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387 Identification of a protein which interacts with the cytoplasmic domain of the Alzheimer amyloid protein Precursor
FIFTH INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE
The highest age-adjusted partial correlations between plasma concentrations of each three antioxidants and the tests were as follows: r=.252 (p=.000) between lycopeae and v F r words, r=.163 (p=.000) between betacarotene and VFT words, and r=.lI9 (p=.010) between a-tocopherel and HVR immediate recall. In two groups by the mean concentrations of antioxidants, the group having higher lycopen concentration managed significantly better in six out of nine cognitive subtests (p=.006 to .019; ANCOVA, adjusted for age and education) and the group with higher betacaroten concentration in five out of the nine subtests (p=.004 to .018). Neither the lag-time nor the ox-LDL-ab titre had any significant association with cognitive performance. Our study shows that neuropsychological tests assessing cognitive functions are positively correlated with the plasma concentrations of antioxidative carotenoids lycopcne and betacarotene.
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phosphorylation of BAPP to the same compartment. Intracellular phosphorylation of BAPP was inhibited by Brefeldin A and by incubating cells at 20oc, thus excluding phosphorylation in the endoplasmic reticulum or trans Golgi network. Ectodomain phosphorylation within the trans cistemae of the Golgi oceured not only with mutant Swedish BAPP, but also with wildtype BAPP and its close homologue APLP2. In addition to phosphorylation within the trans cistemae of the Golgi, BAPP was also found to undergo phosphorylation at the cell-surface by an ecto-protein kinase. Therefore this study revealed two distinct cellular locations for 13APP phosphorylation. Data will also be presented on the identification of the phosphorylation site within the BAPP sequence as well as on candidate protein kinases capable to phosphorylate 13APP and a model peptide substrate in vitro at the correct amino acid.
389
Molecular Pathology H 387 Identification of a Protein which Interacts with the Cytoplasmic Domain of the Alzheimer Amyloid Protein Precursor T. Watanabe*, T. Suzuki, J. Sukegawa, I. Sukegawa, A.C. Naim, and P. Greengard. Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, 1230 York Ave., New York, NY 10021, USA Alzheimer amyloid protein precursor (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids. The amino acid sequences of this cytoplasmic domain are conserved in human and rat suggesting on important function for the domain. Identification of proteins which interact with the cytoplasmic domain would hopefully give us new insights into the physiological function of APP and in turn into the pathogenesis of Alzheimer's disease. To identify proteins which interact with the cytoplasmic domain of APP, we prepared an affinity column by immobilizing a synthetic peptide corresponding to residues 645-694 of APP695. Using this affinity column, we identified a 130 kDa protein (p130) in rat brain cytosol. Polyclonal antibodies were prepared against synthetic peptides based on amino acid sequences of tryptic peptides derived from p130. These antibodies recognized p130 in rat brain cytosol and in tissue culture cell lysates (e.g. PC12 and 293). Cell fractionation of PC12 cells followed by immunoblot analysis indicated that p130 was present mainly in the cytosolic fraction. p130 in the cytosolic fraction of PCI2 cells coprecipitated with a GST-APP cytoplasmic domain (residues 649-695)-fusion protein (GST-APPc) but not with GST. The interaction of p130 with GST-APPc was abolished by addition of the cytoplasmic domain peptide (residues 645-694). Furthermore, fpl30 coprecipitated with APP immunoprecipitated by anti-APP antibody rom PC12 cell lysate. APP also eoprecipitated with p130 immunoprecipitated by anti-p130 antibody. These results indicate that p 130, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain.
388 Phosphorylation of fl-Amyloid Precursor Protein at Two Distinct Cellular Locations J. Walter l*, A. Hung2, G. Thinakaran3, S. Sisodia3, D. Selkoe2 and C. Haass 1 lCentral Institute of Mental Health, Department of Molecular Biology, J5, 68159 Mannheim, Germany. 2Department of Neurology and Program in Neuroscience, Harvard Medical School and Center for Neurologic Diseases, Brigham and Woman's Hospital, Boston, MA 02115, USA. 3Neuropathology Laboratory, The Johns Hopkins University, School of Medicine Baltimore, Maryland 21205-2196, USA. The B-amyloid precursor protein (gAPP) is a transmembrane protein which is exclusively phosphorylated on serine residues within its ectodomain. To identify the cellular site of BAPP phosphorylation, we took advantage of an antibody that specifically detects the free C-terminus of g-secretase cleaved BAPP containing the Swedish missense mutation (APPsSW-I~). This antibody previously established the cellular location of the g-secretase cleavage of Swedish BAPP as a post-Golgi secretory compartment. We have now localized the selective ectodomain
P h o s p h o r y l a t i o n o f A P L P 2 in cell cycle-dependent manner K.Ando m*, Y.Satoh m, T.Isohara~2~, M.OishiC2~, K.lijima°), G.S.Lim c2~, W.Wasco°~, R.E.Tanzi °~, Nairn,A.C ¢2~, Y.Kirinom, P.Greengard ¢2~, S.E.Gandy <4)and T.Suzuki m. ( 1) Lab. of Neurobiophysics, Faculty of Pharmaceutical Sciences, The Univ. of Tokyo, Tokyo 113 Japan. (2) Lab. of Mol. & Cell. Neurescience, The Rockefeller Univ., New York, NY 10021 USA. (3) Lab. of Genetics & Aging, Neuroscience Center, Massachusetts General Hospital, Charlestown, MA 02129 USA. (4) Dept. of Neurology and Neuroscience, Cornell University Medical College, New York. NY 10021 USA. Amyloid precursor-like protein 2, APLP2, is a member of the gene family which includes the Alzheimer amyloid precursor protein (APP). Our previous results demonstrated that APP is phosphorylated by cdc2 kinase in cell cycle-dependent manner in cultured cellsm, and the identical site, Thr 668 (numbering for APP69 s isoform), is phosphorylated in rat brain tissues¢2~.An identical potential phosphorylation site, a consensus sequence for phosphorylation by cdc2 kinase, can also be found in cytoplasmic domain ol APLP2 °). The site in APLP2, Thr736 (numbering for APLP27~~ isoform), was phosphorylated by cdc2 kinase hi vitro as is the analogous site in APP. To analyze the phosphorylation state of APLP2 hi vivo, we prepared anti-APLF'2 antibody which does not cross-react to APP, and we prepared a phosphorylation slate-specific antibody against phospho-Thr736 which does not cross-react with APP phosphorylated at Thr668. Utlizing these antibodies and synchronized cultured cells, we discovered that the phospho-rylation state of APLP2 at Thr736 varied in a cell cycle-dependent manner. Our results suggest that the phosphorylation slate of APP at Thr668 and the phosphorylation state of APLP2 at Thr736 are regulated similarly. (I) Suzatkieta/., (1994)EMBO.J. 13, l 114-1122.(2) Oishiet ol .,(1996) Mol. Medicinein press. (3l Wascoet al., (1993)NatureGenet. 5, 95-100.
390 APP is p h o s p h o r y l a t e d s p e c i f i c a l l y in brain t i s s u e s Y. Satoh°~ *, T. Seki~2),Y. Kirinom and T. Suzukim. (1) Lab. of Neurobiophysics, Faculty of Pharmaceutical Sciences, University of Tokyo. Tokyo 113, Japan. (2) Dep. of Anatomy, Juntendo University School of Medicine. Tokyo.113, Japan. The l%amyloid protein (13A4) is released by proteolytic cleavages from the Alzheimer amyloid protein precursor (APP), which is an integral membrane protein modified by phosphorylation on its cytoplasmic domain°'2~. Phosphorylation of I~APP is a physiological event in brain tissuesc~, although functions of phosphorylation are still unknown. In the present study, we developed a phosphorylation slate-specific antibody to phospho-Thr668 site (numbering for APP,9 s isoform); a site phosphorylated by cdc2 kinase in cultured cellst4~, to analyze the phosphorylation slate of APP in rat tissues.
By utilizing this antibody and
The highest age-adjusted partial correlations between plasma concentrations of each three antioxidants and the tests were as follows: r=.252 (p=.000) between lycopeae and v F r words, r=.163 (p=.000) between betacarotene and VFT words, and r=.lI9 (p=.010) between a-tocopherel and HVR immediate recall. In two groups by the mean concentrations of antioxidants, the group having higher lycopen concentration managed significantly better in six out of nine cognitive subtests (p=.006 to .019; ANCOVA, adjusted for age and education) and the group with higher betacaroten concentration in five out of the nine subtests (p=.004 to .018). Neither the lag-time nor the ox-LDL-ab titre had any significant association with cognitive performance. Our study shows that neuropsychological tests assessing cognitive functions are positively correlated with the plasma concentrations of antioxidative carotenoids lycopcne and betacarotene.
$97
phosphorylation of BAPP to the same compartment. Intracellular phosphorylation of BAPP was inhibited by Brefeldin A and by incubating cells at 20oc, thus excluding phosphorylation in the endoplasmic reticulum or trans Golgi network. Ectodomain phosphorylation within the trans cistemae of the Golgi oceured not only with mutant Swedish BAPP, but also with wildtype BAPP and its close homologue APLP2. In addition to phosphorylation within the trans cistemae of the Golgi, BAPP was also found to undergo phosphorylation at the cell-surface by an ecto-protein kinase. Therefore this study revealed two distinct cellular locations for 13APP phosphorylation. Data will also be presented on the identification of the phosphorylation site within the BAPP sequence as well as on candidate protein kinases capable to phosphorylate 13APP and a model peptide substrate in vitro at the correct amino acid.
389
Molecular Pathology H 387 Identification of a Protein which Interacts with the Cytoplasmic Domain of the Alzheimer Amyloid Protein Precursor T. Watanabe*, T. Suzuki, J. Sukegawa, I. Sukegawa, A.C. Naim, and P. Greengard. Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, 1230 York Ave., New York, NY 10021, USA Alzheimer amyloid protein precursor (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids. The amino acid sequences of this cytoplasmic domain are conserved in human and rat suggesting on important function for the domain. Identification of proteins which interact with the cytoplasmic domain would hopefully give us new insights into the physiological function of APP and in turn into the pathogenesis of Alzheimer's disease. To identify proteins which interact with the cytoplasmic domain of APP, we prepared an affinity column by immobilizing a synthetic peptide corresponding to residues 645-694 of APP695. Using this affinity column, we identified a 130 kDa protein (p130) in rat brain cytosol. Polyclonal antibodies were prepared against synthetic peptides based on amino acid sequences of tryptic peptides derived from p130. These antibodies recognized p130 in rat brain cytosol and in tissue culture cell lysates (e.g. PC12 and 293). Cell fractionation of PC12 cells followed by immunoblot analysis indicated that p130 was present mainly in the cytosolic fraction. p130 in the cytosolic fraction of PCI2 cells coprecipitated with a GST-APP cytoplasmic domain (residues 649-695)-fusion protein (GST-APPc) but not with GST. The interaction of p130 with GST-APPc was abolished by addition of the cytoplasmic domain peptide (residues 645-694). Furthermore, fpl30 coprecipitated with APP immunoprecipitated by anti-APP antibody rom PC12 cell lysate. APP also eoprecipitated with p130 immunoprecipitated by anti-p130 antibody. These results indicate that p 130, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain.
388 Phosphorylation of fl-Amyloid Precursor Protein at Two Distinct Cellular Locations J. Walter l*, A. Hung2, G. Thinakaran3, S. Sisodia3, D. Selkoe2 and C. Haass 1 lCentral Institute of Mental Health, Department of Molecular Biology, J5, 68159 Mannheim, Germany. 2Department of Neurology and Program in Neuroscience, Harvard Medical School and Center for Neurologic Diseases, Brigham and Woman's Hospital, Boston, MA 02115, USA. 3Neuropathology Laboratory, The Johns Hopkins University, School of Medicine Baltimore, Maryland 21205-2196, USA. The B-amyloid precursor protein (gAPP) is a transmembrane protein which is exclusively phosphorylated on serine residues within its ectodomain. To identify the cellular site of BAPP phosphorylation, we took advantage of an antibody that specifically detects the free C-terminus of g-secretase cleaved BAPP containing the Swedish missense mutation (APPsSW-I~). This antibody previously established the cellular location of the g-secretase cleavage of Swedish BAPP as a post-Golgi secretory compartment. We have now localized the selective ectodomain
P h o s p h o r y l a t i o n o f A P L P 2 in cell cycle-dependent manner K.Ando m*, Y.Satoh m, T.Isohara~2~, M.OishiC2~, K.lijima°), G.S.Lim c2~, W.Wasco°~, R.E.Tanzi °~, Nairn,A.C ¢2~, Y.Kirinom, P.Greengard ¢2~, S.E.Gandy <4)and T.Suzuki m. ( 1) Lab. of Neurobiophysics, Faculty of Pharmaceutical Sciences, The Univ. of Tokyo, Tokyo 113 Japan. (2) Lab. of Mol. & Cell. Neurescience, The Rockefeller Univ., New York, NY 10021 USA. (3) Lab. of Genetics & Aging, Neuroscience Center, Massachusetts General Hospital, Charlestown, MA 02129 USA. (4) Dept. of Neurology and Neuroscience, Cornell University Medical College, New York. NY 10021 USA. Amyloid precursor-like protein 2, APLP2, is a member of the gene family which includes the Alzheimer amyloid precursor protein (APP). Our previous results demonstrated that APP is phosphorylated by cdc2 kinase in cell cycle-dependent manner in cultured cellsm, and the identical site, Thr 668 (numbering for APP69 s isoform), is phosphorylated in rat brain tissues¢2~.An identical potential phosphorylation site, a consensus sequence for phosphorylation by cdc2 kinase, can also be found in cytoplasmic domain ol APLP2 °). The site in APLP2, Thr736 (numbering for APLP27~~ isoform), was phosphorylated by cdc2 kinase hi vitro as is the analogous site in APP. To analyze the phosphorylation state of APLP2 hi vivo, we prepared anti-APLF'2 antibody which does not cross-react to APP, and we prepared a phosphorylation slate-specific antibody against phospho-Thr736 which does not cross-react with APP phosphorylated at Thr668. Utlizing these antibodies and synchronized cultured cells, we discovered that the phospho-rylation state of APLP2 at Thr736 varied in a cell cycle-dependent manner. Our results suggest that the phosphorylation slate of APP at Thr668 and the phosphorylation state of APLP2 at Thr736 are regulated similarly. (I) Suzatkieta/., (1994)EMBO.J. 13, l 114-1122.(2) Oishiet ol .,(1996) Mol. Medicinein press. (3l Wascoet al., (1993)NatureGenet. 5, 95-100.
390 APP is p h o s p h o r y l a t e d s p e c i f i c a l l y in brain t i s s u e s Y. Satoh°~ *, T. Seki~2),Y. Kirinom and T. Suzukim. (1) Lab. of Neurobiophysics, Faculty of Pharmaceutical Sciences, University of Tokyo. Tokyo 113, Japan. (2) Dep. of Anatomy, Juntendo University School of Medicine. Tokyo.113, Japan. The l%amyloid protein (13A4) is released by proteolytic cleavages from the Alzheimer amyloid protein precursor (APP), which is an integral membrane protein modified by phosphorylation on its cytoplasmic domain°'2~. Phosphorylation of I~APP is a physiological event in brain tissuesc~, although functions of phosphorylation are still unknown. In the present study, we developed a phosphorylation slate-specific antibody to phospho-Thr668 site (numbering for APP,9 s isoform); a site phosphorylated by cdc2 kinase in cultured cellst4~, to analyze the phosphorylation slate of APP in rat tissues.
By utilizing this antibody and