP phosphorylation were evaluated. Expression of MKK7 alone slightly enhanced the JNK activity, but did not enhance APP phosphorylation (Fig. 4B). Coexpression of MKK7 with JIP-1b enhanced the JNK activity and APP phosphorylation. Coexpression of MKK7 with the ΔPID of JIP-1b, which can interact with MKK7 (15Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (589) Google Scholar) but not with APP, enhanced the JNK activity, but not the APP phosphorylation. Expression of MLK3 alone enhanced the JNK activity and APP phosphorylation (Fig. 4C). Coexpre