Tumor necrosis factor-a converting enzyme is processed by proprotein-convertases to its mature form which is degraded upon phorbol ester stimulation Kristina Endres, Andreas Anders, Elzbieta Kojro, Sandra Gilbert, Falk Fahrenholz and Rolf Postina Institute of Biochemistry, Johannes Gutenberg-University, Mainz, Germany Tumor necrosis factor-a converting enzyme (TACE or ADAM17) is a member of the ADAM (a disintegrin and metalloproteinase) family of type I membrane proteins and mediates the ectodomain shedding of various membrane- anchored signaling and adhesion proteins. TACE is syn- thesized as an inactive zymogen, which is subsequently proteolytically processed to the catalytically active form. We have identified the proprotein-convertases PC7 and furin to be involved in maturation of TACE. This maturation is negatively influenced by the phorbol ester phorbol-12- myristate-13-acetate (PMA), which decreases the cellular amount of the mature form of TACE in PMA-treated HEK293 and SH-SY5Y cells. Furthermore, we found that stimulation of protein kinase C or protein kinase A signaling pathways did not influence long-term degradation of mature TACE. Interestingly, PMA treatment of furin-deficient LoVo cells did not affect the degradation of mature TACE. By examination of furin reconstituted LoVo cells we were able to exclude the possibility that PMA modulates furin activity. Moreover, the PMA dependent decrease of the mature enzyme form is specific for TACE, as the amount of mature ADAM10 was unaffected in PMA-treated HEK293 and SH-SY5Y cells. Our results indicate that the activation of TACE by the proprotein-convertases PC7 and furin is very similar to the maturation of ADAM10 although there is a significant difference in the cellular stability of the mature enzyme forms after phorbol ester treatment. Keywords:ADAM10;Alzheimer’sdisease;furin;PC7; TACE. ADAMs (a disintegrin and metalloproteinases) are a family of integral type I membrane glycoproteins which play an important role in egg-sperm binding and fusion [1,2], muscle cell fusion [3,4] and the development of neuronal and epithelial cells [5,6]. ADAM members are characterized by a well defined domain structure, consisting of a N-terminal prodomain followed by a metalloproteinase domain, a disintegrin domain, a cysteine rich domain, which usually contains an epithelial growth factor repeat, a transmem- brane and a cytoplasmic domain [7,8]. Approximately half of the presently known ADAMs have a catalytic site consensus sequence for zinc-dependent metalloproteinases (HEXGHXXGXXHD) and are therefore predicted to be catalytically active [9]. ADAMs are involved in the release of the extracellular domains of different membrane-anchored signal proteins such as cytokines, growth factors, growth factor receptors and adhesion proteins [10]. The cellular mechanisms and signaling pathways that regulate this ectodomain shedding are gradually being elucidated [11–13]. The most intensively studied inducer of the shedding process is phorbol-12-myristate-13-acetate (PMA), a syn- thetic activator of protein kinase C (PKC). For some of the ADAM proteinases it has been shown that the catalytic site is maintained inactive via a so called cysteine switch mechanism performed by the N-terminal prodomain [14,15]. The essential step for zymogen activa- tion is the proteolytic processing by proprotein-convertases at a characteristic motif, which is located between the prodomain and the metalloproteinase domain. Proprotein- convertases form a family of calcium-dependent endopro- teinases, which presently comprises seven distinct members, including furin, PC2, PC1/PC3, PACE4, PC4, PC5/PC6 and PC7/PC8/LPC [16,17]. A large number of proproteins with various specificities are processed by these subtilisin- like convertases. Typically, cleavage occurs C-terminal to the common consensus sequence RX(K/R)R. Proteolytic activation of substrates, mediated by PC7 or furin, takes place in the trans-Golgi network, in endosomes and at the cell surface [18,19]. Both convertases share an overlapping substrate specificity and therefore the selectivity of substrate proteolysis depends on each ones exact cellular localization. As intracellular trafficking is regulated by their cytosolic domains, which contain different sorting motifs, it is possible that the localization of PC7 is distinct from that of furin [19]. Recently, we have demonstrated that over- expression of the proprotein-convertases PC7 and furin in Correspondence to R. Postina or F. Fahrenholz, Institute of Biochemistry, Johannes Gutenberg-University, Becherweg 30, D-55099 Mainz, Germany. Fax: + 49 6131 3925348, Tel.: + 49 6131 3925833, E-mail: bio.chemie@uni-mainz.de. Abbreviations:Ab, amyloid b peptide; ADAM, a disintegrin and metalloproteinase; APP, amyloid precursor protein; APPsa, a-secre- tase cleaved soluble APP; APPsb, b-secretase cleaved soluble APP; DMEM, Dulbecco’s modified Eagle’s medium; HEK293, human embryonic kidney cells; PC, proprotein-convertase; PMA, phorbol- 12-myristate-13-acetate; TACE, tumor necrosis factor-a converting enzyme; PVDF, poly(vinylidene difluoride). (Received 20 February 2003, revised 31 March 2003, accepted 4 April 2003) Eur. J. Biochem. 270, 2386–2393 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03606.x HEK293 cells leads to an increased maturation of ADAM10, which further results in an enhanced cleavage of the Alzheimer’s amyloid precursor protein (APP) at the a-secretase specific site [20]. Three distinct shedding processes are implicated in the emergence of Alzheimer’s disease [21,22]. The transmem- brane protein APP is processed at first either by the a-secretase or the b-secretase leading to the release of two distinguishable extracellular fragments of APP (APPsa and APPsb, respectively). The generated membrane remaining APP stubs are subsequently cleaved by the c-secretase. Depending on the exact cleavage site of the c-secretase at the APP stubs, which were generated by the b-secretase, amyloid b (Ab) peptides comprising 39–42 amino acids are generated [22]. Ab peptides are the major component of amyloid plaques, which are found in brains of patients suffering from Alzheimer’s disease. As a-secretase mediated processing of APP precludes the formation of the Ab peptides the a-secretase can be considered as a protective factor against the generation of these neurotoxic peptides [23]. Protein kinase C activation by phorbol esters increases the APPsa release and simultaneously reduces the production of Ab peptides [24,25]. Three members of the ADAM family have been shown to act as a-secretases [26–28]. ADAM9 overexpression has been reported to increase the basal and protein kinase C- dependent APPsa release [28], but the purified enzyme failed to cleave a synthetic peptide at the major a-secretase cleavage-site [29]. In contrast, ADAM10 has been found to have constitutive and regulated a-secretase activity as well as many other properties expected for an a-secretase [27]. Additionally, in situ hybridization analysis in human cortical neurons provided evidence for the coexpression of APP with ADAM10 and b-site APP-cleaving enzyme (BACE)suggestingthatADAM10ismostlikelythe physiologically relevant a-secretase [30]. Finally, experi- ments performed with TACE-deficient cells pointed to a participation of TACE in only the regulated, protein kinase C-stimulated a-secretase pathway [26,31]. In another cellular context a constitutive a-secretase activity of TACE was demonstrated [32]. As PC7 and furin act as pro-a-secretase converting enzymes [20], we investigated the proteolytic processing of TACE by overexpression of these convertases in HEK293 cells. We were able to show, that both proprotein-conver- tases contribute to TACE maturation. Moreover, we examined the effect of PMA on the processing of endo- genous TACE and ADAM10 in various mammalian cells and discovered a reduction in the amount of mature TACE compared to ADAM10 after PMA treatment. Experimental procedures Primary antibodies The following antibodies were used: anti-ADAM10, a polyclonal rabbit antibody against endogenous ADAM10 and anti-TACE, and a polyclonal rabbit antibody against endogenous TACE (Chemikon International, Temecula, CA). Both antibodies are directed against the C-terminal part of the proteins and therefore recognize both the full-length as well as the mature enzymes. For detection of secreted APPsa the monoclonal antibody 6E10 (Signet Laboratories) was used. As secondary antibodies alkaline phosphatase-coupled antibodies (Tropix) were used. Cell culture and transfections HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 2m M glutamine, 100 UÆmL )1 penicillin and 100 mgÆmL )1 streptomycin. LoVoandSH-SY5YcellsweregrowninDMEM nutrient mixture F-12 supplemented with 10% fetal bovine serum, 2 m M glutamine, 100 UÆmL )1 penicillin and 100 mgÆmL )1 streptomycin. Transfection of LoVo cells was performed using the calcium phosphate method. Inhibition of TACE processing by decanoyl-RVKR- chloromethylketone HEK293 cells were cultured in the presence of 30 l M decanoyl-RVKR-chloromethylketone (Bachem AG, Swit- zerland) in DMEM containing 25 m M Hepes, pH 7.0 at 37 °C. Inhibitor-containing medium was changed every 6–8 h. After two days of incubation the cells were lyzed and analyzed by Western blotting. Construction of expression vectors Blunt end cDNAs of either bovine furin or rat PC7 were cloned into pIRES1hyg (Clontech), leading to the expression vectors pIRES1hyg-furin or pIRES1hyg-PC7, respectively [20]. Cloning of the furin nucleotide sequence from LoVo cells Total RNA from LoVo cells was isolated using the RNeasy kit (Qiagen, Hilden, Germany). The two-step RT-PCR was carried out using Superscript II (Lifetechnologies) and Taq DNA polymerase (Promega) with the specific primers Fur1_for (5¢-GTGGGCCGGAAAGTGAGCCA-3¢)and Fur2_rev (5¢-CCCTTGTAGGAGATGAGGCC-3¢). The resulting 1058 bp amplificate was isolated, subcloned in pUC57 (MBI Fermentas) and sequenced. Western blot analysis of TACE and ADAM10 Cells were washed and collected with NaCl/P i then cells were suspended in cracking buffer [(5 m M Hepes pH 7,4 containing 2 m M dithiothreitol, 2 m M 1,10-Phenanthroline and a proteinase inhibitor cocktail (complete mini, Roche)]. Cells were disrupted by shock-freezing in liquid nitrogen and after thawing centrifuged in a table top centrifuge to sediment cellular membrane proteins (20 min, 4 °C, 20 000 g). Each pellet was suspended in cracking buffer and lyzed by addition of an equal volume of 2· Laemmli buffer containing 100 m M dithiothreitol, heated to 95 °C for 20 min, separated by SDS/PAGE on 7.5% gels and transferred by electroblotting to poly(vinylidene difluoride) (PVDF) membranes. After blocking with NaCl/P i contain- ing 0.2% I-Block (Tropix, Bedford) and 0.1% Tween 20 for Ó FEBS 2003 TACE maturation and vanishing of its mature form (Eur. J. Biochem. 270) 2387 1 h at room temperature, the primary antibodies against ADAM10 or TACE (anti-TACE, 1 : 2500 or anti- ADAM10, 1 : 1000) were added for 1 h at room tempera- ture. Bound antibodies were detected with an alkaline phosphatase-coupled secondary antibody (Tropix) using the chemiluminescence substrate CDPstar (Tropix). Emitted light was densitometrically analyzed by using a digital camera and the software AIDA 2.0 (Raytest, Straubenhardt, Germany). Isolation and detection of APPsa by Western blot analysis Depending on each cell line an appropriate number of cells was seeded on poly L -lysine coated 10-cm dishes and grown for 20 h close to confluency. Then, cells were washed twice with serum-free culture medium and incuba- ted for 4.5 h in serum-free culture medium containing 2m M glutamine, 100 UÆmL )1 penicillin, 100 mgÆmL )1 streptomycin and 10 lgÆmL )1 fatty acid-free BSA either in the absence or presence of 1 l M PMA. After collection of the cell culture supernatant, proteins were precipitated with a final concentration of 10% trichloroacetic acid by centrifugation. The pellets were washed twice with ice-cold acetone, dried and dissolved in 2· Laemmli buffer containing 100 m M dithiothreitol. The samples were heated to 95 °C for 10 min, separated by SDS/PAGE on 7.5% gels and blotted onto PVDF membranes. The membranes were blocked as described above and then were incubated with antibody 6E10 (1 : 2500) for 1 h at room tempera- ture. Detection of bound antibodies was performed as described above. Results Proteolytic processing of TACE by PC7 and furin TACE has been shown to be synthesized as a zymogen, which is constitutively processed in the secretory pathway. Removal of the prodomain occurs after the protein exits the medial Golgi, but before its arrival on the cell surface [33]. TACE possesses the putative proprotein-convertase recog- nition sequence (RVKR), which is thought to be used to generate the mature enzyme [34,35]. To test the possibility whether proprotein-convertases are involved in the maturation of TACE, the synthetic inhibitor decanoyl-RVKR-chloromethylketone was used. This inhi- bitor prevents the proteolytic activity of proprotein-conver- tases by covalently binding at their catalytic site [36]. Immunoblot analysis of endogenous TACE revealed a clearly lowered amount of the mature enzyme in inhibitor- treated HEK293 cells compared to untreated cells (Fig. 1A). This result confirms that proprotein-convertases are involved in prodomain removal. Next we examined whether PC7 or furin might be proprotein-convertases which are able to cleave the TACE zymogen. Therefore, HEK293 cells were stably transfected with expression vectors containing either the PC7 or the furin cDNA. As a control, HEK293 cells were transfected with the empty expression vector. Cellular membrane proteins were subjected to Western blot analysis and immunologically detected proteins corresponding to imma- ture and mature TACE were densitometrically quantified (Fig. 1B,C). Whereas the ratio of mature TACE relative to the immature form in HEK control cells was 131 ± 24% Fig. 1. Proteolytic processing of TACE by proprotein-convertases. In every case TACE was detected with a rabbit polyclonal antibody. (A) Inhibition of TACE processing by the inhibitor decanoyl-RVKR-chloromethylketone. The inhibitor was added to a final concentration of 30 l M to HEK293 cells. After 48 h the cells were lyzed and membrane proteins were immunoblotted. A representative example of three experiments is shown. (B) Western blot of endogenous TACE in HEK293 cells stably transfected with vector pIRES1hyg alone (HEK293 control), PC7 (HEK293 + PC7) or furin (HEK293 + furin). Blotted cellular membrane proteins were analyzed. (C) Densitometric analysis of TACE pro- cessing. The proform of TACE in each cell line was set to 100%. The mature form is expressed as percentage of the proform and as mean ± SD of three independent experiments. Significance was determined by the t-test (w, P < 0.05). (D) Proteolytic processing of TACE in furin-deficient LoVo cells. Cells were grown in DMEM nutrient mixture F-12 almost to confluency, then lyzed and membrane proteins were analyzed by Western blotting. 2388 K. Endres et al.(Eur. J. Biochem. 270) Ó FEBS 2003 the ratio in HEK cells overexpressing either PC7 or furin was 187 ± 24% and 216 ± 25%, respectively. Thus, increased amounts of the proteolytically processed mature form were detected in PC7 as well as in furin overexpressing cells suggesting that both proprotein-convertases are able to process TACE. As higher amounts of the mature form were detected in furin overexpressing cells, it appears that TACE is a better substrate for furin than for PC7. On basis of this result we investigated the effect of furin- deficiency on TACE maturation. As the human carcinoma cell line LoVo expresses only the proprotein-convertases PACE4 and PC7, but no functionally active furin [37,38], these cells enabled us to study the role of other proprotein- convertases in the processing of TACE. Western blot analysis performed with cellular membrane proteins revealed both the immature and the mature form of endogenous TACE indicating that missing furin activity can be compensated by PC7 and/or PACE4 (Fig. 1D). PMA treatment of HEK293 cells causes the loss of mature TACE but does not affect mature ADAM10 TACE maturation seems to be very similar to the matur- ation of ADAM10, which is also processed by furin and PC7 [20]. For TACE it is known that specifically its mature form disappears from the surface of Jurkat cells after treatment with the phorbol ester PMA [39]. On the basis of this result we examined if the mature form of TACE and that of its closest homologue ADAM10 are also disappear- ing after PMA stimulation in HEK293 cells. Furthermore, we were interested in the time course of the disappearance. HEK293 cells were treated with either 1 l M PMA or dimethylsulfoxide. After 1.5–6 h the cells were harvested and cellular membranes were isolated. Mature and imma- ture forms of endogenous TACE and ADAM10 were detected by Western blot analysis and quantified as described under Experimental procedures. PMA treatment induced a time dependent disappearance of the mature form of TACE, which could clearly be seen 1.5 h after PMA addition and was evident after 3 h (Fig. 2A,B). In contrast to TACE, the mature form of ADAM10 was not degraded within 6 h of PMA treatment (Fig. 2A). This result implicates a different susceptibility of TACE and ADAM10 turnover to PMA induced signal transduction processes. Moreover, the amount of mature TACE apparently decreases linearly with the time of PMA treatment with a halftime of approximately 6 h (Fig. 2B). Stimulation of protein kinases C and A do not affect the amount of mature TACE As the nonphysiological PKC stimulator PMA decreased the amount of mature TACE, we were interested in whether a more physiological pathway for PKC activation causes similar effects. HEK293 cells express G protein-coupled muscarinic receptors and agonist binding results in an intracellular increase of the second messengers inositol 1,4,5-trisphos- phate and diacylglycerol. The latter like PMA binds to the C1b domain of most PKC isoenzymes and activates them. HEK293 cells were treated with 100 l M acetylcholine and harvested after 4 h as described. However, we did not find diminished amounts of mature TACE (Fig. 3) although the used cells responded to the applied ligand with an intracel- lular calcium-efflux (proved by fura-2/Ca 2+ fluorescence measurements; data not shown). Therefore, we conclude Fig. 2. Effect of the phorbol ester PMA on the processing of endogenous TACE and ADAM10 in HEK293 cells. Cells were incubated for 1.5–6 h in DMEM containing either 1 l M PMA dissolved in dimethylsulfoxide or an equivalent volume of dimethylsulfoxide as indicated and further handled as described under Experimental pro- cedures. Cell membrane proteins were separated by SDS/PAGE and blotted onto a PVDF membrane. Detection of TACE and ADAM10 was performed as described under Experimental procedures. (A) A typical Western blot is shown. Open arrows mark the immature enzyme forms and black arrows the mature forms. (B) Quantitative analysis of mature TACE degradation by Western blot. The ratio of mature TACE to immature TACE was determined in the absence (s) or presence of 1 l M PMA (d) for the indicated incubation times (1.5– 6 h). Values are the means of a representative experiment performed in duplicate. An example of three independent experiments is shown. Fig. 3. Stimulation of PKC and PKA signaling pathways. HEK293 cells were treated for 4 h with either 1 l M PMA, 100 l M acethylcholine (Ach) or 0.2 m M dibutyryl-cAMP (dB-cAMP) then cellular membrane proteins were subjected to Western blot analysis. Mature and full- length forms of TACE were detected with an anti-TACE Ig and quantified by using an alkaline phosphatase-coupled secondary anti- body as described under Experimental procedures. Results obtained with unstimulated cells were set to 100%. Values represent mean ± SD from a characteristic experiment using triplicates. A representative example of two experiments is shown. Ó FEBS 2003 TACE maturation and vanishing of its mature form (Eur. J. Biochem. 270) 2389 that the stimulatory effect of acetylcholine was not main- tained long enough by the cells to induce the long-term effect on TACE degradation. As intracellular signaling pathways act as networks and mutually influence each other we investigated whether the PKA signaling pathway might be involved in the reduction of mature TACE. For this purpose we tested the effect of the cAMP-analogon dibutyryl-cAMP (dB-cAMP), which is a strong and long-lasting effector of PKA. HEK293 cells were incubated in medium supplemented with 0.2 m M dB-cAMP for 4 h and membrane proteins were analyzed by immunoblotting. As shown in Fig. 3 dB-cAMP displayed no effect on the expression and on the amount of mature TACE. Similar results were obtained for ADAM10, where also neither dB-cAMP nor acetylcholine affected its maturation (not shown). The effect of PMA on mature TACE and ADAM10 in SH-SY5Y and LoVo cells To demonstrate that the reduction of catalytically active TACE following phorbol ester stimulation is not restricted to HEK293, we tested two other cell lines in respect to APPsa production and TACE as well as ADAM10 maturation: The human SH-SY5Y cell line is of neuronal origin; the other line LoVo (colon carcinoma) was chosen because it was described to be insensitive to PMA in the context of a-secretase activity and APPsa secretion [40]. Each cell line was incubated for 4 h either with 1 l M PMA or dimethylsulfoxide and proteins in the cell culture supernatants as well as cell membrane proteins were analyzed by immunoblotting. In accordance with the result in HEK293 cells, ADAM10 maturation was not affected in LoVo and undifferentiated SH-SY5Y cells after PMA treatment (Fig. 4A). In contrast, a PMA mediated disappearance of the mature form of TACE could be detected in HEK293 and in SH-SY5Y cells but was completely absent in LoVo cells (Fig. 4A). As shown in Fig. 4B, PMA treatment induced the release of APPsa in all tested cell lines. This indicates that at least the common a-secretase stimulatory properties of PMA are retained by all cell lines tested. In the cell culture supernatant of SH-SY5Y cells two forms of APPsa can be detected as these cells express two isoforms of APP, APP751 and the neuronal isoform APP695. PMA-induced release of APPsa by LoVo cells Recently, it has been reported that the furin-deficient cell line LoVo is devoid of PKC-dependent APPsa secretion which was interpreted that furin is involved in regulated APP shedding [40]. In contrast to this result, our experi- ments clearly demonstrate that LoVo cells exhibit an augmented release of endogenous APPsa after treatment with PMA (Fig. 4B). Because of our contradictory finding we considered it necessary to verify the identity of the LoVo cells, which were used in our experiments. The loss of furin activity in LoVo cells is caused by two mutant furin alleles. One mutation is a single nucleotide deletion, leading to an aberrant termination of the furin polypeptide [37], the other is a nucleotide exchange, which leads to the amino acid exchange W547R in the homo B domain of furin [41]. To confirm these mutations, furin mRNA of LoVo cells was amplified by RT-PCR with suitable primers. The obtained nucleotide sequence contained the expected nuc- leotide exchange in the furin mRNA (not shown) confirm- ing the integrity of the LoVo cell line used in our experiments. In conclusion, our results indicate that furin is not necessarily needed for the PMA-induced APP shedding in LoVo cells. The lack of furin is not the key for the persistence of mature TACE in LoVo cells after PMA stimulation In contrast to the other tested cell lines, a PMA mediated decrease of mature TACE was not observed in furin- deficient LoVo cells. To test the possibility that furin participates in mature TACE degradation, we examined whether overexpression of functionally active furin in LoVo cells restores the effect of PMA on the degradation of mature TACE. Therefore, LoVo cells were reconstituted with furin by a transient transfection. After 48 h transfected cells were stimulated with 1 l M PMA and cellular mem- brane proteins were analyzed by immunoblotting. When compared to mock transfected cells (LoVo Hyg) the amount of mature TACE was increased in cells that were transfected with the furin expression vector (LoVo Furin, Fig. 5). This Fig. 4. Effect of PMA on the processing of endogenous TACE and ADAM10 and on the APPsa release from HEK293 LoVo and SHY-5Y cells. (A) Proteolytic processing of endogenous TACE and ADAM10 in PMA-treated cells. Cells were treated for 4 h with either 1 l M PMA or dimethylsulfoxide as control. Then cellular membrane proteins were subjected to Western blot analysis. Mature and full-length forms of TACE and ADAM10 were detected with suitable antibodies. Open arrows mark the immature enzyme forms and black arrows the mature forms in representative experiments. (B) PMA induced APPsa release from cells. Cells were incubated for 4.5 h in fetal bovine serum-free DMEM supplemented with 10 lgÆmL )1 fatty acid-free BSA and either with 1 l M PMA dissolved in dimethylsulfoxide or the equivalent vol- ume of dimethylsulfoxide as control. The cell culture supernatants were collected and proteins were precipitated with trichloroacetic acid. Afterwards, the samples were subjected to Western blot analysis with the primary antibody 6E10 and an alkaline phosphatase-conjugated secondary mouse antibody. A representative example of two experi- ments is shown. 2390 K. Endres et al.(Eur. J. Biochem. 270) Ó FEBS 2003 indicates an effective transfection and confirms that matur- ation of TACE is mediated by furin. Nevertheless, in furin reconstituted LoVo cells no loss of mature TACE occurred after PMA treatment suggesting that furin is not involved in a mechanism which decreases the amount of mature TACE (Fig. 5). Discussion The prodomain of the catalytically active members of the ADAM family is thought to act as an inhibitor of the proteinase via a cysteine switch mechanism [42,43]. There- fore removal of the prodomain is required to obtain the proteolytically active enzyme [14,33,44]. Recently, we have shown that ADAM10 is proteolytically processed by both furin and PC7 and that the removal of the prodomain is accompanied by an enhanced proteolytic activity [20]. For TACE it has been shown that the maturation occurs during the transit of the protein through the late Golgi compart- ment suggesting that prodomain removal is performed by a furin-type proprotein-convertase [33]. Consistent with this model, TACE contains a putative proprotein-convertase cleavage site, which might be used to generate the mature enzyme [34,35]. Here we demonstrate that proprotein- convertases are indeed involved in the maturation of TACE and that pro-TACE is proteolytically processed by both furin and PC7 to its mature form, most likely to increase its proteolytical activity. Because higher amounts of mature TACE could be detected in furin overexpressing cells, it might be that pro-TACE is a better substrate for furin than for PC7. However, this observation may also be due to different expression levels of the proprotein-convertases and is therefore difficult to substantiate. The examination of TACE processing in LoVo cells indicates that there is redundancy in the proteolytic maturation of TACE as other members of the PC family can compensate a lacking furin activity. Therefore, we cannot exclude that additional members of the PC family also contribute to TACE activation. Our results further demonstrate that long-term treatment of HEK293 and SH-SY5Y cells with the phorbol ester PMA negatively regulates the amount of the mature form of TACE. This is in accordance to results obtained with Jurkat cells where the phorbol ester effect on mature TACE reduction was attributed to protein degradation [39]. In contrast to HEK293, SH-SY5Y and Jurkat cells TACE maturation is unaffected by PMA in LoVo cells. Interestingly, the amount of mature ADAM10 is not significantly affected in spite of PMA stimulation in the tested cell lines. Thus, the mature forms of TACE and ADAM10 differ in their cellular stability. While mature TACE is degraded during long-term PMA treatment ADAM10 resists degradation. As TACE possesses an internalization motive (YESL) in its cytoplasmic domain and the effect of PMA on the amount of mature TACE was inhibited by blocking endocytosis [39] it is possible that the effect of phorbol esters on TACE maturation depends on vesicle formation and endocytosis. PMA is known to bind to the C1b domain of PKC and to activate its activity. To elucidate whether activation of PKC indeed mediates mature TACE disappearance we stimula- ted PKC via the G protein-coupled muscarinic acetylcholine receptor. However, long-term treatment of cells with a receptor-saturating concentration of acetylcholine did not influence mature TACE degradation although the cells used in our study responded on ligand application with a fast Ca 2+ efflux. The calcium efflux is mediated by the second messenger inositol 1,4,5-trisphoshate, which is generated together with diacylglycerol from phosphatidyl inositol 4,5-bisphosphate by PLCb. Obviously, receptor mediated increase of diacylglycerol and activation of PKC does not affect the degradation of mature TACE. An agonist-induced activation of cellular signaling pathways is a short-term effect. G protein-coupled receptors are desensitized upon permanent agonist avail- ability and therefore do not respond any longer to effector protein activation. As mature TACE degradation is a long-term effect, the short-term activation of PKC by diacylglycerol might not cause a similar effect. Alternatively, our results with acetylcholine stimulation of cells which had no effect on TACE maturation indicates that the effect of PMA may be independent of PKC and may include other PMA binding molecules such as the Munc proteins, which are involved in vesicle formation [45]. Intracellular signaling pathways act as networks and are mutually influenced. Therefore we investigated the effect of a long-term PKA activation on mature TACE disappear- ance. Activation of PKA by dibutyryl-cAMP, a more stable cAMP analogue, did not influence the degradation of mature TACE indicating that this effect is not dependent on PKA. A PMA mediated decrease in the amount of mature TACE did not occur in the furin-deficient cell line LoVo. Therefore, we investigated the role of furin in the PMA mediated decrease of mature TACE. Furin cycles between the trans-Golgi network and the cell surface and its localization depends on phosphorylation of its C-terminus. Whereas casein kinase II mediated furin phosphorylation is important for its localization to the trans-Golgi network, unphosphorylated furin is found in Fig. 5. Reconstitution of furin activity in LoVo cells. LoVo cells were transiently transfected with a furin cDNA containing expression vector (LoVo Furin) or with the empty vector as control (LoVo Hyg). Treatment with 1 l M PMA or dimethylsulfoxide as control was per- formed for 4 h. Subsequently, TACE proteins were detected and quantified in cell membrane fractions as described in Experimental procedures. The proform of TACE in each cell line was set to 100%. The mature form is expressed as percentage of the proform and as mean ± SD of three independent experiments. Ó FEBS 2003 TACE maturation and vanishing of its mature form (Eur. J. Biochem. 270) 2391 secretory granules [46]. Furthermore, the activity of the furin phosphorylating casein kinase II can be increased by PKC [47]. Thus, decreased amounts of mature TACE after PMA treatment might be the result of a PKC-induced colocalization of TACE and furin in a cellular compartment where TACE is degraded. There furin probably acts as a cofactor which activates the TACE degrading cascade. Reconstitution of furin activity in LoVo cells, however, did not rescue the PMA induced degradation of mature TACE although the cells were able to respond on PMA treatment with APPsa secretion. This indicates that the enzymatic activity of furin may not be required for the PMA induced disappearance of mature TACE. Nevertheless, we cannot exclude the possibility, that the increased maturation of TACE in furin transfected LoVo cells compensates to some extent the effect of a PMA-induced degradation of mature TACE. As LoVo cells are of carcinoma origin, another mutation or a chromosomal rearrangement event could be responsible for the inactivation of the mature TACE degrading machinery, which is sensitive to phorbol esters. Taken together, both TACE and ADAM10 possess a-secretase activity and are proteolytically activated by PC7 and furin. Furthermore, a furin-independent and PMA induced disappearance of mature TACE takes place which is not evident for mature ADAM10. Thus, mature forms of TACE and ADAM10 differ in their cellular stability, which may affect their a-secretase activity in vivo. 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PMA, phorbol- 12-myristate-13-acetate; TACE, tumor necrosis factor-a converting enzyme; PVDF, poly(vinylidene difluoride). (Received 20 February 2003, revised