Proc. Nall. Acad. Sci. USA Vol. 89, pp. 3055-3059, April 1992
Medical Sciences
Protein phosphorylation regulates secretion of Alzheimer fi/A4 amyloid precursor protein (proteolytic processing/protein kinase/protein phosphatase/phorbol ester/okadaic acid)
GREGG L. CAPORASO*, SAMUEL E. GANDY*tt, JOSEPH D. BUXBAUM*, TRIPRAYAR V. RAMABHADRAN*, AND PAUL GREENGARD*§ *Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021;
tThe Nathan S. Kline Institute for Psychiatric
Research, Orangeburg, NY 10962; and tDepartment of Psychiatry, New York University Medical Center, New York, NY 10016
Contributed by Paul Greengard, December 26, 1991
A
Extracellular deposition of the (3/A4 amyloid ABSTRACT peptide is a characteristic feature of the brain in patients with Alzheimer disease. ,f/A4 amyloid is derived from the amyloid precursor protein (APP), an integral membrane protein that exists as three major isoforms (APP695, APP75l, and APP770). Secreted forms of APP found in blood plasma and cerebrospinal fluid arise by proteolytic cleavage of APP within the 13/A4 amyloid domain, precluding the possibility of amyloidogenesis for that population of molecules. In the present study, we have demonstrated that treatment of PC12 cells with phorbol ester produces a severalfold increase in secretion of APP6s, APP751, and APP770. This increase is augmented by simultaneous treatment with the protein phosphatase inhibitor okadaic acid. These data indicate that protein phosphorylation regulates intra-3/A4 amyloid cleavage and APP secretion. These and other results suggest that APP molecules can normally follow either of two processing pathways: regulated secretion or proteolytic degradation unassociated with secretion.
KPI OX-2 T--
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hs am _~d
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FIG. 1. (A) Schematic diagram of the APP molecule showing extracellular, transmembrane, and cytoplasmic regions: 3/A4 amyloid domain (shaded); normal intra-P/A4 amyloid cleavage site (15) (arrow); domain inserts for APP751 (KPI only) and APP770 (both KPI and OX-2) isoforms; and putative N-glycosylation sites (CHO). (B) Autoradiogram of APP species immunoprecipitated from cell lysates (lanes 1-5) or conditioned medium (lanes 6-9) of [35S]methioninelabeled PC12 cells chased for 1 h in the absence of phorbol 12,13dibutyrate (PDBu) (lanes 1-5) or for 4 h in the presence of PDBu (lanes 6-9). Four immunoprecipitating antibodies were used to identify the various APP species. Lanes: 1 and 6, anti-amino-terminal monoclonal antibody 22C11; 2 and 7, anti-KPI domain monoclonal antibody 56.1; 3 and 8, anti-carboxyl-terminal affinity-purified polyclonal antibody 369A; 4, antibody 369A preincubated with synthetic peptide corresponding to the cytoplasmic domain of APP (residues 645-694); 5 and 9, whole serum from a guinea pig injected with unglycosylated APP695 produced in Escherichia coli. Autoradiograms are from a single experiment analyzed on a 4-15% continuous gradient SDS/polyacrylamide gel. Molecular masses (kDa) are indicated.
A characteristic pathological feature of Alzheimer disease is the extracellular deposition of 8/A4 amyloid in parenchymal brain tissues and in the walls of cerebral and meningeal blood vessels (1-3). This -4-kDa peptide is derived from the amyloid precursor protein (APP), a transmembrane glycoprotein that exists as three major isoforms (APP695, APP751, and APP770; Fig. 1 A), which result from the alternative splicing of mRNA from a single gene (4-10). The larger APP751 and APP770 isoforms contain a Kunitz-type protease inhibitor (KPI) insert in their extracellular domains (8-10). Secreted amino-terminal fragments of APP have been found in blood plasma and cerebrospinal fluid (11-13). These secreted fragments result from cleavage of APP within the ,f/A4 amyloid domain (14, 15). The process of APP secretion, therefore, precludes the cerebral amyloidogenesis associated with Alzheimer disease. We have previously demonstrated that activation of protein kinase C (PKC) in PC12 cells by treatment with phorbol ester results in more rapid intracellular turnover of APP and increased production of carboxylterminal APP fragments (16). In the present report, we describe the regulation by protein phosphorylation of APP secretion in PC12 cells.
was added at the start of the chase period. Control cells were treated with drug vehicle alone (0.05-0.15% dimethyl sulfoxide, final concentration). The amounts of labeled APP holoproteins and APP fragments were quantitated as described (17). It should be noted that synthesis of labeled protein continued after the start of the chase and, therefore, that the values for the recovery of mature APP, secreted APP, and the carboxyl-terminal APP fragment are overestimated in absolute terms but accurate in relative terms.
MATERIALS AND METHODS The materials and methods used in this report, including the [35S]methionine pulse-chase procedure, have been reported (17). PDBu, 4a-PDBu, and okadaic acid were purchased from LC Services (Woburn, MA). When the effects of phorbol esters (1 ,uM) or okadaic acid (1 ,uM) were examined, drug
RESULTS Identification of APP Species. We employed domainspecific antibodies to identify the APP species present in cell lysates and conditioned medium from metabolically labeled Abbreviations: APP, amyloid precursor protein; PDBu, phorbol 12,13-dibutyrate; PKC, protein kinase C; KPI, Kunitz-type protease inhibitor. §To whom reprint requests should be addressed.
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.
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PC12 cells. Antibodies directed against the amino terminus (22C11) or against the carboxyl terminus (369A) of APP immunoprecipitated, from cell lysates, proteins with molecular masses of 106, 112, 116, 125, 139, and 146 kDa (Fig. 1B, lanes 1 and 3). The 112-, 116-, 139-, and 146-kDa proteins were also immunoprecipitated with antibody 56.1 (18), which was raised against the KPI domain of APP751 and APP770 (Fig. 1B, lane 2). An antibody raised against unglycosylated APP695 produced in E. coli was found to immunoprecipitate mainly the 106-, 112-, and 116-kDa proteins (Fig. 1B, lane 5). In addition, antibody 369A, but not the other antibodies, immunoprecipitated a 16.3-kDa peptide (data not shown), which was found by radiosequence analysis to be the carboxyl-terminal fragment resulting from normal intra-P/A4 amyloid cleavage of APP (15) (unpublished results).¶ Preincubation of antibody 369A with a synthetic peptide corresponding to the cytoplasmic domain of APP abolished recovery of all cell-associated APP species (Fig. 1B, lane 4). Proteins with molecular masses of 109, 123, and 129 kDa were immunoprecipitated from conditioned medium using antibody 22C11 (Fig. 1B, lane 6), but no protein was recovered using antibody 369A (Fig. 1B, lane 8), indicating that only amino-terminal APP fragments were present in the medium. The 123- and 129-kDa species could also be immunoprecipitated using antibody 56.1 (Fig. 1B, lane 7). Based upon earlier reports (11, 16) and the current results, we conclude that the 106-, 112-, and 116-kDa proteins are immature (partially glycosylated) APP695, APP751, and APP770; that the 125-, 139-, and 146-kDa proteins are mature (fully glycosylated) APP695, APP751, and APP770; and that the 109-, 123-, and 129-kDa proteins are the secreted aminoterminal fragments of APP695, APP751, and APP770, respec-
tively. Effects of Phorbol Ester on Maturation and Turnover ofAPP Holoprotein and on Recovery of the Carboxyl-Terminal APP Fragment. We have demonstrated (16) that treatment ofPC12 cells with PDBu has no effect on APP maturation but results in more rapid turnover of mature APP and increased production of a carboxyl-terminal APP fragment. Those results were extended to differentiate among APP isoforms in the present study. Recovery of mature APP in intact cells was maximal at 30 min of chase and decreased thereafter (Fig. 2). The recovery of mature APP in the presence of PDBu at 30 min of chase represented -73% of that seen in control cells. By 8 h of the chase period, the amounts of labeled mature APP isoforms had returned to the levels present at the start of chase. Maximal recovery of the 16.3-kDa carboxylterminal APP fragment occurred at 1 h of chase and was increased 90% over basal levels when cells were treated with PDBu (Fig. 3). Effect of Phorbol Ester on APP Secretion. As phorbol esters were found to cause an increase in mature APP turnover, we examined whether this effect was associated with an alteration in the rate and/or amount of APP secretion. In the present study, PDBu treatment caused a dramatic increase in APP secretion (Fig. 4). When the amounts of secreted APP were quantitated, the relative phorbol ester effects were approximately the same among APP isoforms (Fig. 5). In both untreated and treated cells, APP secretion continued up to 4 h of the chase period. From 4 to 8 h, the amount of secreted APP fragments recovered from the medium remained unchanged or decreased slightly. The amount of secreted APP in the medium decreased with time when the 4-h conditioned medium was incubated in the absence of cells (data not shown), indicating that the disappearance was attributable to proteolysis.
$The 16.3-kDa APP species was previously reported by our group as 15 kDa
(16).
Proc. Natl. Acad Sci. USA 89 (1992) 40 30 20
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FIG. 2. Recovery of mature APP in the absence (open circles) or presence (solid diamonds) of PDBu. (A) Mature APP695. (B) Mature APP751. (C) Mature APP-no. Results are the mean ± SEM of seven experiments (n = 3 to 7 for individual time points). Statistical significance between untreated and treated samples for individual time points was determined by Student's unpaired t test (*, P < 0.05).
There was a 3-fold accumulation of secreted APP in the medium of PDBu-treated cells relative to control levels. Phorbol ester caused an increase in the rate of accumulation and in the absolute amount of secreted APP. At 4 h of chase, =14% of the labeled APP present at the start of chase was recovered as secreted amino-terminal APP fragments from the medium of control cells, whereas =43% was recovered from the medium of PDBu-treated cells. This indicates that under control conditions, a relatively small pool of APP was cleaved within the f3/A4 amyloid domain to produce secreted APP. The majority of APP molecules were presumably degraded by an intracellular proteolytic pathway unassociated with secretion (17). When the difference between mature APP holoprotein recovered from PDBu-treated and untreated cells (at 30 min of chase) was compared to the difference between secreted APP fragments recovered from the medium of PDBu-treated and untreated cells (at 4 h of chase), a close correlation was found (27 ± 6 versus 31 ± 3 relative units, respectively). Thus, the phorbol ester-stimulated turnover of mature APP
Medical Sciences:
Caporaso et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
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FIG. 3. Recovery of the 16.3-kDa carboxyl-terminal APP fragment in the absence (open circles) or presence (solid diamonds) of PDBu. Results are the mean ± SEM of seven experiments (n = 3 to 7 for individual time points). Statistical significance between untreated and treated samples for individual time points was determined by Student's unpaired t test (*, P < 0.05).
holoprotein could be quantitatively accounted for by enhanced APP secretion. Absence of Effect of Inactive Phorbol Ester on APP Secretion. As a control for the specificity of PDBu activation of PKC, cells were also treated with the inactive analogue, 4a-PDBu. There was virtually no effect on APP maturation or holoprotein turnover (data not shown) or on APP secretion (Fig. 6) in the presence of 4a-PDBu relative to cells treated with vehicle alone. Effect of Combined Treatment with Phorbol Ester and Okadaic Acid on APP Secretion. Combined treatment with PDBu and okadaic acid, an inhibitor of protein phosphatases 1 and 2A, was previously found to increase recovery of APP carboxyl-terminal fragments (16). Therefore, we examined the effect of combined treatment on APP secretion (Fig. 7). Cells treated for 1 h with okadaic acid alone secreted more APP than untreated cells but less APP than cells treated with PDBu. When cells were treated with both PDBu and okadaic acid, APP secretion at 1 h was greater than APP secretion in cells treated with PDBu or okadaic acid alone. Although combined treatment with PDBu and okadaic acid produces a carboxyl-terminal fragment that migrates on SDS/polyacrylamide gels more slowly than the 16.3-kDa species (16), no evidence of truncated secreted APP fragments was found. Furthermore, no treatments resulted in the detection of cleaved amino-terminal APP fragments in cell lysates.
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FIG. 4. Autoradiograms of APP species immunoprecipitated from conditioned medium of [35S]methionine-labeled PC12 cells incubated during the chase period for the indicated times in the absence (A) or presence (B) of PDBu. Autoradiograms are from a single experiment analyzed on 6% SDS/polyacrylamide gels.
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FIG. 5. Recovery of secreted amino-terminal APP fragments in the absence (open circles) or presence (solid diamonds) of PDBu (see Fig. 4). (A) Secreted APP695. (B) Secreted APP751. (C) Secreted APP-n0. Results are the mean SEM of seven experiments (n = 3 to 7 for individual time points). Statistical significance between untreated and treated samples for individual time points was determined by Student's unpaired t test (*, P < 0.05). ±
DISCUSSION We have demonstrated in PC12 cells that, under basal conditions, a relatively small pool of APP was cleaved within the f3/A4 amyloid domain to produce secreted APP, indicating that the majority of APP molecules was degraded by a proteolytic pathway unassociated with secretion. The intra,B/A4 amyloid cleavage and secretion of amino-terminal APP fragments were stimulated severalfold by PDBu treatment, presumably by the activation of PKC. These data provide a possible mechanism for regulation of APP function. Regulation by PKC of the conversion of integral proteins from membrane-bound to soluble forms with a subsequent alteration in their function has been reported for tumor necrosis factor receptor (19), colony-stimulating factor 1 receptor (20), murine neutrophil MEL-14 antigen (21), and pro-transforming growth factor a (22). On the basis of the resemblance of the APP topology to that of certain cellsurface receptors, APP was postulated to function as a cell-surface binding protein for an as yet unidentified ligand
3058
Medical Sciences: Caporaso et al. 8
Proc. Natl. Acad. Sci. USA 89 (1992) N-terminal fragment
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FIG. 8. Proposed scheme for the cellular trafficking and proteolytic processing of APP.
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FIG. 6. Recovery of secreted amino-terminal APP fragments in the absence (open circles) or presence (solid diamonds) of 4a-PDBu. (A) Secreted APP695. (B) Secreted APP751. (C) Secreted APP-no. Results are the mean + SEM of four experiments (n = 2 to 4 for individual time points). There was no statistical significance between untreated and treated samples for any time points as determined by Student's unpaired t test.
(5), an interpretation supported by the localization of some APP molecules to the plasma membrane (11). Regulated proteolytic cleavage and secretion of APP by activation of PKC could serve two purposes: down-regulation of APP from the cell surface and release of a functional APP fragment into the surrounding environment (23). Although the physiological function of APP is not known, secreted amino-terminal
FIG. 7. Autoradiogram of APP species immunoprecipitated from conditioned medium of [35S]methionine-labeled PC12 cells incubated during the chase period for 1 h in the absence or presence of agents that regulate protein phosphorylation. Lanes: 1, vehicle alone; 2, PDBu; 3, okadaic acid; 4, PDBu plus okadaic acid.
fragments of APP751 and APP770 have been identified as protease nexin II (24, 25), a soluble protein that forms complexes with epidermal growth factor-binding protein, the y subunit of nerve growth factor, and trypsin (26). The physiological significance of these interactions is not understood, but protease nexin II released from platelets has been found to be a potent inhibitor of coagulation factor XIa (27). We have not yet determined whether the phorbol esterinduced effects on APP secretion are due to phosphorylation of APP by PKC ("substrate activation"), activation of an APP "secretase" ("enzyme activation"), or redirected cellular trafficking that brings APP in contact with its protease(s) (23). Support for the "substrate activation" model comes from studies in our laboratory demonstrating that PKC can phosphorylate synthetic APP carboxyl-terminal peptides (28) and from other studies that demonstrate that mature APP is phosphorylated in cultures of intact cells (29). In a system that might be analogous, phosphorylation of the polymeric immunoglobulin receptor results in acceleration of its transcytosis and proteolytic cleavage to produce secretory component (30). The "enzyme activation" model is supported by studies on the colony-stimulating factor 1 receptor, in which PKC has been suggested to act on the protease responsible for the release of soluble receptor rather than on the membrane-bound receptor itself (20). The majority of APP molecules are degraded in unstimulated PC12 cells by a chloroquine-sensitive endosomal/ lysosomal proteolytic pathway; a small fraction undergoes intra-(3/A4 amyloid cleavage by a chloroquine-insensitive pathway to produce secreted amino-terminal APP fragments and a 16.3-kDa carboxyl-terminal APP fragment (17). These earlier observations, combined with the results of the present report, suggest a scheme for the cellular trafficking and proteolytic processing of APP (Fig. 8). Our results indicate that PKC stimulates the nonamyloidogenic secretory pathway of APP. At present, the possibility cannot be excluded that PKC also regulates the alternative, and possibly amyloidogenic, APP degradative (endosomal/lysosomal) pathway. The existence of distinct physiological and amyloidogenic pathways could facilitate the development of antiamyloidogenic drugs. Note Added in Proof. During the preparation of this manuscript, we learnzd of other work in progress which supports the hypothesis that APP secretion is regulated by PKC. By using 1 AM PDBu to treat 293 (human embryonic kidney) or K562 (human mononuclear leukemia) cells transfected with human APP695, APP751, or APP,,o expression constructs, S. Gillespie, T. Golde, and S. Younkin (personal communication) have observed increases in the secreted forms of
Medical Sciences:
Caporaso et al.
APP695, APP751, or APP-no and decreases in the corresponding mature holoproteins, similar to the results reported here. Thus, it appears that regulation of APP secretion by PKC occurs independent of species and cell type. We thank Drs. A. J. Czernik, F. S. Gorelick, K. Iverfeldt, A. C. Nairn, C. Nordstedt, and Y. L. Siow for their suggestions and advice, and we thank Dr. A. J. Czernik for his review of this manuscript. This research was sponsored by U.S. Public Health Service Medical Scientist Training Program Grant GM07739 (to G.L.C.), Clinical Investigator Development Award NS-01095 (to S.E.G.), Individual National Research Service Award NS-08523 (to J.D.B.), and U.S. Public Health Service Grant AG-09464 (to P.G.). 1. Tomlinson, B. E. & Corsellis, J. A. N. (1984) in Greenfield's Neuropathology, eds. Adams, J. H., Corsellis, J. A. N. & Duchen, L. W. (Arnold, London), pp. 909-918. 2. Glenner, G. G. & Wong, C. W. (1984) Biochem. Biophys. Res. Commun. 120, 885-890. 3. Masters, C. L., Simms, G., Weinmann, N. A., Multhaup, G.,
McDonald, B. L. & Beyreuther, K. (1985) Proc. Natl. Acad.
Sci. USA 82, 4245-4249. 4. Goldgaber, D., Lerman, M. I., McBride, 0. W., Saffiotti, U. & Gajdusek, D. C. (1987) Science 235, 877-880. 5. Kang, J., Lemaire, H. G., Unterbeck, A., Salbaum, J. M., Masters, C. L., Grzeschik, K. H., Multhaup, G., Beyreuther, K. & Muller-Hill, B. (1987) Nature (London) 325, 733-736. 6. Tanzi, R. E., Gusella, J. F., Watkins, P. C., Bruns, G. A. P., St. George-Hyslop, P., van Keuren, M. L., Patterson, D., Pagan, S., Kurnit, D. M. & Neve, R. L. (1987) Science 235, 880-883. 7. Robakis, N. K., Ramakrishna, N., Wolfe, G. & Wisniewski, H. M. (1987) Proc. Natl. Acad. Sci. USA 84, 4190-4194. 8. Ponte, P., Gonzalez-DeWhitt, P., Schilling, J., Miller, J., Hsu, D., Greenberg, B., Davis, K., Wallace, W., Lieberburg, I., Fuller, F. & Cordell, B. (1988) Nature (London) 331, 525-527. 9. Tanzi, R. E., McClatchey, A. I., Lamperti, E. D., VillaKomaroff, L., Gusella, J. F. & Neve, R. L. (1988) Nature (London) 331, 528-530. 10. Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S. & Ito, H. (1988) Nature (London) 331, 530-532. 11. Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J. M., Masters, C. L. & Beyreuther, K. (1989) Cell 57, 115126. 12. Palmert, M. R., Podlisny, M. B., Witker, D. S., Oltersdorf, T., Younkin, L. H., Selkoe, D. J. & Younkin, S. G. (1989) Proc. Natl. Acad. Sci. USA 86, 6338-6342.
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13. Bush, A. I., Martins, R. N., Rumble, B., Moir, R., Fuller, S., Milward, E., Currie, J., Ames, D., Weidemann, A., Fischer, P., Multhaup, G., Beyreuther, K. & Masters, C. L. (1990) J. Biol. Chem. 265, 15977-15983. 14. Sisodia, S. S., Koo, E. H., Beyreuther, K., Unterbeck, A. & Price, D. L. (1990) Science 248, 492-495. 15. Esch, F. S., Keim, P. S., Beattie, E. C., Blacher, R. W., Culwell, A. R., Oltersdorf, T., McClure, D. & Ward, P. J. (1990) Science 248, 1122-1124. 16. Buxbaum, J. D., Gandy, S. E., Cicchetti, P., Ehrlich, M. E., Czernik, A. J., Fracasso, R. P., Ramabhadran, T. V., Unterbeck, A. J. & Greengard, P. (1990) Proc. Natl. Acad. Sci. USA 87, 6003-6006. 17. Caporaso, G. L., Gandy, S. E., Buxbaum, J. D. & Greengard, P. (1992) Proc. Natl. Acad. Sci. USA 89, 2252-2256. 18. Wunderlich, D., Lee, A., Fracasso, R. P., Mierz, D. V., Bayney, R. M. & Ramabhadran, T. V. (1992) J. Immunol. Methods 14, 1-11. 19. Lantz, M., Gullberg, U., Nilsson, E. & Olsson, I. (1990) J. Clin. Invest. 86, 13%-1402. 20. Downing, J. R., Roussel, M. F. & Sherr, C. J. (1989) Mol. Cell. Biol. 9, 2890-28%. 21. Kishimoto, T. K., Jutila, M. A., Berg, E. L. & Butcher, E. C. (1989) Science 245, 1238-1241. 22. Pandiella, A. & Massagud, J. (1991) Proc. NatI. Acad. Sci. USA 88, 1726-1730.
23. Ehlers, M. R. W. & Riordan, J. F. (1991) Biochemistry 30, 10065-10074. 24. Oltersdorf, T., Fritz, L. C., Schenk, D. B., Lieberburg, I., Johnson-Wood, K. L., Beattie, E. C., Ward, P. J., Blacher, R. W., Dovey, H. F. & Sinha, S. (1989) Nature (London) 341, 144-147. 25. Van Nostrand, W. E., Wagner, S. L., Suzuki, M., Choi, B. H., Farrow, J. S., Geddes, J. W., Cotman, C. W. & Cunningham, D. D. (1989) Nature (London) 341, 546-549. 26. Van Nostrand, W. E. & Cunningham, D. D. (1989) J. Biol. Chem. 262, 8508-8514. 27. Smith, R. P., Higuchi, D. A. & Broze, G. J., Jr. (1990) Science 248, 1126-1128. 28. Gandy, S. E., Czernik, A. J. & Greengard, P. (1988) Proc.
NatI. Acad. Sci. USA 85, 6218-6221.
29. Oltersdorf, T., Ward, P. J., Henriksson, T., Beattie, E. C., Neve, R., Lieberburg, I. & Fritz, L. C. (1990) J. Biol. Chem. 265, 4492-4497. 30. Casanova, J. E., Breitfeld, P. P., Ross, S. A. & Mostov, K. E. (1990) Science 248, 742-745.
Medical Sciences
Protein phosphorylation regulates secretion of Alzheimer fi/A4 amyloid precursor protein (proteolytic processing/protein kinase/protein phosphatase/phorbol ester/okadaic acid)
GREGG L. CAPORASO*, SAMUEL E. GANDY*tt, JOSEPH D. BUXBAUM*, TRIPRAYAR V. RAMABHADRAN*, AND PAUL GREENGARD*§ *Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021;
tThe Nathan S. Kline Institute for Psychiatric
Research, Orangeburg, NY 10962; and tDepartment of Psychiatry, New York University Medical Center, New York, NY 10016
Contributed by Paul Greengard, December 26, 1991
A
Extracellular deposition of the (3/A4 amyloid ABSTRACT peptide is a characteristic feature of the brain in patients with Alzheimer disease. ,f/A4 amyloid is derived from the amyloid precursor protein (APP), an integral membrane protein that exists as three major isoforms (APP695, APP75l, and APP770). Secreted forms of APP found in blood plasma and cerebrospinal fluid arise by proteolytic cleavage of APP within the 13/A4 amyloid domain, precluding the possibility of amyloidogenesis for that population of molecules. In the present study, we have demonstrated that treatment of PC12 cells with phorbol ester produces a severalfold increase in secretion of APP6s, APP751, and APP770. This increase is augmented by simultaneous treatment with the protein phosphatase inhibitor okadaic acid. These data indicate that protein phosphorylation regulates intra-3/A4 amyloid cleavage and APP secretion. These and other results suggest that APP molecules can normally follow either of two processing pathways: regulated secretion or proteolytic degradation unassociated with secretion.
KPI OX-2 T--
Exterior Cytoplasm CHO CHO!
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FIG. 1. (A) Schematic diagram of the APP molecule showing extracellular, transmembrane, and cytoplasmic regions: 3/A4 amyloid domain (shaded); normal intra-P/A4 amyloid cleavage site (15) (arrow); domain inserts for APP751 (KPI only) and APP770 (both KPI and OX-2) isoforms; and putative N-glycosylation sites (CHO). (B) Autoradiogram of APP species immunoprecipitated from cell lysates (lanes 1-5) or conditioned medium (lanes 6-9) of [35S]methioninelabeled PC12 cells chased for 1 h in the absence of phorbol 12,13dibutyrate (PDBu) (lanes 1-5) or for 4 h in the presence of PDBu (lanes 6-9). Four immunoprecipitating antibodies were used to identify the various APP species. Lanes: 1 and 6, anti-amino-terminal monoclonal antibody 22C11; 2 and 7, anti-KPI domain monoclonal antibody 56.1; 3 and 8, anti-carboxyl-terminal affinity-purified polyclonal antibody 369A; 4, antibody 369A preincubated with synthetic peptide corresponding to the cytoplasmic domain of APP (residues 645-694); 5 and 9, whole serum from a guinea pig injected with unglycosylated APP695 produced in Escherichia coli. Autoradiograms are from a single experiment analyzed on a 4-15% continuous gradient SDS/polyacrylamide gel. Molecular masses (kDa) are indicated.
A characteristic pathological feature of Alzheimer disease is the extracellular deposition of 8/A4 amyloid in parenchymal brain tissues and in the walls of cerebral and meningeal blood vessels (1-3). This -4-kDa peptide is derived from the amyloid precursor protein (APP), a transmembrane glycoprotein that exists as three major isoforms (APP695, APP751, and APP770; Fig. 1 A), which result from the alternative splicing of mRNA from a single gene (4-10). The larger APP751 and APP770 isoforms contain a Kunitz-type protease inhibitor (KPI) insert in their extracellular domains (8-10). Secreted amino-terminal fragments of APP have been found in blood plasma and cerebrospinal fluid (11-13). These secreted fragments result from cleavage of APP within the ,f/A4 amyloid domain (14, 15). The process of APP secretion, therefore, precludes the cerebral amyloidogenesis associated with Alzheimer disease. We have previously demonstrated that activation of protein kinase C (PKC) in PC12 cells by treatment with phorbol ester results in more rapid intracellular turnover of APP and increased production of carboxylterminal APP fragments (16). In the present report, we describe the regulation by protein phosphorylation of APP secretion in PC12 cells.
was added at the start of the chase period. Control cells were treated with drug vehicle alone (0.05-0.15% dimethyl sulfoxide, final concentration). The amounts of labeled APP holoproteins and APP fragments were quantitated as described (17). It should be noted that synthesis of labeled protein continued after the start of the chase and, therefore, that the values for the recovery of mature APP, secreted APP, and the carboxyl-terminal APP fragment are overestimated in absolute terms but accurate in relative terms.
MATERIALS AND METHODS The materials and methods used in this report, including the [35S]methionine pulse-chase procedure, have been reported (17). PDBu, 4a-PDBu, and okadaic acid were purchased from LC Services (Woburn, MA). When the effects of phorbol esters (1 ,uM) or okadaic acid (1 ,uM) were examined, drug
RESULTS Identification of APP Species. We employed domainspecific antibodies to identify the APP species present in cell lysates and conditioned medium from metabolically labeled Abbreviations: APP, amyloid precursor protein; PDBu, phorbol 12,13-dibutyrate; PKC, protein kinase C; KPI, Kunitz-type protease inhibitor. §To whom reprint requests should be addressed.
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.
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PC12 cells. Antibodies directed against the amino terminus (22C11) or against the carboxyl terminus (369A) of APP immunoprecipitated, from cell lysates, proteins with molecular masses of 106, 112, 116, 125, 139, and 146 kDa (Fig. 1B, lanes 1 and 3). The 112-, 116-, 139-, and 146-kDa proteins were also immunoprecipitated with antibody 56.1 (18), which was raised against the KPI domain of APP751 and APP770 (Fig. 1B, lane 2). An antibody raised against unglycosylated APP695 produced in E. coli was found to immunoprecipitate mainly the 106-, 112-, and 116-kDa proteins (Fig. 1B, lane 5). In addition, antibody 369A, but not the other antibodies, immunoprecipitated a 16.3-kDa peptide (data not shown), which was found by radiosequence analysis to be the carboxyl-terminal fragment resulting from normal intra-P/A4 amyloid cleavage of APP (15) (unpublished results).¶ Preincubation of antibody 369A with a synthetic peptide corresponding to the cytoplasmic domain of APP abolished recovery of all cell-associated APP species (Fig. 1B, lane 4). Proteins with molecular masses of 109, 123, and 129 kDa were immunoprecipitated from conditioned medium using antibody 22C11 (Fig. 1B, lane 6), but no protein was recovered using antibody 369A (Fig. 1B, lane 8), indicating that only amino-terminal APP fragments were present in the medium. The 123- and 129-kDa species could also be immunoprecipitated using antibody 56.1 (Fig. 1B, lane 7). Based upon earlier reports (11, 16) and the current results, we conclude that the 106-, 112-, and 116-kDa proteins are immature (partially glycosylated) APP695, APP751, and APP770; that the 125-, 139-, and 146-kDa proteins are mature (fully glycosylated) APP695, APP751, and APP770; and that the 109-, 123-, and 129-kDa proteins are the secreted aminoterminal fragments of APP695, APP751, and APP770, respec-
tively. Effects of Phorbol Ester on Maturation and Turnover ofAPP Holoprotein and on Recovery of the Carboxyl-Terminal APP Fragment. We have demonstrated (16) that treatment ofPC12 cells with PDBu has no effect on APP maturation but results in more rapid turnover of mature APP and increased production of a carboxyl-terminal APP fragment. Those results were extended to differentiate among APP isoforms in the present study. Recovery of mature APP in intact cells was maximal at 30 min of chase and decreased thereafter (Fig. 2). The recovery of mature APP in the presence of PDBu at 30 min of chase represented -73% of that seen in control cells. By 8 h of the chase period, the amounts of labeled mature APP isoforms had returned to the levels present at the start of chase. Maximal recovery of the 16.3-kDa carboxylterminal APP fragment occurred at 1 h of chase and was increased 90% over basal levels when cells were treated with PDBu (Fig. 3). Effect of Phorbol Ester on APP Secretion. As phorbol esters were found to cause an increase in mature APP turnover, we examined whether this effect was associated with an alteration in the rate and/or amount of APP secretion. In the present study, PDBu treatment caused a dramatic increase in APP secretion (Fig. 4). When the amounts of secreted APP were quantitated, the relative phorbol ester effects were approximately the same among APP isoforms (Fig. 5). In both untreated and treated cells, APP secretion continued up to 4 h of the chase period. From 4 to 8 h, the amount of secreted APP fragments recovered from the medium remained unchanged or decreased slightly. The amount of secreted APP in the medium decreased with time when the 4-h conditioned medium was incubated in the absence of cells (data not shown), indicating that the disappearance was attributable to proteolysis.
$The 16.3-kDa APP species was previously reported by our group as 15 kDa
(16).
Proc. Natl. Acad Sci. USA 89 (1992) 40 30 20
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FIG. 2. Recovery of mature APP in the absence (open circles) or presence (solid diamonds) of PDBu. (A) Mature APP695. (B) Mature APP751. (C) Mature APP-no. Results are the mean ± SEM of seven experiments (n = 3 to 7 for individual time points). Statistical significance between untreated and treated samples for individual time points was determined by Student's unpaired t test (*, P < 0.05).
There was a 3-fold accumulation of secreted APP in the medium of PDBu-treated cells relative to control levels. Phorbol ester caused an increase in the rate of accumulation and in the absolute amount of secreted APP. At 4 h of chase, =14% of the labeled APP present at the start of chase was recovered as secreted amino-terminal APP fragments from the medium of control cells, whereas =43% was recovered from the medium of PDBu-treated cells. This indicates that under control conditions, a relatively small pool of APP was cleaved within the f3/A4 amyloid domain to produce secreted APP. The majority of APP molecules were presumably degraded by an intracellular proteolytic pathway unassociated with secretion (17). When the difference between mature APP holoprotein recovered from PDBu-treated and untreated cells (at 30 min of chase) was compared to the difference between secreted APP fragments recovered from the medium of PDBu-treated and untreated cells (at 4 h of chase), a close correlation was found (27 ± 6 versus 31 ± 3 relative units, respectively). Thus, the phorbol ester-stimulated turnover of mature APP
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Proc. Natl. Acad. Sci. USA 89 (1992)
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FIG. 3. Recovery of the 16.3-kDa carboxyl-terminal APP fragment in the absence (open circles) or presence (solid diamonds) of PDBu. Results are the mean ± SEM of seven experiments (n = 3 to 7 for individual time points). Statistical significance between untreated and treated samples for individual time points was determined by Student's unpaired t test (*, P < 0.05).
holoprotein could be quantitatively accounted for by enhanced APP secretion. Absence of Effect of Inactive Phorbol Ester on APP Secretion. As a control for the specificity of PDBu activation of PKC, cells were also treated with the inactive analogue, 4a-PDBu. There was virtually no effect on APP maturation or holoprotein turnover (data not shown) or on APP secretion (Fig. 6) in the presence of 4a-PDBu relative to cells treated with vehicle alone. Effect of Combined Treatment with Phorbol Ester and Okadaic Acid on APP Secretion. Combined treatment with PDBu and okadaic acid, an inhibitor of protein phosphatases 1 and 2A, was previously found to increase recovery of APP carboxyl-terminal fragments (16). Therefore, we examined the effect of combined treatment on APP secretion (Fig. 7). Cells treated for 1 h with okadaic acid alone secreted more APP than untreated cells but less APP than cells treated with PDBu. When cells were treated with both PDBu and okadaic acid, APP secretion at 1 h was greater than APP secretion in cells treated with PDBu or okadaic acid alone. Although combined treatment with PDBu and okadaic acid produces a carboxyl-terminal fragment that migrates on SDS/polyacrylamide gels more slowly than the 16.3-kDa species (16), no evidence of truncated secreted APP fragments was found. Furthermore, no treatments resulted in the detection of cleaved amino-terminal APP fragments in cell lysates.
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FIG. 4. Autoradiograms of APP species immunoprecipitated from conditioned medium of [35S]methionine-labeled PC12 cells incubated during the chase period for the indicated times in the absence (A) or presence (B) of PDBu. Autoradiograms are from a single experiment analyzed on 6% SDS/polyacrylamide gels.
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FIG. 5. Recovery of secreted amino-terminal APP fragments in the absence (open circles) or presence (solid diamonds) of PDBu (see Fig. 4). (A) Secreted APP695. (B) Secreted APP751. (C) Secreted APP-n0. Results are the mean SEM of seven experiments (n = 3 to 7 for individual time points). Statistical significance between untreated and treated samples for individual time points was determined by Student's unpaired t test (*, P < 0.05). ±
DISCUSSION We have demonstrated in PC12 cells that, under basal conditions, a relatively small pool of APP was cleaved within the f3/A4 amyloid domain to produce secreted APP, indicating that the majority of APP molecules was degraded by a proteolytic pathway unassociated with secretion. The intra,B/A4 amyloid cleavage and secretion of amino-terminal APP fragments were stimulated severalfold by PDBu treatment, presumably by the activation of PKC. These data provide a possible mechanism for regulation of APP function. Regulation by PKC of the conversion of integral proteins from membrane-bound to soluble forms with a subsequent alteration in their function has been reported for tumor necrosis factor receptor (19), colony-stimulating factor 1 receptor (20), murine neutrophil MEL-14 antigen (21), and pro-transforming growth factor a (22). On the basis of the resemblance of the APP topology to that of certain cellsurface receptors, APP was postulated to function as a cell-surface binding protein for an as yet unidentified ligand
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Proc. Natl. Acad. Sci. USA 89 (1992) N-terminal fragment
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FIG. 6. Recovery of secreted amino-terminal APP fragments in the absence (open circles) or presence (solid diamonds) of 4a-PDBu. (A) Secreted APP695. (B) Secreted APP751. (C) Secreted APP-no. Results are the mean + SEM of four experiments (n = 2 to 4 for individual time points). There was no statistical significance between untreated and treated samples for any time points as determined by Student's unpaired t test.
(5), an interpretation supported by the localization of some APP molecules to the plasma membrane (11). Regulated proteolytic cleavage and secretion of APP by activation of PKC could serve two purposes: down-regulation of APP from the cell surface and release of a functional APP fragment into the surrounding environment (23). Although the physiological function of APP is not known, secreted amino-terminal
FIG. 7. Autoradiogram of APP species immunoprecipitated from conditioned medium of [35S]methionine-labeled PC12 cells incubated during the chase period for 1 h in the absence or presence of agents that regulate protein phosphorylation. Lanes: 1, vehicle alone; 2, PDBu; 3, okadaic acid; 4, PDBu plus okadaic acid.
fragments of APP751 and APP770 have been identified as protease nexin II (24, 25), a soluble protein that forms complexes with epidermal growth factor-binding protein, the y subunit of nerve growth factor, and trypsin (26). The physiological significance of these interactions is not understood, but protease nexin II released from platelets has been found to be a potent inhibitor of coagulation factor XIa (27). We have not yet determined whether the phorbol esterinduced effects on APP secretion are due to phosphorylation of APP by PKC ("substrate activation"), activation of an APP "secretase" ("enzyme activation"), or redirected cellular trafficking that brings APP in contact with its protease(s) (23). Support for the "substrate activation" model comes from studies in our laboratory demonstrating that PKC can phosphorylate synthetic APP carboxyl-terminal peptides (28) and from other studies that demonstrate that mature APP is phosphorylated in cultures of intact cells (29). In a system that might be analogous, phosphorylation of the polymeric immunoglobulin receptor results in acceleration of its transcytosis and proteolytic cleavage to produce secretory component (30). The "enzyme activation" model is supported by studies on the colony-stimulating factor 1 receptor, in which PKC has been suggested to act on the protease responsible for the release of soluble receptor rather than on the membrane-bound receptor itself (20). The majority of APP molecules are degraded in unstimulated PC12 cells by a chloroquine-sensitive endosomal/ lysosomal proteolytic pathway; a small fraction undergoes intra-(3/A4 amyloid cleavage by a chloroquine-insensitive pathway to produce secreted amino-terminal APP fragments and a 16.3-kDa carboxyl-terminal APP fragment (17). These earlier observations, combined with the results of the present report, suggest a scheme for the cellular trafficking and proteolytic processing of APP (Fig. 8). Our results indicate that PKC stimulates the nonamyloidogenic secretory pathway of APP. At present, the possibility cannot be excluded that PKC also regulates the alternative, and possibly amyloidogenic, APP degradative (endosomal/lysosomal) pathway. The existence of distinct physiological and amyloidogenic pathways could facilitate the development of antiamyloidogenic drugs. Note Added in Proof. During the preparation of this manuscript, we learnzd of other work in progress which supports the hypothesis that APP secretion is regulated by PKC. By using 1 AM PDBu to treat 293 (human embryonic kidney) or K562 (human mononuclear leukemia) cells transfected with human APP695, APP751, or APP,,o expression constructs, S. Gillespie, T. Golde, and S. Younkin (personal communication) have observed increases in the secreted forms of
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APP695, APP751, or APP-no and decreases in the corresponding mature holoproteins, similar to the results reported here. Thus, it appears that regulation of APP secretion by PKC occurs independent of species and cell type. We thank Drs. A. J. Czernik, F. S. Gorelick, K. Iverfeldt, A. C. Nairn, C. Nordstedt, and Y. L. Siow for their suggestions and advice, and we thank Dr. A. J. Czernik for his review of this manuscript. This research was sponsored by U.S. Public Health Service Medical Scientist Training Program Grant GM07739 (to G.L.C.), Clinical Investigator Development Award NS-01095 (to S.E.G.), Individual National Research Service Award NS-08523 (to J.D.B.), and U.S. Public Health Service Grant AG-09464 (to P.G.). 1. Tomlinson, B. E. & Corsellis, J. A. N. (1984) in Greenfield's Neuropathology, eds. Adams, J. H., Corsellis, J. A. N. & Duchen, L. W. (Arnold, London), pp. 909-918. 2. Glenner, G. G. & Wong, C. W. (1984) Biochem. Biophys. Res. Commun. 120, 885-890. 3. Masters, C. L., Simms, G., Weinmann, N. A., Multhaup, G.,
McDonald, B. L. & Beyreuther, K. (1985) Proc. Natl. Acad.
Sci. USA 82, 4245-4249. 4. Goldgaber, D., Lerman, M. I., McBride, 0. W., Saffiotti, U. & Gajdusek, D. C. (1987) Science 235, 877-880. 5. Kang, J., Lemaire, H. G., Unterbeck, A., Salbaum, J. M., Masters, C. L., Grzeschik, K. H., Multhaup, G., Beyreuther, K. & Muller-Hill, B. (1987) Nature (London) 325, 733-736. 6. Tanzi, R. E., Gusella, J. F., Watkins, P. C., Bruns, G. A. P., St. George-Hyslop, P., van Keuren, M. L., Patterson, D., Pagan, S., Kurnit, D. M. & Neve, R. L. (1987) Science 235, 880-883. 7. Robakis, N. K., Ramakrishna, N., Wolfe, G. & Wisniewski, H. M. (1987) Proc. Natl. Acad. Sci. USA 84, 4190-4194. 8. Ponte, P., Gonzalez-DeWhitt, P., Schilling, J., Miller, J., Hsu, D., Greenberg, B., Davis, K., Wallace, W., Lieberburg, I., Fuller, F. & Cordell, B. (1988) Nature (London) 331, 525-527. 9. Tanzi, R. E., McClatchey, A. I., Lamperti, E. D., VillaKomaroff, L., Gusella, J. F. & Neve, R. L. (1988) Nature (London) 331, 528-530. 10. Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S. & Ito, H. (1988) Nature (London) 331, 530-532. 11. Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J. M., Masters, C. L. & Beyreuther, K. (1989) Cell 57, 115126. 12. Palmert, M. R., Podlisny, M. B., Witker, D. S., Oltersdorf, T., Younkin, L. H., Selkoe, D. J. & Younkin, S. G. (1989) Proc. Natl. Acad. Sci. USA 86, 6338-6342.
Proc. Natl. Acad. Sci. USA 89 (1992)
3059
13. Bush, A. I., Martins, R. N., Rumble, B., Moir, R., Fuller, S., Milward, E., Currie, J., Ames, D., Weidemann, A., Fischer, P., Multhaup, G., Beyreuther, K. & Masters, C. L. (1990) J. Biol. Chem. 265, 15977-15983. 14. Sisodia, S. S., Koo, E. H., Beyreuther, K., Unterbeck, A. & Price, D. L. (1990) Science 248, 492-495. 15. Esch, F. S., Keim, P. S., Beattie, E. C., Blacher, R. W., Culwell, A. R., Oltersdorf, T., McClure, D. & Ward, P. J. (1990) Science 248, 1122-1124. 16. Buxbaum, J. D., Gandy, S. E., Cicchetti, P., Ehrlich, M. E., Czernik, A. J., Fracasso, R. P., Ramabhadran, T. V., Unterbeck, A. J. & Greengard, P. (1990) Proc. Natl. Acad. Sci. USA 87, 6003-6006. 17. Caporaso, G. L., Gandy, S. E., Buxbaum, J. D. & Greengard, P. (1992) Proc. Natl. Acad. Sci. USA 89, 2252-2256. 18. Wunderlich, D., Lee, A., Fracasso, R. P., Mierz, D. V., Bayney, R. M. & Ramabhadran, T. V. (1992) J. Immunol. Methods 14, 1-11. 19. Lantz, M., Gullberg, U., Nilsson, E. & Olsson, I. (1990) J. Clin. Invest. 86, 13%-1402. 20. Downing, J. R., Roussel, M. F. & Sherr, C. J. (1989) Mol. Cell. Biol. 9, 2890-28%. 21. Kishimoto, T. K., Jutila, M. A., Berg, E. L. & Butcher, E. C. (1989) Science 245, 1238-1241. 22. Pandiella, A. & Massagud, J. (1991) Proc. NatI. Acad. Sci. USA 88, 1726-1730.
23. Ehlers, M. R. W. & Riordan, J. F. (1991) Biochemistry 30, 10065-10074. 24. Oltersdorf, T., Fritz, L. C., Schenk, D. B., Lieberburg, I., Johnson-Wood, K. L., Beattie, E. C., Ward, P. J., Blacher, R. W., Dovey, H. F. & Sinha, S. (1989) Nature (London) 341, 144-147. 25. Van Nostrand, W. E., Wagner, S. L., Suzuki, M., Choi, B. H., Farrow, J. S., Geddes, J. W., Cotman, C. W. & Cunningham, D. D. (1989) Nature (London) 341, 546-549. 26. Van Nostrand, W. E. & Cunningham, D. D. (1989) J. Biol. Chem. 262, 8508-8514. 27. Smith, R. P., Higuchi, D. A. & Broze, G. J., Jr. (1990) Science 248, 1126-1128. 28. Gandy, S. E., Czernik, A. J. & Greengard, P. (1988) Proc.
NatI. Acad. Sci. USA 85, 6218-6221.
29. Oltersdorf, T., Ward, P. J., Henriksson, T., Beattie, E. C., Neve, R., Lieberburg, I. & Fritz, L. C. (1990) J. Biol. Chem. 265, 4492-4497. 30. Casanova, J. E., Breitfeld, P. P., Ross, S. A. & Mostov, K. E. (1990) Science 248, 742-745.
