strates in various experimental systems (25). Therefore, the effect of okadaic ... Tomlinson, B. E. & Corsellis, J. A. N. (1984) in Greenfield's. Neuropathology, eds.
Proc. Natl. Acad. Sci. USA Vol. 87, pp. 6003-6006, August 1990
Neurobiology
Processing of Alzheimer ,l/A4 amyloid precursor protein: Modulation by agents that regulate protein phosphorylation (dementia/phosphoprotein/endocytosis/intelization/proteolysis)
JOSEPH D. BUXBAUM*t, SAMUEL E. GANDY*t§, PIERA CICCHErrI*, MICHELLE E. EHRLICH*t, ANDREW J. CZERNIK*, R. PAUL FRACASSO¶, TRIPRAYAR V. RAMABHADRAN¶, AXEL J. UNTERBECK¶, AND PAUL GREENGARD* *Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021; 1Molecular Therapeutics, Inc., West Haven, CT 06516; tDepartment of Psychiatry, New York University, New York, NY 10016; and §The Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY 10962
Contributed by Paul Greengard, May 29, 1990
receptor (17, 18). Phosphorylation by PKC of these two receptors at these sites targets them for internalization (15, 17). By analogy, phosphorylation of 3APP may regulate its internalization and catabolism. It seems possible, therefore, that abnormal phosphorylation associated with AD might alter the metabolism ofj3APP and lead to deposition of 83/A4 amyloid protein (13). The metabolism of f3APP has been studied in PC12 rat pheochromocytoma cells (19). In the present study, we have examined the effects of agents that regulate protein phosphorylation on the metabolism of f3APP in these cells. 11
ABSTRACT The turnover and processing of the Alzheimer f/A4 amyloid precursor protein (JIAPP) has been studied in PC12 cells after treatment with agents that regulate protein phosphorylation. Phorbol 12,13-dibutyrate, an agent that stinmulates protein kinase C, decreased the levels of mature fiAPP and increased the levels of 15- and 19-kDa peptides. These peptides appeared to be COOH-terminal fragments of PAPP, which arose when phorbol 12,13-dibutyrate increased the rate of proteolytic processing of mature forms of .8APP. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, also led to decreased levels of mature I3APP and increased levels of the 15and 19-kDa peptides. H-7, an inhibitor of protein kinase C and of several other protein kinases, apparently decreased the rate of proteolytic processing of mature fiAPP. The sizes of the putative COOH-terminal fragments observed after treatment with either phorbol 12,13-dibutyrate or okadaic acid suggest that one or both may contain the entire ,B/A4 region of (3APP and thus be amyloidogenic. Our results support the hypothesis that abnormal protein phosphorylation may play a role in the development of the cerebral amyloidosis that accompanies Alzheimer disease.
MATERIALS AND METHODS Phorbol 12,13-dibutyrate (PDBu), 4-a-phorbol 12,13dibutyrate, and phorbol 12-myristate 13-acetate were purchased from LC Services (Woburn, MA). Okadaic acid was purchased from Moana BioProducts (Honolulu). H-7 was purchased from Seikagaku Kogyo (Tokyo). A synthetic peptide (J3APP`5-694) corresponding to the COOH-terminal region of ,3APP [the numbering of amino acids corresponds to those of human PAPP695 (6)] was prepared by the Yale Protein and Nucleic Acid Chemistry Facility (New Haven, CT). Antibodies were prepared by immunizing rabbits with this peptide. Sera were screened by immunoblot analysis of lysates of Escherichia coli that expressed a fusion protein including amino acids 19-695 of human pAPP695. Sera that were immunoreactive against the recombinant fusion protein were further screened for immunoprecipitating activity against [35S]methionine-labeled /3APP695, which was produced from 83APP695 cDNA by successive in vitro transcription (kit purchased from Stratagene) and translation (reticulocyte lysate kit purchased from Promega). Immunoprecipitation was performed as described (20). Sera showing high-efficiency (>90%o) immunoprecipitating activity for recombinant /3APP were affinity purified on columns of Sepharose 4B pAPP645-694 (21). PC12 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) fetal calf serum and 5% (vol/vol) heat-inactivated horse serum (GIBCO). For metabolic labeling, the cells were washed twice in phosphatebuffered saline, removed from the dish by trituration, and preincubated at 6 x 106 cells per ml in methionine-free DMEM, supplemented with 1 mCi of [35S]methionine per ml (1200 Ci/mmol; 1 Ci = 37 GBq; NEN) and 20 mM Hepes (pH
Alzheimer disease (AD) is characterized by the presence of certain neuropathological features, including intracellular neurofibrillary tangles and extracellular parenchymal and cerebrovascular amyloid (1). Abnormalities of protein phosphorylation are characteristic of AD (2-5). Two components of neurofibrillary tangles, the microtubule-associated protein tau and neurofilament proteins, are abnormally phosphorylated (2, 3). Moreover, protein kinase C (PKC) activity has been reported to be altered in AD brain as well as in fibroblasts derived from patients with sporadic AD, familial AD, and Down syndrome (4, 5). The predominant constituent of cerebral plaques and cerebrovascular amyloid is the ,B/A4 amyloid protein, which is derived from the large transmembrane ,B/A4 amyloid precursor protein (,BAPP) (6-12). PAPP is composed of three major isoforms, 3APP695, ,(APP751, and BAPP-n0, which arise from alternate splicing of a single gene. (The subscripts denote the lengths of the ,BAPP primary sequences.) ,3APP is a phosphoprotein, and a synthetic peptide corresponding to a sequence present in the ,BAPP transmembrane and cytoplasmic domains serves as an effective substrate for PKC (13, 14). This phosphorylation site, common to all three isoforms of ,BAPP and located seven residues from the predicted transmembrane/cytoplasmic border of the protein, is similar in position to PKC phosphorylation sites found in the epidermal growth factor receptor (15, 16) and in the interleukin 2
Abbreviations: AD, Alzheimer disease; ,BAPP, ,B/A4 amyloid preprotein ki-
cursor protein; PDBu, phorbol 12,13-dibutyrate; PKC, nase C.
tTo whom reprint requests should be addressed. IlThis work was presented at the Presidential Symposium of the 143rd Annual Meeting of the American Psychiatric Association, New York, May 16, 1990.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6003
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7.4) for 20 min. The cells were then diluted with 5-6 vol of aerated DMEM, containing excess methionine and 20 mM Hepes (pH 7.4), and were incubated for chase periods from 0 to 135 min with additions as described. Test substances, dissolved in dimethyl sulfoxide as vehicle, were added either at the start of the chase period (time zero) or 35 min later. Vehicle alone had no effect at the concentrations used (0.1-1%) in these experiments. Cell viability, as determined by trypan blue exclusion, was >90% at the start of the experiments and decreased
