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Inhibition of Protein Phosphatase 1 Stimulates Secretion of Alzheimer Amyloid Precursor Protein

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Inhibition of Protein Phosphatase 1 Stimulates Secretion of Alzheimer Amyloid Precursor Protein Inhibition of Protein Phosphatase 1 Stimulates Secretion of Alzheimer Amyloid Precursor Protein Edgar F. da Cruz e Silva, Odete A. B. da Cruz e Silva, Cassia Thais B. V. Zaia, and Paul Greengard The Rockefeller University, Laboratory of Molecular and Cellular Neuroscience, New York, New York, U.S.A. ABSTRACT Background: Aberrant metabolism of the Alzheimer Results: The availability of protein phosphatase inhibi- amyloid precursor protein (APP) or its amyloidogenic A,B tors with different relative potencies against the different fragment is thought to be centrally involved in Alzhei- types of serine/threonine-specific protein phosphatase mer's disease. Nonamyloidogenic processing of APP in- allowed us to examine which of the four known types of volves its cleavage within the AP3 domain by a protease, protein phosphatase might be involved in the regulation termed a-secretase, and release of the large extracellular of APP secretion. Both okadaic acid and calyculin A domain, termed APPs. Secretion of ApPs can be stimu- stimulated the secretion of APP from COS-1 cells in a lated by phorbol esters, activators of protein kinase C, dose-dependent manner. The half-maximal dose for with concurrent inhibition of AP production. While the stimulation of APP secretion was approximately 100-fold role of protein kinases on APP metabolism has been higher with okadaic acid than with calyculin A. investigated, considerably less effort has been devoted to Conclusions: The nearly 100-fold difference in the ob- elucidating the role played by protein phosphatases. served IC50 values for okadaic acid and calyculin A im- Okadaic acid, a protein phosphatase inhibitor, has been plicates a type 1 protein phosphatase in the control of shown to stimulate secretion of APPs, but the identity of APPs production. Protein phosphatase 1 (PP1) is known the phosphatase involved has not been investigated. to be highly expressed in adult mammalian brain, both Materials and Methods: The secretion of APP5 from in neurons and glia. The identification of a specific phos- COS-1 cells was measured in the absence or presence of phatase type in the control of APP secretion opens new various doses of serine/threonine-specific phosphatase avenues to the development of rational therapeutic in- inhibitors. Quantitation of the derived IC50 values was tervention strategies aimed at the prevention and/or used to determine the identity of the phosphatase in- treatment of Alzheimer's Disease. volved in the control of APP secretion. INTRODUCTION genic pathway by a protease (a-secretase) that Alzheimer's disease (AD) is the most common of cleaves APP within the AP3 domain and releases the neurodegenerative diseases, probably affect- the large extracellular domain (APPS). ing more than 50% of individuals over the age of Previous work has shown that treatment of a 80. Central to the pathology of the disease is the variety of cells with phorbol esters stimulates the formation of senile plaques in the brains of af- release of APPs, presumably via protein kinase fected individuals. The amyloid core of these C-mediated phosphorylation of a target protein plaques is comprised largely of a small 39-43 (2-5). A similar effect can be obtained using amino acid peptide (1), termed AP3, derived from okadaic acid (an inhibitor of serine/threonine- the Alzheimer amyloid precursor protein (APP). specific protein phosphatases), possibly via in- APP can also be processed via a nonamyloido- creased phosphorylation of the same target pro- tein due to phosphatase inhibition. In addition, Address correspondence and reprint requests to: Edgar F. protein kinase C (PKC) was shown to phosphor- da Cruz e Silva, The Rockefeller University, Box 296, 1230 York Avenue, New York, NY 10021, U.S.A. ylate the cytoplasmic domain of APP (6,7) at a Copyright 1995, Molecular Medicine, 1076-1551/95/$10.50/0 535 Molecular Medicine, Volume 1, Number 5, July 199 5 5 3 5 - 541 536 Molecular Medicine, Volume 1, Number 5, July 1995 site that is also phosphorylated in vivo (M. Oishi (DMEM), and fetal bovine serum were obtained and P. Greengard, unpublished observations). from Life Technologies Inc. (Gaithersburg, MD, However, APPs secretion can still be stimulated U.S.A.). Cantharidin was purchased from Sigma by phorbol esters or okadaic acid in the absence Chemical Co. (St. Louis, MO, U.S.A.). Murine of the cytoplasmic domain of APP (4,5). Thus, monoclonal antibody 22C 11, prepared against although the identity of the target protein has the amino terminus of APP (12), was purchased not been elucidated, it is unlikely to be the APP from Boehringer Mannheim Corporation (Indi- protein itself. Phorbol ester stimulation of APPs anapolis, IN, U.S.A.). Murine monoclonal anti- secretion implicates PKC in APP processing. Like- body 6E10, whose epitope is located within res- wise, the stimulation of APPs secretion by oka- idues 1-17 of AP3, was obtained from Drs. H. M. daic acid implicates a phosphatase. However, the Wisniewski and K. S. Kim (New York State In- question of which type of serine/threonine-spe- stitute for Basic Research in Developmental cific protein phosphatase is involved in APP se- Disabilities). cretion has not been addressed previously. Four major types of serine/threonine-spe- cific protein phosphatase have been identified in Cell Culture, Drug Treatment, and eukaryotic cells (termed 1, 2A, 2B, and 2C), Detection of Secreted APP which can be distinguished by a variety of bio- COS- 1 cells were grown in DMEM supplemented chemical and enzymatic properties (reviewed in with 10% fetal bovine serum at 370C and 5% Ref. 8). Of the four types of protein phosphata- CO2. The cells were subcultured in triplicate into ses, PP1 and PP2A exhibit broad and overlapping six-well plates and grown to confluency. After substrate specificities in vitro, accounting for al- two washes with phosphate buffered saline, the most all measurable activity towards a variety of cells were incubated in the absence or presence phosphoproteins regulating cellular processes. of varying concentrations of either PDBu (for 60 PP2B (also known as calcineurin) is active on a min) or the indicated phosphatase inhibitors (for much more restricted range of substrates, and 90 min). When a combination of drugs was used, PP2C is structurally unrelated to the other types. the time of exposure was kept to 60 min. All Recent recombinant DNA approaches identified drugs were prepared by diluting the stock solu- the existence of several isoforms for each phos- tions into serum-free DMEM. Following incuba- phatase type and revealed that types 1, 2A, and tion as described above, the conditioned medium 2B belong to a gene family distinct from that of was collected into tubes containing SDS (1% phosphatase 2C. A number of "novel" phos- final concentration), and boiled for 5 min. The phatases have also been identified in a variety of samples, normalized for protein content (13), organisms, the first of which was termed protein were subjected to SDS-polyacrylamide gel elec- phosphatase X (9). trophoresis on 7.5% gels (14). The separated A variety of compounds is now available ca- proteins were then electrophoretically trans- pable of specifically inhibiting serine/threonine- ferred onto nitrocellulose and probed with anti- specific protein phosphatases. Two of the best bodies 22C 11 or 6E10, as described in the figure characterized are okadaic acid and calyculin A legends. The immunoblots were washed and (10). Since they readily enter intact cells, they then incubated successively with rabbit anti- can be used to determine which phosphatase is mouse IgG (Cappel, Durham, NC) and 125I-pro- active in a particular process by virtue of their tein A (Amersham, Arlington Heights, IL, different relative potencies against the various U.S.A.). Immunoreactive bands were detected by phosphatase types. In this study we have used autoradiography and quantitated using a Phos- them to identify the protein phosphatase in- phorlmager (Molecular Dynamics, Sunnyvale, volved in the control of APPs production. The CA, U.S.A.). data presented here were the subject of a prelim- inary report (1 1). RESULTS MATERIALS AND METHODS Several previous studies showed that APP secre- tion can be stimulated in a variety of cells using Materials phorbol esters. Indeed, in a previous report we Calyculin A, okadaic acid, PDBu (phorbol 12,13- demonstrated that PDBu (phorbol 12,13-dibu- dibutyrate), Dulbecco's modified Eagle's medium tyrate) could stimulate the secretion of both en- E. F. da Cruz e Silva et al.: PP1 Inhibition Stimulates APP Secretion 537 TABLE 1. Concentration of test substance PDBu C -9 -8 -7 -6 required to achieve half-maximal secretion of APPS EC50 (nM) Test Substance 22C11 6E10 Okadaic Acid 80 110 Calyculin A 1 1 Cantharidin 500 ND FIG. 1. Stimulation of APPs production from PDBu 54 ND COS-1 cells in response to PDBu treatment Cells were treated with the PDBu concentrations in- The range of concentrations used for each test substance dicated (10-9_10-6 M), and the APPs released into was as follows: 10-10_10-6 M okadaic acid, 10-1"-10-7 M 5 the medium was quantitated by immunoblotting calyculin A, 10-9_10 M cantharidin, and 10-10_10-6 M PDBu. The amount of APP secreted into the medium was with antibody 22C 11. C, control without PDBu. expressed as a percentage of the maximal amount, as ex- emplified in Fig. 2B. Immunoreactive bands were quanti- tated by immunoblotting using a Phosphorlmager. The drug concentration required to achieve half-maximal secre- tion of APP from COS-1 cells (EC50) was calculated from dogenous APP and transfected wild-type and the average results obtained in several experiments, each performed in triplicate, and are tabulated above. ND, not mutant APP expressed in COS-1 cells (4). In the determined.
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