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W2166888773 abstract "β-Secretase (BACE) is a transmembrane aspartyl protease, which generates the N terminus of Alzheimer's disease amyloid β-peptide. Here, we report that BACE can be phosphorylated within its cytoplasmic domain at serine residue 498 by casein kinase 1. Phosphorylation exclusively occurs after full maturation of BACE by propeptide cleavage and complex N-glycosylation. Phosphorylation/dephosphorylation affects the subcellular localization of BACE. BACE wild type and an S498D mutant that mimics phosphorylated BACE are predominantly located within juxtanuclear Golgi compartments and endosomes, whereas nonphosphorylatable BACE S498A accumulates in peripheral EEA1-positive endosomes. Antibody uptake assays revealed that reinternalization of BACE from the cell surface is independent of its phosphorylation state. After reinternalization, BACE wild type as well as BACE S498D are efficiently retrieved from early endosomal compartments and further targeted to later endosomal compartments and/or the trans-Golgi network. In contrast, nonphosphorylatable BACE S498A is retained within early endosomes. Our results therefore demonstrate regulated trafficking of BACE within the secretory and endocytic pathway. β-Secretase (BACE) is a transmembrane aspartyl protease, which generates the N terminus of Alzheimer's disease amyloid β-peptide. Here, we report that BACE can be phosphorylated within its cytoplasmic domain at serine residue 498 by casein kinase 1. Phosphorylation exclusively occurs after full maturation of BACE by propeptide cleavage and complex N-glycosylation. Phosphorylation/dephosphorylation affects the subcellular localization of BACE. BACE wild type and an S498D mutant that mimics phosphorylated BACE are predominantly located within juxtanuclear Golgi compartments and endosomes, whereas nonphosphorylatable BACE S498A accumulates in peripheral EEA1-positive endosomes. Antibody uptake assays revealed that reinternalization of BACE from the cell surface is independent of its phosphorylation state. After reinternalization, BACE wild type as well as BACE S498D are efficiently retrieved from early endosomal compartments and further targeted to later endosomal compartments and/or the trans-Golgi network. In contrast, nonphosphorylatable BACE S498A is retained within early endosomes. Our results therefore demonstrate regulated trafficking of BACE within the secretory and endocytic pathway. amyloid β-peptide brefeldin A β-amyloid precursor protein casein kinase cytoplasmic tail early endosome antigen 1 glutathione S-transferase hymenialdisine human embryonic kidney polyvinylidene difluoride polyacrylamide gel electrophoresis β-secretase endoglycosidase H phosphate-buffered saline endoplasmic reticulum trans-Golgi network wild type peptide:N-glycanase F. Alzheimer's disease is the most common form of dementia and is pathologically characterized by the invariant accumulation of senile plaques and neurofibrillary tangles in certain areas of the brains of Alzheimer's disease patients (1Selkoe D.J. Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1519) Google Scholar). The major constituent of senile plaques is the amyloid β-peptide (Aβ),1 which derives from the β-amyloid precursor protein (βAPP) by endoproteolytic processing (2Haass C. Selkoe D.J. Cell. 1993; 75: 1039-1042Abstract Full Text PDF PubMed Scopus (736) Google Scholar). β-Secretase cleaves βAPP at the N terminus of the Aβ-domain, resulting in the generation of soluble APPS-β and a membrane-associated C-terminal fragment bearing the complete Aβ-domain. Subsequent cleavage of this fragment by γ-secretase, which appears to be identical with the presenilins, results in the release and secretion of Aβ (3Haass C. De Strooper B. Science. 1999; 286: 916-919Crossref PubMed Scopus (364) Google Scholar).Recently, an aspartyl protease with β-secretase activity was identified in human embryonic kidney (HEK) 293 cells and was initially called BACE (β-site APP-cleaving enzyme, Asp2, or memapsin 2) (4Hussain I. Powell D. Howlett D.R. Tew D.G. Meek T.D. Chapman C. Gloger I.S. Murphy K.E. Southan C.D. Ryan D.M. Smith T.S. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (997) Google Scholar, 5Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (737) Google Scholar, 6Sinha 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. John V. et al.Nature. 1999; 402: 537-540Crossref PubMed Scopus (1472) Google Scholar, 7Vassar 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 (3256) Google Scholar, 8Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1328) Google Scholar) (Fig. 1 A). A close homologue was also identified and termed as BACE-2, Asp1, DRAP, or memapsin 1 (4Hussain I. Powell D. Howlett D.R. Tew D.G. Meek T.D. Chapman C. Gloger I.S. Murphy K.E. Southan C.D. Ryan D.M. Smith T.S. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (997) Google Scholar, 5Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (737) Google Scholar,9Acquati F. Accarino M. Nucci C. Fumagalli P. Jovine L. Ottolenghi S. Taramelli R. FEBS Lett. 2000; 468: 59-64Crossref PubMed Scopus (121) Google Scholar). 2For reasons of simplicity, we use the terms BACE and BACE-2 in this study.2For reasons of simplicity, we use the terms BACE and BACE-2 in this study. Both enzymes are type I membrane proteins sharing significant homology with other members of the aspartyl protease family (5Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (737) Google Scholar, 6Sinha 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. John V. et al.Nature. 1999; 402: 537-540Crossref PubMed Scopus (1472) Google Scholar, 7Vassar 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 (3256) Google Scholar, 8Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1328) Google Scholar). While BACE-2 is predominantly expressed in peripheral tissues, BACE is highly expressed in neurons, the major site of Aβ generation. However, it appears that βAPP is not the only substrate for BACE. In fact, mouse βAPP is a very poor substrate for β-secretase activity (10De Strooper B. Simons M. Multhaup G. Van Leuven F. Beyreuther K. Dotti C.G. EMBO J. 1995; 14: 4932-4938Crossref PubMed Scopus (161) Google Scholar). “Humanizing” the Aβ domain of mouse βAPP by three amino acid substitutions resulted in efficient cleavage by β-secretase (10De Strooper B. Simons M. Multhaup G. Van Leuven F. Beyreuther K. Dotti C.G. EMBO J. 1995; 14: 4932-4938Crossref PubMed Scopus (161) Google Scholar), suggesting that βAPP may not be the exclusive substrate for BACE. However, other physiological substrates of BACE remain to be identified.BACE is cotranslationally modified by N-glycosylation and further maturates by complex glycosylation as well as proteolytic removal of its prodomain by a furin-like protease (11Bennett B.D. Denis P. Haniu M. Teplow D.B. Kahn S. Louis J.C. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 37712-37717Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 13Creemers J.W. Dominguez D.I. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2001; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 14Huse J.T. Pijak D.S. Leslie G.J. Lee V.M. Doms R.W. J. Biol. Chem. 2000; 275: 33729-33737Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). The majority of BACE molecules are localized within Golgi and endosomal compartments, where they colocalize with βAPP (4Hussain I. Powell D. Howlett D.R. Tew D.G. Meek T.D. Chapman C. Gloger I.S. Murphy K.E. Southan C.D. Ryan D.M. Smith T.S. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (997) Google Scholar, 5Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (737) Google Scholar, 7Vassar 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 (3256) Google Scholar, 12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 14Huse J.T. Pijak D.S. Leslie G.J. Lee V.M. Doms R.W. J. Biol. Chem. 2000; 275: 33729-33737Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). The acidic pH optimum of BACE (5Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (737) Google Scholar, 6Sinha 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. John V. et al.Nature. 1999; 402: 537-540Crossref PubMed Scopus (1472) Google Scholar, 7Vassar 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 (3256) Google Scholar, 8Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1328) Google Scholar) indicates that it is predominantly active within late Golgi compartments and/or endosomes/lysosomes. This is consistent with previous findings demonstrating that β-secretase cleavage of βAPP can occur in all of these acidic compartments (15Haass C. Koo E.H. Mellon A. Hung A.Y. Selkoe D.J. Nature. 1992; 357: 500-503Crossref PubMed Scopus (768) Google Scholar, 16Haass C. Lemere C.A. Capell A. Citron M. Seubert P. Schenk D. Lannfelt L. Selkoe D.J. Nat. Med. 1995; 1: 1291-1296Crossref PubMed Scopus (437) Google Scholar, 17Koo E.H. Squazzo S.L. J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar, 18Koo E.H. Squazzo S.L. Selkoe D.J. Koo C.H. J. Cell Sci. 1996; 109: 991-998Crossref PubMed Google Scholar).It has recently been reported that BACE is reinternalized from the cell surface to early endosomes and can recycle back to the cell surface, a process that depends on a dileucine motif in the cytoplasmic tail of BACE (14Huse J.T. Pijak D.S. Leslie G.J. Lee V.M. Doms R.W. J. Biol. Chem. 2000; 275: 33729-33737Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). This signal is located close to a negatively charged domain, which contains a potential phosphorylation site. We found that BACE is indeed phosphorylated within its C-terminal domain and that the biological function of BACE phosphorylation resides in the regulation of the retrieval of reinternalized BACE from endosomes.DISCUSSIONIn this study, we analyzed the subcellular trafficking of BACE dependent on its phosphorylation state. A single phosphorylation site was identified by mutagenesis of serine residue 498 in the C-terminal domain of BACE (Fig. 1 A). By testing several protein kinasesin vitro, we found that CK-1 can phosphorylate BACE at thein vivo phosphorylation site at serine residue 498. HD, which preferentially inhibits CK-1 beside glycogen synthase kinase 3β and cyclin-dependent kinases (29Meijer L. Thunnissen A.M. White A.W. Garnier M. Nikolic M. Tsai L.H. Walter J. Cleverley K.E. Salinas P.C. Wu Y.Z. Biernat J. Mandelkow E.M. Kim S.H. Pettit G.R. Chem. Biol. 2000; 7: 51-63Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar), significantly reduced phosphorylation of the cytoplasmic domain of BACE in cellular extracts. The phosphorylation site identified at serine residue 498 is preceded by a stretch of acidic amino acid residues (Fig. 1 A) representing a canonical recognition motif for CK-1, but not for glycogen synthase kinase 3β or cyclin-dependent kinases. Taken together, these data indicate that CK-1 or a CK-1-like kinase is involved in the phosphorylation of BACE in vivo. Phosphorylation occurs exclusively on the fully maturated BACE after propeptide removal and complexN-glycosylation. This indicates that phosphorylation of BACE takes place selectively after its exit from the ER, presumably in Golgi or post-Golgi compartments. CK-1 occurs in several isoforms, and some have been shown to be associated with the plasma membrane and synaptic vesicles and to selectively phosphorylate a subset of membrane proteins (36Gross S.D. Anderson R.A. Cell Signal. 1998; 10: 699-711Crossref PubMed Scopus (266) Google Scholar). CK-1 is implicated in the regulation of vesicular trafficking in yeast presumably by phosphorylating components of clathrin adaptor proteins (37Faundez V.V. Kelly R.B. Mol. Biol. Cell. 2000; 11: 2591-2604Crossref PubMed Scopus (65) Google Scholar, 38Panek H.R. Stepp J.D. Engle H.M. Marks K.M. Tan P.K. Lemmon S.K. Robinson L.C. EMBO J. 1997; 16: 4194-4204Crossref PubMed Scopus (128) Google Scholar). Our data suggest that a CK-1 isoform with a particular subcellular distribution (i.e. in late Golgi and/or endosomal compartments) might be responsible for the highly selective phosphorylation of mature BACE. It has been reported that CK-1 is significantly elevated in Alzheimer's disease brains (39Schwab C. DeMaggio A.J. Ghoshal N. Binder L.I. Kuret J. McGeer P.L. Neurobiol. Aging. 2000; 21: 503-510Crossref PubMed Scopus (124) Google Scholar, 40Yasojima K. Kuret J. DeMaggio A.J. McGeer E. McGeer P.L. Brain Res. 2000; 865: 116-120Crossref PubMed Scopus (91) Google Scholar). Moreover, Aβ has been shown to activate CK-1 in vitro(41Chauhan A. Chauhan V.P. Murakami N. Brockerhoff H. Wisniewski H.M. Brain Res. 1993; 629: 47-52Crossref PubMed Scopus (44) Google Scholar). However, it remains to be determined if phosphorylation of BACE is altered during pathogenesis of Alzheimer's disease.In order to investigate the cellular function of phosphorylation/dephosphorylation on the trafficking of BACE, we generated mutants in which the phosphorylation site at serine residue 498 has been substituted by an alanine or an aspartate residue to mimic nonphosphorylated and phosphorylated forms of BACE, respectively. The major advantage of this strategy is to circumvent the use of agents modulating kinase or phosphatase activities that might cause nonspecific or indirect effects (42Egelhoff T.T. Lee R.J. Spudich J.A. Cell. 1993; 75: 363-371Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 43Walter J. Schindzielorz A. Grunberg J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1391-1396Crossref PubMed Scopus (109) Google Scholar). BACE carrying the S498D substitution was predominantly localized in juxtanuclear compartments, where it partially colocalizes with the Golgi marker protein giantin. A very similar distribution was observed for BACE WT, which is consistent with previous results (7Vassar 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 (3256) Google Scholar, 12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 14Huse J.T. Pijak D.S. Leslie G.J. Lee V.M. Doms R.W. J. Biol. Chem. 2000; 275: 33729-33737Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). In contrast, the nonphosphorylated form BACE S498A showed less localization in juxtanuclear structures but pronounced localization in vesicular compartments including EEA1 positive early endosomes. It was demonstrated previously that the complete deletion of the cytoplasmic domain of BACE results in its retention within the ER and in impaired maturation (12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). However, the phosphorylation site mutants BACE S498A and BACE S498D mature normally by complex N-glycosylation and proteolytic removal of the prodomain as compared with BACE WT. Therefore, the significant differences in subcellular localization of the mutant variants of BACE are due to distinct sorting. Consistent with previous results (12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 14Huse J.T. Pijak D.S. Leslie G.J. Lee V.M. Doms R.W. J. Biol. Chem. 2000; 275: 33729-33737Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar), we found that BACE is transported to the cell surface and that BACE is internalized into EEA1-positive early endosomal compartments. BACE WT as well as the mutant derivatives S498A and S498D were efficiently endocytosed, indicating that phosphorylation of BACE does not determine its reinternalization from the cell surface. Rather, we found that phosphorylation of BACE is functionally required for efficient retrieval of the enzyme from early endosomes to later endosomal and/or TGN compartments from which BACE might be recycled into the secretory pathway.Interestingly, phosphorylation of reinternalized BACE affected its colocalization with its protein substrate βAPP in juxtanuclear structures. However, overexpressing BACE WT or the phosphorylation site mutants led to increased secretion of Aβ as compared with untransfected control cell lines (data not shown). This may reflect that expression of exogenous BACE leads to saturated levels of Aβ generation, regardless of the differences in subcellular localization of the BACE variants. Indeed, previous studies demonstrated that βAPP could be cleaved by β-secretase activity in distinct compartments, including endosomes and late Golgi compartments (16Haass C. Lemere C.A. Capell A. Citron M. Seubert P. Schenk D. Lannfelt L. Selkoe D.J. Nat. Med. 1995; 1: 1291-1296Crossref PubMed Scopus (437) Google Scholar, 17Koo E.H. Squazzo S.L. J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar, 44Lo A.C. Haass C. Wagner S.L. Teplow D.B. Sisodia S.S. J. Biol. Chem. 1994; 269: 30966-30973Abstract Full Text PDF PubMed Google Scholar). We also demonstrate that reinternalization of BACE was not affected by the S498A or S498D mutation. In addition, all variants of BACE undergo normal maturation by complex N-glycosylation. These data indicate that at least two major sites of β-secretase activity, endosomes and secretory vesicles, can be reached by BACE independent of its phosphorylation state. However, it might be possible that regulation of subcellular trafficking of endogenously expressed BACE by phosphorylation affects proteolytic processing of other, yet unknown, protein substrates. In addition, we cannot yet exclude the possibility that phosphorylation may have subtle effects on βAPP processing.The regulatory mechanism of subcellular trafficking of BACE is highly reminiscent to that of furin. Retrieval of furin from endosomal to TGN compartments was also shown to be dependent on phosphorylation/dephosphorylation of its cytoplasmic domain (45Molloy S.S. Thomas L. Kamibayashi C. Mumby M.C. Thomas G. J. Cell Biol. 1998; 142: 1399-1411Crossref PubMed Scopus (83) Google Scholar). Therefore, the subcellular localization of BACE is regulated in a remarkably similar fashion like furin, a protease that has recently been demonstrated to be required for propeptide cleavage of BACE (11Bennett B.D. Denis P. Haniu M. Teplow D.B. Kahn S. Louis J.C. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 37712-37717Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 13Creemers J.W. Dominguez D.I. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2001; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Alzheimer's disease is the most common form of dementia and is pathologically characterized by the invariant accumulation of senile plaques and neurofibrillary tangles in certain areas of the brains of Alzheimer's disease patients (1Selkoe D.J. Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1519) Google Scholar). The major constituent of senile plaques is the amyloid β-peptide (Aβ),1 which derives from the β-amyloid precursor protein (βAPP) by endoproteolytic processing (2Haass C. Selkoe D.J. Cell. 1993; 75: 1039-1042Abstract Full Text PDF PubMed Scopus (736) Google Scholar). β-Secretase cleaves βAPP at the N terminus of the Aβ-domain, resulting in the generation of soluble APPS-β and a membrane-associated C-terminal fragment bearing the complete Aβ-domain. Subsequent cleavage of this fragment by γ-secretase, which appears to be identical with the presenilins, results in the release and secretion of Aβ (3Haass C. De Strooper B. Science. 1999; 286: 916-919Crossref PubMed Scopus (364) Google Scholar). Recently, an aspartyl protease with β-secretase activity was identified in human embryonic kidney (HEK) 293 cells and was initially called BACE (β-site APP-cleaving enzyme, Asp2, or memapsin 2) (4Hussain I. Powell D. Howlett D.R. Tew D.G. Meek T.D. Chapman C. Gloger I.S. Murphy K.E. Southan C.D. Ryan D.M. Smith T.S. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (997) Google Scholar, 5Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (737) Google Scholar, 6Sinha 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. John V. et al.Nature. 1999; 402: 537-540Crossref PubMed Scopus (1472) Google Scholar, 7Vassar 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 (3256) Google Scholar, 8Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1328) Google Scholar) (Fig. 1 A). A close homologue was also identified and termed as BACE-2, Asp1, DRAP, or memapsin 1 (4Hussain I. Powell D. Howlett D.R. Tew D.G. Meek T.D. Chapman C. Gloger I.S. Murphy K.E. Southan C.D. Ryan D.M. Smith T.S. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (997) Google Scholar, 5Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (737) Google Scholar,9Acquati F. Accarino M. Nucci C. Fumagalli P. Jovine L. Ottolenghi S. Taramelli R. FEBS Lett. 2000; 468: 59-64Crossref PubMed Scopus (121) Google Scholar). 2For reasons of simplicity, we use the terms BACE and BACE-2 in this study.2For reasons of simplicity, we use the terms BACE and BACE-2 in this study. Both enzymes are type I membrane proteins sharing significant homology with other members of the aspartyl protease family (5Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (737) Google Scholar, 6Sinha 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. John V. et al.Nature. 1999; 402: 537-540Crossref PubMed Scopus (1472) Google Scholar, 7Vassar 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 (3256) Google Scholar, 8Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1328) Google Scholar). While BACE-2 is predominantly expressed in peripheral tissues, BACE is highly expressed in neurons, the major site of Aβ generation. However, it appears that βAPP is not the only substrate for BACE. In fact, mouse βAPP is a very poor substrate for β-secretase activity (10De Strooper B. Simons M. Multhaup G. Van Leuven F. Beyreuther K. Dotti C.G. EMBO J. 1995; 14: 4932-4938Crossref PubMed Scopus (161) Google Scholar). “Humanizing” the Aβ domain of mouse βAPP by three amino acid substitutions resulted in efficient cleavage by β-secretase (10De Strooper B. Simons M. Multhaup G. Van Leuven F. Beyreuther K. Dotti C.G. EMBO J. 1995; 14: 4932-4938Crossref PubMed Scopus (161) Google Scholar), suggesting that βAPP may not be the exclusive substrate for BACE. However, other physiological substrates of BACE remain to be identified. BACE is cotranslationally modified by N-glycosylation and further maturates by complex glycosylation as well as proteolytic removal of its prodomain by a furin-like protease (11Bennett B.D. Denis P. Haniu M. Teplow D.B. Kahn S. Louis J.C. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 37712-37717Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 13Creemers J.W. Dominguez D.I. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2001; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 14Huse J.T. Pijak D.S. Leslie G.J. Lee V.M. Doms R.W. J. Biol. Chem. 2000; 275: 33729-33737Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). The majority of BACE molecules are localized within Golgi and endosomal compartments, where they colocalize with βAPP (4Hussain I. Powell D. Howlett D.R. Tew D.G. Meek T.D. Chapman C. Gloger I.S. Murphy K.E. Southan C.D. Ryan D.M. Smith T.S. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (997) Google Scholar, 5Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (737) Google Scholar, 7Vassar 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 (3256) Google Scholar, 12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 14Huse J.T. Pijak D.S. Leslie G.J. Lee V.M. Doms R.W. J. Biol. Chem. 2000; 275: 33729-33737Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). The acidic pH optimum of BACE (5Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (737) Google Scholar, 6Sinha 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. John V. et al.Nature. 1999; 402: 537-540Crossref PubMed Scopus (1472) Google Scholar, 7Vassar 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 (3256) Google Scholar, 8Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1328) Google Scholar) indicates that it is predominantly active within late Golgi compartments and/or endosomes/lysosomes. This is consistent with previous findings demonstrating that β-secretase cleavage of βAPP can occur in all of these acidic compartments (15Haass C. Koo E.H. Mellon A. Hung A.Y. Selkoe D.J. Nature. 1992; 357: 500-503Crossref PubMed Scopus (768) Google Scholar, 16Haass C. Lemere C.A. Capell A. Citron M. Seubert P. Schenk D. Lannfelt L. Selkoe D.J. Nat. Med. 1995; 1: 1291-1296Crossref PubMed Scopus (437) Google Scholar, 17Koo E.H. Squazzo S.L. J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar, 18Koo E.H. Squazzo S.L. Selkoe D.J. Koo C.H. J. Cell Sci. 1996; 109: 991-998Crossref PubMed Google Scholar). It has recently been reported that BACE is reinternalized from the cell surface to early endosomes and can recycle back to the cell surface, a process that depends on a dileucine motif in the cytoplasmic tail of BACE (14Huse J.T. Pijak D.S. Leslie G.J. Lee V.M. Doms R.W. J. Biol. Chem. 2000; 275: 33729-33737Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). This signal is located close to a negatively charged domain, which contains a potential phosphorylation site. We found that BACE is indeed phosphorylated within its C-terminal domain and that the biological function of BACE phosphorylation resides in the regulation of the retrieval of reinternalized BACE from endosomes. DISCUSSIONIn this study, we analyzed the subcellular trafficking of BACE dependent on its phosphorylation state. A single phosphorylation site was identified by mutagenesis of serine residue 498 in the C-terminal domain of BACE (Fig. 1 A). By testing several protein kinasesin vitro, we found that CK-1 can phosphorylate BACE at thein vivo phosphorylation site at serine residue 498. HD, which preferentially inhibits CK-1 beside glycogen synthase kinase 3β and cyclin-dependent kinases (29Meijer L. Thunnissen A.M. White A.W. Garnier M. Nikolic M. Tsai L.H. Walter J. Cleverley K.E. Salinas P.C. Wu Y.Z. Biernat J. Mandelkow E.M. Kim S.H. Pettit G.R. Chem. Biol. 2000; 7: 51-63Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar), significantly reduced phosphorylation of the cytoplasmic domain of BACE in cellular extracts. The phosphorylation site identified at serine residue 498 is preceded by a stretch of acidic amino acid residues (Fig. 1 A) representing a canonical recognition motif for CK-1, but not for glycogen synthase kinase 3β or cyclin-dependent kinases. Taken together, these data indicate that CK-1 or a CK-1-like kinase is involved in the phosphorylation of BACE in vivo. Phosphorylation occurs exclusively on the fully maturated BACE after propeptide removal and complexN-glycosylation. This indicates that phosphorylation of BACE takes place selectively after its exit from the ER, presumably in Golgi or post-Golgi compartments. CK-1 occurs in several isoforms, and some have been shown to be associated with the plasma membrane and synaptic vesicles and to selectively phosphorylate a subset of membrane proteins (36Gross S.D. Anderson R.A. Cell Signal. 1998; 10: 699-711Crossref PubMed Scopus (266) Google Scholar). CK-1 is implicated in the regulation of vesicular trafficking in yeast presumably by phosphorylating components of clathrin adaptor proteins (37Faundez V.V. Kelly R.B. Mol. Biol. Cell. 2000; 11: 2591-2604Crossref PubMed Scopus (65) Google Scholar, 38Panek H.R. Stepp J.D. Engle H.M. Marks K.M. Tan P.K. Lemmon S.K. Robinson L.C. EMBO J. 1997; 16: 4194-4204Crossref PubMed Scopus (128) Google Scholar). Our data suggest that a CK-1 isoform with a particular subcellular distribution (i.e. in late Golgi and/or endosomal compartments) might be responsible for the highly selective phosphorylation of mature BACE. It has been reported that CK-1 is significantly elevated in Alzheimer's disease brains (39Schwab C. DeMaggio A.J. Ghoshal N. Binder L.I. Kuret J. McGeer P.L. Neurobiol. Aging. 2000; 21: 503-510Crossref PubMed Scopus (124) Google Scholar, 40Yasojima K. Kuret J. DeMaggio A.J. McGeer E. McGeer P.L. Brain Res. 2000; 865: 116-120Crossref PubMed Scopus (91) Google Scholar). Moreover, Aβ has been shown to activate CK-1 in vitro(41Chauhan A. Chauhan V.P. Murakami N. Brockerhoff H. Wisniewski H.M. Brain Res. 1993; 629: 47-52Crossref PubMed Scopus (44) Google Scholar). However, it remains to be determined if phosphorylation of BACE is altered during pathogenesis of Alzheimer's disease.In order to investigate the cellular function of phosphorylation/dephosphorylation on the trafficking of BACE, we generated mutants in which the phosphorylation site at serine residue 498 has been substituted by an alanine or an aspartate residue to mimic nonphosphorylated and phosphorylated forms of BACE, respectively. The major advantage of this strategy is to circumvent the use of agents modulating kinase or phosphatase activities that might cause nonspecific or indirect effects (42Egelhoff T.T. Lee R.J. Spudich J.A. Cell. 1993; 75: 363-371Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 43Walter J. Schindzielorz A. Grunberg J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1391-1396Crossref PubMed Scopus (109) Google Scholar). BACE carrying the S498D substitution was predominantly localized in juxtanuclear compartments, where it partially colocalizes with the Golgi marker protein giantin. A very similar distribution was observed for BACE WT, which is consistent with previous results (7Vassar 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 (3256) Google Scholar, 12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 14Huse J.T. Pijak D.S. Leslie G.J. Lee V.M. Doms R.W. J. Biol. Chem. 2000; 275: 33729-33737Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). In contrast, the nonphosphorylated form BACE S498A showed less localization in juxtanuclear structures but pronounced localization in vesicular compartments including EEA1 positive early endosomes. It was demonstrated previously that the complete deletion of the cytoplasmic domain of BACE results in its retention within the ER and in impaired maturation (12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). However, the phosphorylation site mutants BACE S498A and BACE S498D mature normally by complex N-glycosylation and proteolytic removal of the prodomain as compared with BACE WT. Therefore, the significant differences in subcellular localization of the mutant variants of BACE are due to distinct sorting. Consistent with previous results (12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 14Huse J.T. Pijak D.S. Leslie G.J. Lee V.M. Doms R.W. J. Biol. Chem. 2000; 275: 33729-33737Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar), we found that BACE is transported to the cell surface and that BACE is internalized into EEA1-positive early endosomal compartments. BACE WT as well as the mutant derivatives S498A and S498D were efficiently endocytosed, indicating that phosphorylation of BACE does not determine its reinternalization from the cell surface. Rather, we found that phosphorylation of BACE is functionally required for efficient retrieval of the enzyme from early endosomes to later endosomal and/or TGN compartments from which BACE might be recycled into the secretory pathway.Interestingly, phosphorylation of reinternalized BACE affected its colocalization with its protein substrate βAPP in juxtanuclear structures. However, overexpressing BACE WT or the phosphorylation site mutants led to increased secretion of Aβ as compared with untransfected control cell lines (data not shown). This may reflect that expression of exogenous BACE leads to saturated levels of Aβ generation, regardless of the differences in subcellular localization of the BACE variants. Indeed, previous studies demonstrated that βAPP could be cleaved by β-secretase activity in distinct compartments, including endosomes and late Golgi compartments (16Haass C. Lemere C.A. Capell A. Citron M. Seubert P. Schenk D. Lannfelt L. Selkoe D.J. Nat. Med. 1995; 1: 1291-1296Crossref PubMed Scopus (437) Google Scholar, 17Koo E.H. Squazzo S.L. J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar, 44Lo A.C. Haass C. Wagner S.L. Teplow D.B. Sisodia S.S. J. Biol. Chem. 1994; 269: 30966-30973Abstract Full Text PDF PubMed Google Scholar). We also demonstrate that reinternalization of BACE was not affected by the S498A or S498D mutation. In addition, all variants of BACE undergo normal maturation by complex N-glycosylation. These data indicate that at least two major sites of β-secretase activity, endosomes and secretory vesicles, can be reached by BACE independent of its phosphorylation state. However, it might be possible that regulation of subcellular trafficking of endogenously expressed BACE by phosphorylation affects proteolytic processing of other, yet unknown, protein substrates. In addition, we cannot yet exclude the possibility that phosphorylation may have subtle effects on βAPP processing.The regulatory mechanism of subcellular trafficking of BACE is highly reminiscent to that of furin. Retrieval of furin from endosomal to TGN compartments was also shown to be dependent on phosphorylation/dephosphorylation of its cytoplasmic domain (45Molloy S.S. Thomas L. Kamibayashi C. Mumby M.C. Thomas G. J. Cell Biol. 1998; 142: 1399-1411Crossref PubMed Scopus (83) Google Scholar). Therefore, the subcellular localization of BACE is regulated in a remarkably similar fashion like furin, a protease that has recently been demonstrated to be required for propeptide cleavage of BACE (11Bennett B.D. Denis P. Haniu M. Teplow D.B. Kahn S. Louis J.C. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 37712-37717Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 13Creemers J.W. Dominguez D.I. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2001; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). In this study, we analyzed the subcellular trafficking of BACE dependent on its phosphorylation state. A single phosphorylation site was identified by mutagenesis of serine residue 498 in the C-terminal domain of BACE (Fig. 1 A). By testing several protein kinasesin vitro, we found that CK-1 can phosphorylate BACE at thein vivo phosphorylation site at serine residue 498. HD, which preferentially inhibits CK-1 beside glycogen synthase kinase 3β and cyclin-dependent kinases (29Meijer L. Thunnissen A.M. White A.W. Garnier M. Nikolic M. Tsai L.H. Walter J. Cleverley K.E. Salinas P.C. Wu Y.Z. Biernat J. Mandelkow E.M. Kim S.H. Pettit G.R. Chem. Biol. 2000; 7: 51-63Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar), significantly reduced phosphorylation of the cytoplasmic domain of BACE in cellular extracts. The phosphorylation site identified at serine residue 498 is preceded by a stretch of acidic amino acid residues (Fig. 1 A) representing a canonical recognition motif for CK-1, but not for glycogen synthase kinase 3β or cyclin-dependent kinases. Taken together, these data indicate that CK-1 or a CK-1-like kinase is involved in the phosphorylation of BACE in vivo. Phosphorylation occurs exclusively on the fully maturated BACE after propeptide removal and complexN-glycosylation. This indicates that phosphorylation of BACE takes place selectively after its exit from the ER, presumably in Golgi or post-Golgi compartments. CK-1 occurs in several isoforms, and some have been shown to be associated with the plasma membrane and synaptic vesicles and to selectively phosphorylate a subset of membrane proteins (36Gross S.D. Anderson R.A. Cell Signal. 1998; 10: 699-711Crossref PubMed Scopus (266) Google Scholar). CK-1 is implicated in the regulation of vesicular trafficking in yeast presumably by phosphorylating components of clathrin adaptor proteins (37Faundez V.V. Kelly R.B. Mol. Biol. Cell. 2000; 11: 2591-2604Crossref PubMed Scopus (65) Google Scholar, 38Panek H.R. Stepp J.D. Engle H.M. Marks K.M. Tan P.K. Lemmon S.K. Robinson L.C. EMBO J. 1997; 16: 4194-4204Crossref PubMed Scopus (128) Google Scholar). Our data suggest that a CK-1 isoform with a particular subcellular distribution (i.e. in late Golgi and/or endosomal compartments) might be responsible for the highly selective phosphorylation of mature BACE. It has been reported that CK-1 is significantly elevated in Alzheimer's disease brains (39Schwab C. DeMaggio A.J. Ghoshal N. Binder L.I. Kuret J. McGeer P.L. Neurobiol. Aging. 2000; 21: 503-510Crossref PubMed Scopus (124) Google Scholar, 40Yasojima K. Kuret J. DeMaggio A.J. McGeer E. McGeer P.L. Brain Res. 2000; 865: 116-120Crossref PubMed Scopus (91) Google Scholar). Moreover, Aβ has been shown to activate CK-1 in vitro(41Chauhan A. Chauhan V.P. Murakami N. Brockerhoff H. Wisniewski H.M. Brain Res. 1993; 629: 47-52Crossref PubMed Scopus (44) Google Scholar). However, it remains to be determined if phosphorylation of BACE is altered during pathogenesis of Alzheimer's disease. In order to investigate the cellular function of phosphorylation/dephosphorylation on the trafficking of BACE, we generated mutants in which the phosphorylation site at serine residue 498 has been substituted by an alanine or an aspartate residue to mimic nonphosphorylated and phosphorylated forms of BACE, respectively. The major advantage of this strategy is to circumvent the use of agents modulating kinase or phosphatase activities that might cause nonspecific or indirect effects (42Egelhoff T.T. Lee R.J. Spudich J.A. Cell. 1993; 75: 363-371Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 43Walter J. Schindzielorz A. Grunberg J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1391-1396Crossref PubMed Scopus (109) Google Scholar). BACE carrying the S498D substitution was predominantly localized in juxtanuclear compartments, where it partially colocalizes with the Golgi marker protein giantin. A very similar distribution was observed for BACE WT, which is consistent with previous results (7Vassar 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 (3256) Google Scholar, 12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 14Huse J.T. Pijak D.S. Leslie G.J. Lee V.M. Doms R.W. J. Biol. Chem. 2000; 275: 33729-33737Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). In contrast, the nonphosphorylated form BACE S498A showed less localization in juxtanuclear structures but pronounced localization in vesicular compartments including EEA1 positive early endosomes. It was demonstrated previously that the complete deletion of the cytoplasmic domain of BACE results in its retention within the ER and in impaired maturation (12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). However, the phosphorylation site mutants BACE S498A and BACE S498D mature normally by complex N-glycosylation and proteolytic removal of the prodomain as compared with BACE WT. Therefore, the significant differences in subcellular localization of the mutant variants of BACE are due to distinct sorting. Consistent with previous results (12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 14Huse J.T. Pijak D.S. Leslie G.J. Lee V.M. Doms R.W. J. Biol. Chem. 2000; 275: 33729-33737Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar), we found that BACE is transported to the cell surface and that BACE is internalized into EEA1-positive early endosomal compartments. BACE WT as well as the mutant derivatives S498A and S498D were efficiently endocytosed, indicating that phosphorylation of BACE does not determine its reinternalization from the cell surface. Rather, we found that phosphorylation of BACE is functionally required for efficient retrieval of the enzyme from early endosomes to later endosomal and/or TGN compartments from which BACE might be recycled into the secretory pathway. Interestingly, phosphorylation of reinternalized BACE affected its colocalization with its protein substrate βAPP in juxtanuclear structures. However, overexpressing BACE WT or the phosphorylation site mutants led to increased secretion of Aβ as compared with untransfected control cell lines (data not shown). This may reflect that expression of exogenous BACE leads to saturated levels of Aβ generation, regardless of the differences in subcellular localization of the BACE variants. Indeed, previous studies demonstrated that βAPP could be cleaved by β-secretase activity in distinct compartments, including endosomes and late Golgi compartments (16Haass C. Lemere C.A. Capell A. Citron M. Seubert P. Schenk D. Lannfelt L. Selkoe D.J. Nat. Med. 1995; 1: 1291-1296Crossref PubMed Scopus (437) Google Scholar, 17Koo E.H. Squazzo S.L. J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar, 44Lo A.C. Haass C. Wagner S.L. Teplow D.B. Sisodia S.S. J. Biol. Chem. 1994; 269: 30966-30973Abstract Full Text PDF PubMed Google Scholar). We also demonstrate that reinternalization of BACE was not affected by the S498A or S498D mutation. In addition, all variants of BACE undergo normal maturation by complex N-glycosylation. These data indicate that at least two major sites of β-secretase activity, endosomes and secretory vesicles, can be reached by BACE independent of its phosphorylation state. However, it might be possible that regulation of subcellular trafficking of endogenously expressed BACE by phosphorylation affects proteolytic processing of other, yet unknown, protein substrates. In addition, we cannot yet exclude the possibility that phosphorylation may have subtle effects on βAPP processing. The regulatory mechanism of subcellular trafficking of BACE is highly reminiscent to that of furin. Retrieval of furin from endosomal to TGN compartments was also shown to be dependent on phosphorylation/dephosphorylation of its cytoplasmic domain (45Molloy S.S. Thomas L. Kamibayashi C. Mumby M.C. Thomas G. J. Cell Biol. 1998; 142: 1399-1411Crossref PubMed Scopus (83) Google Scholar). Therefore, the subcellular localization of BACE is regulated in a remarkably similar fashion like furin, a protease that has recently been demonstrated to be required for propeptide cleavage of BACE (11Bennett B.D. Denis P. Haniu M. Teplow D.B. Kahn S. Louis J.C. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 37712-37717Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 12Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 13Creemers J.W. Dominguez D.I. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2001; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). We are grateful to Drs. H.-P. Hauri for anti-giantin and E. H. Koo for anti-βAPP antibodies and to Dr. V. Kinzel for purified protein kinase A. We thank Drs. L. Meijer and G. Pettit for hymenialdisine. We also thank Drs. M. Sastre and H. Steiner for helpful discussions and critically reading the manuscript and L. Meyn for expert technical assistance." @default.
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W2166888773 title "Phosphorylation Regulates Intracellular Trafficking of β-Secretase" @default.
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W2166888773 doi "https://doi.org/10.1074/jbc.m011116200" @default.
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