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• W2056510710 abstract "The Alzheimer's disease-associated presenilin (PS) 1 is intimately involved in γ-secretase cleavage of β-amyloid precursor protein and other proteins. In addition, PS1 plays a role in β-catenin signaling and in the regulation of apoptosis. Here we demonstrate that phosphorylation of PS1 is regulated by two independent signaling pathways involving protein kinase (PK) A and PKC and that both kinases can directly phosphorylate the large hydrophilic domain of PS1 in vitro and in cultured cells. A phosphorylation site at serine residue 346 was identified that is selectively phosphorylated by PKC but not by PKA. This site is localized within a recognition motif for caspases, and phosphorylation strongly inhibits proteolytic processing of PS1 by caspase activity during apoptosis. Moreover, PS1 phosphorylation reduces the progression of apoptosis. Our data indicate that phosphorylation/dephosphorylation at the caspase recognition site provides a mechanism to reversibly regulate properties of PS1 in apoptosis. The Alzheimer's disease-associated presenilin (PS) 1 is intimately involved in γ-secretase cleavage of β-amyloid precursor protein and other proteins. In addition, PS1 plays a role in β-catenin signaling and in the regulation of apoptosis. Here we demonstrate that phosphorylation of PS1 is regulated by two independent signaling pathways involving protein kinase (PK) A and PKC and that both kinases can directly phosphorylate the large hydrophilic domain of PS1 in vitro and in cultured cells. A phosphorylation site at serine residue 346 was identified that is selectively phosphorylated by PKC but not by PKA. This site is localized within a recognition motif for caspases, and phosphorylation strongly inhibits proteolytic processing of PS1 by caspase activity during apoptosis. Moreover, PS1 phosphorylation reduces the progression of apoptosis. Our data indicate that phosphorylation/dephosphorylation at the caspase recognition site provides a mechanism to reversibly regulate properties of PS1 in apoptosis. Neuronal cell death underlies the pathogenesis of Alzheimer's disease, and it has been suggested that apoptotic mechanisms are involved in this process (1.Mattson M.P. Nat. Rev. Mol. Cell Biol. 2000; 1: 120-129Crossref PubMed Scopus (1247) Google Scholar). Classical features of apoptosis, including DNA fragmentation, activation of caspases, and cleavage of poly(ADP-ribose) polymerase (PARP) 1The abbreviations used are: PARP, poly(ADP-ribose) polymerase; βAPP, β-amyloid precursor protein; PS, presenilin; NTF, N-terminal fragment; CTF, C-terminal fragment; HEK, human embryonic kidney; PKA, protein kinase A; PKC, protein kinase C; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PDBU, phorbol 12,13-dibutyrate; STS, staurosporine; OA, okadaic acid; WT, wild type; Aβ, amyloid β-peptide.1The abbreviations used are: PARP, poly(ADP-ribose) polymerase; βAPP, β-amyloid precursor protein; PS, presenilin; NTF, N-terminal fragment; CTF, C-terminal fragment; HEK, human embryonic kidney; PKA, protein kinase A; PKC, protein kinase C; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PDBU, phorbol 12,13-dibutyrate; STS, staurosporine; OA, okadaic acid; WT, wild type; Aβ, amyloid β-peptide. can be detected in Alzheimer's disease brains, and it has been shown that the amyloid β-peptide (Aβ), a major constituent of β-amyloid plaques, can induce apoptosis of neuronal cells (1.Mattson M.P. Nat. Rev. Mol. Cell Biol. 2000; 1: 120-129Crossref PubMed Scopus (1247) Google Scholar).Aβ derives from the larger β-amyloid precursor protein (βAPP) by sequential cleavages mediated by β- and γ-secretases (2.Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5121) Google Scholar, 3.Walter J. Kaether C. Steiner H. Haass C. Curr. Opin. Neurobiol. 2001; 11: 585-590Crossref PubMed Scopus (158) Google Scholar). The presenilin (PS) 1 and PS2 proteins are critically involved in γ-secretase activity (4.Herreman A. Serneels L. Annaert W. Collen D. Schoonjans L. de Strooper B. Nat. Cell Biol. 2000; 2: 461-462Crossref PubMed Scopus (450) Google Scholar) and might represent members of a family of aspartic proteases that catalyze cleavage of proteins within their transmembrane domains (5.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1676) Google Scholar, 6.Steiner H. Haass C. Nat. Rev. Mol. Cell Biol. 2000; 1: 217-224Crossref PubMed Scopus (144) Google Scholar, 7.Zhang Z. Nadeau P. Song W. Donoviel D. Yuan M. Bernstein A. Yankner B.A. Nat. Cell Biol. 2000; 2: 463-465Crossref PubMed Scopus (357) Google Scholar). Pathogenic mutations in the PS genes that cause familial forms of early onset Alzheimer's disease lead to increased production of the 42 amino acid form of Aβ that aggregates much faster than Aβ40 to form insoluble fibrils (8.Younkin S.G. J. Physiol. (Paris). 1998; 92: 289-292Crossref PubMed Scopus (247) Google Scholar). PS are multi-pass transmembrane proteins that undergo endoproteolytic processing to generate stable N-terminal (NTF) and C-terminal fragments (CTF) (9.Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar). The fragments assemble to a high molecular weight complex with other proteins including nicastrin (10.Yu G. Nishimura M. Arawaka S. Levitan D. Zhang L. Tandon A. Song Y.Q. Rogaeva E. Chen F. Kawarai T. Supala A. Levesque L. Yu H. Yang D.S. Holmes E. Milman P. Liang Y. Zhang D.M. Xu D.H. Sato C. Rogaev E. Smith M. Janus C. Zhang Y. Aebersold R. Farrer L.S. Sorbi S. Bruni A. Fraser P. St George-Hyslop P. Nature. 2000; 407: 48-54Crossref PubMed Scopus (820) Google Scholar), APH-1 a/b (11.Gu Y. Chen F. Sanjo N. Kawarai T. Hasegawa H. Duthie M. Li W. Ruan X. Luthra A. Mount H.T. Tandon A. Fraser P.E. St George-Hyslop P. J. Biol. Chem. 2003; 278: 7374-7380Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), and PEN-2 (12.Steiner H. Winkler E. Edbauer D. Prokop S. Basset G. Yamasaki A. Kostka M. Haass C. J. Biol. Chem. 2002; 277: 39062-39065Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar) that are also essential for γ-secretase activity and Notch signaling (13.Haass C. Steiner H. Trends Cell Biol. 2002; 12: 556-562Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 14.Francis R. McGrath G. Zhang J. Ruddy D.A. Sym M. Apfeld J. Nicoll M. Maxwell M. Hai B. Ellis M.C. Parks A.L. Xu W. Li J. Gurney M. Myers R.L. Himes C.S. Hiebsch R. Ruble C. Nye J.S. Curtis D. Dev. Cell. 2002; 3: 85-97Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar).In addition to the involvement in intramembranous proteolysis, other functions have been attributed to PS proteins, including the regulation of calcium homeostasis (15.Mattson M.P. Physiol. Rev. 1997; 77: 1081-1132Crossref PubMed Scopus (873) Google Scholar, 16.Yoo A.S. Cheng I. Chung S. Grenfell T.Z. Lee H. Pack-Chung E. Handler M. Shen J. Xia W. Tesco G. Saunders A.J. Ding K. Frosch M.P. Tanzi R.E. Kim T.W. Neuron. 2000; 27: 561-572Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 17.Leissring M.A. Akbari Y. Fanger C.M. Cahalan M.D. Mattson M.P. LaFerla F.M. J. Cell Biol. 2000; 149: 793-798Crossref PubMed Scopus (285) Google Scholar), cell adhesion (18.Georgakopoulos A. Marambaud P. Efthimiopoulos S. Shioi J. Cui W. Li H.C. Schutte M. Gordon R. Holstein G.R. Martinelli G. Mehta P. Friedrich Jr., V.L. Robakis N.K. Mol. Cell. 1999; 4: 893-902Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 19.Baki L. Marambaud P. Efthimiopoulos S. Georgakopoulos A. Wen P. Cui W. Shioi J. Koo E. Ozawa M. Friedrich Jr., V.L. Robakis N.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2381-2386Crossref PubMed Scopus (156) Google Scholar), and the subcellular trafficking of several membrane proteins, like TrkB, telencephalin, and βAPP (20.Annaert W.G. Esselens C. Baert V. Boeve C. Snellings G. Cupers P. Craessaerts K. de Strooper B. Neuron. 2001; 32: 579-589Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 21.Kaether C. Lammich S. Edbauer D. Ertl M. Rietdorf J. Capell A. Steiner H. Haass C. J. Cell Biol. 2002; 158: 551-561Crossref PubMed Scopus (168) Google Scholar, 22.Naruse S. Thinakaran G. Luo J.J. Kusiak J.W. Tomita T. Iwatsubo T. Qian X. Ginty D.D. Price D.L. Borchelt D.R. Wong P.C. Sisodia S.S. Neuron. 1998; 21: 1213-1221Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Moreover, PS1 binds to β-catenin and negatively regulates Wnt signaling (23.Killick R. Pollard C.C. Asuni A.A. Mudher A.K. Richardson J.C. Rupniak H.T. Sheppard P.W. Varndell I.M. Brion J.P. Levey A.I. Levy O.A. Vestling M. Cowburn R. Lovestone S. Anderton B.H. J. Biol. Chem. 2001; 276: 48554-48561Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 24.Kang D.E. Soriano S. Xia X. Eberhart C.G. de Strooper B. Zheng H. Koo E.H. Cell. 2002; 110: 751-762Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 25.Xia X. Qian S. Soriano S. Wu Y. Fletcher A.M. Wang X.J. Koo E.H. Wu X. Zheng H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10863-10868Crossref PubMed Scopus (205) Google Scholar). Several lines of evidence indicate that PS proteins are also involved in apoptosis (1.Mattson M.P. Nat. Rev. Mol. Cell Biol. 2000; 1: 120-129Crossref PubMed Scopus (1247) Google Scholar). Initially, a gene trap analysis for anti-apoptotic factors revealed a C-terminal sequence of PS2 (26.Vito P. Lacana E. D'Adamio L. Science. 1996; 271: 521-525Crossref PubMed Scopus (453) Google Scholar). It was also demonstrated that inhibition of PS1 expression in cultured tumor cells or mice strains with spontaneous tumor development induces a higher rate of apoptosis (27.Roperch J.P. Alvaro V. Prieur S. Tuynder M. Nemani M. Lethrosne F. Piouffre L. Gendron M.C. Israeli D. Dausset J. Oren M. Amson R. Telerman A. Nat. Med. 1998; 4: 835-838Crossref PubMed Scopus (155) Google Scholar). Both PS1 and PS2 are also substrates for caspases in vitro and in cultured cells (28.Grunberg J. Walter J. Loetscher H. Deuschle U. Jacobsen H. Haass C. Biochemistry. 1998; 37: 2263-2270Crossref PubMed Scopus (59) Google Scholar, 29.Loetscher H. Deuschle U. Brockhaus M. Reinhardt D. Nelboeck P. Mous J. Grunberg J. Haass C. Jacobsen H. J. Biol. Chem. 1997; 272: 20655-20659Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 30.Kim T.W. Pettingell W.H. Jung Y.K. Kovacs D.M. Tanzi R.E. Science. 1997; 277: 373-376Crossref PubMed Scopus (326) Google Scholar), and the C-terminal cleavage product of PS2 has been shown to inhibit Fas-induced apoptosis (31.Vito P. Ghayur T. D'Adamio L. J. Biol. Chem. 1997; 272: 28315-28320Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). On the other hand, proapoptotic activities of PS proteins have also been reported (32.Alves D.C. Paitel E. Mattson M.P. Amson R. Telerman A. Ancolio K. Checler F. Mattson M.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4043-4048Crossref PubMed Scopus (111) Google Scholar, 33.Araki W. Yuasa K. Takeda S. Takeda K. Shirotani K. Takahashi K. Tabira T. J. Neurochem. 2001; 79: 1161-1168Crossref PubMed Scopus (45) Google Scholar, 34.Janicki S. Monteiro M.J. J. Cell Biol. 1997; 139: 485-495Crossref PubMed Scopus (114) Google Scholar). Notably, FAD-associated mutations in the PS genes have been shown to sensitize cells to several apoptotic stimuli (35.Wolozin B. Iwasaki K. Vito P. Ganjei J.K. Lacana E. Sunderland T. Zhao B. Kusiak J.W. Wasco W. D'Adamio L. Science. 1996; 274: 1710-1713Crossref PubMed Scopus (390) Google Scholar, 36.Yankner B.A. Neuron. 1996; 16: 921-932Abstract Full Text Full Text PDF PubMed Scopus (910) Google Scholar, 37.Mattson M.P. Guo Q. Furukawa K. Pedersen W.A. J. Neurochem. 1998; 70: 1-14Crossref PubMed Scopus (230) Google Scholar).In this study we investigated whether phosphorylation of PS1 modulates its role in apoptosis. PS1 was found to be phosphorylated at serine residue 346 by PKC. The phosphorylation at this site regulates the caspase-mediated cleavage of PS1 and inhibits the progression of apoptosis.MATERIALS AND METHODScDNAs and Fusion Proteins—The phosphorylation site mutants of PS1 were generated by PCR techniques using appropriate oligonucleotides. The resulting PCR fragments were subcloned into the EcoRI/XhoI restriction sites of pcDNA3.1 containing a zeocine resistance gene (Invitrogen). The fusion proteins of the maltose-binding protein and the hydrophilic loop domain of PS1 have been described earlier (38.Walter J. Grunberg J. Schindzielorz A. Haass C. Biochemistry. 1998; 37: 5961-5967Crossref PubMed Scopus (57) Google Scholar).Cell Culture and Transfection—Human embryonic kidney (HEK) 293 cells, green monkey kidney Cos-7 cells, human cervix carcinoma cells (HeLa), and human mammary carcinoma fibroblasts (MCF-7 cells) were cultured in Dulbecco's modified Eagle's medium with Glutamax (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen). The HEK293 cell line stably overexpressing βAPP695 (39.Haass C. Schlossmacher M.G. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B.L. Lieberburg I. Koo E.H. Schenk D. Teplow D.B. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1749) Google Scholar) and the MCF-7 cells stably expressing Fas (40.Jaattela M. Benedict M. Tewari M. Shayman J.A. Dixit V.M. Oncogene. 1995; 10: 2297-2305PubMed Google Scholar) have been described previously. Transfection of cells with PS1 cDNAs was carried out using FuGENE 6 reagent (Roche Applied Science) according to the supplier's instructions. Single cell clones were generated by selection in 200 μg/ml zeocine (Invitrogen).Antibodies, Immunoprecipitation, and Immunoblotting—The antibodies 3027, 2953 (41.Walter J. Grunberg J. Capell A. Pesold B. Schindzielorz A. Citron M. Mendla K. St George-Hyslop P. Multhaup G. Selkoe D.J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5349-5354Crossref PubMed Scopus (101) Google Scholar), 3711 (38.Walter J. Grunberg J. Schindzielorz A. Haass C. Biochemistry. 1998; 37: 5961-5967Crossref PubMed Scopus (57) Google Scholar), APS18 (42.Capell A. Grunberg J. Pesold B. Diehlmann A. Citron M. Nixon R. Beyreuther K. Selkoe D.J. Haass C. J. Biol. Chem. 1998; 273: 3205-3211Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar), NT1 (43.Mathews P.M. Cataldo A.M. Kao B.H. Rudnicki A.G. Qin X. Yang J.L. Jiang Y. Picciano M. Hulette C. Lippa C.F. Bird T.D. Nochlin D. Walter J. Haass C. Levesque L. Fraser P.E. Andreadis A. Nixon R.A. Mol. Med. 2000; 6: 878-891Crossref PubMed Google Scholar), and BI.HF5C (44.Steiner H. Duff K. Capell A. Romig H. Grim M.G. Lincoln S. Hardy J. Yu X. Picciano M. Fechteler K. Citron M. Kopan R. Pesold B. Keck S. Baader M. Tomita T. Iwatsubo T. Baumeister R. Haass C. J. Biol. Chem. 1999; 274: 28669-28673Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar), have been described previously. Monoclonal antibody against Fas was kindly provided by Drs. P. Krammer and M. E. Peter (German Cancer Research Center, Heidelberg, Germany), and polyclonal anti-PARP antibody was obtained from Roche Applied Science. The proteins were detected by Western immunoblotting using an enhanced chemiluminescence technique (Amersham Biosciences).Phosphorylation of PS1—Phosphorylation of PS1 in cultured cells was carried out as described earlier (41.Walter J. Grunberg J. Capell A. Pesold B. Schindzielorz A. Citron M. Mendla K. St George-Hyslop P. Multhaup G. Selkoe D.J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5349-5354Crossref PubMed Scopus (101) Google Scholar). Protein kinase or phosphatase activities were modulated by the addition of the activators or inhibitors as indicated in the respective experiment. In vitro phosphorylation assays with purified PKA from bovine heart (kindly provided by Dr. V. Kinzel) or with rat brain PKC (Biomol) were carried out as described previously (38.Walter J. Grunberg J. Schindzielorz A. Haass C. Biochemistry. 1998; 37: 5961-5967Crossref PubMed Scopus (57) Google Scholar). Recombinant protein representing the loop region of PS1 (amino acids 263–407) was used as substrate. Phosphorylation reactions were started by the addition of 10 μm [γ-32P]ATP and allowed to proceed for 20 min at 32 °C. To control the kinase activities, parallel phosphorylation reactions were carried out using histone (0.5 mg/ml; Sigma) as protein substrate. The reactions were stopped by the addition of SDS sample buffer.Two-dimensional Phosphopeptide Mapping—32P-Labeled PS1 was analyzed by two-dimensional phosphopeptide mapping according to Boyle et al. (45.Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1273) Google Scholar) after digestion with trypsin (sequencing grade; Roche Applied Science). In some experiments synthetic peptides representing phosphorylated PS1 were added to the digest as indicated in the respective experiments. The peptides were dissolved in 300 μl of pH 1.9 buffer (7.8% (v/v) glacial acetic acid, 2.5% (v/v) formic acid (88%)) and spotted onto a cellulose-coated TLC plate (Merck). After thin layer electrophoresis for 20 min at 1 kV (first dimension), chromatography with phosphochromatography buffer (37.5% (v/v) n-butanol, 25% (v/v) pyridine, 6.1% (v/v) glacial acetic acid) was carried out (second dimension). The radiolabeled peptides were detected by autoradiography. To visualize synthetic peptides, TLC plates were stained with ninhydrin.In Vitro Cleavage by Caspases—One μg of the respective fusion proteins or 5 μg of synthetic peptides were incubated at 37 °C for 4 h in 25 μl of cleavage assay buffer (20 mm Hepes, pH 7.2, 100 mm sodium chloride, 10 mm magnesium chloride, 1 mm EDTA, 0.1% CHAPS, 10% sucrose) in the presence or absence of 20 ng of recombinant active caspase-3 (PharMingen). The reactions were terminated by shock freezing in liquid nitrogen. Cleavage of fusion proteins was analyzed by Western immunoblotting, and cleavage of synthetic peptides was analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.Matrix-assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry—Synthetic PS1 peptides, after incubation in the presence or absence of recombinant caspase-3, were desalted and purified by microbore reversed phase high pressure liquid chromatography. The peptides were eluted with an acetonitrile gradient in 0.1% trifluoroacetic acid on a 150 × 1.0 mm Vydac C18 column. 0.5 μl of selected peak fractions were applied onto the dried matrix spot. The matrix consisted of 13 mg of nitrocellulose (Bio-Rad) and 20 mg of α-cyano 4-hydroxy-cinnamic acid (Sigma) dissolved in 1 ml of acetone:isopropanol 2:3 (v/v). 0.5 μl of the matrix solution was applied onto the sample target. The samples were analyzed with a Perseptive Biosystems (Framingham, MA) Voyager Elite delayed extraction time-of-flight reflectron mass spectrometer at an acceleration voltage of 20 kV. Calibration was internal to the samples with Des-Arg-Bradykinin (Sigma) and adreno-corticotropic hormone (18.Georgakopoulos A. Marambaud P. Efthimiopoulos S. Shioi J. Cui W. Li H.C. Schutte M. Gordon R. Holstein G.R. Martinelli G. Mehta P. Friedrich Jr., V.L. Robakis N.K. Mol. Cell. 1999; 4: 893-902Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 19.Baki L. Marambaud P. Efthimiopoulos S. Georgakopoulos A. Wen P. Cui W. Shioi J. Koo E. Ozawa M. Friedrich Jr., V.L. Robakis N.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2381-2386Crossref PubMed Scopus (156) Google Scholar, 20.Annaert W.G. Esselens C. Baert V. Boeve C. Snellings G. Cupers P. Craessaerts K. de Strooper B. Neuron. 2001; 32: 579-589Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 21.Kaether C. Lammich S. Edbauer D. Ertl M. Rietdorf J. Capell A. Steiner H. Haass C. J. Cell Biol. 2002; 158: 551-561Crossref PubMed Scopus (168) Google Scholar, 22.Naruse S. Thinakaran G. Luo J.J. Kusiak J.W. Tomita T. Iwatsubo T. Qian X. Ginty D.D. Price D.L. Borchelt D.R. Wong P.C. Sisodia S.S. Neuron. 1998; 21: 1213-1221Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 23.Killick R. Pollard C.C. Asuni A.A. Mudher A.K. Richardson J.C. Rupniak H.T. Sheppard P.W. Varndell I.M. Brion J.P. Levey A.I. Levy O.A. Vestling M. Cowburn R. Lovestone S. Anderton B.H. J. Biol. Chem. 2001; 276: 48554-48561Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 24.Kang D.E. Soriano S. Xia X. Eberhart C.G. de Strooper B. Zheng H. Koo E.H. Cell. 2002; 110: 751-762Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 25.Xia X. Qian S. Soriano S. Wu Y. Fletcher A.M. Wang X.J. Koo E.H. Wu X. Zheng H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10863-10868Crossref PubMed Scopus (205) Google Scholar, 26.Vito P. Lacana E. D'Adamio L. Science. 1996; 271: 521-525Crossref PubMed Scopus (453) Google Scholar, 27.Roperch J.P. Alvaro V. Prieur S. Tuynder M. Nemani M. Lethrosne F. Piouffre L. Gendron M.C. Israeli D. Dausset J. Oren M. Amson R. Telerman A. Nat. Med. 1998; 4: 835-838Crossref PubMed Scopus (155) Google Scholar, 28.Grunberg J. Walter J. Loetscher H. Deuschle U. Jacobsen H. Haass C. Biochemistry. 1998; 37: 2263-2270Crossref PubMed Scopus (59) Google Scholar, 29.Loetscher H. Deuschle U. Brockhaus M. Reinhardt D. Nelboeck P. Mous J. Grunberg J. Haass C. Jacobsen H. J. Biol. Chem. 1997; 272: 20655-20659Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 30.Kim T.W. Pettingell W.H. Jung Y.K. Kovacs D.M. Tanzi R.E. Science. 1997; 277: 373-376Crossref PubMed Scopus (326) Google Scholar, 31.Vito P. Ghayur T. D'Adamio L. J. Biol. Chem. 1997; 272: 28315-28320Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 32.Alves D.C. Paitel E. Mattson M.P. Amson R. Telerman A. Ancolio K. Checler F. Mattson M.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4043-4048Crossref PubMed Scopus (111) Google Scholar, 33.Araki W. Yuasa K. Takeda S. Takeda K. Shirotani K. Takahashi K. Tabira T. J. Neurochem. 2001; 79: 1161-1168Crossref PubMed Scopus (45) Google Scholar, 34.Janicki S. Monteiro M.J. J. Cell Biol. 1997; 139: 485-495Crossref PubMed Scopus (114) Google Scholar, 35.Wolozin B. Iwasaki K. Vito P. Ganjei J.K. Lacana E. Sunderland T. Zhao B. Kusiak J.W. Wasco W. D'Adamio L. Science. 1996; 274: 1710-1713Crossref PubMed Scopus (390) Google Scholar, 36.Yankner B.A. Neuron. 1996; 16: 921-932Abstract Full Text Full Text PDF PubMed Scopus (910) Google Scholar, 37.Mattson M.P. Guo Q. Furukawa K. Pedersen W.A. J. Neurochem. 1998; 70: 1-14Crossref PubMed Scopus (230) Google Scholar, 38.Walter J. Grunberg J. Schindzielorz A. Haass C. Biochemistry. 1998; 37: 5961-5967Crossref PubMed Scopus (57) Google Scholar) (Sigma).Induction and Analysis of Apoptosis—HeLa, HEK293, or Cos-7 cells were treated with 1 μm staurosporine (STS) for the time points indicated. Apoptosis of MCF-7 cells overexpressing Fas was induced by incubation with monoclonal anti-Fas antibody (2 μg/ml). Progression of apoptosis was monitored by the following parameters: (a) Cleavage of poly(ADP-ribose) polymerase (PARP) was analyzed by immunoblotting (46.Walter J. Schindzielorz A. Grunberg J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1391-1396Crossref PubMed Scopus (109) Google Scholar). (b) Early changes in membrane permeability were detected by cell staining with 1 μg/ml Hoechst 33342 (Sigma) (47.Elstein K.H. Zucker R.M. Exp. Cell Res. 1994; 211: 322-331Crossref PubMed Scopus (125) Google Scholar). The accumulation of the dye in apoptotic cells was analyzed using an inverted fluorescence microscope (Leica DMIL, Wetzlar, Germany) equipped with a band pass excitation filter of 340–380 nm and a long pass emission filter of 425 nm. (c) Caspase-3 activity was measured as described previously (48.Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T.-T. Videta L.Y. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3777) Google Scholar). In brief, the cells were exposed to 1 μm STS for 2 or 6 h, respectively. After determination of cell numbers, the cells were homogenized in 750 μl of caspase-3 assay buffer (10 mm Hepes/KOH, pH 7.4, 2 mm EDTA, 0,1% CHAPS, 5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin A, 20 μg/ml leupeptin, 10 μg/ml aprotinin). The homogenate was centrifuged at 100,000 × g at 4 °C, and the supernatant was incubated with the fluorogenic caspase-3 tetrapeptide-substrate Ac-DEVD-amino-4-methylcoumarin (Calbiochem, Bad Soden, Germany) at a final concentration of 20 μm. The cleavage of the caspase-3 substrate was followed by determination of emission at 460 nm after excitation at 390 nm using a fluorescence plate reader.RESULTSPhosphorylation of PS1 by PKA and PKC—Previously, we and others have shown that phosphorylation of the PS1 CTF increases upon activation of PKC or PKA (41.Walter J. Grunberg J. Capell A. Pesold B. Schindzielorz A. Citron M. Mendla K. St George-Hyslop P. Multhaup G. Selkoe D.J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5349-5354Crossref PubMed Scopus (101) Google Scholar, 49.Seeger M. Nordstedt C. Petanceska S. Kovacs D.M. Gouras G.K. Hahne S. Fraser P. Levesque L. Czernik A.J. St George-Hyslop P. Sisodia S.S. Thinakaran G. Tanzi R.E. Greengard P. Gandy S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5090-5094Crossref PubMed Scopus (133) Google Scholar). To investigate whether PKC and PKA independently regulate phosphorylation of PS1, we pharmacologically activated or inactivated these kinases in HEK293 cells during incubation with [32P]orthophosphate. Activation of PKC with phorbol 12,13-dibutyrate (PDBu) results in strong increase of phosphate incorporation into the PS1 CTF (Fig. 1A, upper panel). Consistent with previous results (41.Walter J. Grunberg J. Capell A. Pesold B. Schindzielorz A. Citron M. Mendla K. St George-Hyslop P. Multhaup G. Selkoe D.J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5349-5354Crossref PubMed Scopus (101) Google Scholar, 49.Seeger M. Nordstedt C. Petanceska S. Kovacs D.M. Gouras G.K. Hahne S. Fraser P. Levesque L. Czernik A.J. St George-Hyslop P. Sisodia S.S. Thinakaran G. Tanzi R.E. Greengard P. Gandy S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5090-5094Crossref PubMed Scopus (133) Google Scholar), phosphorylation of the PS1 CTF induces a slower migration in SDS gels resulting in an apparent molecular mass shift from ∼ 20 to ∼ 23 kDa (Fig. 1A, lower panel). Cell treatment with the PKC inhibitor GF109203X selectively suppresses the PDBu-induced phosphorylation of PS1 CTF. In contrast, the PKA inhibitor H-89 had no significant effect on PDBu induced phosphorylation, indicating that PDBu selectively increases PKC-dependent phosphorylation of PS1. Activation of PKA by cell treatment with forskolin also increases phosphorylation of PS1. In contrast to the PDBu-induced phosphorylation, the forskolin-induced phosphorylation of PS1 is strongly suppressed by the PKA inhibitor H-89 but not by the PKC inhibitor GF109203X (Fig. 1A). These results demonstrate that phosphorylation of the PS1 CTF can be regulated by two independent signaling pathways involving PKA or PKC, respectively.The hydrophilic loop region between TM6 and TM7 of PS1 contains several consensus recognition sites for these kinases (Fig. 2A). We therefore tested whether both kinases can phosphorylate this domain in vitro using a recombinant protein that represents amino acids 263–407 of PS1 as a substrate. Both PKA and PKC readily phosphorylate the recombinant PS1 sequence in a time-dependent manner (Fig. 1B), indicating that the hydrophilic loop region of PS1 is a substrate for both kinases.Fig. 2PS1 is phosphorylated at Ser346 by PKC. A, alignment of PS1 loop amino acid sequences from several mammalian species. Potential phosphorylation sites within the minimal consensus sequence for PKC ((R/K)XS) are indicated by arrowheads. The amino acid sequence of human PS1 also contains a recognition motif for PKA (RRX(S/T)) at Ser310. Note that the recognition motif at Ser346 is conserved in all mammalian species (boxed). The cleavage site for caspase is indicated by an arrow. B, recombinant proteins representing amino acids 263–407 of PS1 WT, S310A and S346A were incubated with PKA or PKC in the presence of [γ-32P]ATP for 20 min. The radiolabeled proteins were detected by autoradiography (upper panels), and the total proteins were visualized by Coomassie staining (lower panels). Note that PKA-mediated phosphorylation of PS1 containing the S310A mutation is completely abolished. C, HEK293 cells were labeled with [32P]orthophosphate in the presence of PDBu, and PS1 CTF was isolated by immunoprecipitation with antibody 3027 and SDS-PAGE. After blotting to polyvinylidene difluoride membrane, radiolabeled PS1 CTF was digested with trypsin in the presence of the synthetic phosphopeptide DS(p)HLGPLR, which represents a tryptic digestion product of PS1 (amino acids 345–352). After two-dimensional separation of the digestion products, radiolabeled peptides were detected by autoradiography (left panel). The synthetic peptide was visualized by staining the plate with ninhydrin (middle panel). Overlay of the autoradiogram with the stained TLC plate demonstrated co-migration of one in vivo phosphorylated peptide with the synthetic peptide (indicated by dashed circle (right panel)).View Large Image Figure ViewerDownload Hi-res image Download (PPT)It has been" @default.
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• W2056510710 title "Phosphorylation of Presenilin 1 at the Caspase Recognition Site Regulates Its Proteolytic Processing and the Progression of Apoptosis" @default.
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