Title: Differential Roles of JIP Scaffold Proteins in the Modulation of Amyloid Precursor Protein Metabolism
Abstract: We previously found that the JNK-interacting proteins JIP1b and JIP2 associate with the cytoplasmic domain of the Alzheimer's amyloid precursor protein (APP) (Taru, H., Iijima, K., Hase, M., Kirino, Y., Yagi, Y., and Suzuki, T. (2002) J. Biol. Chem. 277, 20070–20078). This interaction involves the carboxyl-terminal phosphotyrosine interaction (PI) domain of JIP1b or JIP2 and the GYENPTY motif in the APP cytoplasmic domain. The expression of JIP1b stabilizes immature APP and suppresses the production of a secreted large extracellular amino-terminal domain of APP, the generation of a cleaved intracellular carboxyl-terminal fragment of APP, and the secretion of β-amyloid 40 and 42. Deletion of the PI domain or alteration of PI amino acid residues prevents JIP1b from interacting with APP and affecting its metabolism, but deletion of the JNK-binding domain of JIP1b has no effect. JIP2, a weaker APP-binding protein, does not influence the processing of APP, although it is known that both JIP1b and JIP2 equally regulate the JNK signaling cascade. The present results suggest that JIP1b can directly modulate APP metabolism by interacting with the APP cytoplasmic domain, independent of its regulation of the JNK signaling cascade. We previously found that the JNK-interacting proteins JIP1b and JIP2 associate with the cytoplasmic domain of the Alzheimer's amyloid precursor protein (APP) (Taru, H., Iijima, K., Hase, M., Kirino, Y., Yagi, Y., and Suzuki, T. (2002) J. Biol. Chem. 277, 20070–20078). This interaction involves the carboxyl-terminal phosphotyrosine interaction (PI) domain of JIP1b or JIP2 and the GYENPTY motif in the APP cytoplasmic domain. The expression of JIP1b stabilizes immature APP and suppresses the production of a secreted large extracellular amino-terminal domain of APP, the generation of a cleaved intracellular carboxyl-terminal fragment of APP, and the secretion of β-amyloid 40 and 42. Deletion of the PI domain or alteration of PI amino acid residues prevents JIP1b from interacting with APP and affecting its metabolism, but deletion of the JNK-binding domain of JIP1b has no effect. JIP2, a weaker APP-binding protein, does not influence the processing of APP, although it is known that both JIP1b and JIP2 equally regulate the JNK signaling cascade. The present results suggest that JIP1b can directly modulate APP metabolism by interacting with the APP cytoplasmic domain, independent of its regulation of the JNK signaling cascade. β-amyloid peptide Alzheimer's disease β-amyloid precursor protein the cytoplasmic domain of APP 3-[(3-cholamidpropyl)dimethylammonio]-1-propane-sulfonic acid c-Jun amino-terminal kinase JNK-interacting protein phosphotyrosine interaction glutathione S-transferase mouse neuroblastoma Neuro-2a cells intracellular carboxyl-terminal fragment of APP truncated at α- (CTFα) or β-site (CTFβ) the secreted large amino-terminal ectodomain of APP cleaved at the α- or β-site Dulbecco's modified Eagle's medium fetal bovine serum enzyme-linked immunosorbent assay N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine APP-like hemagglutinin The β-amyloid peptide (Aβ)1 is a component of amyloid plaques, one of the major hallmarks of Alzheimer's disease (AD), and is implicated in the pathology of AD (reviewed in Ref. 1Selkoe D.J. Nature. 1999; 399: 23-31Crossref PubMed Scopus (1519) Google Scholar). Aβ is generated by proteolytic cleavage of the β-amyloid precursor protein (APP), an integral membrane protein with a short intracellular carboxyl terminus (2Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3916) Google Scholar). Following translation, APP is subjected toN-glycosylation (immature APP) in the endoplasmic reticulum and additional O-glycosylation (mature APP) in the Golgi complex, transported to the plasma membrane, internalized by endocytosis, and delivered to endosomes and lysosomes (3Weidemann A. Konig G. Bunke D. Fischer P. Salbaum J.M. Masters C.L. Beyreuther K. Cell. 1989; 57: 115-126Abstract Full Text PDF PubMed Scopus (1035) Google Scholar, 4Haass C. Koo E.H. Mellon A. Hung A.Y. Selkoe D.J. Nature. 1992; 357: 500-503Crossref PubMed Scopus (768) Google Scholar, 5Tomita S. Kirino Y. Suzuki T. J. Biol. Chem. 1998; 273: 6277-6284Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). As it travels within the cell, APP is subject to proteolytic cleavage by α- or β- and γ-secretases. In the minor, or amyloidogenic, processing pathway, the β-secretase, an aspartic protease BACE (6Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3255) Google Scholar, 7Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. Jewett N. Keim P. Knops J. Lieberburg I. Power M. Tan H. Tatsuno G. Tung J. Schenk D. Seubert P. Suomensaari S.M. Wang S. Walker D. Zhau J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1472) Google Scholar), cleaves APP into two fragments, a large amino-terminal ectodomain (sAPPβ) and a truncated carboxyl-terminal fragment (CTFβ); cleavage takes place mainly in compartments of the secretory pathway (4Haass C. Koo E.H. Mellon A. Hung A.Y. Selkoe D.J. Nature. 1992; 357: 500-503Crossref PubMed Scopus (768) Google Scholar, 8Golde T.E. Estus S. Younkin L.H. Selkoe D.J. Younkin S.G. Science. 1992; 255: 728-730Crossref PubMed Scopus (618) Google Scholar, 9Koo E.H. Squazzo S.L. J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar, 10Thinakaran G. Teplow D.B. Siman R. Greenberg B. Sisodia S.S. J. Biol. Chem. 1996; 271: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). The CTFβ is further cleaved by γ-secretase, which results in the release of the Aβ peptides 40 (Aβ40) or 42 (Aβ42) (reviewed in Refs. 1Selkoe D.J. Nature. 1999; 399: 23-31Crossref PubMed Scopus (1519) Google Scholar and11Haass C. De Strooper B. Science. 1999; 286: 916-919Crossref PubMed Scopus (364) Google Scholar). In contrast, the majority of APP is cleaved by α-secretase within the Aβ domain to produce sAPPα and CTFα, either at the plasma membrane or in the secretary pathway (4Haass C. Koo E.H. Mellon A. Hung A.Y. Selkoe D.J. Nature. 1992; 357: 500-503Crossref PubMed Scopus (768) Google Scholar, 12Sisodia S.S. Koo E.H. Beyreuther K. Unterbeck A. Price D.L. Science. 1990; 248: 492-495Crossref PubMed Scopus (738) Google Scholar, 13Sisodia S.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6075-6079Crossref PubMed Scopus (627) Google Scholar). This non-amyloidogenic processing of APP results not in the secretion of Aβ peptides but rather of a small peptide (p3) that consists of the carboxyl-terminal half of Aβ generated by further cleavage of CTFα at the γ-site. Like the full-length APP, the proteolytic fragments generated by either cleavage pathway are also proposed to play numerous roles in cell physiology. Aβ is widely known to be toxic for cells (14Yankner B.A. Dawes L.R. Fisher S. Villa-Komaroff L. Oster-Granite M.L. Neve R.L. Science. 1989; 245: 417-420Crossref PubMed Scopus (810) Google Scholar) and to be tightly associated with the progression of AD (reviewed in Ref. 1Selkoe D.J. Nature. 1999; 399: 23-31Crossref PubMed Scopus (1519) Google Scholar). The overexpression of human CTFβ in mice causes neuronal and synaptic degeneration (15Oster-Granite M.L. McPhie D.L. Greenan J. Neve R.L. J. Neurosci. 1996; 16: 6732-6741Crossref PubMed Google Scholar), the impairment of learning ability, and the suppression of long term potentiation (16Nalbantoglu J. Tirado-Santiago G. Lahsaini A. Poirier J. Goncalves O. Verge G. Momoli F. Welner S.A. Massicotte G. Julien J.P. Shapiro M.L. Nature. 1997; 387: 500-505Crossref PubMed Scopus (305) Google Scholar). The secreted sAPP is thought to modulate neuronal function and cell survival (reviewed in Ref. 17Mattson M.P. Physiol. Rev. 1997; 77: 1081-1132Crossref PubMed Scopus (873) Google Scholar). Recent reports suggest that the cytoplasmic tail fragment, which is generated from CTF by γ-cleavage, may be implicated in transcriptional regulation (18Cao X. Südhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1045) Google Scholar). Therefore, an analysis of the intracellular mechanism of APP processing is important for understanding the pathogenesis of AD as well as the physiological function(s) of APP in normal tissues.The cytoplasmic domain of APP (APPcyt) has been suggested to be important for the regulation of intracellular trafficking and metabolism of APP (19Lai A. Sisodia S.S. Trowbridge I.S. J. Biol. Chem. 1995; 270: 3565-3573Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Substitutions of certain amino acids in APPcyt are known to affect the processing of APP. Substitution of Ala for Tyr-682 (with respect to the numbering convention for the APP 695 isoform), for Asn-684, and for Pro-685 in the681GYENPTY687 motif results in the impaired internalization of APP from the plasma membrane and in a decrease in the secretion of sAPP and Aβ (20Perez 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 (341) Google Scholar). Substitution of Ala for Arg-672 in the 667VTPEER672 motif increases the production of intracellular CTFβ (21Tomita S. Kirino Y. Suzuki T. J. Biol. Chem. 1998; 273: 19304-19310Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). These mutations either directly prevent the interaction of APP with a class of cytoplasmic proteins termed APP binders or they cause a conformational change in APPcyt that in turn prevents interaction with an APP binder (22Ramelot T.A. Nicholson L.K. J. Mol. Biol. 2001; 307: 871-884Crossref PubMed Scopus (129) Google Scholar, 23Ando 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). Several APP binders such as members of FE65 and X11 families of proteins have been reported to interact with APPcyt and to regulate APP metabolism (24Sabo S.L. Lanier L.M. Ikin A.F. Khorkova O. Sahasrabudhe S. Greengard P. Buxbaum J.D. J. Biol. Chem. 1999; 274: 7952-7957Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 25Borg J.P. Yang Y., De Taddeo-Borg M. Margolis B. Turner R.S. J. Biol. Chem. 1998; 273: 14761-14766Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 26Sastre M. Turner R.S. Levy E. J. Biol. Chem. 1998; 273: 22351-22357Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 27Tomita 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).We have found that JIP1b and JIP2, proteins isolated originally as mammalian counterparts of the Drosophila melanogaster APP-like (APPL)-binding protein APLIP1, can bind APPcyt (28Taru 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 (114) Google Scholar). JIP1 and JIP2 were independently identified as scaffold proteins that selectively bind components of the JNK signaling pathway and have been shown to be abundantly expressed in neural tissues (29Dickens 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, 30Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (583) Google Scholar, 31Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (405) Google Scholar). JIP1b is also known as islet brain 1 (IB1) and was independently cloned as a transactivator of the GLUT2gene, which encodes a facilitated glucose transporter (32Bonny C. Nicod P. Waeber G. J. Biol. Chem. 1998; 273: 1843-1846Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Recent findings also suggest that JIP1 and JIP2 may be among the cargo of the motor protein kinesin (33Verhey 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 (494) Google Scholar). But the relevance of the interaction of JIP1 and JIP2 with APP remains unclear. In this study, we found that the co-expression of JIP1b and APP modulates the processing of APP in an interaction-dependent manner in mouse neuroblastoma Neuro-2a (N2a) cells. On the other hand, we did not observe an influence on APP processing by JIP2 or JIP1a, which are weaker APP binders but which regulate the JNK signaling cascade in the same manner as JIP1b (29Dickens 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, 30Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (583) Google Scholar, 31Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (405) Google Scholar). The present results suggest that the regulation of APP metabolism by JIP1b is dependent on its direct interaction with APPcyt rather than on an interaction with JNK, with a subsequent activation of the JNK signaling cascade. These observations suggest that JIP family proteins are not functionally equivalent with respect to APP metabolism, although they act on the JNK system in an identical manner.DISCUSSIONIn the present study, we observed several changes in cellular APP metabolism as a result of the co-expression of JIP1b as follows: the persistence of the immature form of APP, a suppression of the secretion of sAPP and Aβ, and a decrease in the production of intracellular CTFα. For these modulations of APP metabolism by JIP1b, a direct interaction of JIP1b with APPcyt should be responsible, because JIP1a and the JIP1b mutants ΔPI, F642V, and F687V, which bind APP very weakly or not at all, do not affect APP metabolism as JIP1b does. On the other hands a shorter JIP1b mutant SH3-PI that could interact with APP failed to affect APP processing, with respect to the stabilization of immature APP and the decrease of CTFα production. Therefore, the effects of JIP1b may not be attributed to the competitive inhibition of other unknown endogenous protein(s) that interact with APP and regulate APP metabolism because of the property of JIP1b itself.As a first interpretation for the presented results, we mentioned that the binding of JIP1b to APPcyt may interfere only with the maturation of APP. Defects in the maturation of APP caused by pharmacological treatment with reagents such as brefeldin A or by the alteration of the ectodomain of APP are associated with a decrease in the secretion of sAPP and Aβ (5Tomita S. Kirino Y. Suzuki T. J. Biol. Chem. 1998; 273: 6277-6284Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 44Caporaso G.L. Gandy S.E. Buxbaum J.D. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2252-2256Crossref PubMed Scopus (174) Google Scholar, 45Haass C. Hung A.Y. Schlossmacher M.G. Teplow D.B. Selkoe D.J. J. Biol. Chem. 1993; 268: 3021-3024Abstract Full Text PDF PubMed Google Scholar). These observations suggest that if APP processing could be inhibited before the maturation step by the binding of JIP1b to the APPcyt, the amount of secreted sAPP and Aβ might be decreased as a result. However, another interpretation is more conceivable that JIP1b may not only interfere with the maturation but also take part in prevention of secretion by affecting the export of vesicles including intact mature APP or its cleaved derivatives, for example because JIP1b interacts with mature APP and CTFs as with immature APP. Actually in the cells co-expressing the SH3-PI mutant, although there was no difference in the ratio of immature APP to mature APP when compared with the cells expressing APP alone, the levels of Aβ secreted into the medium were suppressed as in the cells expressing JIP1b. This suggests that JIP1b can affect APP processing in multiple ways rather than can only stabilize the immature APP. Comparing the effect of ΔN with that of SH3-PI mutant, the amino acid sequence of JIP1b adjacent to the SH3 domain (amino acids 264–459) was responsible for stabilizing the immature APP and decreasing the amount of CTFα. It is conceivable that this region of JIP1b was important for putative protein interaction, which was responsible for the modulation of APP processing. Among the previously reported binding proteins to JIP1b, mitogen-activated protein kinase kinase 7 is likely to interact with the amino acid sequence 264–459 of JIP1b (30Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (583) Google Scholar). Alternatively, the region (amino acids 264–459) may contribute to stabilize the conformation of the carboxyl-terminal region (amino acids 460–707) of JIP1b and may be responsible for the protein interaction of the carboxyl-terminal region. The molecular motor kinesin interacts with the carboxyl-terminal region of JIP1b. This interaction mediates the association of kinesin with membrane proteins such as ApoER2 (33Verhey 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 (494) Google Scholar). X11, an APP-binding protein that has a similar effect on APP metabolism to that of JIP1b (25Borg J.P. Yang Y., De Taddeo-Borg M. Margolis B. Turner R.S. J. Biol. Chem. 1998; 273: 14761-14766Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 26Sastre M. Turner R.S. Levy E. J. Biol. Chem. 1998; 273: 22351-22357Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), also interacts with kinesin (46Setou M. Nakagawa T. Seog D.H. Hirokawa N. Science. 2000; 288: 1796-1802Crossref PubMed Scopus (601) Google Scholar). Although kinesin has been involved in axonal transport of APP (47Kamal A. Stokin G.B. Yang Z. Xia C.H. Goldstein L.S. Neuron. 2001; 28: 449-459Abstract Full Text Full Text PDF Scopus (444) Google Scholar), it is still unclear whether JIP1b and X11 mediate the APP transport by kinesin. Further analysis of the interaction of JIP1b with such proteins may be needed to understand the detailed mechanism to modulate APP metabolism.JIP family proteins selectively bind several components of JNK signaling cascades and facilitate the activation of JNK (30Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (583) Google Scholar, 31Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (405) Google Scholar). However, in this study, the contribution of JNK activation to the modulation of APP metabolism by JIP1b may be small. First the JIP1b mutants ΔPI, F642V, and F687V and JIP1a, which possess binding regions for JNK signaling molecules (29Dickens 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, 30Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (583) Google Scholar) and may function as same as JIP1b in JNK pathway, did not alter APP metabolism as JIP1b did, suggesting that the ability to affect the JNK pathway is not enough to alter APP processing. Conversely, the ΔN mutant, which cannot bind JNK and facilitate the activation of JNK, still affected the processing of APP as JIP1b, indicating that the direct interaction of JIP1b with JNK was not necessary to modulate APP processing. Recently it was reported (43Mudher A. Chapman S. Richardson J. Asuni A. Gibb G. Pollard C. Killick R. Iqbal T. Raymond L. Varndell I. Sheppard P. Makoff A. Gower E. Soden P.E. Lewis P. Murphy M. Golde T.E. Rupniak H.T. Anderton B.H. Lovestone S. J. Neurosci. 2001; 21: 4987-4995Crossref PubMed Google Scholar) that the expression or the subsequent activation of JNK is involved in the increase of α-secretion of APP in HEK293 cells. It seems possible that the overexpression of JIP1b or ΔN could be attributed to inhibit putative activity of endogenous JNK on APP processing. However, JIP1b or ΔN affected not only α-secretion but also β-secretion and the stabilization of cellular APP in our study, whereas the activation of JNK was reported to alter only α-secretion (43Mudher A. Chapman S. Richardson J. Asuni A. Gibb G. Pollard C. Killick R. Iqbal T. Raymond L. Varndell I. Sheppard P. Makoff A. Gower E. Soden P.E. Lewis P. Murphy M. Golde T.E. Rupniak H.T. Anderton B.H. Lovestone S. J. Neurosci. 2001; 21: 4987-4995Crossref PubMed Google Scholar). In addition, even in the cells overexpressing JNK3, we could not observe apparent differences in APP processing and in the effects of JIP1b and ΔN on APP processing from the cells not expressing JNK exogenously (data not shown). Therefore, as far as we observed in this report, JNK may not be implicated in the effects of JIP1b to modulate APP processing.We have recently reported (28Taru 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 (114) Google Scholar) that the expression of JIP1 proteins affects the phosphorylation of APP accompanied with JNK activation, but the contribution of APP phosphorylation to the modulation of APP metabolism by JIP1b may also be small because APP was hardly phosphorylated in this study. In neurons, however, the activity of JNK was constitutively higher than in cultured cells (48Xu X. Raber J. Yang D., Su, B. Mucke L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12655-12660Crossref PubMed Scopus (108) Google Scholar, 49Coffey E.T. Hongisto V. Dickens M. Davis R.J. Courtney M.J. J. Neurosci. 2000; 20: 7602-7613Crossref PubMed Google Scholar), and we do not rule out the possibility that the JNK signaling cascade involves APP metabolism in neurons. In addition, JNK may phosphorylate APPin vivo and in vitro (28Taru 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 (114) Google Scholar, 50Standen C.L. Brownlees J. Grierson A.J. Kesavapany S. Lau K.F. McLoughlin D.M. Miller C.C. J. Neurochem. 2001; 76: 316-320Crossref PubMed Scopus (108) Google Scholar). Therefore, it is conceivable that JIP1b modulates APP metabolism both by directly interacting with APP and by facilitating JNK activity in neurons.JIP2 cannot modulate APP processing as can JIP1b. As demonstrated by several JIP1b mutants, this may reflect the differential APP-binding properties of JIP1b and JIP2. We observed that the interaction of JIP2 with APP is weaker than that of JIP1b with APP (Fig. 1, Dand E) (28Taru 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 (114) Google Scholar), although JIP1b and JIP2 share a highly conserved domain structure, including the PI domain (31Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (405) Google Scholar). In addition, we found that JIP1a, like JIP2, also has no significant effect on APP processing in the present study, whereas the level of JIP1a seems to be much lower than that of JIP1b in neuronal cells (49Coffey E.T. Hongisto V. Dickens M. Davis R.J. Courtney M.J. J. Neurosci. 2000; 20: 7602-7613Crossref PubMed Google Scholar). 3H. Taru and T. Suzuki, unpublished observations. JIP3, another member of the JIP family of proteins, does not possess a PI domain (51Ito M. Yoshioka K. Akechi M. Yamashita S. Takamatsu N. Sugiyama K. Hibi M. Nakabeppu Y. Shiba T. Yamamoto K.I. Mol. Cell. Biol. 1999; 19: 7539-7548Crossref PubMed Scopus (225) Google Scholar, 52Kelkar N. Gupta S. Dickens M. Davis R.J. Mol. Cell. Biol. 2000; 20: 1030-1043Crossref PubMed Scopus (249) Google Scholar) and may not affect APP processing via a direct interaction. Taken together, the JIP family proteins JIP2, JIP1a, and JIP3 may have roles different from that of JIP1b, with respect to the regulation of APP metabolism.Although the physiological function(s) of APP are not sufficiently understood, there are several reports that cell-associated and/or soluble-secreted APP are involved in neuroprotection, cell growth, cell adhesion, and neurite outgrowth (reviewed in Ref. 1Selkoe D.J. Nature. 1999; 399: 23-31Crossref PubMed Scopus (1519) Google Scholar). JIP1b may be implicated in these events via modulating APP processing. In AD, the generation and deposition of Aβ are thought to be closely related to pathogenesis. JIP1b is particularly enriched in regions of the brain vulnerable to AD (53Kim I.J. Lee K.W. Park B.Y. Lee J.K. Park J. Choi I.Y. Eom S.J. Chang T.S. Kim M.J. Yeom Y.I. Chang S.K. Lee Y.D. Choi E.J. Han P.L. J. Neurochem. 1999; 72: 1335-1343Crossref PubMed Scopus (48) Google Scholar) and could alter the production of both Aβ40 and 42 as reported here, whereas the effect of JIP1b on the accumulation of Aβ is unclear. Therefore, a further analysis of the interaction of APP with proteins in the JIP family in neurons may contribute to an understanding of the molecular mechanism of AD pathogenesis and to propositions for novel therapeutic strategies. The β-amyloid peptide (Aβ)1 is a component of amyloid plaques, one of the major hallmarks of Alzheimer's disease (AD), and is implicated in the pathology of AD (reviewed in Ref. 1Selkoe D.J. Nature. 1999; 399: 23-31Crossref PubMed Scopus (1519) Google Scholar). Aβ is generated by proteolytic cleavage of the β-amyloid precursor protein (APP), an integral membrane protein with a short intracellular carboxyl terminus (2Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3916) Google Scholar). Following translation, APP is subjected toN-glycosylation (immature APP) in the endoplasmic reticulum and additional O-glycosylation (mature APP) in the Golgi complex, transported to the plasma membrane, internalized by endocytosis, and delivered to endosomes and lysosomes (3Weidemann A. Konig G. Bunke D. Fischer P. Salbaum J.M. Masters C.L. Beyreuther K. Cell. 1989; 57: 115-126Abstract Full Text PDF PubMed Scopus (1035) Google Scholar, 4Haass C. Koo E.H. Mellon A. Hung A.Y. Selkoe D.J. Nature. 1992; 357: 500-503Crossref PubMed Scopus (768) Google Scholar, 5Tomita S. Kirino Y. Suzuki T. J. Biol. Chem. 1998; 273: 6277-6284Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). As it travels within the cell, APP is subject to proteolytic cleavage by α- or β- and γ-secretases. In the minor, or amyloidogenic, processing pathway, the β-secretase, an aspartic protease BACE (6Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3255) Google Scholar, 7Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. Jewett N. Keim P. Knops J. Lieberburg I. Power M. Tan H. Tatsuno G. Tung J. Schenk D. Seubert P. Suomensaari S.M. Wang S. Walker D. Zhau J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1472) Google Scholar), cleaves APP into two fragments, a large amino-terminal ectodomain (sAPPβ) and a truncated carboxyl-terminal fragment (CTFβ); cleavage takes place mainly in compartments of the secretory pathway (4Haass C. Koo E.H. Mellon A. Hung A.Y. Selkoe D.J. Nature. 1992; 357: 500-503Crossref PubMed Scopus (768) Google Scholar, 8Golde T.E. Estus S. Younkin L.H. Selkoe D.J. Younkin S.G. Science. 1992; 255: 728-730Crossref PubMed Scopus (618) Google Scholar, 9Koo E.H. Squazzo S.L. J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar, 10Thinakaran G. Teplow D.B. Siman R. Greenberg B. Sisodia S.S. J. Biol. Chem. 1996; 271: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). The CTFβ is further cleaved by γ-secretase, which results in the release of the Aβ peptides 40 (Aβ40) or 42 (Aβ42) (reviewed in Refs. 1Selkoe D.J. Nature. 1999; 399: 23-31Crossref PubMed Scopus (1519) Google Scholar and11Haass C. De Strooper B. Science. 1999; 286: 916-919Crossref PubMed Scopus (364) Google Scholar). In contrast, the majority of APP is cleaved by α-secretase within the Aβ domain to produce sAPPα and CTFα, either at the plasma membrane or in the secretary pathway (4Haass C. Koo E.H. Mellon A. Hung A.Y. Selkoe D.J. Nature. 1992; 357: 500-503Crossref PubMed Scopus (768) Google Scholar, 12Sisodia S.S. Koo E.H. Beyreuther K. Unterbeck A. Price D.L. Science. 1990; 248: 492-495Crossref PubMed Scopus (738) Google Scholar, 13Sisodia S.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6075-6079Crossref PubMed Scopus (627) Google Scholar). This non-amyloidogenic processing of APP results not in the secretion of Aβ peptides but rather of a small peptide (p3) that consists of the carboxyl-terminal half of Aβ generated by further cleavage of CTFα at the γ-site. Like the full-length APP, the prote