(Received for publication, July 3, 1995)
From the
Phorbol esters, activators of protein kinase C (PKC), regulate
the relative utilization of alternative processing pathways for the
Alzheimer -amyloid precursor protein (
-APP) in intact cells,
increasing the production of nonamyloidogenic soluble
-APP
(s
-APP) and decreasing that of neurotoxic
-amyloid (A
)
peptide. The molecular and cellular bases of PKC-regulated
-APP
cleavage are poorly understood. Here we demonstrate in a reconstituted
cell-free system that activation of endogenous PKC increases formation
from the trans-Golgi network of secretory vesicles containing
-APP and that this effect can be mimicked by purified PKC. The
results demonstrate directly that PKC is involved in regulation of
secretory vesicle formation and provide a mechanism by which PKC may
reduce the formation of the A
peptide characteristic of Alzheimer
disease.
Alzheimer disease (AD) ()is a common
neurodegenerative disorder characterized by dementia and by
accumulation in the brain of extracellular deposits composed of
-amyloid peptide (A
) (Glenner and Wong, 1984). Signal
transduction via protein phosphorylation governs the relative
utilization of competing pathways for the metabolism of the
-amyloid precursor protein (
-APP) (Kang et al.,
1987), the type 1 integral glycoprotein from which A
is derived by
proteolytic processing. Activated protein kinase C (PKC) stimulates
nonamyloidogenic
-APP cleavage (Buxbaum et al., 1990;
Caporaso et al., 1992), generating soluble
-APP
(s
-APP) at the expense of other pathways, including that involved
in the formation of A
(Hung et al., 1993). PKC-regulated
-APP cleavage does not require changes in the phosphorylation
state of the
-APP cytoplasmic tail (da Cruz e Silva et
al., 1993), suggesting that one or more molecules of the
-APP
trafficking and processing apparatus are PKC substrate phosphoproteins
involved in the mechanism by which PKC regulates
-APP cleavage.
Most -APP resides intracellularly, codistributing with TGN38, a
marker of the trans-Golgi network (TGN) (Caporaso et
al., 1994). Thus, it seemed possible that PKC might exert its
actions on regulated
-APP cleavage by redistributing
-APP out
of its usual residence in the TGN and toward post-TGN compartments
where it can undergo processing. This possibility is supported by
studies demonstrating stimulation by phorbol esters of the release of
glycosaminoglycans, intraluminal molecules of the constitutive
secretory pathway (De Matteis et al., 1993; Ohashi and
Huttner, 1994). Therefore, we have tested the possibility that an
important component of regulated
-APP cleavage is PKC-stimulated
formation from TGN of constitutive secretory vesicles containing and
transporting mature
-APP.
Quantification and
pairwise analyses were carried out using a Bio-Rad phosphor imaging
system (Molecular Analyst version 2.0 software). For each
experiment, the level of budding observed in the presence of an aliquot
of a standard preparation of cytosol was taken as 1 arbitrary unit of
budding efficiency, and other levels within a given experiment were
normalized to this value.
Figure 1:
PKC
activation in intact PC12 cells leads to redistribution of
holo--APP from a Golgi-rich fraction to s
-APP in conditioned
culture medium. Cells were metabolically labeled with
[
S]sulfate, followed by analysis of
-APP in
a Golgi-rich membrane fraction from the cells and of s
-APP in the
culture medium. The autoradiograms are from one of two experiments,
which yielded virtually identical results.
Cytosol stimulated the formation of nascent
vesicles containing -APP (Fig. 2, autoradiogram, lane1versuslane 6; Fig. 3A, lane1versuslane 2). This stimulatory effect was mimicked by purified
PKC (Woodgett and Hunter, 1987), with a half-maximal effect at 9
µg/ml and a maximal effect at 25 µg/ml (Fig. 2, graph; Fig. 3A, lane1versuslane 6). In the presence of cytosol, the
addition of PDBu increased vesicle budding, presumably due to
activation of endogenous PKC (Fig. 3A, lane2versuslane5). The rate of
vesicle budding observed in the presence of cytosol alone or of cytosol
plus PDBu/PKC was greatly reduced in the presence of PKC inhibitor
peptide-(19-36) (House and Kemp, 1987) (Fig. 3A, lane2versuslane 3, lane 7versuslane 8). Cytosol caused a significant
increase in vesicle budding from the TGN even in the presence of an
optimally effective amount of PDBu/PKC (Fig. 3A, lane6versuslane7),
suggesting the presence of a cytosolic factor(s) in addition to PKC. A
similar additional stimulatory effect of cytosol was observed when
cytosol was added to stripped Golgi membranes (see below) in the
presence of optimally effective PDBu/PKC (not shown). The effect of
cytosol alone was abolished in the absence of an energy-regenerating
system or when incubation was carried out at 20 °C, a temperature
that blocks TGN exit (Fig. 3B).
Figure 2:
Stimulation of vesicle budding from TGN
by cytosol and by purified PKC. A TGN-rich fraction derived from
[S]sulfate-labeled cells was incubated under
standard conditions in the presence of cytosol or of various
concentrations of purified PKC followed by analysis of vesicle budding
(see ``Materials and Methods'').
Figure 3:
Regulation of vesicle budding from TGN. A, effect of cytosol (1 mg/ml), PDBu (1 µM), PKC
(25 µg/ml), and PKC inhibitor peptide-(19-36) (200
µM) on vesicle budding. B, reduction of vesicle
budding by removal of energy regenerating system (-ERS),
incubation at 20 °C, or addition of either GTPS (30
µM) or (AlF
)
(80
µM Al
plus 6 mM F
). Means ± S.E. for three experiments
are shown. asterisk, different from cytosol alone (p < 0.01); dagger, different from PDBu/PKC alone (p < 0.01).
Figure 4:
PKC stimulates vesicle budding from washed
TGN-rich membranes. TGN fractions derived from
[S]sulfate-labeled cells were washed with low
(control) or high salt and analyzed for vesicle budding in the absence
or presence of PDBu (1 µM)/purified PKC (25 µg/ml) or
cytosol (1 mg/ml).
In Alzheimer
patients of the Swedish familial AD type, there is an abnormally low
ratio of processing of -APP via the non-amyloidogenic s
-APP
pathway relative to the amyloidogenic A
pathway (Felsenstein et al., 1994a). This ratio is normalized by activation of PKC,
which enhances processing via the non-amyloidogenic pathway while
decreasing processing via the amyloidogenic pathway (Felsenstein et
al., 1994b; Citron et al., 1994). The present study
reveals one cellular mechanism by which PKC produces these effects. In
addition, the recent discovery that a major familial AD gene
(Sherrington et al., 1995) encodes a protein homologous to the Caenorhabditis elegans sperm molecule spe4, which
plays a role in membrane protein sorting (L'Hernault and
Arduengo, 1992), raises the possibility that missorting of
-APP
may contribute to some forms of AD. A more complete understanding of
the molecules that control
-APP trafficking and processing events,
as well as an understanding of how these molecules are regulated,
should lead to new insights into the etiology, pathogenesis, and
therapy of AD.
Note Added in Proof-After this study was finished, additional evidence supporting the existence of a role for PKC in the regulation of TGN vesicle formation was described by Buccione et al. (R. Buccione, S. Bannykh, I. Santone, M. Baldassarre, F. Facchiano, Y. Bozzi, G. Di Tullio, A. Mironov, A. Luini, and M. De Matteis, submitted for publication) as well as by Simon et al. (Simon, J.-P., Ivanov, I. E., Shopsin, B., Hersh, D., Adesnik, M., and Sabatini, D. D. (1995) Cold Spring Harbor Symp. Quant. Biol.60, 222).