Báo cáo khoa học: Tumor necrosis factor-a converting enzyme is processed by proprotein-convertases to its mature form which is degraded upon phorbol ester stimulation pptx

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 ind

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’s disease; 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-secrea-secre-tase 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)

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) suggesting that ADAM10 is most likely the

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,

2 mMglutamine, 100 UÆmL)1penicillin and 100 mgÆmL)1 streptomycin

LoVo and SH-SY5Y cells were grown in DMEM nutrient mixture F-12 supplemented with 10% fetal bovine serum, 2 mM 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 lM decanoyl-RVKR-chloromethylketone (Bachem AG, Swit-zerland) in DMEM containing 25 mM Hepes, pH 7.0 at

37C 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-stepRT-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 bpamplificate was isolated, subcloned in pUC57 (MBI Fermentas) and sequenced

Western blot analysis of TACE and ADAM10 Cells were washed and collected with NaCl/Pi then cells were suspended in cracking buffer [(5 mM Hepes pH 7,4 containing 2 mMdithiothreitol, 2 mM1,10-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 topcentrifuge to sediment cellular membrane proteins (20 min, 4C,

20 000 g) Each pellet was suspended in cracking buffer and lyzed by addition of an equal volume of 2· Laemmli buffer containing 100 mM dithiothreitol, heated to 95C 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/Pi contain-ing 0.2% I-Block (Tropix, Bedford) and 0.1% Tween 20 for

1 h at room temperature, the primary antibodies against

ADAM10 or TACE (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 softwareAIDA2.0 (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

2 mM 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 lMPMA 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 mMdithiothreitol The samples were heated

to 95C 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 uninhibitor-treated 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 maimma-ture 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.

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 lM 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 lMacetylcholine 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/Ca2+ fluorescence measurements; data not shown) Therefore, we conclude

Fig 2 Effect of the phorbol ester PMA on the processing of endogenous TACE andADAM10 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 andPKA 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.

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 mM

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 lM

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 lM 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 andon the APPsa release from HEK293 LoVo andSHY-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)1fatty 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.

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

Ca2+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.

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

Acknowledgements

This work was supported by grants from the Hirnliga e.V.,

the Deutsche Forschungsgemeinschaft (FA-122/4: DFG Priority

Program – Cellular mechanisms of Alzheimer’s disease) and Fonds

der Chemischen Industrie.

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