INSERM EPI 99-36, Laboratoire d'Hématologie, Faculté de Médecine, 27 Bd Jean Moulin, Marseilles 13385 Cedex 5, France
* Author for correspondence (e-mail: Franck.Peiretti{at}medecine.univ-mrs.fr)
Accepted 6 February 2003
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Summary |
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Key words: TACE, SAP97, TNF, PDZ3, Protein interaction
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Introduction |
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Tumor necrosis factor alpha converting enzyme (TACE) is an ADAM (ADAM17)
originally described as the main enzyme responsible for tumor necrosis factor
(TNF) release from membranes (Black
et al., 1997
; Moss et al.,
1997
). Subsequently, TACE was implicated in the ectodomain
shedding of TNF receptors p55 (TNFR1), p75 (TNFR2)
(Peschon et al., 1998
;
Reddy et al., 2000
) and
several other proteins (Moss et al.,
2001
).
The regulation of TACE activity is poorly understood. On the basis of the
effect of phorbol esters on the internalization and degradation of
cell-surface TACE (Doedens and Black,
2000), a multilevel regulation of TACE activity involving changes
in membrane targeting or changes in the interaction of the enzyme with
regulatory proteins was proposed (Reddy et
al., 2000
). The cytoplasmic domain of TACE contains several
signaling motifs such as SH3 ligand domains and a protein kinase C
phosphorylation site (Black et al.,
1997
). Mitotic arrest deficient 2 protein (MAD2) was identified as
a partner of TACE cytoplasmic tail (Nelson
et al., 1999
), and this finding suggests a potential relationship
between TACE and the cell cycle, but does not provide information on the
regulation of TACE activity. The extracellular signal-regulated kinase binds
to the cytoplasmic tail of TACE and phosphorylates threonine 735
(Diaz-Rodriguez et al., 2002
).
Recently, the protein-tyrosine phosphatase PTPH1 was shown to interact with
the C-terminus of TACE and was suggested to be a negative regulator of TACE
levels and function (Zheng et al.,
2002
).
To gain insights into the regulation of TACE activity, we searched for binding partners that interact with its cytoplasmic domain. Yeast two-hybrid experiments allowed us to identify the synapse associated protein 97 (SAP97) as a potential binding partner of the TACE cytoplasmic tail. This interaction was confirmed biochemically in vitro and demonstrated in COS-7 cells by co-immunoprecipitation and immunofluorescence microscopy. Confocal microscopy revealed some overlapping clusters of endogenous TACE and SAP97 in COS-7 cells and in CACO-2 cells. A functional implication of this interaction was suggested by the fact that overexpression of SAP97 decreased the release of TACE-processed substrates, whereas overexpression of a mutant form of SAP97 that did not bind TACE did not have an effect.
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Materials and Methods |
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All SAP97-cDNA-containing plasmid vectors were generated from pEGFP SAP97
as described elsewhere (Wu et al.,
1998). For in vitro translation, the sequence of the pET28b
(Novagen) containing the T7 promoter and the ribosome-binding site was
introduced in the pEGFP-containing SAP97 sequence. For two-hybrid experiments,
SAP97 sequences were introduced into pGADC1 plasmid vector
(James et al., 1996
),
generating SAP97 fused to the GAL4 activation domain.
Complete coding regions of TNF, TNFR1 and TNFR2 cDNAs were introduced into pcDNA3 plasmid vector (Invitrogen).
Yeast two-hybrid screen
The yeast reporter strain PJ69a (James
et al., 1996) was co-transformed with a human placental cDNA
library cloned into the pACT2 vector (Clontech) and pAS-TACE (described
above).
Selection was made by growth on histidine-, adenine-, leucine- and tryptophane-free media. LacZ gene expression was determined with a colorimetric filter assay. cDNA clones from positive colonies were isolated, transferred into XL1 Blue bacteria and identified by cDNA sequencing.
Binding assays in vitro
GST fusion proteins were overexpressed in BL21 Escherichia coli.
Cells were lysed by sonication and GST fusion proteins were purified using
glutathione-Sepharose 4B beads as recommended by the manufacturer (Amersham
Pharmacia Biotech). The amount of cell lysate incubated with the beads was
adjusted so that the level of the different GST fusion proteins eluted from
the glutathione-agarose beads was similar. After extensive washes with PBS,
the beads were washed once and suspended in 500 µl of binding buffer (75 mM
NaCl, 20 mM Tris-HCl pH 7.4, 0.1 mM EDTA, 2.5 mM MgCl2, 0.75 mg/ml
BSA, 0.1% Tween 20, 1 mM DTT).
In vitro transcriptions and translations experiments were performed using T7 TNT Quick Coupled Reticulocyte Lysate System (Promega). Biotinylation of the neosynthesized protein was performed using TranscendTM (Promega).
10 µl of reticulocyte lysate (containing the biotinylated protein) was
added to the glutathione-agarose bound GST fusion protein in binding buffer
and incubated for 4 hours at 4°C on a rotator, then washed four times with
PBS and incubated in 20 µl 1x SDS sample buffer for 5 minutes at
95°C. The supernatant was submitted to a SDS polyacrylamide gel
electrophoresis according to Laemmli
(Laemmli, 1970). After
electrotransfer onto a PVDF membrane, biotinylated proteins were detected
using a streptavidin alkaline phosphatase conjugate.
Cell culture and transfection
COS-7 cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% (v/v) fetal bovine serum, 2 mM glutamine, 100 i.u./ml
penicillin and 100 µg/ml streptomycin. CACO-2 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 20% (v/v) fetal bovine
serum, 0.1 mM non-essential amino acids, 2 mM glutamine, 100 i.u./ml
penicillin and 100 µg/ml streptomycin. Transient transfections were
performed with Polyfect reagent (Qiagen). Analyses were made 48 hours after
transfection.
Immunoprecipitation of overexpressed proteins
Cells were lysed with 1% (v/v) Nonidet P40, 20 mM Tris-HCl, pH 7.4, 75 mM
NaCl, protease inhibitors and 10 µM of the metalloprotease inhibitor active
on TACE RU36156. Lysates were preclarified by centrifugation and
incubated for 1 hour with Protein A/G plus agarose (Santa Cruz Biotechnology)
and for the next 2 hours with Protein A/G plus agarose coated with antibodies
(anti-HA epitope or anti-GFP from Santa Cruz Biotechnology). Beads were washed
four times with lysis buffer and incubated in 20 µl of 1x SDS sample
buffer for 5 minutes at 95°C. The supernatant was submitted to western
blot analysis to detect either overexpressed SAP97 using the anti-SAP97
monoclonal antibody (kindly provided by C. Garner, University of Alabama,
Birmingham) or the endogenous TACE using the cytoplasmic-domain-specific TACE
antibody from Santa Cruz Biotechnology (# C-15).
Immunoprecipitation of endogenous proteins
The interaction between TACE and SAP97 was maintained by lowering the pH of
the lysis buffer to 6.8. To decrease non-specific binding, the preclarified
lysate was concentrated using a Centricon YM-100, which removes proteins with
a mass below 100 kDa and the retentat was diluted to the initial volume with
lysis buffer. The lysate was then treated as described above except that the
antibody used for immunoprecipitation was anti-SAP97 (Santa Cruz
Biotechnology).
Detection was made using either the cytoplasmic-domain-specific TACE antibody or the ectodomain-specific TACE antibody form R&D Systems Europe (Lille, France) (clone: 111633).
Immunocytochemistry
Localization of haTACE was performed in COS-7 cells. Cells were washed
three times with PBS (with calcium and magnesium), fixed for 5 minutes in 1%
paraformaldehyde, permeabilized with 0.2% Triton-X 100 in PBS for 20 minutes,
washed with PBS 1% BSA and incubated with anti-HA epitope antibody for 1 hour.
After extensive washes, rhodamine-labeled secondary antibody was incubated for
1 hour. Cells were washed and observed by epifluorescence microscopy.
Localization of endogenous TACE and SAP97 was performed in COS-7 and CACO-2
cells. Cells were fixed, permeabilized and incubated with anti-SAP97 for 1
hour, washed and incubated for 1 hour with a fluorescein isothiocyanate
(FITC)-conjugated secondary antibody, washed again and incubated for 1 hour
with a phycoerithrin (PE)-conjugated antibody against TACE (R&D Systems,
clone: 111633). Analyses were made by confocal microscopy using a Leica PCS
SP2 microscope.
Flow cytometry analysis
Surface expression of the overexpressed form of TACE (haTACE) on COS-7
cells was analyzed by flow cytometry using FITC-conjugated monoclonal antibody
against HA epitope (clone BMG-3F10 from Roche). Cells assigned to
intracellular staining were treated according to the specification of the
Intrastain kit from Dako Cytomation (Trappes, France). FITC-labeled cells were
analyzed on a XL-cytofluorograph (Coulter Electronics Inc.) at 488 and 525 nm,
which correspond to excitation and detection wavelengths, respectively.
Enzymatic deglycosylation
N-linked carbohydrate residues were removed using either
peptide:N-glycosidase F (PNGase F) or endoglycosidase H (EndoH) (New
England Biolabs) as described by the manufacturer. COS-7 cells were directly
lysed in the reaction buffer furnished with the enzyme. Lysates were heated at
100°C for 10 minutes; aliquots were adjusted for deglycosylation and
treated with or without 1000 units of PNGase F or EndoH at 37°C for 8
hours. Samples were then separated by SDS-PAGE and analyzed by western
blotting.
Protein assays
Total protein of cell lysates was assayed using the bicinchoninic acid
protein assay kit from Sigma. Total amount (free and bound forms) of TNF,
TNFR1 and TNFR2 were assayed in cell lysates and in culture media after 24
hours of accumulation, according to the specification of their respective
enzyme-linked immunosorbent assays kits (R&D Systems).
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Results |
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Analysis of the interaction of TACE with SAP97
Our two-hybrid screens showed that the sequence of SAP97 from amino acid
422 to amino acid 904 is involved in the binding with the cytoplasmic tail of
TACE. A mutant form of SAP97 deleted for the last 165 amino acids (85% of the
GK domain) was also able to interact with TACE as suggested by the result of a
two-hybrid screen (data not shown). Thus, the cytoplasmic tail of TACE binds
SAP97 between its amino acids 422 and 739. This region of SAP97 contains the
PDZ3 domain, the SH3 domain and 25 amino acids of the GK domain. We narrowed
down this region by deleting the PDZ3 (SAP97DPDZ3) or
SH3 (SAP97DSH3) domains from full-length SAP97 and
testing the interaction of these mutants forms with TACE cytoplasmic tail
using two-hybrid system (Fig.
1A). Mutant forms of SAP97 were correctly produced in yeast
(Fig. 1B), and both deletions
slowed down the growth of yeast on selective media. However, PDZ3 domain
deletion was the most effective in reducing the growth of yeast
(Fig. 1A). The involvement of
the PDZ3 domain of SAP97 in the binding to TACE cytoplasmic tail was also
investigated using an in vitro biochemical binding assay. In vitro translated
and biotinylated fragments of SAP97 (Fig.
1C, lanes 1 and 2) were incubated with glutathione-agarose bound
GST-TACE cytoplasmic tail and submitted to pull-down experiments. The GST-TACE
cytoplasmic tail retained more SAP97DPDZ1-2 than
SAP97DPDZ1-3 (Fig.
1C, compare lanes 3 and 4). None of these forms binds the GST
alone (Fig. 1C, lanes 5 and 6).
These results suggest a privileged role of the PDZ3 domain of SAP97 in the
interaction with TACE.
PDZ domains are multifunctional protein-protein recognition modules
involved in the clustering of signaling molecules and play an important role
in organizing protein networks on membranes
(Fujita and Kurachi, 2000;
Harris and Lim, 2001
). In many
cases, PDZ domains specifically bind a motif occurring at the C-terminus of
target proteins (Harris et al.,
2001
). To test if the extreme C-terminal sequence of TACE could be
responsible for the interaction with SAP97, we modified this sequence and we
analyzed the binding of TACE to SAP97 using the two-hybrid system. Yeast were
co-transformed with pGADC1 coding for the full-length SAP97 and with pAS2-1
TACE (coding for the wild-type cytoplasmic tail of TACE:
821E-T-E-C824) or with pAS2-1 TACEm (coding for a
mutated version of the cytoplasmic tail of TACE:
821D-A-E-C824). Yeast expressing TACE and SAP97 were
able to grow during nutritional selection unlike those expressing TACEm and
SAP97 (Fig. 2A). However, TACEm
fused to GAL4 DNA-binding domain was produced in yeast as efficiently as TACE
(Fig. 2B). This finding
suggests that the C-terminal extremity of TACE is involved in the interaction
with SAP97.
Interaction of TACE and SAP97 in mammalian cells
COS-7 cells constitutively express detectable amounts of TACE and SAP97
(see below). By size exclusion chromatography, SAP97 and TACE (immature and
mature forms) co-eluted in a range of molecular masses between 250 kDa and 150
kDa (data not shown). As the molecular mass of each protein is between 90 and
120 kDa, this suggests that these proteins exist in complexes. We used a
specific antibody to immunoprecipitate SAP97 from COS-7 cells lysate, and the
presence of endogenous TACE in the resulting immune complex was assessed by
western blots with two different antibodies. The cytoplasmic-domain-specific
TACE antibody allowed us to detect proteins migrating at the level of the
immature (with the prodomain) and mature (without the prodomain) forms of TACE
(Fig. 3A); an unidentified fast
migrating protein was also detected. The ectodomain-specific TACE antibody,
which mainly recognizes the mature form of TACE in NP-40-based cell lysate,
allowed us to detect a protein that migrated at the level of the mature form
of TACE (Fig. 3B). This result
suggests that endogenous TACE and SAP97 are able to interact. Moreover,
immature and mature forms of TACE were co-immunoprecipitated with
overexpressed GFP-SAP97 but not with GFP-SAP97DPDZ3,
suggesting that in mammalian cells, immature and mature forms of TACE interact
with the PDZ3 domain of SAP97 (Fig.
3C). When a HA-tagged membrane-anchored cytoplasmic tail of TACE
(haTACE) was overexpressed together with the full-length GFP-SAP97, both
proteins were immunoprecipitated with an anti-HA epitope antibody
(Fig. 3D). Mutations of the
extreme C-terminal part of haTACE (haTACEm) abolished the
co-immunoprecipitation, suggesting that in mammalian cells, the C-terminal
extremity of TACE is engaged in the interaction with SAP97. Altogether, these
data are strongly favor of an intracellular interaction between human TACE
(immature and mature forms) and SAP97. This interaction involves the extreme
C-terminal part of TACE and the PDZ3 domain of SAP97.
Intracellular localization of SAP97 and TACE
Localization of overexpressed SAP97 and TACE in COS-7 cells was assessed by
epifluorescence microscopy. GFP-SAP97 exhibited a diffuse pattern
(Fig. 4A), and this
distribution was comparable to that of GFP-SAP97DPDZ3
(data not shown). haTACE showed also a diffuse staining pattern in COS-7 cells
(Fig. 4B), which has already
been described (Schlondorff et al.,
2000). This distribution is similar to that of haTACEm (data not
shown) and is, a priori, consistent with endoplasmic reticulum and/or surface
protein localization. When overexpressed with haTACE, GFP-SAP97 accumulated as
round aggregates (Fig. 4C).
This particular distribution was clearly evident in more than 70% of the
transfected cells and was not observed when GFP-SAP97 was coexpressed with
haTACEm (Fig. 4D) or when the
PDZ3 domain of GFP-SAP97 was deleted (Fig.
4E). This result suggests that the interaction between
overexpressed GFP-SAP97 and haTACE, which involves their PDZ3 domain and the
extreme C-terminal part, respectively, is responsible for their intracellular
aggregation. To support this result, we examined, simultaneously,
intracellular distributions of overexpressed GFP-SAP97 and haTACE and noticed
that they mostly colocalized in so-called aggregates
(Fig. 5). Altogether, these
data show that association of overexpressed haTACE with GFP-SAP97 results in
the alteration of their intracellular distribution.
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The form of the human TACE that was overexpressed in COS-7 cells did not
contain the prodomain and the catalytic domain (Materials and Methods). Since
the prodomain was described to be necessary for the targeting of TACE
(Milla et al., 1999) we
investigated the cellular distribution of haTACE by analyzing its sensitivity
to N-glycosidase F (PNGase F) and endoglycosydase H (EndoH).
N-linked sugars are sensitive to PNGase F but most of them become
resistant to EndoH after they are modified in the medial Golgi. We found that
both enzymes were equally efficient in deglycosylating the overexpressed
haTACE (Fig. 6A). This suggests
that haTACE is present in the early secretory pathway (endoplasmic reticulum
and/or proximal Golgi). By comparison, deglycosylation of the endogenous TACE
showed, as already described (Schlondorff
et al., 2000
), that both immature and mature forms of TACE were
sensitive to PNGase F. By contrast, only the immature form of TACE was
sensitive to EndoH (Fig. 6B),
suggesting that the immature form is present in the endoplasmic reticulum and
proximal Golgi, whereas the mature form traverses the medial Golgi. Detection
of the overexpressed haTACE by flow cytometry favours the hypothesis that the
overexpressed haTACE cannot go through the Golgi apparatus. Less than 10% of
the cells expressed a very small amount of haTACE at the cell surface
(Fig. 6C), whereas, virtually
all transfected cells (around 60%) expressed haTACE intracellularly
(Fig. 6D). When co-transfected
with SAP97 or SAP97DPDZ3, haTACE was always sensitive to
deglycosylation by EndoH (data not shown), suggesting that haTACE is always
present in the early secretory pathway. Taken together these data allowed us
to conclude that the interaction between haTACE and GFP-SAP97, which can be
visualized by the aggregation of these proteins, takes place in the early
secretory pathway. However, since overexpressed haTACE cannot traverse the
medial Golgi, we hypothesized that endogenous TACE and SAP97 interact in other
cell compartments. In favor of this hypothesis are the results shown on
Fig. 3A-C, which demonstrate
that endogenous or overexpressed SAP97 can co-immunoprecipitate both immature
(present in the early secretory pathway) and mature (that had traversed the
medial Golgi) forms of TACE. To confirm the physiological relevance of this
interaction, we localized endogenous TACE and SAP97 by immunofluorescence
confocal microscopy. In COS-7 cells, the staining associated with the
detection of each of these two proteins showed a diffuse pattern that
significantly overlapped in some intracellular areas and at the cell lateral
membrane (Fig. 7A). In the
epithelial cell line CACO-2, SAP97 and TACE were produced
(Fig. 7B) and colocalized at
the lateral membrane at sites of cell-cell contacts
(Fig. 7A).
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Functional implication of TACE/SAP97 interaction
We investigated whether the overexpression of SAP97 affected the ability of
endogenous TACE to release its substrates. COS-7 cells were co-transfected
with TACE substrates (TNF, TNFR1 and TNFR2) together with different forms of
SAP97, and the amount of substrates (both released and cell-associated) was
measured. We first verified that the releases of TNF, TNFR1 and TNR2 from
COS-7 cells were mainly due to TACE activity. The release of TACE substrates
was reduced by treatment with the metalloprotease inhibitor RU36156
active on TACE (Gallea-Robache et al.,
1997) and by overexpression of a dominant-negative form of TACE
(haTACE mentioned above) (Table
1), which suggests that TACE is responsible for the shedding of
the overexpressed TNF, TNFR1 and TNFR2 from COS-7 cells
(Table 1). The coexpression of
GFP-SAP97 together with TACE substrates did not alter the original
intracellular distribution of GFP-SAP97 (data not shown) but reduced the
amount of released TACE-processed substrates
(Table 1) at least as
efficiently as the dominant-negative form of TACE. This inhibitory property
was lost when cells were co-transfected with TACE substrates and
SAP97DPDZ3 (Table
1). Accordingly, cells co-transfected with SAP97 expressed more
cell-associated TNF and TNFR2 (370±90 pg/µg and 55±5 pg/µg
of cellular proteins, respectively) than cells co-transfected with
SAP97DPDZ3 (190±20 pg/µg and 26±3
pg/µg, respectively). The amount of cell-associated TNFR1 assayed in cell
lysates was below the detection limit of the assay. This result suggests that,
when cells were co-transfected with SAP97, the low release of TACE-processed
substrates is not due to an inhibition of their synthesis but rather to a
downregulation of their cleavage. These data emphasize the specific inhibitory
effect of overexpressed SAP97 on the release of TACE-processed substrates.
Because deletion of the region of SAP97 involved in the binding with TACE (the
PDZ3 domain) abrogates this inhibitory effect, it can be reasonably concluded
that the interaction of overexpressed SAP97 with the endogenous TACE is
responsible for the inhibitory effect on the release of TACE-processed
substrates. Dual color cytometry analysis with a PE-labeled anti-TACE
ectodomain antibody that detects TACE at the surface of GFP-positive cells did
not show any significant difference in the amount of endogenous TACE exposed
at the surface of GFP-SAP97- and
GFP-SAP97DPDZ3-overexpressing cells (data not shown).
This result suggests that the reduced TACE activity measured in SAP97
overexpressing cells was not due to a decreased amount of active TACE exposed
at the cell surface.
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Discussion |
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The interaction between TACE and SAP97 and the identification of the elements responsible for this interaction were confirmed in COS-7 cells by co-immunoprecipitation of endogenous and overexpressed proteins (Fig. 3). Microscopic observation of overexpressed haTACE and GFP-SAP97 distribution into COS-7 cells confirmed the demonstration of a molecular interaction between these overexpressed proteins as both proteins were found colocalized in cells as round aggregates (Figs 4, 5). This particular aggregation was not observed when GFP-SAP97DPDZ3 was expressed or when the C-terminal extremity of the overexpressed haTACE was mutated. However, deglycosylation experiments suggest that, in contrast to the endogenous TACE, the overexpressed form of TACE does not traverse the medial Golgi (Fig. 6A). Therefore, the colocalization of overexpressed TACE and SAP97 reasonably argues for their interaction but remains uninformative as to where endogenous TACE and SAP97 interact. The fact that both endogenous immature (present in the early secretory pathway) and mature (that had traversed the medial Golgi) forms of TACE were co-immunoprecipitated with endogenous and overexpressed SAP97 (Fig. 3) suggests that the interaction also takes place after TACE had traversed the medial Golgi.
In COS-7 cells, immunofluorescence confocal microscopy revealed a diffuse
pattern for both endogenous SAP97 and TACE that overlapped in some
intracellular areas and at the cell lateral membrane. Consistent with previous
results (Wu et al., 1998), we
found that in the epithelial cell line CACO-2, SAP97 localized at the lateral
membrane at cell-cell adhesion sites where it was reported to be associated
with the cortical cytoskeleton (Reuver and
Garner, 1998
). Furthermore, in our study we showed a lateral
membrane localization pattern of TACE that significantly overlapped that of
SAP97.
Coexpression of SAP97 with potassium channel protein (Kv1) results in the
formation of intracellular aggregates containing these two proteins and blocks
Kv1 channels surface expression (Kim and
Sheng, 1996; Tiffany et al.,
2000
). Endogenous SAP97 associates with a subset of GluRI early in
the secretory pathway (in the endoplasmic reticulum or cis-Golgi) and
dissociates at the plasma membrane (Sans
et al., 2001
); these data show that interactions involving SAP97
in the early secretory pathway are of physiological importance. Interestingly,
the observed colocalization of overexpressed GFP-SAP97 and haTACE in the early
secretory pathway is very similar to that described for SAP97 and Kv1
channels. Overexpression of SAP97 (which binds TACE) reduced the release of
three different TACE-processed substrates, TNF, TNFR1 and TNFR2, whereas that
of SAP97DPDZ3 (that does not bind TACE) did not have an effect
(Table 1). By contrast, cells
co-transfected with SAP97 expressed more cell-associated TNF and TNFR2 than
cells co-transfected with SAP97DPDZ3. These data
strongly suggest that the interaction between endogenous TACE and
overexpressed SAP97 is involved in the downregulation of TACE-processed
substrates release (reinforcing the idea that TACE and SAP97 interact) and
that in our conditions the above mentioned substrates are essentially
processed by TACE. This finding also suggests a probable functional
implication of the association between endogenous TACE and SAP97.
In light of the literature mentioned above, it is tempting to envisage that the downregulation of the release of TACE-processed substrates triggered by the overexpression of SAP97 could be the consequence of an intracellular sequestration of endogenous TACE. However, using dual color cytometry, we did not measure significant change in the amount of endogenous TACE exposed at the surface of cells overexpressing GFP-SAP97 and GFP-SAP97DPDZ3, ruling out the possibility that this hypothetical intracellular retention of TACE modifies the amount of cell-surface active TACE.
The C-terminal sequence of TACE binds to the PDZ domain of the
protein-tyrosine phosphatase PTPH1 (Zheng
et al., 2002). However, this interaction was not necessary to
allow overexpressed PTPH1 to decrease the level of TACE, which can result in a
reduced amount of TNF accumulated in the cell culture media. The absence of
phosphorylated tyrosine in the cytoplasmic sequence of TACE leads the authors
to suggest that PTPH1 acts as a negative regulator of TACE levels and
function, most probably, by dephosphorylating a yet to be defined effector.
These and our results underline the ability of the C-terminal sequence of TACE
to bind PDZ sequences.
The need for an appropriate organization of the cytoskeleton for the
correct positioning of both the shedding machinery and the substrates at the
cell membrane has been evoked (Mullberg et
al., 2000) in two situations. One is related to the effect of cell
adhesion and spreading on the shedding of membrane-anchored heparin-binding
epidermal-like growth factor (Gechtman et
al., 1999
) and the other is related to the effect of inhibitors
that block calmodulin binding to the cytoplasmic tail of L-selectin and
accelerate the release of L-selectin from cells
(Kahn et al., 1998
). By
analogy, we speculate that the already described scaffolding role of SAP97 is
involved in the transport and/or the organization of TACE, which should have
an impact on TACE activity. Further investigations to evaluate the
physiological implication of the interaction between TACE and SAP97 on TACE
activity will be of particular interest.
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Acknowledgments |
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