Differential involvement of protein kinase C alpha and epsilon
in the regulated secretion of soluble amyloid precursor protein
Cristina Lanni, Michela Mazzucchelli, Emanuela Porrello, Stefano Govoni and Marco Racchi
Department of Experimental and Applied Pharmacology, Centre of Excellence in Applied Biology and School of Pharmacy,
University of Pavia, Viale Taramelli 14, 27100 Pavia, Italy
We investigated the differential role of protein k inase C
(PKC) isoforms in the regulated proteolytic release of
soluble amyloid precursor protein (sAPPa)inSH-SY5Y
neuroblastoma cells. We used cells stably transfected with
cDNAs encoding either PKCa or PKCe in the antisense
orientation, producing a reduction of the expression of
PKCa and PKCe, respectively. Reduced expression of
PKCa and/or PKCe did not modify the response of the
kinase to phorbol ester stimulation, demonstrating translo-
cation of the respective isoforms from the cytosolic fraction
to specific intracellular compartments with an interesting
differential l ocalization of PKCa to the plasma me mbrane
and P KCe to Golgi-like structures. Reduced expression of
PKCa significantly impaired the secretion of s APPa induced
by treatment with phorbol esters. Treatment o f PKCa-
deficient cells with carbachol induced a s ignificant release of
sAPPa. T hese results s uggest that the i nvolvement of PKC a
in carbachol-induced sAPPa release is negligible. The
response to carbachol is instead completely blocked in
PKCe-deficient cells suggesting the importance o f PKCe in
coupling cholinergic receptors with APP metabolism.
Keywords: A lzheimer’s disease; cholinergic receptors; neuro-
blastoma; phorbol esters; signal transduction.
Alzheimer’s disease (AD), the most common type of
dementia, is characterized by deposition in the brain of
fibrillar aggregates of a peptide named beta-amyloid (Ab),

of PKCa an d PKCb isoforms in guinea pig brain were
shown to increase sAPPa production.
In this work we sought to differentiate the role played by
PKCa and PKCe in the r egulated processing of APP. There
is substantial evidence in the literature for a significant role
of PKCe both in the regulation of APP metabolism [11–14]
and in the pharmacology of muscarinic receptor signalling
[15]. PKCe is one of the most e xtensively studied Ca
2+
-
independent isoenzymes of the PKC family. PKCe may
participate in the regulation of diverse functions in cells of
various origin, including the modulation o f gene expression
[16], Raf-1 mitogenicity [17], neoplastic transformation
[18,19], cell adhesion [20], extension and maintenance of
motile cellular protrusions [21], contraction in smooth
muscle cells [22] and cardiomyocytes [23], and finally
secretory vesicle trafficking [24]. PKCe is a typical multi-
domain protein in which the overall struc tural organization
has been conserved in orthologous genes from yeast to
mammals. H owever, i n mammals, PKCe has acquired short
sequence m otifs i n t he regulatory N-terminal region that are
not evident in invertebrates (AplII of Aplysia and PKC
d98F of Drosophila [25]) and are postulated to function
as localization signals in the subcellular targeting of this
protein kinase.
The a im of our study was t o characterize and differentiate
theroleofPKCa and PKCe in the r egulated secretion o f
Correspondence to M. Racchi, Department of Experimental and
Applied Pharmacology, Viale Taramelli 14, 27100, P a via, Italy.

sodium pyruvate (1 m
M
)at37°Cin5%CO
2
/95% air.
The cell line with s table antisense downregulation of PKCe
was provided by T . B. Shea (McLean Hospital, Boston,
MA, USA) and was grown in the same medium with the
addition of the s electing agent G418 (Gibco Life Technol-
ogies) at 400 lgÆmL
)1
.Fortheexperiments,4· 10
6
cells
were seeded in 60-mm dishes and c ultured for 48 h. Prior to
the experiment confluent monolayers of cells were washed
twice with NaCl/P
i
and once with serum-free culture
medium. Experimental treatments for the detection of
sAPPa released into the conditioned medium were per-
formed in serum-free MEM w ith incubation for 2 h at
37 °C. Experiments for the detection of activated MEK
were performed with incubations of 10 min. In all experi-
ments involving the use of inhibitors such as PD98059, the
compounds were preincubated for 30 s prior to the addition
of PMA or carbachol.
Immunodetection of sAPPa and PKC
Conditioned medium w as collected after 2 h of incubation
and centrifuged at 13 000 g for5mintoremovedetached

sAPPa visualized using an enhanced chemiluminescent
methods ( Pierce, Rockford, IL, USA). For the detection of
PKC, cells were homogenized in a buffer containing 20 m
M
Tris/HCl pH 7.5, 2 m
M
EDTA, 0 .2 m
M
phenylmethylsulfo-
nyl fluoride, 20 lgÆmL
)1
leupeptin, 25 lgÆmL
)1
aprotinin
and 0.5% Triton X-100. Proteins were measured as
described earlier and subjected to Western blot analysis
with the method indicated previously using i soform-specific
mAb from Transduction Labo ratories (Lexington, KY,
USA) and from Santa Cruz Biotechnology.
Western blot for ERK phosphorylation
SH-SY5Y cells were cultured in serum-free medium over-
night before stimulation with agonists for 10 min with or
without 30 min of preincubation with PD 98059. After
stimulation, the cells were lysed in lysis buffer (62.5 m
M
Tris/HCl pH 6.8, 2% SDS, 10% glycerol, 50 m
M
dithio-
threitol, 0 .1% Bromphenol blue). Cells lysates were boiled
for 5 min and the n ce ntrifuged at 10 0 00 g at room

i
containing 3% h ydrogen peroxide
and 10% methanol for 15 min; nonspecific binding with
PKCa and PKCe was blocked by incubation for 30 min
with NaCl/P
i
containing 1% BSA. Cells were incubated for
1 h with antibodies specific for PKCa or PKCe, diluted
1:50inNaCl/P
i
/1% BSA s olution. Cells wer e washed with
NaCl/P
i
and then incubated for 1 h at room temperature
with an antirabbit I gG antibody conjugated with fluorescein
isothiocyanate (FITC; Calbiochem, Inalco S.p.A., Milan,
Italy) diluted 1 : 4500 in NaCl/Pi/1% BSA. After the
Ó FEBS 2004 Differential role of PKC isoforms in APP processing (Eur. J. Biochem. 271) 3069
fluorescent labelling proc edures, cells were finally counter-
stainedforDNAwithfor5minwitha0.1lgÆmL
)1
HOECST 33342 solution in NaCl/P
i
, and mounted upside
down on glass slides, in a drop of Mowiol (Calbiochem).
Images were obtained with a confocal microscope Leica
DM IRBE with a software Leica TCS SP.
Densitometry and statistics
Following acquisition of the Western blot image through an
AGFA scanner and analysis by means o f the

reactivity is significantly reduced ()66.4% ± 3.4;
mean ± SD o f t riplicate samples) compared to t h e parental
cell line (Fig. 1A). Differences were not observed in the
expression of PKCd, bI, bII and e isoforms between SY-wt
and SYa4 cells (Fig. 1A). Similarly, immunoblot analysis of
SYDe neuroblastoma cells showed a significant reduction in
the e xpre ssion of PKCe ()69.4% ± 10.7; mean ± SD of
triplicate samples), compared to the parental cell line
(Fig. 1 B). No differences were found in the expression of
PKCd, bIandbII; however, a decrease in the expression of
PKCa ()57.3% ± 15.5; mean ± SD of triplicate samples)
was observed (Fig. 1B).
Activation of PKC was determined by examining
translocation of cytosolic PKC t o a particulate membrane
fraction, because PKC activation involves a stable associ-
ation of PKC with membranes [4,7,8]. In order to show
also the subcellular compartment where translocation
takes place we subjected the cells to immunocytochemical
analysis and confocal microscopy. PKCa (Fig. 2 ) and
PKCe (Fig. 3) were detected predominantly in the cyto-
plasm of untreated cells; stimulation of the cells with
PMA induced a translocation of cytosolic PKCa to
structures probably corresponding to the plasma mem-
brane (Fig. 2B,C,E,F). Although the reduced immuno-
reactivity of PKCa in the SYa4 cells is e vident a lso i n the
immunocytochemical images (Fig. 2D) the phorbol ester
stimulation contributes to the translocation of the residual
immunoreactive PKCa tothesameplasmamem-
brane compartment as shown in the parental cell line
(Fig. 2 E,F). The translocation of PKCe was followed in

processing of APP. Parental SYwt and SYDe cells were
treated with increasing concentrations of PMA (10 n
M
–
1 l
M
)for2handsAPPa was measured in conditioned
medium by Western blot. As shown in Fig. 4, SYwt
Fig. 2. Fluorescence micrographs o f SYwt and SYa4 cells after treatment with PMA 100 n
M
for 5 or 15 min. FITC-immunolabelling for PKCa;
nuclear DNA was counterstained with Hoechst 33342 (magnification, · 63).
Fig. 3. Flu oresc ence micrographs of SYwt and SY De cells after treatment with PMA 100 n
M
for 5 or 15 min. FITC-immunolabelling for PKCe;
nuclear DNA was counterstained with Hoechst 33342 (magnification, · 63).
Ó FEBS 2004 Differential role of PKC isoforms in APP processing (Eur. J. Biochem. 271) 3071
stimulated with PMA, showed a significant increase in
sAPPa release compared to basal levels and reached a
maximum of approximately threefold increase at 1 00 n
M
PMA. In contrast SYDe showed a slight and not significant
increase in sAPPa release at all concentrations of PMA
tested. This pattern is similar to that observed in SY a4 cells
[9] and may be due not only to PKCe down regulation but
also to the fact that SYDe cells show reduced expression of
PKCa in addition to PKCe.
The cellular model of SH-SY5Y cells was chosen in
particular because of endogenous expression of muscarinic
receptors, the stimulation of which is coupled to increased

Fig. 4. Se cret ion of sAPP a following PM A treatment of SYwt, SYa4
and SYDe neuroblastoma cells. Incubation of the cells for 2 h in the
presence of in creasing con centrations of PMA (10 n
M
,100n
M
,1l
M
)
was followed by Western blot of proteins co llected from th e condi-
tioned media. Data are expressed as percentage of basal r elease and are
representative of three to four independent experiments. *P <0.05
compared to the same data for SYwt cells.
Fig. 5. Secretion of sAPPa following carbachol treatment in SYwt,
SYa4 and SY De neuroblastoma c ells. Incubation of the cells for 2 h in
the presence of increasing concentratio ns of carbachol (10 l
M
,100 l
M
,
1m
M
) was followed by Western blot of proteins collected from t he
conditio ned media. Data are exp ressed as percentage of basal relea se
and are representative of thre e to four in dependent e xperiments.
*P < 0 .05 compared to the same data for SYwt cells. The inset
Western b lot represent an example of the pattern of sAPP a release
obtained by treatment with carbachol 1 m
M
.

PKC was one o f the first signal transduction-related
molecules t o be implicated in the regulation of APP
metabolism [2,3] suggesting, in particular, that the nona-
myloidogenic a-secretase pathway is activated by PKC.
This simplification may not reflect the f ull c omplexity of t he
system, yet it is inte re sting to note t hat d efective PKC i s one
of the most consistent findings i n AD brain an d peripheral
tissues [29,33]. In fibroblasts from AD patients defective
APP metabolism is paralleled by a specific downregulation
of PKCa [26]. The same extent of protein expression
reduction was reproduced when using a neuroblastoma cell
line stably e xpressing the cDNA for PKCa in the antisense
orientation (SYa4). We have shown that the pattern of
response to phorbol ester s hown by the SYa4cell line [9] is
remarkably similar to that of AD fibroblasts [26], support-
ing the suggestion that the loss of a high-affinity bindin g site
for phorbol esters due to downregulation of PKCa reduces
the sensitivity of the cells to direct PKC activation. Higher
concentrations of PMA are necessary to elicit a significant
secretion of sAPPa in SYa4 cells, perhaps necessary for the
activation of Ca2
+
-independent PKCs such as PKCe.
In fact, the pattern of response to phorbol ester in a
neuroblastoma cell line stably expressing the cDNA for
PKCe in antisense orientation (SYDe) is different from that
showninSYa4 cells in that the response is completely
abolished. It should be noticed that in SYDe the antisense
strategy has resulted not only in reduced expression of
PKCe but also to reduced expression of PKCa, perhaps

PKCa and PKCe isoforms could be correlated to a
differential involvement in the regulation of APP process-
ing. In cell-free systems it has been shown that activation of
endogenous PKC increases formation from the trans-Golgi
network (TGN) of secretory vesicles containing APP,
suggesting a role for PKC in the regulation of secretory
vesicle formation [34]. Furthermore Skovronsky and col-
leagues [35] have shown that r egulated a-secretase APP
cleavage can occur in the TGN by specific detection of T GN
resident a-secretase a ctivity following PKC activation.
In addition to the reports suggesting a s ignifcant role for
PKCa in phorbol ester r egulated sAPP a release a number o f
reports in the literature indicate that PKCe is equally, if not
exclusively, involved. Kinouchi et al. showed initially that
an increased release of sAPP a could be induced by
overexpression of PKCe in 3Y1 cells. These results were
also obtained by overexpression of PKCa but not by
overexpression of PKCd [12]. Inhibition of PKCe was
instead obtained with strategies involving the overexpres-
sion of the PKCe V1 region, which binds specifically to the
receptor for activated C-kinase (RACK), blocking the
activation of the kinase specifically [13]. These experiments
resulted in a reduced release of sAPPa following phorbol
ester treatment; however, the data were obtained in B103
neuroblastoma cells overexpressing APP. Those cells
reportedly do not express endogenous APP and therefore
may not includ e the completely physiological machiner y for
APP p rocessing. F inally, expression of a peptide inhibitor of
PKCe resulted in the inhibition of phorbol ester-induced
sAPPa release [14]. It is worth mentioning that the

This suggestio n is consistent with data in the literature that
indicate PKCe as the only protein kinase isoform involved
in the s ignalling pathway downstream of muscarinic m3
receptors in SK-N-BE(C) neuroblasto ma cells [15]. The
Ó FEBS 2004 Differential role of PKC isoforms in APP processing (Eur. J. Biochem. 271) 3073
signalling pathways downstream of muscarinic receptors
involve both PKC-dependent and -independent mecha-
nisms c oupled to the activation o f the MAP-kinase pathway
[39]. It w as shown that MAP-kinase a ctivation c an be
obtained downstream of muscarinic receptors by a mech-
anism involving the activation of Src tyrosine kinase [39]
without involving PKC activity. The fact that downregula-
tion of PKC a [9] and PKCe do not modify the possibility t o
activate MAP-kinase following carbachol treatment is
consistent with the presence of a redundant signalling
pathway downstream of the cholinergic receptor. In addi-
tion, the f act that sAPPa release followin g treatment w ith
carbachol is completely blocked i n SYDe regardless of a full
activation of MAP-kinase demonstrates that the latter
signalling system is not involved in the carbachol-mediated
regulated processing of APP in these cells.
In summary, the results indicate that PKCa and PKCe
have differential roles in the regulation of APP processing
and sAPPa release in SH-SY5Y cells – the former being
involved predominantly i n the response to direct activation
of the kinase and the latter being involved exclusively in
muscarinic re ceptor r egulated sAP Pa release, a r ole possibly
extended to other G-protein coupled receptors.
Acknowledgements
We are grateful to Dr Thomas B. Shea of the McLean Hospital,

of the amyloid precursor protein. M ol. Psychiatry 8, 209–216.
10. Rossner, S., Mendla, K., Schliebs, R. & Bigl, V. (2001) Protein
kinase Calpha and beta1 isoforms are regulators of alpha-secre-
tory proteolytic processing of amyloid precursor protein in vivo.
Eur. J. Neurosci. 13, 1644–1648.
11. Rossner,S.,Beck,M.,Stahl,T.,Mendla,K.,Schliebs,R.&Bigl,
V. (2000) Constitutiv e overactivation of protein kina se C in guinea
pig brain increases alpha-secretory APP processing without
decreasing beta-amyloid generation. Eur. J. Neurosci. 12, 3191–
3200.
12. Kinouchi, T., Sorimachi, H., Maruyama, K ., Mizuno, K., O hno,
S., Ishiura, S . & Suz u ki, K. (1995) Conventional pr otein kinase C
(PKC)-alpha and novel PKC epsilon, but not -delta, increase the
secretion of an N-terminal fragment of A lzheimer’s disease amy-
loid precursor p rotein from PKC cDNA transfected 3Y1 fib ro-
blasts. FEBS Lett. 364, 203–206.
13. Yeon, S.W., Jung, M.W., Ha, M .J., Kim, S.U., Huh, K., Sav age,
M.J., Masliah, E. & Mook-Jung, I. (2001) Blockade of PKC
epsilon activation at tenuates p horbol este r-induced in crease of
alpha-secretase-derived secreted form of amyloid precursor p ro-
tein. Biochem. Bioph ys. Res. Comm. 280, 782–787.
14.Zhu,G.,Wang,D.,Lin,Y.H.,McMahon,T.,Koo,E.H.&
Messing, R.O. (2001) Protein kinase C epsilon suppresses Abeta
production and promotes activation of alp ha-secretase. Biochem.
Biophys. Res. Comm. 285, 997–1006.
15. Kim,J.Y.,Yang,M.S.,Oh,C.D.,Kim,K.T.,Ha,M.J.,Kang,
S.S. & Chun, J.S. (1999) Signalling pathway leading to an acti-
vation of mitogen-activated protein kinase by stimulating M3
muscarinic receptor. Biochem. J. 337, 275–280.
16. Reifel-Miller, A. E., Conarty, D.M., Valas ek, K.M., Iversen, P.W.,

23. Huang,X.P.,Pi,Y.,Lokuta,A.J.,Greaser,M.L.&Walker,J.W.
(1997) Arachidonic acid stimulates protein kinase C-epsilon
redistribution in heart cells. J. Cell Sci. 110, 1625–1634.
24. Prekeris, R., Mayhew, M.W., Cooper, J.B. & Terrian, D.M.
(1996) Identification and localization of an actin-binding motif
that is unique to the epsilon isoform of protein kinase C and
participates in the regulation of synaptic function. J. Ce ll Biol. 132,
77–90.
25. Sossin, W.S., Diaz-Arrastia, R. & Schwartz, J.H. (1993) Char-
acterization of tw o isoforms of protein kinase C in t he nervous
system of Aplysia californica. J. Biol. Chem. 268, 5763–5768.
3074 C. Lanni et al. (Eur. J. Biochem. 271) Ó FEBS 2004
26. Bergamaschi, S., Binetti, G., Govoni, S., Wetsel, W.C., Battaini,
F., Trabucchi, M. & Racchi, M . ( 1995) D efective phorbol ester-
stimulated secretion of beta-amyloid precursor protein from Alz-
heimer’s disease fibroblasts. Neurosci. Lett. 201,1–5.
27. Boyce, J.J. & Shea, T.B. (1997) Phosphorylation events mediated
by protein kinase C alpha and epsilon p articipate in regulation of
tau steady-state levels and generation of certain ÔAlzheimer-likeÕ
phospho-epitopes. Int. J. Dev Neurosci. 15, 295–307.
28. Lehel, C., Olah, Z., Jakab, G. & Anderson, W.B. (1995) Protein
kinase C e psilon is localized to the Golgi via i ts zinc-finger domain
and modulates Golgi function. Proc. Natl Acad. Sci. USA 92,
1406–1410.
29. Gasparini, L., Racchi, M., Binetti, G., Trabuc chi, M., Solerte,
S.B.,Alkon,D.,Etcheberrigaray, R., Gibson, G., Blass, J.,
Paoletti, R. & Govoni, S. (1998) Peripheral markers in testing
pathophysiological hypotheses and diagnosing Alzheimer’s dis-
ease. FASEB J. 12, 17–34.
30. Vestling, M., Cedazo-Minguez, A., Adem, A., Wiehager, B.,

amyloid precursor protein secretion by the m3 muscarinic acetyl-
choline receptor. J. Biol. Chem. 270, 8337–8344.
38.Racchi,M.,Sironi,M.,Caprera,A.,Konig,G.&Govoni,S.
(2001) Short- and long-term effect of acetylcholinesterase inhibi-
tion on the expression and metabolism of the amyloid precursor
protein. Mol. Psychiatry 6, 520–528.
39. Slack, B.E. (2000) The m3 muscarinic acetylcholine recept or is
coupled to mitogen-activated protein kinase v ia protein kinase C
and epidermal growth factor receptor kinase. Biochem. J. 348,
381–387.
Ó FEBS 2004 Differential role of PKC isoforms in APP processing (Eur. J. Biochem. 271) 3075