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), derived from p roteolytic 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 fragmentofAPP(sAPPa) that is secreted in the c onditioned medium of cell cultures, human plasma and in the cerebrospinal fluid. Other enzymes, b-andc-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 sign al transduc- tion pathways [4]. Although these isoenzymes share a similar structural domain organization, differences in their substrate specificity, cofactor requirements, tissue and cellular d istribution, and subcellular l ocalization s uggest that each of the different PKC isoenzymes p lays a specific and distinct regulatory role in cellular signal transduction [4–8]. The role of i ndividual 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 sAPP a release [9], further supporting a series of reports that pointed to a specific role for PKCa in APP p rocessing in v itro (for review see [2]), and most recently a lso in vivo [1 0,11] where constitutive overactivation 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. Fax: +39 0382507405, Tel.: +39 038250 7738, E-mail: racchi@unipv.it Abbreviations:Ab, beta-amyloid; AD, Alzheimer’s disease; APP, amyloid precursor protein; P K C, protein kinase C; P MA, 4b-phorbol 12-myristate 13-acetate. Note:C.LanniandM.Mazzucchellicontributed equ ally to this work. (Received 2 March 2004, revised 6 May 2004, accepted 1 June 2004) Eur. J. Biochem. 271, 3068–3075 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04240.x sAPPa. We have therefore studied the d irect and receptor- mediated activation of PKC in a cellular model of downregulation o f 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 c ulture media, supplements and foetal c alf serum were from Gibco Life T echnologies. Electrophoresis reagents were from Bio-Rad. All other reagents were of t he highest grade available and were purchased from Sigma Chemical Co. unless o therwise indicated. 4b-Phorbol 12-myristate 13-acetate (PMA), PD98059 (Alexis Biochemicals, San Diego, CA, USA) w ere dissolved in dimethyl sulfoxide and stored at )20 °C. Stock s 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 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 cells and debris. Proteins in the medium were p recipitated quantitatively b y t he deoxychol ate/trichloroacetic acid pro- cedure as described previously [26]. Cell monolayers were washed twice with ice-cold NaCl/P i and lysed on the tissue culture dish b y a ddition of ice -cold lysi s buffer ( 50 m M Tris/ HClpH7.5,150m M NaCl, 5 m M EDTA, 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 p rotein loading on each blot was obtained by load ing a volume o f sample of conditioned medium standardized to total cell lysate protein concentra- tion. Proteins were subjected to SDS/PAGE (10%) and then tran sferred 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 o f s APPa, 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 antibod y. 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 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 temperature for 5 min; then 25 lLofthelysatewere 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 a t a density of 5 · 10 5 viable cells per well in a 24-well plate. Cells on coverslips were treated with PMA 100 n M in Krebs buffer for 5 or 15 m in, whereas control cells were incubated w ith vehicle (dimethyl sulfoxide) a lone in Krebs buffer for 5 m in. After treatment, cells were fixed in ethanol 70% at )20 °C, washed with NaCl/P i and permeabilized for 15 min at room temperature with 0.01% Triton X-100 in NaCl/P i . To quench e ndogenous peroxidase activity, cells were treatedwithNaCl/P 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 NIH IMAGE 1.47 program (Wayne Rasband, NIH, Research Services Branch, N IMH, B ethesda, M D, 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. Pre liminary experiments with serial diluitions of secreted protein allowed determination of optimal linear range for chemiluminescen ce reaction. Data were analysed using t he analysis of variance test followed, when significant, by an appropriate post hoc comparison such as the Dunnett’s or Student’s t-t est; 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 M 1 and M 3 muscarinic receptors, making them particularly suitable for the characterization of PKC- dependent and r eceptor-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 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 SYDe cells in comparison with the parental SYwt cells. PKCe isoform appears to translocate, following challenge with phorbol ester, to ÔGolgi-lik eÕ 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, d id not modify the ability of the residual kinase t o t ranslocate to the same i ntracellular compartments. Fig. 1. Evaluation by Western blotting of the expression o f PKC isoforms in S Ya4 and SYDe as compared to that in SYwt neuroblastoma cells. Cell lysate proteins were p robed with mouse anti-PKC mAb. Samples of rat cere- bellum homogenate were included as positive controls and for molecular s ize identifi cation (data not shown). (A) Comparison of the pattern of e xpression o f PKC i n S Ya4tothat in SYwt. PKCa is the only iso form with re- duced expression while PKCe, d, bIandbII show no differences. ( B) In SYDe, in addition to the e xpected reduced expression of PKCe, reduced expression of PKCa was also ob- served, with no changes in the other isofo rms of the kinase. Tubulin Western blot images are included as loading controls. 3070 C. Lanni et al. (Eur. J. Biochem. 271) Ó FEBS 2004 We demonstrated previously that downregulation of PKCa significantly affects PMA-induced sAPPa release [9], without affecting carbachol-regulated APP processing. We now eval uated how downregulation o f P KCe may affect the 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 release of sAPPa. In our p revious 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 i soform 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 m M (Fig. 5). The W estern 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 a nd the MAP-kinase pathway we studied whether t he downregula- tion of PKCe influences the activation 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 ce lls with carbachol for 10 min resulted in a significant increase in the phosphorylation of E rk-1 and E rk-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. 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 . Fig. 6. Erk1/Erk2 p hosphorylation following tr eatment with c arabachol in SYwt and SYDe cells. As indicated b y equal activation of Erk1/Erk2 phosphorylation the MAP-kinase pathway is not affe cted by PK Ce and PKCa downregulation. Cells were preincubated overnight with serum-free MEM and then treated f or 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 indicatedinMaterialsandmethodsand probed on a Western blot with phospho-specific antibodies (upper panel) and antibodies to Erk1/Erk2 (lo wer panel) to correct for protein loading. 3072 C. Lanni et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Discussion Here 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 o f PKC by phorbol esters or by indirect receptor-mediated activation. It is known that among multiple signal transduction molecules, different isoforms o f PKC may be involved and can specifically contribute to t he complex regulation of A PP metabolism. Many of the signal t ransduction 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 d efects have been associated with aberrant APP metabolism [26,30–32]. 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 because of common overlapping sequences. The significant downregulation of t he 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 C a2 + -dependent PKC isoforms, PKC bIandbII, a nd at least one Ca2 + -independent isoform, PKCd. The downstream e ffect o f d ifferent PKC isoforms is o ften dependent upon redistribution o f 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 t heir m icroenvironment a t t he 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 ce lls that the zinc-finger domain of PKCe was found to contain all of the informa- tion necessary for exclusive localization 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 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 involvement of PKCa in these experiments has been ruled out b ecause of the lack of inhibition by Go ¨ 6976, which is a specific inhibitor o f C a 2+ -dependent isoforms. In our hands the inhibitor Go ¨ 6976 can always block the phorbol ester induced sAPP a release to a significant extent [9,36] yet it w as of particular interest that Go ¨ 6976 did not block the carbachol-mediated release of sAPPa in SYwt and SYa4 cells [9], an in dication that while PKC a was clearly involved in p horbol ester-mediated APP processing it was not necessary for receptor-mediated activation of sAPP a release. The s timulation 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 C a 2+ -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 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, Boston, MA, USA for the gift of SYa4andSYDe ce lls. This work w as made p ossible through grants from the I talian MIUR (prot. # 2003057355–2 003 a nd pr ot # MM05221899–2000 to S. G.), from the University of Pavia ( FAR 2003 to M. R .; Progetto Giovani Rice rcatori to M. M., C. L. and E. P.) a nd from the Italian Ministry of H ealth (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 p harmacology of amyloid precursor protein processing. Exp. Gerontol. 38, 145–157. 3. Racchi, M. & Govoni, S. (1999) Rationalizing a pharmacological intervention on th e amyloid p recursor protein metabolism. Trends Pharmacol. Sci. 20, 418–423. 4. Nishizuka, Y. (1995) Protein kinase C and lipid signaling for sustained cellular r esponses. FASEB J. 9, 484–496. 5. Dekker, L.V. & Parker, P.J. (1994) Protein kinase C -a qu estion of specificity. Trends Biochem. Sci. 19, 73–77. 6. Kampfer, S., U berall, F., Giselbrecht,S.,Hellbert,K.,Baier,G.& Grunicke, H.H. (1998) Characterization of PKC isozyme specific functions in cellular s ignaling. Adv. Enzyme Regul. 38, 35–48. 7. Csukai, M. & Mochly-Rosen, D. (1999) Pharmacologic modula- tion of protein kin ase C isozym es: 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. (200 3) Role of prote in kinase C alpha in the regulated secretion 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., 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 e nha ncing 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 N IH 3T3 cells in duc es op posite e ffec ts o n g rowth, morphology, anchorage depende nce, and tumorige nicity. J. Biol. Chem. 268, 6090–6096. 19. Perletti, G., Tessitore, L., Sesc a, E., Pani, P., Dianzani, M.U. & Piccinini, F. (1996) Epsilon PKC acts like a marker of progressive malignancy in rat liver, b ut 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)Differentialtrans- 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 C a (2+)-independent contractio n in vascular smooth m uscle. 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. 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., Racchi, M., Lannfelt, L. & Cowburn, R.F. (1999) Protein kinase C and amylo id pre curso r prote in p roce ssin g in s kin fi br oblas ts from sporadic and familial Alzheimer’s disease cases. Bioc him. 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. 77 7, 332–337. 32. Govoni, S., Bergamaschi, S., Racchi, M., Battaini, F., B inetti, 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: ph ysiological and pathological examples. Mol. Neurobiol. 16, 49–62. 34. Xu, H ., Greengard, P . & Gandy, S. (1995) Regulated f ormation of Golgi secretory vesicles containing A lzhe imer 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 o f amyloid-be ta precursor protein i n t he trans-golgi n etwork. J. Biol. Chem. 275, 2 568–2575. 36. Benussi, L., Govoni, S., Gasparin i, 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 prote in 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 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 . Differential involvement of protein kinase C alpha and epsilon in the regulated secretion of soluble amyloid precursor protein Cristina Lanni, Michela. (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.