Differential involvement of protein kinase C alpha and epsilonin the regulated secretion of soluble amyloid precursor protein Cristina Lanni, Michela Mazzucchelli, Emanuela Porrello, Ste
Trang 1Differential 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 kinase C
(PKC) isoforms in the regulated proteolytic release of
soluble amyloid precursor protein (sAPPa) in SH-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 localization of PKCa to the plasma membrane
and PKCe to Golgi-like structures Reduced expression of PKCa significantly impaired the secretion of sAPPa induced
by treatment with phorbol esters Treatment of PKCa-deficient cells with carbachol induced a significant release of sAPPa These results suggest that the involvement of PKCa
in carbachol-induced sAPPa release is negligible The response to carbachol is instead completely blocked in PKCe-deficient cells suggesting the importance of PKCe in coupling cholinergic receptors with APP metabolism Keywords: Alzheimer’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),
derived from proteolytic processing of a larger precursor
called amyloid precursor protein (APP) [1] APP is
meta-bolized by several alternative pathways: in the secretory
pathway, it is cleaved extracellularly within the Ab domain
by a-secretase to generate a soluble nonamyloidogenic
fragment of APP (sAPPa) that is secreted in the conditioned
medium of cell cultures, human plasma and in the
cerebrospinal fluid Other enzymes, b- and c-secretase,
cleave APP at the N and C termini of Ab, respectively,
releasing the amyloidogenic peptide [2,3]
APP processing by a-secretase occurs via a constitutive
pathway and by receptor-mediated activation of multiple
signal trasduction pathways among which protein kinase C
(PKC) is a major player
PKC is a family of at least 12 isoenzymes of serine/
threonine protein kinases, central to many signal
transduc-tion pathways [4] Although these isoenzymes share a
similar structural domain organization, differences in their
substrate specificity, cofactor requirements, tissue and
cellular distribution, and subcellular localization suggest
that each of the different PKC isoenzymes plays a specific
and distinct regulatory role in cellular signal transduction [4–8]
The role of individual PKC isoforms in the regulation of APP proteolytic processing is not yet understood Recently
we demonstrated that PKCa was specifically involved in phorbol ester-induced sAPPa release [9], further supporting
a series of reports that pointed to a specific role for PKCa
in APP processing in vitro (for review see [2]), and most recently also in vivo [10,11] where constitutive overactivation
of PKCa and 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 regulated 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 extensively studied Ca2+ -independent isoenzymes of the PKC family PKCe may participate in the regulation of diverse functions in cells of various origin, including the modulation of 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 structural organization has been conserved in orthologous genes from yeast to mammals However, in mammals, PKCe has acquired short sequence motifs in the 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 aim of our study was to characterize and differentiate the role of PKCa and PKCe in the regulated secretion of
Correspondence to M Racchi, Department of Experimental and
Applied Pharmacology, Viale Taramelli 14, 27100, Pavia, Italy.
Fax: +39 0382507405, Tel.: +39 0382507738,
E-mail: racchi@unipv.it
Abbreviations: Ab, beta-amyloid; AD, Alzheimer’s disease; APP,
amyloid precursor protein; PKC, protein kinase C; PMA, 4b-phorbol
12-myristate 13-acetate.
Note: C Lanni and M Mazzucchelli contributed equally to this work.
(Received 2 March 2004, revised 6 May 2004, accepted 1 June 2004)
Trang 2sAPPa We have therefore studied the direct and
receptor-mediated activation of PKC in a cellular model of
downregulation of PKCe and/or PKCa to understand the
respective roles of these specific isoforms of PKC in the
activation of APP proteolytic processing
Materials and methods
Materials
All culture media, supplements and foetal calf serum were
from Gibco Life Technologies Electrophoresis reagents
were from Bio-Rad All other reagents were of the highest
grade available and were purchased from Sigma Chemical
Co unless otherwise indicated 4b-Phorbol 12-myristate
13-acetate (PMA), PD98059 (Alexis Biochemicals, San
Diego, CA, USA) were dissolved in dimethyl sulfoxide and
stored at)20 C Stocks were diluted in serum-free medium
before the experiments Carbachol was dissolved and
diluted to working concentration in serum-free minimum
essential medium (MEM) at the moment of use
Cell cultures and experimental treatments
SH-SY5Y neuroblastoma cells were cultured in Eagle’s
MEM supplemented with 10% foetal bovine serum,
penicillin/streptomycin, nonessential amino acids and
sodium pyruvate (1 mM) at 37C in 5% CO2/95% air
The cell line with stable 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 selecting agent G418 (Gibco Life
Technol-ogies) at 400 lgÆmL)1 For the experiments, 4· 106 cells
were seeded in 60-mm dishes and cultured for 48 h Prior to
the experiment confluent monolayers of cells were washed
twice with NaCl/Pi 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 with incubation for 2 h at
37C 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 was collected after 2 h of incubation
and centrifuged at 13 000 g for 5 min to remove detached
cells and debris Proteins in the medium were precipitated
quantitatively by the deoxycholate/trichloroacetic acid
pro-cedure as described previously [26] Cell monolayers were
washed twice with ice-cold NaCl/Piand lysed on the tissue
culture dish by addition of ice-cold lysis buffer (50 mMTris/
HCl pH 7.5, 150 mMNaCl, 5 mMEDTA, 1% Triton
X-100) An aliquot of the cell lysate was used for protein
analysis with the Bio-Rad Bradford kit for protein
quan-tification Normalization of protein loading on each blot
was obtained by loading a volume of sample of conditioned
medium standardized to total cell lysate protein
concentra-tion Proteins were subjected to SDS/PAGE (10%) and
then transferred onto poly(vinylidene difluoride) (PVDF)
membrane (DuPont NEN) The membrane was blocked for
1 h with 10% nonfat dry milk in Tris-buffered saline containing 1% Tween-20 For the detection of sAPPa, membranes were immunoblotted with the antibody 6E10 (Chemicon-Prodotti Gianni, Milan, Italy) Detection was carried out by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (Kirkegaard and Perry Laboratories, Gaithersburgh, MD, USA) for 1 h as secon-dary antibody The blots were then washed extensively and sAPPa visualized using an enhanced chemiluminescent methods (Pierce, Rockford, IL, USA) For the detection of PKC, cells were homogenized in a buffer containing 20 mM Tris/HCl pH 7.5, 2 mMEDTA, 0.2 mM phenylmethylsulfo-nyl fluoride, 20 lgÆmL)1leupeptin, 25 lgÆmL)1aprotinin and 0.5% Triton X-100 Proteins were measured as described earlier and subjected to Western blot analysis with the method indicated previously using isoform-specific mAb from Transduction Laboratories (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 mM Tris/HCl pH 6.8, 2% SDS, 10% glycerol, 50 mM dithio-threitol, 0.1% Bromphenol blue) Cells lysates were boiled for 5 min and then centrifuged at 10 000 g at room temperature for 5 min; then 25 lL of the lysate were separated by SDS/PAGE on 10% acrylamide and proteins subjected to electrophoretic transfer to PVDF membranes Blots were probed with either a rabbit polyclonal antibody specific for ERK (p44/p42 MAP kinase) (New England Biolabs) or a mAb for phosphorylated ERK (phospho-44/
42 MAP kinase) (Upstate Biotec Inc., Lake Placid, NY, USA), and developed by chemiluminescence following incubation with the appropriate horseradish-peroxidase conjugated secondary antibody
Immunocytochemical analysis of PKC translocation SH-SY5Y neuroblastoma cells were seeded on glass cover-slips at a density of 5· 105viable cells per well in a 24-well plate Cells on coverslips were treated with PMA 100 nMin Krebs buffer for 5 or 15 min, whereas control cells were incubated with vehicle (dimethyl sulfoxide) alone in Krebs buffer for 5 min
After treatment, cells were fixed in ethanol 70% at )20 C, washed with NaCl/Piand permeabilized for 15 min
at room temperature with 0.01% Triton X-100 in NaCl/Pi
To quench endogenous peroxidase activity, cells were treated with NaCl/Pi containing 3% hydrogen peroxide and 10% methanol for 15 min; nonspecific binding with PKCa and PKCe was blocked by incubation for 30 min with NaCl/Picontaining 1% BSA Cells were incubated for
1 h with antibodies specific for PKCa or PKCe, diluted
1 : 50 in NaCl/Pi/1% BSA solution Cells were washed with NaCl/Piand then incubated for 1 h at room temperature with an antirabbit IgG antibody conjugated with fluorescein isothiocyanate (FITC; Calbiochem, Inalco S.p.A., Milan, Italy) diluted 1 : 4500 in NaCl/Pi/1% BSA After the
Trang 3fluorescent labelling procedures, cells were finally
counter-stained for DNA with for 5 min with a 0.1 lgÆmL)1
HOECST 33342 solution in NaCl/Pi, 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 of the NIH IMAGE
1.47 program (Wayne Rasband, NIH, Research Services
Branch, NIMH, Bethesda, MD, USA), the relative densities
of the bands were expressed as arbitrary units and
normalized to data obtained from control sample run
under the same conditions Controls were processed in
parallel with stimulated samples and always included in the
same blot Preliminary experiments with serial diluitions of
secreted protein allowed determination of optimal linear
range for chemiluminescence reaction Data were analysed
using the analysis of variance test followed, when significant,
by an appropriate post hoc comparison such as the
Dunnett’s or Student’s t-test; a P value < 0.05 was
considered significant The data reported are expressed as
mean ± SD of at least three independent experiments
Results
SH-SY5Y human neuroblastoma cells spontaneously
express M1 and M3 muscarinic receptors, making them
particularly suitable for the characterization of
PKC-dependent and receptor-mediated APP metabolism Besides
the parental SH-SY5Y cells (SY-wt), we obtained a cell line
transfected with an expression plasmid containing PKCa
antisense cDNA (SYa4) and a cell line transfected with an
expression plasmid containing PKCe antisense cDNA
(SYDe) [27] Western blot analysis showed that in SYa4
neuroblastoma cells, the expression of PKCa
immuno-reactivity is significantly reduced ()66.4% ± 3.4; mean ± SD of triplicate samples) compared to the 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 expression of PKCe ()69.4% ± 10.7; mean ± SD of triplicate samples), compared to the parental cell line (Fig 1B) No differences were found in the expression of PKCd, bI and bII; 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 to 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 evident also in the immunocytochemical images (Fig 2D) the phorbol ester stimulation contributes to the translocation of the residual immunoreactive PKCa to the same plasma mem-brane compartment as shown in the parental cell line (Fig 2E,F) The translocation of PKCe was followed in SYDe cells in comparison with the parental SYwt cells PKCe isoform appears to translocate, following challenge with phorbol ester, to Golgi-like structures (Fig 3B,C,E,F), consistent with its putative role as regulator of Golgi functions [28] As discussed before for PKCa, the reduced expression of PKCe in SYDe cells, did not modify the ability
of the residual kinase to translocate to the same intracellular compartments
Fig 1 Evaluation by Western blotting of the expression of PKC isoforms in SYa4 and SYDe
as compared to that in SYwt neuroblastoma cells Cell lysate proteins were probed with mouse anti-PKC mAb Samples of rat cere-bellum homogenate were included as positive controls and for molecular size identification (data not shown) (A) Comparison of the pattern of expression of PKC in SYa4 to that
in SYwt PKCa is the only isoform with re-duced expression while PKCe, d, bI and bII show no differences (B) In SYDe, in addition
to the expected reduced expression of PKCe, reduced expression of PKCa was also ob-served, with no changes in the other isoforms
of the kinase Tubulin Western blot images are included as loading controls.
Trang 4We demonstrated previously that downregulation of
PKCa significantly affects PMA-induced sAPPa release [9],
without affecting carbachol-regulated APP processing We
now evaluated how downregulation of PKCe may affect the
processing of APP Parental SYwt and SYDe cells were treated with increasing concentrations of PMA (10 nM–
1 lM) for 2 h and sAPPa was measured in conditioned medium by Western blot As shown in Fig 4, SYwt
Fig 2 Fluorescence micrographs of 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 Fluorescence micrographs of SYwt and SYDe 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).
Trang 5stimulated with PMA, showed a significant increase in
sAPPa release compared to basal levels and reached a
maximum of approximately threefold increase at 100 nM
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 SYa4 cells
[9] and may be due not only to PKCe downregulation 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
release of sAPPa In our previous experiments, as well as
in the current set of data, in spite of reduced expression of
PKCa, SYa4 cells demonstrated a complete response to
carbachol stimulation in terms of sAPPa release [9] (Fig 5)
suggesting that the defective isoform was not involved in the
receptor-mediated activation of APP processing
Treatment of SYDe with increasing concentrations of
carbachol did not elicit a significant release of sAPPa, in
contrast to parallel experiments conducted on SYwt and
SYa4 cells which responded to carbachol with a
concen-tration-dependent increase in sAPPa release with a
maxi-mally effective concentration of 1 mM(Fig 5) The Western
blot inset in Fig 5 is an example showing the complete lack
of response to carbachol of SYDe cells
As the pathway downstream of muscarinic receptors is
complex and involves the activation of ERKs and the
MAP-kinase pathway we studied whether the
downregula-tion of PKCe influences the activadownregula-tion of MEK in our
cellular models We previously demonstrated that in
SH-SY5Y cells the activation of the MAP-kinase pathway
is not significantly involved in the carbachol-regulated sAPPa release and it is not affected by PKCa downregu-lation [9] Similarly the treatment of SYDe cells with carbachol for 10 min resulted in a significant increase in the phosphorylation of Erk-1 and Erk-2 (Fig 6) in a way quantitatively similar to that of the parental SYwt cell line
In addition stimulation of Erks phosphorylation was inhibited by PD-98059 in both cell lines
Fig 4 Secretion of sAPPa following PMA treatment of SYwt, SYa4
and SYDe neuroblastoma cells Incubation of the cells for 2 h in the
presence of increasing concentrations of PMA (10 n M , 100 n M , 1 l M )
was followed by Western blot of proteins collected from the
condi-tioned media Data are expressed as percentage of basal release 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 SYDe neuroblastoma cells Incubation of the cells for 2 h in the presence of increasing concentrations of carbachol (10 l M , 100 l M ,
1 m M ) was followed by Western blot of proteins collected from the conditioned media Data are expressed as percentage of basal release and are representative of three to four independent experiments.
*P < 0.05 compared to the same data for SYwt cells The inset Western blot represent an example of the pattern of sAPPa release obtained by treatment with carbachol 1 m M
Fig 6 Erk1/Erk2 phosphorylation following treatment with carabachol
in SYwt and SYDe cells As indicated by equal activation of Erk1/Erk2 phosphorylation the MAP-kinase pathway is not affected by PKCe and PKCa downregulation Cells were preincubated overnight with serum-free MEM and then treated for 10 min with carbachol (1 m M ) following a 30-min pretreatment with vehicle or PD 98059 (50 l M ) Cell lysates were collected as indicated in Materials and methods and probed on a Western blot with phospho-specific antibodies (upper panel) and antibodies to Erk1/Erk2 (lower panel) to correct for protein loading.
Trang 6Here we demonstrate that PKCe is specifically involved
in carbachol-mediated activation of sAPPa release in
SH-SY5Y cells Our goal was to demonstrate the
differen-tial involvement of PKC isoforms in APP processing
resulting either from direct activation of PKC by phorbol
esters or by indirect receptor-mediated activation
It is known that among multiple signal transduction
molecules, different isoforms of PKC may be involved and
can specifically contribute to the complex regulation of APP
metabolism Many of the signal transduction mechanisms,
neurotransmitter receptors and other receptor ligands
involved in APP processing regulation, have been described
as defective in AD [29] and in some cases these defects have
been associated with aberrant APP metabolism [26,30–32]
PKC was one of the first signal transduction-related
molecules to 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 full complexity of the
system, yet it is interesting to note that defective PKC is one
of the most consistent findings in AD brain and 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 expressing the cDNA for PKCa in the antisense
orientation (SYa4) We have shown that the pattern of
response to phorbol ester shown 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 bindsupport-ing 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
shown in SYa4 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
because of common overlapping sequences The significant
downregulation of the two isoforms is however, sufficient to
abolish completely the effect of phorbol esters on sAPPa
release in spite of the presence of unchanged levels of two
other Ca2+-dependent PKC isoforms, PKC bI and bII, and
at least one Ca2+-independent isoform, PKCd
The downstream effect of different PKC isoforms is often
dependent upon redistribution of the kinase to specific
intracellular compartments Inactive cytosolic-resident
pro-tein kinases may be recruited to perform distinct functions
based on the localization signals that they have received,
and their microenvironment at the time of activation In this
study we show a different PMA-induced redistribution of
PKCa and PKCe isoforms in SH-SY5Y cells While PKCa
translocated from the cytosolic compartment to the plasma
membrane, PKCe translocation was evident from the
cytosolic fraction to Golgi-like structures as early as 5 min
after PMA treatment This data is interesting and is
consistent with reports that suggested that PKCe may be involved in regulating Golgi-related processes [28] Lehel
et al demonstrated in NIH-3T3 cells that the zinc-finger domain of PKCe was found to contain all of the informa-tion necessary for exclusive localizainforma-tion to the Golgi network and that both the holoenzyme and its zinc finger region modulate Golgi function It is interesting also to observe that a different translocation and redistribution of 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 regulated a-secretase APP cleavage can occur in the TGN by specific detection of TGN resident a-secretase activity following PKC activation
In addition to the reports suggesting a signifcant role for PKCa in phorbol ester regulated sAPPa release a number of reports in the literature indicate that PKCe is equally, if not exclusively, involved Kinouchi et al showed initially that
an increased release of sAPPa 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 include the completely physiological machinery for APP processing Finally, expression of a peptide inhibitor of PKCe resulted in the inhibition of phorbol ester-induced sAPPa release [14] It is worth mentioning that the involvement of PKCa in these experiments has been ruled out because of the lack of inhibition by Go¨6976, which is a specific inhibitor of Ca2+-dependent isoforms In our hands the inhibitor Go¨6976 can always block the phorbol ester induced sAPPa release to a significant extent [9,36] yet it was
of particular interest that Go¨6976 did not block the carbachol-mediated release of sAPPa in SYwt and SYa4 cells [9], an indication that while PKCa was clearly involved
in phorbol ester-mediated APP processing it was not necessary for receptor-mediated activation of sAPPa release The stimulation of G-protein coupled receptors by neurotransmitters can regulate APP processing by PKC-dependent signalling pathways In our cell system, as in others in the literature, the cholinergic receptor stimulation
of sAPPa release can be blocked by GF109203X [9,37,38] suggesting clear involvement of PKC-dependent mechanism although not related to Ca2+-dependent isoforms of the kinase Our experiments show that response to carbachol is completely blocked in SYDe, clearly indicating that PKCe may play a crucial role in receptor-mediated sAPPa release This suggestion is consistent with data in the literature that indicate PKCe as the only protein kinase isoform involved
in the signalling pathway downstream of muscarinic m3 receptors in SK-N-BE(C) neuroblastoma cells [15] The
Trang 7signalling pathways downstream of muscarinic receptors
involve both PKC-dependent and -independent
mecha-nisms coupled to the activation of the MAP-kinase pathway
[39] It was shown that MAP-kinase activation can 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 PKCa [9] and PKCe do not modify the possibility to
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 fact that sAPPa release following treatment with
carbachol is completely blocked in 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 in the response to direct activation
of the kinase and the latter being involved exclusively in
muscarinic receptor regulated sAPPa release, a role possibly
extended to other G-protein coupled receptors
Acknowledgements
We are grateful to Dr Thomas B Shea of the McLean Hospital,
Boston, MA, USA for the gift of SYa4 and SYDe cells This work was
made possible through grants from the Italian MIUR (prot #
2003057355–2003 and prot # MM05221899–2000 to S G.), from the
University of Pavia (FAR 2003 to M R.; Progetto Giovani Ricercatori
to M M., C L and E P.) and from the Italian Ministry of Health
(Progetto Finalizz Alzheimer to S G and to M R.).
References
1 Hardy, J & Selkoe, D.J (2002) The amyloid hypothesis of
Alz-heimer’s disease: progress and problems on the road to
thera-peutics Science 297, 353–356.
2 Racchi, M & Govoni, S (2003) The pharmacology of amyloid
precursor protein processing Exp Gerontol 38, 145–157.
3 Racchi, M & Govoni, S (1999) Rationalizing a pharmacological
intervention on the amyloid precursor protein metabolism Trends
Pharmacol Sci 20, 418–423.
4 Nishizuka, Y (1995) Protein kinase C and lipid signaling for
sustained cellular responses FASEB J 9, 484–496.
5 Dekker, L.V & Parker, P.J (1994) Protein kinase C-a question of
specificity Trends Biochem Sci 19, 73–77.
6 Kampfer, S., Uberall, F., Giselbrecht, S., Hellbert, K., Baier, G &
Grunicke, H.H (1998) Characterization of PKC isozyme specific
functions in cellular signaling Adv Enzyme Regul 38, 35–48.
7 Csukai, M & Mochly-Rosen, D (1999) Pharmacologic
modula-tion of protein kinase C isozymes: the role of RACKs and
sub-cellular localisation Pharmacol Res 39, 253–259.
8 Kanashiro, C.A & Khalil, R.A (1998) Signal transduction by
protein kinase C in mammalian cells Clin Exp Pharmacol.
Physiol 25, 974–985.
9 Racchi, M., Mazzucchelli, M., Pascale, A., Sironi, M & Govoni,
S (2003) Role of protein kinase C alpha in the regulated secretion
of the amyloid precursor protein Mol 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) Constitutive overactivation of protein kinase 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., Ohno, S., Ishiura, S & Suzuki, K (1995) Conventional protein kinase C (PKC)-alpha and novel PKC epsilon, but not -delta, increase the secretion of an N-terminal fragment of Alzheimer’s disease amy-loid precursor protein from PKC cDNA transfected 3Y1 fibro-blasts FEBS Lett 364, 203–206.
13 Yeon, S.W., Jung, M.W., Ha, M.J., Kim, S.U., Huh, K., Savage, M.J., Masliah, E & Mook-Jung, I (2001) Blockade of PKC epsilon activation attenuates phorbol ester-induced increase of alpha-secretase-derived secreted form of amyloid precursor pro-tein Biochem Biophys 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 alpha-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., Valasek, K.M., Iversen, P.W., Burns, D.J & Birch, K.A (1996) Protein kinase C isozymes dif-ferentially regulate promoters containing PEA-3/12-O-tetradeca-noylphorbol-13-acetate response element motifs J Biol Chem.
271, 21666–21671.
17 Cacace, A.M., Ueffing, M., Philipp, A., Han, E.K., Kolch, W & Weinstein, I.B (1996) PKC epsilon functions as an oncogene
by enhancing activation of the Raf kinase Oncogene 13, 2517– 2526.
18 Mischak, H., Goodnight, J.A., Kolch W., Martiny-Baron G., Schaechtle, C., Kazanietz, M.G., Blumberg, P.M., Pierce, J.H & Mushinski, J.F (1993) Overexpression of protein kinase C-delta and -epsilon in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity J Biol Chem 268, 6090–6096.
19 Perletti, G., Tessitore, L., Sesca, E., Pani, P., Dianzani, M.U & Piccinini, F (1996) Epsilon PKC acts like a marker of progressive malignancy in rat liver, but fails to enhance tumorigenesis in rat hepatoma cells in culture Biochem Biophys Res Commun 221, 688–691.
20 Chun, J.S., Ha, M.J & Jacobson, B.S (1996) Differential trans-location of protein kinase C epsilon during HeLa cell adhesion to a gelatin substratum J Biol Chem 271, 13008–13012.
21 Fagerstrom, S., Pahlman, S., Gestblom, C & Nanberg, E (1996) Protein kinase C-epsilon is implicated in neurite outgrowth in differentiating human neuroblastoma cells Cell Growth Differ 7, 775–785.
22 Horowitz, A., Clement-Chomienne, O., Walsh, M.P & Morgan, K.G (1996) Epsilon-isoenzyme of protein kinase C induces a Ca (2+)-independent contraction in vascular smooth muscle Am J Physiol 271, 589–594.
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 Cell Biol 132, 77–90.
25 Sossin, W.S., Diaz-Arrastia, R & Schwartz, J.H (1993) Char-acterization of two isoforms of protein kinase C in the nervous system of Aplysia californica J Biol Chem 268, 5763–5768.
Trang 826 Bergamaschi, S., Binetti, G., Govoni, S., Wetsel, W.C., Battaini,
F., Trabucchi, M & Racchi, M (1995) Defective 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 participate in regulation of
tau steady-state levels and generation of certain Alzheimer-like
phospho-epitopes Int J Dev Neurosci 15, 295–307.
28 Lehel, C., Olah, Z., Jakab, G & Anderson, W.B (1995) Protein
kinase C epsilon is localized to the Golgi via its zinc-finger domain
and modulates Golgi function Proc Natl Acad Sci USA 92,
1406–1410.
29 Gasparini, L., Racchi, M., Binetti, G., Trabucchi, 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.,
Racchi, M., Lannfelt, L & Cowburn, R.F (1999) Protein kinase
C and amyloid precursor protein processing in skin fibroblasts
from sporadic and familial Alzheimer’s disease cases Biochim.
Biophys Acta 1453, 341–350.
31 Govoni, S., Racchi, M., Bergamaschi, S., Trabucchi, M., Battaini,
F., Bianchetti, A & Binetti, G (1996) Defective protein kinase C
alpha leads to impaired secretion of soluble beta-amyloid
pre-cursor protein from Alzheimer’s disease fibroblasts Ann NY
Acad Sci 777, 332–337.
32 Govoni, S., Bergamaschi, S., Racchi, M., Battaini, F., Binetti, G.,
Bianchetti, A & Trabucchi, M (1993) Cytosol protein kinase C
downregulation in fibroblasts from Alzheimer’s disease patients Neurology 43, 2581–2586.
33 Pascale, A., Govoni, S & Battaini, F (1998) Age-related altera-tion of PKC, a key enzyme in memory processes: physiological and pathological examples Mol Neurobiol 16, 49–62.
34 Xu, H., Greengard, P & Gandy, S (1995) Regulated formation of Golgi secretory vesicles containing Alzheimer beta-amyloid pre-cursor protein J Biol Chem 270, 23243–23245.
35 Skovronsky, D.M., Moore, D.B., Milla, M.E., Doms, R.W., Lee
& V.M (2000) Protein kinase C-dependent alpha-secretase com-petes with beta-secretase for cleavage of amyloid-beta precursor protein in the trans-golgi network J Biol Chem 275, 2568–2575.
36 Benussi, L., Govoni, S., Gasparini, L., Binetti, G., Trabucchi, M., Bianchetti, A & Racchi, M (1998) Specific role for protein kinase
C alpha in the constitutive and regulated secretion of amyloid precursor protein in human skin fibroblasts Neurosci Lett 240, 97–101.
37 Slack, B.E., Breu, J., Petryniak, M.A., Srivastava, K & Wurtman, R.J (1995) Tyrosine phosphorylation-dependent stimulation of 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 receptor is coupled to mitogen-activated protein kinase via protein kinase C and epidermal growth factor receptor kinase Biochem J 348, 381–387.