THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 269, No. 11, Issue of March 18, pp. 8376-8382, 1994 Printed in U.S.A. The Release of Alzheimer’s Disease p Amyloid Peptide Is Reduced by Phorbol Treatment* (Received for publication, October 25, 1993) J. StevenJacobsenSO, Michael A. SpruytS, Abraham M. Brown$, Sudhir R. Sahasrabudhen, Arthur J. Blumell, Michael P. Vitek**, Helen A. Muenkelll, and June SonnenbergReinesS From the Departments of $Central Nervous System Biological Research and [MolecularPharmacology, Medical Research Division, Lederle Laboratories Division, American Cyanamid Company, Pearl River, New York 10965 Amyloid precursor protein (APP) is cleaved predomi- nantly within the p amyloid peptide (BAP) domain to release a non-amyloidogenic amino-terminal PN2 frag- ment. Treatment of cells with phorbol dibutyrate, an agent which activates protein kinase C, has been shown to increase the release of an amino-terminal fragment. A panel of mutant APP reporter constructs was expressed in which each of the potential phosphorylation sites lo- cated within the cytoplasmic domain of APP was re- placed with alanine residues. Phorbol response patterns were unchanged for each of these mutants, suggesting that induced cleavage occurs independently of APP sub- strate phosphorylation. We find that phorbol (a) in- creases the release of a PN2 fragment thatis consistent with the normal secretase activity, (b) decreases the re- lease of a shorter amino-terminal APP fragment thatis cleaved near the amino terminus of BAP, and (c) de- creases the release of BAP which was identified based on electrophoretic mobility, epitope mapping, and radio- sequencing. These data demonstrate that pharmaco- logical treatment can reduce the formation of BAP and suggests that protein kinase C activators could be devel- oped as therapeutic agents to block BAP formation. Abnormal extracellular accumulation of amyloid and senile plaques observed in the brain of Alzheimer’s disease patients consists principally of p amyloid peptide (BAP)’ which is thought to be central to the pathogenesis and memory loss. The 39-42-amino acid (aa) BAP is derived by proteolytic cleavage from a larger amyloid precursor protein (APP), a transmem- brane receptor-like glycoprotein which is expressed as three major isoforms containing either 695, 751, or 770 aa (1-9). The major proteolytic cleavage of APP occurs within the jw- tamembranous ectodomain by “secretase” leading to the secre- tion2 of an amino-terminal (NH2-terminal) APP fragment * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.Section 1734 solelyto indicate this fact. Dr. J. Steven Jacobsen, Bldg. 200, Rm. 3615A, Lederle Laboratories, 5 To whom correspondence and reprint requests should be addressed: Pearl River, NY 10965. Tel.: 914-732-3087 Fax: 914-732-5537. ll Present address: Dept. of Molecular Neurobiology, Hoechst-Roussel Pharmaceuticals, Inc., Routes 202-206, P. 0. Box 2500, Somerville, NJ 08876. ** Present address: The Picower Inst. for Medical Research, 350 Community Dr., Manhasset, NY 11030. The abbreviations used are: BAP, p amyloid peptide; PN2, NH2- terminal amyloid precursor protein fragment; CM, conditioned me- dium; aa, amino acid(s1; APP, amyloid precursor protein; PKC, protein kinase C; PDBu, phorbol dibutyrate;APP-REP, APP reporter; SP, sub- stance P; Tricine, N-~2-hydroxy-l,l-bis~hydroxymethyl~ethyllglycine. “Secretion” is used onlyto describe the proteolytic process which results in the release of an NH,-terminal APP (or APP-REP) fragment (PN2) into conditioned medium (CM). This cleavage takes place within the BAP sequence (betweenBAP aa residues 16 and 17) and precludes the proteolytic generation of BAP from APP (10- 12) which is thought to involve an alternative pathway (13). Release of a shorter NHz-terminal APP fragment following cleavage at the amino terminus of BAP (13), a longer NH2- terminal fragmentfollowing cleavage at a distal site carboxyl- terminal (COOH-terminal) to the secretase site (14), as well as BAP (15, 161, into CM have all been observed. An endosomal- lysosomal pathway hasbeen suggested to generate potentially amyloidogenic fragments (17-19). Enhancement of the release of BAP into CM is observed by expressing constructs containing the Swedish KM-to-NL mutation, which flanks the NH2 termi- nus of BAP (20, 21), or 99 aa (Ctss) derived from COOH-termi- nal APP sequences, which includes BAP and cytoplasmic APP domains (161, Activation of protein kinase C (PKC) is known to regulate the secretion (viaproteolytic cleavage) or internalization of a num- ber of membrane proteins (22-25). Phosphorylation could be involved in regulation of APP processing and the generation of BAP and amyloidogenic fragments, since the APP holoprotein is phosphorylated (26-30). Treatment of cells with phorbol dibutyrate (PDBu), an agent which activates PKC, increases the release of NHz-terminal APP fragment(s), increases the generation of cell-associated COOH-terminal APP fragments and decreases the amount of mature full-length APP forms, suggesting that substrate APP phosphorylation is involved (26- 31). To better characterize the mechanisms ofAPP proteolysis, we have developed an APP reporter (APP-REP) as a model system for the expression and cleavage of APP molecules (Ref. 32; Fig. IA). APP-REP is distinguished from endogenously expressed APP by the deletion of 276 central aa of APP and insertionof the Substance P (SP) reporter epitope in the NH2-terminal ectodomain to enable the immunological detection of PN2 frag- ments released into CM following proteolytic cleavage of sub- strate. APP-REP contains 113 aa derived from the COOH-ter- minal portion of APP and includes intact BAP and flanking sequences, the transmembrane spanning region and the cyto- plasmic COOH terminus ofAPP. In this paper we use APP-REP to determine (a) the nature of the NHz-terminal fragmentb) released from cells after treatment by PDBu, (b) whether in- creasedPN2 release is correlatedwith a reduction in BAP release, and (c) whether modulation of PN2 release operates by APP substrate phosphorylation. We find that PDBu treatment specifically increases the release of PN2 with a corresponding decrease in both the release of a shorter PN2-like NHz-terminal fragment and BAP. These events occur independently of sub- into the extracellular environment and is not intended to imply or infer any other mechanism. 8376 This is an Open Access article under the CC BY license.