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INTRODUCTION |
Various cell surface proteins with diverse functions
undergo regulated proteolytic cleavage of their extracellular
domains, a process that results in ectodomain shedding (for review see Refs. 1-4). The subsequent release of the polypeptide from the cell
can have profound consequences not only at the cellular physiological level but also at a more tissue-wide or even systemic level. For example, transmembrane L-selectin is an adhesion protein for
leukocytes, and ectodomain cleavage of L-selectin may therefore
down-regulate inflammation by limiting neutrophil accumulation and
lymphocyte activation (5-8). Because shedding plays such an important
role in the regulation of cellular functions, its dysregulation can contribute to disease conditions. Thus, ectodomain cleavage of transmembrane tumor necrosis factor-
(TNF-
)1 and consequent
release of soluble TNF-
is thought to contribute to cachexia and
arthritis, which cannot be induced by transmembrane TNF-
(9, 10). In
addition, families carrying a non-cleavable TNF-
receptor are
presented with severe autoimmune reactions (11). However, despite its
importance, the molecular mechanisms underlying ectodomain processes
are poorly understood.
Ectodomain shedding also regulates the activities and roles of
transmembrane growth factors such as transforming growth factor-
(TGF-
) and its family members. These growth factors are expressed at
the cell surface as transmembrane proteins, yet can also be released as
soluble growth factors as a result of their ectodomain shedding
(12-17). Both the soluble and the transmembrane forms of these growth
factors can activate the receptors, i.e. the EGF/TGF-
receptor or related transmembrane tyrosine kinases (13, 18-21). Thus,
transmembrane TGF-
or related growth factors only exert autocrine
activity and stimulate receptors on adjacent cells and are most likely
unable to induce receptor internalization. In contrast, release of
soluble ligand following ectodomain shedding allows the cell to induce
biological effects, e.g. TGF-
-mediated proliferation, on
non-adjacent cells and allows for ligand-induced internalization of the
receptors. In addition to these two modes of ligand signaling depending
on ectodomain shedding of the transmembrane growth factor, their
receptors can also be subject to ectodomain cleavage, thereby
down-regulating ligand-induced receptor activation (22-24).
Diverse stimuli are known to activate ectodomain shedding (25-29).
These inducers are often artificial compounds that are thought to
correlate with natural signaling pathways. For example, the phorbol
ester PMA, which activates protein kinase C, is known as a potent
activator of ectodomain shedding of various transmembrane proteins (27,
30, 31). In contrast, little is as yet known about natural inducers and
signaling pathways that activate ectodomain shedding under
physiological conditions. Recent studies (32-36) have uncovered the
roles of mitogen-activated protein (MAP) kinase signaling pathways in
the activation of ectodomain shedding. Thus, growth factors and
activated tyrosine kinase receptors rapidly induce ectodomain shedding
of TGF-
and other transmembrane proteins through induction of the
Erk MAP kinase signaling pathway without the need for new protein
synthesis (32). PMA-induced ectodomain shedding of TGF-
or its
family member heparin binding-epidermal growth factor-like growth
factor (HB-EGF) and TNF-
is also mediated through activation of the
Erk signaling pathway (32, 33, 35). In addition, activation of the p38
MAP kinase pathway, often a result of inflammatory mediators or
physiological stress, also leads to ectodomain shedding (32, 37). Thus,
inhibition of both Erk and/or p38 MAP kinase signaling pathways
strongly suppresses ectodomain shedding of diverse transmembrane
proteins in response to various stimuli (32, 37).
Inhibitor studies and functional experiments have revealed that the
cleavage of TGF-
and various other transmembrane proteins is
mediated by cell surface metalloprotease(s) (30, 38-40). Functional inactivation through gene targeting has subsequently implicated a role
for the transmembrane protease TACE in ectodomain shedding of not only
TNF-
but also L-selectin and TGF-
in vivo (31). Accordingly, cells deficient in functional TACE expression display impaired release of soluble TGF-
as well as the TGF-
-related amphiregulin, HB-EGF, and neuregulins (17, 41). Finally, TACE has also
been implicated in shedding of various other proteins, e.g.
ErbB4, TNF receptors,
-amyloid precursor protein, and Notch, thus
illustrating its role in diverse physiological contexts (31, 42-44).
TACE, a member of the adamalysin (ADAM) family of transmembrane
metalloproteases and also known as ADAM 17, is a prototype "sheddase." Its characterization and studies on the mechanisms of
activation may therefore provide models for how other adamalysins function. The protein sequence of TACE consists of an extracellular metalloprotease domain flanked upstream by a prodomain and downstream by a cysteine-rich disintegrin domain, a single transmembrane domain
and a cytoplasmic domain (45, 46). The prodomain serves as an inhibitor
of the protease in its zymogen state and is removed at the late Golgi
processing stage (47). Although it was originally thought to play a
role in substrate recognition, the disintegrin domain has been shown to
be required for shedding of an interleukin 1 receptor only among
several TACE substrates tested (42).
The regulation of ectodomain shedding by TACE and the ability of MAP
kinase signaling pathways to activate shedding without the need for new
protein synthesis suggest that the cytoplasmic domain may act as a
signal transducer that regulates shedding by the protease domain of
TACE in response to intracellular activities. Interestingly, the
130-amino acid cytoplasmic domain of TACE, like several other ADAM
members, has a potential Src homology 3 ligand protein-binding motif
(45, 46). This sequence or a proximal region of TACE interacts with
mitotic arrest deficient 2, although the physiological relevance of
this interaction is unknown (48). More recently, the PDZ
domain-containing protein PTPH1 has been shown to interact with the
carboxyl terminus of the cytoplasmic domain and to possibly
down-regulate TACE function (49).
Phosphorylation plays a critical role in intracellular signaling. It
has been reported that PMA, an artificial shedding activator, can
induce TACE phosphorylation (45, 50). Interestingly, MDC9, which shares
sequence similarity with TACE in its cytoplasmic domain, is
phosphorylated by protein kinase C
(51). In one study this
phosphorylation was shown to regulate the induction of HB-EGF shedding
by PMA (51), whereas other findings indicate that PMA may activate
HB-EGF shedding through the Erk MAP kinase signaling pathway instead
(33, 35). We now show that growth factor or PMA stimulation leads to
enhanced phosphorylation of TACE through activation of the Erk MAP
kinase signaling pathway. We also identified an alternative translation
product of TACE that we name SPRACT. SPRACT corresponds to most of the
cytoplasmic domain of TACE and similarly to TACE undergoes regulated
phosphorylation. We show that TACE is phosphorylated on serines upon
growth factor or serum stimulation. We identified the major site of
phosphorylation in response to growth factor stimulation, as well as
another serine, which shows decreased phosphorylation upon stimulation.
The function of these phosphorylation sites and the role of SPRACT
remain to be characterized.
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EXPERIMENTAL PROCEDURES |
Reagents and Expression Vectors--
Recombinant acidic
fibroblast growth factor (FGF) and EGF were purchased from Calbiochem.
Dialyzed fetal bovine serum (FBS) was purchased from Invitrogen. The
MEK1 inhibitor U0126 and sequencing grade modified trypsin were
purchased from Promega (San Luis Obispo, CA). TAPI-1, an inhibitor of
metalloproteases, including TACE, was a gift from Dr. T. K. Kishimoto (Boehringer Ingelheim) (30, 39). Phenanthroline, another
metalloprotease/TACE inhibitor, was purchased from Sigma (47).
Four polyclonal anti-TACE antibodies were used throughout the course of
this study. A rabbit antiserum from QED Bioscience, Inc. (San Diego,
CA), and a goat antiserum from Santa Cruz Biotechnology (Santa Cruz,
CA) both recognize a carboxyl-terminal sequence. The antibody from QED
Bioscience was found preferable for analyzing endogenous TACE because
of a higher affinity to protein A-agarose beads in immunoprecipitation
and a cleaner background in Western blotting as compared with the
antibody from Santa Cruz Biotechnology. The blocking peptide for the
antisera as well as normal rabbit IgG was purchased from Santa Cruz
Biotechnology. A rabbit polyclonal antibody raised against the
recombinant TACE cytoplasmic domain fused to glutathione
S-transferase, and a polyclonal antiserum against the
extracellular domain of TACE were generous gifts from Dr. Blobel
(Sloan-Kettering Institute, New York) (52, 53).
The expression vector for the wild type TACE was constructed by PCR
amplification of the human TACE cDNA cloned in a bacteriophage
vector that was provided by Dr. Roy Black (Immunex, Seattle, WA). The
PCR-amplified cDNA was inserted into the pRK5 vector, which uses
the human cytomegalovirus promoter to drive expression of an inserted
gene in mammalian cells. Deletion and single or multiple amino acid
substitution mutants of the TACE coding sequence were constructed using
PCR-based approaches. The sequences of wild type and mutant TACE coding
sequences in all expression plasmids were confirmed by automated DNA
sequencing performed at the University of California, San Francisco,
Biomolecular Resource Center. Information on the plasmid design and
construction will be provided upon request. Expression plasmids for
constitutively activated MEK1 (
N-S218E-S222D) (54) and
constitutively activated Erk2 (MCMV5-Erk2-MEK1 LA) (55) were provided
by Dr. Nathalie G. Ahn (University of Colorado) and Dr. Melanie H. Cobb
(University of Texas Southwestern Medical Center), respectively.
Cell Lines, Culture Conditions, and Transfection--
The HeLa
S3 cell line and HEK293 cell line were maintained in Dulbecco's
modified minimal essential medium (DMEM) supplemented with 7% fetal
bovine serum. CHO cells were cultured in DMEM supplemented with 2 mM proline and 10% fetal bovine serum. Transfection of CHO
and HEK293 cells was achieved using the LipofectAMINE reagent (Invitrogen). The EC2 cell line that lacks the expression of
catalytically active TACE as a result of targeted gene inactivation,
i.e. the tace
Zn/
Zn mutation,
was kindly provided by Dr. Roy Black (Immunex). EC2 cells were
maintained in DMEM containing 10% fetal bovine serum and were
transfected using LipofectAMINE and the Plus reagent (Invitrogen).
K562, THP-1, and U937 cell lines were obtained from the University of
California San Francisco Tissue Culture Facility and cultured in RPMI
medium supplement with 10% FBS.
In Vivo 35S and 32P Labeling of
TACE--
CHO cells, grown in 6-well plates, were transfected with
expression vectors for wild type or mutated TACE and cultured in serum-free medium overnight (32). Metabolic labeling of TACE proteins
with [35S]cysteine/methionine was performed as described
previously (32). To detect phosphorylation of TACE in vivo,
HeLa S3 cells at 90% confluency in 6- or 10-cm dishes or CHO cells in
6-well plates transiently transfected with a TACE expression vector
were subjected to overnight serum starvation before 32P
labeling, washed with phosphate-free DMEM, and cultured with the DMEM
supplemented with 10% (v/v) [32P]orthophosphate
(PerkinElmer Life Sciences or Amersham Biosciences) for 2 h. To
observe induced TACE phosphorylation, FGF (final concentration, 10 ng/ml), EGF (final concentration, 10 ng/ml), dialyzed fetal bovine
serum (FBS) (final concentration, 20%) or PMA (final concentration, 20 nM), with or without the MEK inhibitor U0126 (final
concentration, 10 µM), was added into the labeling
medium. When FBS was used, an equal volume of phosphate-free DMEM was
added to control wells. The cells were cultured for an additional 15 min, and labeling was terminated by placing the culture plates on ice
and washing with ice-cold phosphate-buffered saline.
Immunoprecipitations--
Cells were lysed in phosphate-buffered
saline containing 1% Nonidet P-40, 2 mM sodium
orthovanadate, 1 mM NaF, 1 µM
4-(2-aminoethyl)benzenesulfonyl fluoride, 5 mM EDTA, 10 mM phenanthroline as well as "Complete" protease
inhibitor mixture (Roche Applied Science). Immunoprecipitation of TACE
in the cell extract with antibodies from rabbit and protein A-conjugated agarose beads was carried out by rotating on a
platform at 4 °C for 2 h, followed by three washes with the
above lysis buffer. Protein G beads were used for immunoprecipitation
using an anti-TACE antibody from goat. In selected experiments
analyzing the nature of SPRACT, 10 µM TAPI-1 was included
in the steps of cell lysate preparation and immunoprecipitation. The
immunoprecipitated proteins, including TACE and SPRACT, were separated
by SDS-polyacrylamide discontinuous gradient gel electrophoresis using
a Hoefer SE 400 vertical electrophoresis apparatus. The 16-cm gel
consisted of 9 cm of 13.5% gel at the bottom, 3-4.5 cm of 7.5% gel
in middle, and regular 4.5% stacking gel on the top. Electrophoresis
was done at 50 V for about 18 h, using a prestained broad range
molecular standard (Bio-Rad). In most experiments, the 52-kDa ovalbumin entered the 13.5% gel, and the 85-96-kDa bovine serum albumin stayed
in the 7.5% gel. Occasionally, the bovine serum albumin ran into the
7.5% gel. For 35S- or 32P-labeled samples, the
gel was dried, and radiolabeled TACE and SPRACT were visualized
by autoradiography. The phosphorylation levels of these two proteins in
selected autoradiograms were quantitated by the NIH Image 1.61 software
using a Scion 1.62a image acquiring system (Scion Corp., Frederick, MD).
Phosphoamino Acid Analysis--
For phosphoamino acid analysis
of overexpressed TACE, CHO cells grown on 10-cm culture plates were
transfected with the TACE expression plasmid. 32P labeling
and immunoprecipitation of TACE were performed as described above. The
precipitated TACE was resolved with 7.5% SDS-PAGE using a Bio-Rad
mini-protein II electrophoresis apparatus and was transferred onto PVDF
membrane. The radioactive band as visualized by autoradiography was cut
out and incubated for 45 min at 120 °C in the presence of 6.0 N HCl. The resulting samples were dried and redissolved in
a solution containing unlabeled phosphorylated serine, threonine, and
tyrosine standards. Separation of the amino acids by thin layer
electrophoresis and staining of the amino acid standards with ninhydrin
were done as described previously (56). Visualization of TACE-derived
32P-phosphorylated amino acid(s) was accomplished by autoradiography.
To perform phosphoamino acid analysis on endogenous TACE and SPRACT,
HeLa S3 cells grown on 15-cm dishes were labeled with [32P]orthophosphate. TACE and SPRACT were
immunoprecipitated and resolved by discontinuous gel electrophoresis as
described above. Autoradiography was carried out with dried gel, and
radioactive TACE bands were cut out and treated with 6 N
HCl at 100 °C for 90 min. The TACE-derived amino acids in the
solution were dried, reconstituted in a solution containing unlabeled
phosphorylated serine, threonine, and tyrosine, and separated by thin
layer chromatography (57). Location of the amino acid standards was
visualized by ninhydrin staining, and TACE-derived
32P-phosphorylated amino acids were visualized following
autoradiography (57).
CNBr Mapping--
32P-Labeled TACE and SPRACT were
immunoprecipitated from transiently transfected 10-cm plates of CHO
cells, resolved by discontinuous SDS-PAGE as described above, and
transferred onto nitrocellulose membranes. The radioactive bands were
cut out and incubated with 5 µM CNBr in 70% formic acid
(56). The resulting peptides were resolved by 18% SDS-PAGE, and their
phosphorylation was revealed by autoradiography.
Tryptic Peptide Mapping--
32P-Labeled TACE was
prepared as for phosphoamino acid analysis, and phosphorylated TACE was
visualized by direct autoradiography of the wet gels. Gel bands
containing radiolabeled TACE and SPRACT were excised, cut into 6-8
pieces, and subjected to two 45-min washes at 37 °C with a solution
containing equal volumes of acetonitrile and 20 mM ammonium
carbonate, followed by overnight in-gel digestion with modified
trypsin. The resulting peptides were separated by thin layer
electrophoresis followed by thin layer chromatography (56) and
visualized by autoradiography.
Western Blotting--
For detection of TACE and SPRACT from
transfected CHO cell lines, cells grown on 6-well plates, cells were
dissolved by adding SDS-PAGE sample buffer (500 µl/well), removed by
scraping, and sonicated for 10 s to shear the genomic DNA. Samples
of 20 µl were heated to 100 °C for 5 min and subjected to gel
electrophoresis. Proteins were transferred onto PVDF membrane with
0.2-µm pore size (Bio-Rad). We found that SPRACT is relatively poorly
retained by PVDF membranes, especially by those with 0.45-µm pore
size, and that the retention was sensitive to the amounts of the
detergent Tween 20 in the washing buffer and the duration of washes.
The final conditions we adopted for the detection of TACE and SPRACT are as follows. The PVDF membrane was blocked with 5% bovine serum albumin for 2 h, reacted with polyclonal antibodies to the
cytoplasmic domain of TACE or its carboxyl-terminal sequence at the
dilution of 1:1000 for 2 h, washed three times each for 10 min
with Tris-buffered saline containing 0.05% Tween 20 (TBST), and then
reacted to horseradish peroxidase-conjugated goat anti-rabbit IgG
(Sigma) or mouse anti-goat IgG (Santa Cruz Biotechnology) for 1 h.
After three washes as described above, visualization of TACE and SPRACT
was achieved by chemiluminescence with the ECL kit (Amersham
Biosciences).
Endogenous TACE and SPRACT could also be detected in a similar manner
as described above using larger amounts of cell extracts and the more
sensitive ECL Plus kit for visualization. However, the increased total
cell extracts distorted the gel leading to poor resolution of the
protein bands. We found that in HeLa S3 and HEK293 cells this problem
could be circumvented by modifying the sample preparation. Confluent
cells grown on 6-well plates were extracted with the lysis buffer used
for immunoprecipitation (200 µl/well). The lysates were mixed with
0.2 volume of the 5× concentrated sample buffer, and 50 µl of
the mixtures were subjected to gel electrophoresis. The remaining steps
of the Western blotting to detect the endogenous proteins were as
described for transfected CHO cells, except for the use of the ECL Plus
kit (Amersham Biosciences).
Evaluation of TGF-
Ectodomain Shedding--
TGF-
ectodomain shedding was assessed using a pulse-chase assay with
transiently transfected CHO cells, as described (32, 58). The same
assay was also adapted for the use of EC2 cells that lack the
catalytically active TACE.
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RESULTS |
TACE Is Phosphorylated in Response to Growth Factor and
PMA--
Growth factor stimulation induces ectodomain shedding of
transmembrane proteins, including TGF-
, TNF-
, and L-selectin,
through activation of the Erk MAP kinase signaling pathway (32).
Because the transmembrane metalloprotease TACE has been implicated in the cleavage of these transmembrane proteins, we assessed the phosphorylation state of TACE. In extracts prepared from
[32P]orthophosphate-labeled HeLa S3 cells, two
radioactive TACE bands were detected, a 128- and a 100-kDa band (Fig.
1A). Both TACE bands were also
apparent in parallel Western blots of these lysates (Fig.
1B). According to previous studies (47), the 100-kDa band is
the mature TACE that is derived from the 128-kDa glycosylated TACE band
after the removal of the 196-amino acid prodomain (Fig. 1A).

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Fig. 1.
Stimulation of TACE phosphorylation by
activators of ectodomain shedding and detection of SPRACT.
A, HeLa S3 cells were serum-starved, labeled with
[32P]orthophosphate, and treated with the indicated
stimuli. Cells were then lysed, and 32P-labeled TACE was
immunoprecipitated using an antibody recognizing the cytoplasmic domain
of TACE obtained from QED Bioscience or normal rabbit IgG. The 128-kDa
larger TACE form is the glycosylated TACE zymogen with its prodomain,
whereas the 100-kDa band corresponds to the mature form without the
prodomain. These TACE forms are marked as T in all figures.
The 20-kDa protein is SPRACT, which is marked as S in all
figures. B, stimulation with growth factors and PMA did
not lead to significant changes of the level of TACE protein. HeLa S3
cells were serum-starved, treated with indicated stimuli, and lysed.
Western blotting analysis was performed with the same antibody or
control rabbit IgG as in A. C, the blocking
peptide specifically interferes with the detection of phosphorylated
TACE and SPRACT. 32P labeling of HeLa S3 cells and
immunoprecipitation of TACE were performed as described in
A. For peptide competition, the estimated molar ratio of
peptide to antibody is 1000 to 1, based on the average molecular mass
of IgG as 180 kDa. Note only the TACE and SPRACT bands but not the
nonspecific bands were blocked by the peptide in the cell lysate
prepared from PMA-stimulated cells.
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Treatment of serum-starved cells with FGF, EGF, fetal bovine serum, or
PMA for 15 min significantly increased the TACE phosphorylation level
(Fig. 1A). Western blot analysis showed that the stimuli did
not show a significant effect on the level of the total TACE protein
(Fig. 1B). Although some nonspecific bands also underwent increased phosphorylation (Fig. 1A), they were also present
in the IgG control immunoprecipitations and were not detected in Western blots using the TACE antiserum (Fig. 1B).
Furthermore, they were not competed out in immunoprecipitations in the
presence of excess peptide immunogen (Fig. 1C). These
results indicate that both the glycosylated pro-TACE and mature TACE
are phosphorylated and that EGF, FGF, serum, and PMA, which are known
to activate ectodomain shedding, rapidly induce phosphorylation of the
TACE cytoplasmic domain.
Growth factor- or PMA-induced phosphorylation of TACE was also examined
using CHO cells, transfected to express the cloned human TACE cDNA.
CHO cells express a very low level of TACE, but transfection of TACE
readily allowed detection of TACE by immunoprecipitation of
35S-labeled (Fig.
2A) or 32P-labeled
(Fig. 2B) proteins. Besides the 126-kDa precursor form of
TACE and the 100-kDa mature TACE, we also detected a 112-kDa form (Fig.
2A). Pulse-chase experiments suggested that the intermediate 112-kDa band is likely to be the unglycosylated TACE precursor (data
not shown). Previous studies (47) suggest that TACE matures rather
inefficiently. This may explain why the major TACE form detected in
transfected CHO cells is the glycosylated full-length TACE precursor.
FGF treatment for 15 min significantly increased the TACE
phosphorylation level (Fig. 2B), without an effect on the
level of TACE protein (Fig. 2A). In these experiments, the phosphorylation was restricted to the larger, glycosylated TACE form
(Fig. 2B). Fetal bovine serum and PMA, which also induce ectodomain shedding, also stimulated the level of TACE phosphorylation after 15 min (Fig. 2C). These results indicate that TACE,
encoded by the characterized cDNA, is phosphorylated in response to
FGF, serum, or PMA as observed with endogenous TACE.

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Fig. 2.
Regulated phosphorylation of TACE and SPRACT
in transfected CHO cells. A, detection of
35S-labeled TACE and SPRACT in transfected CHO cells
expressing TACE. Cells were serum-starved and metabolically labeled
with [35S]methionine/cysteine, and fresh medium or FGF
was added into the labeling medium for 15 min. Cells were then lysed,
and 35S-labeled TACE was immunoprecipitated using a goat
antibody recognizing a carboxyl-terminal sequence of TACE. The 128-kDa
larger TACE form is the glycosylated TACE zymogen with its prodomain,
whereas the 100-kDa band corresponds to the mature form without the
prodomain. The intermediate 112-kDa band is likely to be the
unglycosylated TACE precursor. pRK5 is the control vector without
cDNA insert. B, stimulation of in vivo
TACE phosphorylation by FGF. Serum-starved, TACE-transfected CHO cells
were labeled with [32P]orthophosphate and treated with
FGF, and 32P-labeled TACE was immunoprecipitated as
described in A. Only the 128-kDa TACE zymogen among the
three TACE forms and SPRACT were visibly phosphorylated.
C, stimulation of in vivo TACE
phosphorylation by dialyzed FBS and PMA. The experiments
were carried out as in B, except that cells were treated
with medium only (M) or FBS or PMA for 15 min.
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SPRACT, a Polypeptide That Corresponds to the TACE Cytoplasmic
Domain--
Immunoprecipitation and Western blot analyses of HeLa S3
cell lysates using an antibody against the carboxyl-terminal sequence of TACE detected not only the two TACE forms mentioned above but also a
20-kDa protein, which we named SPRACT for "small
protein reactive with antibody
against the cytoplasmic domain of TACE" (Fig.
1). SPRACT was also detected in 293 human embryonic kidney cells and
the U937, THP-1, and K562 hematopoietic cell lines by direct Western
blotting or immunoprecipitation followed by Western blotting, using two
additional antibodies against the cytoplasmic domain of TACE (data not
shown). Additionally, SPRACT was expressed from the cloned TACE
cDNA in transfected CHO cells (Fig. 2). Similarly to the TACE
forms, SPRACT showed rapidly increased phosphorylation in response to
EGF, FGF, serum, or PMA stimulation (Fig. 1A and Fig. 2,
B and C), whereas its protein levels remained
constant (Figs. 1B and 2A). The detection of
SPRACT by antibodies against the cytoplasmic domain of TACE in Western
blot analyses rules out the possibility that SPRACT is a TACE-binding
protein and suggests that SPRACT may correspond to a cytoplasmic
segment of TACE. Consistent with this interpretation, SPRACT was not
detected by an antibody that was raised against the extracellular
domain of TACE (47) (data not shown). Furthermore, excess peptide to which the anti-TACE antiserum was raised competed out the signals of
32P-labeled TACE and SPRACT in the extracts of
PMA-stimulated HeLa S3 cells (Fig. 1C). It should be noted
that SPRACT is poorly retained by the PVDF membrane. Up to 90% of
SPRACT can be lost depending on the stringency of the wash condition
(data not shown), and therefore the level of SPRACT, detected by
Western blotting, may be underestimated.
SPRACT Is an Alternative Translation Product--
It has been
suggested that the active form, but not the full-length precursor form,
of TACE can release its cytoplasmic domain through autolysis (47). We
therefore included phenanthroline (10 µM), which has been
demonstrated to inhibit TACE autolysis (47) and yet also inhibits other
metalloproteases, in the cell lysis buffer and throughout the
subsequent immunoprecipitation and washes used to generate the results
in Fig. 1. The detection of both the mature 100-kDa TACE and 20-kDa
SPRACT under these conditions suggests that SPRACT is unlikely to be
generated by autolysis during sample preparation. Previous studies (30,
39) have shown that the metalloprotease inhibitor TAPI-1 inhibits the
cleavage activity of TACE. Inclusion of TAPI-1 (10 µM)
and phenanthroline (10 mM) in the cell lysis buffer and
throughout subsequent washes in immunoprecipitation did not decrease
the level of SPRACT in such experiments (data not shown). Additionally, transfection with an expression vector encoding a catalytically inactive TACE mutant, due to replacement of Glu406 with
alanine, generated the same level of SPRACT as the expression vector
for the wild type catalytically active TACE (Fig.
3A). Furthermore, inclusion of
numerous other types of protease inhibitors in the cell lysis and
during processing of samples, as well as cell lysis using boiling lysis
buffer, did not decrease the SPRACT level either (data not shown),
further suggesting that SPRACT is unlikely to be derived through
autolysis or proteolysis by other proteases during sample
preparation.

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Fig. 3.
SPRACT is generated by alternative
translation. A, SPRACT is expressed from wild type
(WT) TACE or the catalytically inactive E406A mutant TACE
(cat ). CHO cells were transfected with the TACE plasmids
or control vector pRK5 and directly lysed in SDS-PAGE sample buffer and
subjected to Western blot analysis using the same antibody as in Fig.
1. B, schematic presentation of the mutations
introduced in the coding sequence of TACE cDNA. The solid
box represents the transmembrane domain coding sequence. The
proposed internal translation initiation codons for Met in the
cytoplasmic domain were mutated to GCG to encode Ala. The deleted
cytosine in the codon for the transmembrane Ser681 in
pT-shift is underscored in the wild type sequence. The premature stop
codon as a result of the deletion and consequent frameshift in pT-shift
is shown. The dotted area in pT-shift represents a
carboxyl-terminal FLAG epitope tag. C, detection of
truncated TACE proteins produced by pT-shift and CD. CHO
cells transfected with expression vectors for the mutated TACE forms or
wild type TACE or the control pRK5 plasmid were metabolically labeled
with [35S]cysteine/methionine, and the TACE forms were
immunoprecipitated with an antibody against the extracellular domain of
TACE (47) and visualized by autoradiography. These truncated TACE forms
are denoted as t. Although three forms of wild type TACE
were seen, only one prominent band for the truncated TACE forms was
detected. D, analyses of TACE and SPRACT expression by
vectors shown in B. CHO cells transfected with the mutated
expression plasmids or the pRK5 vector were lysed and subjected to
Western blotting as described in A. Note that the
carboxyl-terminal FLAG epitope tag caused a slower migration of SPRACT
produced from pT-shift.
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During the construction of TACE expression plasmids, we unintentionally
obtained an expression vector, now named pT-shift, which contained a
cytosine deletion, in the codon for Ser681 located in the
transmembrane domain, thus causing a frameshift and an immediate
premature translation termination (Fig. 3B). The resulting
truncated protein lacks the carboxyl-terminal portion of the
transmembrane domain and the entire cytoplasmic domain and was detected
by immunoprecipitation using an antibody raised against the
extracellular domain of TACE (Fig. 3C). Nevertheless, the
antibody against the TACE cytoplasmic domain demonstrated that SPRACT
was also expressed from pT-shift, albeit with a somewhat larger
molecular weight due to an added carboxyl-terminal FLAG epitope (Fig.
3D). We therefore conclude that translation of SPRACT did
not depend on the synthesis of full-size TACE. In contrast, SPRACT was
not expressed from a plasmid that encodes a cytoplasmically truncated
TACE (the
CD mutant) (Fig. 3, B and D). These
data suggest that SPRACT is translated from the sequence encoding the cytoplasmic domain of TACE and argue further against the possibility that SPRACT is derived through proteolytic cleavage of TACE. In keeping
with this, SPRACT is expressed by a plasmid that encodes the
cytoplasmic domain of TACE without its extracellular and transmembrane domains, i.e. the
ED/TM mutant (Fig. 3, B and
D).
The cytoplasmic domain of TACE has two juxtamembrane methionines
(Met715 and Met719, Fig. 3B). The
corresponding ATG triplets are both located within a sequence, which,
based on the consensus rules of Kozak (59), should provide efficient
translational initiation. To test if these ATGs could serve as
initiation sites for the translation of SPRACT, we constructed
expression plasmids for full-size TACE with either or both ATG codons
mutated to GCG codons for alanine (Fig. 3B). Whereas SPRACT
is still expressed from TACE expression plasmids lacking either ATG, it
is no longer produced when both juxtamembrane ATGs are mutated to
encode alanines (Fig. 3D). Collectively, these data indicate
that SPRACT is an alternative translation product that can be initiated
at either juxtamembrane ATG codon in the sequence encoding TACE
cytoplasmic domain.
Correlation of TACE and SPRACT Phosphorylation with the Erk MAP
Kinase Signaling Pathway--
We have shown previously that activation
of the Erk MAP kinase pathway mediates growth factor- and PMA-induced
ectodomain shedding of TGF-
, TNF-
, and L-selectin. Accordingly,
ectodomain shedding in response to these inducers is inhibited by
U0126, an inhibitor of MEK1/2 that consequently prevents activation of Erk MAP kinase (32). Our data now show that U0126 also inhibits growth
factor-induced phosphorylation of TACE and SPRACT in HeLa S3 cells to a
level that is similar to the basal phosphorylation level in the absence
of stimulation (Fig. 4A). A
similar inhibition of phosphorylation by U0126 was also apparent in CHO
cells transfected to express TACE (data not shown). Thus, growth
factor-induced phosphorylation of TACE and SPRACT is mediated through
activation of the Erk MAP kinase pathway. U0126 also inhibited
PMA-induced phosphorylation of TACE, but only minimally decreased the
phosphorylation of SPRACT (Fig. 4A). This suggests that
PMA-induced phosphorylation of SPRACT, and possibly TACE, utilizes an
additional kinase pathway(s) different from the MEK/Erk MAP kinase
signaling pathway.

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Fig. 4.
Shedding activators induce phosphorylation of
TACE and SPRACT through the Erk MAP kinase signaling pathway.
A, the MEK inhibitor U0126 inhibits the phosphorylation
of endogenous TACE and SPRACT in HeLa S3 cells in response to EGF,
serum (FBS), or PMA. The samples from cells treated with medium only
were marked as M. HeLa S3 cells were labeled with
[32P]orthophosphate. Indicated shedding activators were
added either alone or together with U0126 at 15 min before termination
of the in vivo labeling. 32P-Phosphorylated TACE
and SPRACT were immunoprecipitated as described in the legend to Fig.
1. B, stimulation of TACE and SPRACT phosphorylation by
expression of activated MEK or Erk MAP kinase. Duplicate CHO cells were
transfected with TACE alone or together with an expression vector for
constitutively activated MEK1 (caMEK1) or Erk2
(caErk2) and labeled in vivo with
[35S]cysteine/methionine or
[32P]orthophosphate. Radioactive TACE and SPRACT were
detected by immunoprecipitation using an antibody against the TACE
cytoplasmic domain (52, 53). The amounts of 35S-labeled
TACE detected in the samples were first quantitated by PhosphorImager
analysis and then used to correct the amounts of
32P-labeled cell extracts for immunoprecipitation. This
normalization was required since cotransfection of the activated
kinases increased the TACE expression (data not shown).
C, U0126 inhibits the induction of the phosphorylation
in TACE and SPRACT encoded by expression vectors for wild type TACE and
the T735A mutant. In vivo 32P labeling and
detection of phosphorylated proteins were performed as A.
The top panel shows the autoradiogram; the bottom
panel shows relative intensities of the 32P-labeled
TACE and SPRACT bands as quantitated by densitometry. The intensities
of the bands were normalized against the intensity of the TACE or
SPRACT bands under non-stimulated conditions.
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The effect of U0126 on growth factor-induced phosphorylation also led
us to evaluate the effects of constitutively activated forms of MEK1
and ERK2. For this, we cotransfected the TACE expression plasmid
together with an expression plasmid for a constitutively active form of
MEK1 or Erk2 into CHO cells. As shown in Fig. 4B, overexpression of either enzyme resulted in increased phosphorylation of TACE and SPRACT.
Erk is a proline-directed Ser/Thr kinase that can directly
phosphorylate Thr-Pro and Ser-Pro with increased preference when a
proline is located at the
2 position (60, 61). Interestingly, Thr735-Pro736 is found as part of a
Pro-Gln-Thr735-Pro sequence in the cytoplasmic domain of
TACE, raising the possibility that TACE might be a direct target for
Erk MAP kinase. To test this, we transfected CHO cells with an
expression vector for wild type TACE or a mutant in which the
Thr735 was substituted by alanine, and we assessed the
phosphorylation of TACE and SPRACT by gel electrophoresis and
subsequent quantitation (Fig. 4C). Replacement of
Thr735 by alanine did not abolish the growth factor- and
PMA-induced phosphorylation of TACE and SPRACT. This induction of
phosphorylation of the T735A mutant TACE was blocked by the MEK
inhibitor U0126 (Fig. 4C), similarly to wild type TACE. This
suggests that the phosphorylation of TACE and SPRACT does not result
from a direct phosphorylation of TACE by Erk MAP kinase but is more
likely mediated by another protein kinase downstream from the Erk MAP
kinase. This result does not exclude the possibility that Erk MAP
kinase has the ability to directly phosphorylate this or another
sequence in TACE.
Phosphorylation of Serines in the TACE Cytoplasmic Domain--
The
cytoplasmic domain of TACE contains 16 serines, 7 threonines, including
Thr735, mentioned above, and 1 tyrosine residue (Fig.
5A). Phosphoamino acid
analysis of hydrolyzed protein showed that the endogenous forms of TACE
isolated from EGF-treated HeLa S3 cells were phosphorylated on serine
but not on threonine or tyrosine (Fig. 5B). In addition, only phosphorylated serine was detected in phosphoamino acid analyses of TACE in FBS-stimulated, transfected CHO cells (Fig. 5C).
The absence of threonine phosphorylation further supports our
conclusion that growth factor-induced phosphorylation does not result
from direct phosphorylation of Thr735 by Erk1/2 MAP
kinase.

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Fig. 5.
Phosphoamino acid analysis and CNBr mapping
of TACE phosphorylation. A, amino acid sequence of
the carboxyl-terminal segment of TACE, composing the transmembrane and
cytoplasmic domain. The transmembrane domain is underlined,
and the methionine residues whose carboxyl peptide bond is targeted by
CNBr are numbered according to their positions in the full-length TACE
precursor and shown in boldface. All cytoplasmic serine
residues are shown in boldface. In addition, the
Ser819 phosphorylation site and the Ser791
dephosphorylation site, identified later, and Thr735 are
marked. B, EGF induces only serine but not threonine or
tyrosine phosphorylation in both the zymogen (left) and
mature (right) forms of TACE. In vivo
32P labeling and precipitation of endogenous TACE were
performed as described in Fig. 2A. TACE forms purified by
SDS-PAGE were subjected to HCl hydrolysis. The resulting
32P-phosphoamino acids were resolved by thin layer
chromatography using phosphoserine (PS), phosphothreonine
(PT), and phosphotyrosine (PY) as markers.
C, serum induces the phosphorylation of overexpressed
TACE on serine residue(s) only. In vivo
32P-labeled TACE was immunoprecipitated from
serum-stimulated CHO cells transfected to express TACE, purified by
SDS-PAGE, and subjected to HCl hydrolysis. The resulting
32P-phosphoamino acids were resolved by thin layer
electrophoresis using the phosphoserine, phosphothreonine, and
phosphotyrosine markers. D, CNBr mapping of in
vivo 32P-phosphorylated TACE and SPRACT. In
vivo 32P-phosphorylated TACE was immunoprecipitated
from serum-stimulated, transfected CHO cells, purified by SDS-PAGE, and
subjected to CNBr hydrolysis. The resulting 32P-peptides
were resolved by SDS-PAGE and visualized by autoradiography. Goat
anti-TACE polyclonal antibody was used for C and
D.
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We next set out to define which serines are phosphorylated in response
to growth factor stimulation. The cytoplasmic domain of TACE contains 4 methionines including the 2 methionines at positions 715 and 719, which
we propose to serve as the first amino acids of SPRACT (Fig.
5A). Cyanogen bromide cleavage, which occurs following a
methionine, is expected to cleave following the cytoplasmic methionines
at positions 715 and 719, 759, and 772. This should result in
cytoplasmic fragments of 4 (Leu716-Met719), 40 (Asp720-Met759), 13 (Asp760-Met772), and 51 (Asp773-Cys824) amino acids, in addition to a
77-amino acid segment (Asn638-Met715) that
spans the transmembrane segment.
CHO cells transfected with the wild type TACE expression plasmid were
32P-labeled in vivo in the absence or presence
of serum, and 32P-labeled TACE and SPRACT were isolated by
preparative gel electrophoresis. Both TACE and SPRACT were digested
using CNBr, and the 32P-labeled digestion products were
separated by gel electrophoresis (Fig. 5D). One or possibly
two 32P-labeled fragments with an apparent molecular
mass of 4-6 kDa were identified. These 32P-labeled
fragments were the same following digestion of TACE or SPRACT. We did
not detect a fragment that was compatible with the 77-amino acid
segment upstream from Met715 and would only be present as a
TACE and not a SPRACT digestion product. These results strongly suggest
that the phosphorylation occurs similarly in both TACE and SPRACT and
that no phosphorylation of serines occurs upstream from
Met715. Additionally, no fragment that could correspond to
the 13-amino acid fragment was detected, even though this fragment was
expected to be resolved on gel. Thus, the
Asp760-Met772 segment is unlikely to be
phosphorylated. The 4-amino acid Leu716-Met719
fragment is not expected to be resolved on the gel. Taken together, these results suggest that serine phosphorylation occurs in either or
both the Asp720-Met759 and
Asp773-Cys824 segments.
Identification of Phosphorylated Serines in the TACE Cytoplasmic
Domain--
To identify the phosphorylation sites, we first
gel-purified in vivo 32P-labeled, full size
TACE, obtained from starved or FGF- or serum-treated, transfected
cells, and we compared their phosphopeptide maps following trypsin
digestion. Comparison of FGF-treated with untreated cells revealed
several 32P-labeled peptides, one of which increased and
another one decreased in intensity following FGF treatment (Fig.
6A). Serum stimulation resulted in enhanced or decreased 32P-labeled intensities
of the same two peptides, whereas some increases in intensities of
other minor peptides were noted as well (Fig. 6A). A similar
analysis of the corresponding gel segment from mock-transfected cells
yielded only one spot, i.e. the one at the start position,
as marked in Fig. 6A (data not shown). Together with
phosphoamino acid analysis data shown in Fig. 5, B and
C, these results suggest that growth factors induce
phosphorylation and dephosphorylation of serine residues.

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Fig. 6.
Tryptic peptide analysis of TACE
phosphorylation. A, FGF or serum stimulation
resulted in phosphorylation and dephosphorylation of different TACE
peptides. In vivo 32P-phosphorylated TACE from
transfected CHO cells was immunoprecipitated, purified by SDS-PAGE, and
subjected to in gel digestion with trypsin. The resulting
32P-phosphorylated peptides were resolved by thin layer
electrophoresis followed by thin layer chromatography and visualized by
autoradiography. The origins of the samples are marked by dashed
circles. The solid arrow points to the peptide whose
phosphorylation level was increased after stimulation with FGF or
serum, and the open arrow points to the peptide whose
phosphorylation level was decreased after stimulation. The
dephosphorylated peptide is rather "mobile" in the electric field,
and we noted that its apparent isoelectric point is significantly
affected by the temperature of the buffer for thin layer
electrophoresis. B, phosphorylation, but not the
dephosphorylation, of TACE peptides is inhibited by U0126, a MEK1/2
inhibitor. Samples were processed as in A except that U0126
was used to block serum-induced MEK activation.
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Because the U0126 inhibitor of MEK1/2 inhibited growth factor-induced
shedding (32) and TACE phosphorylation (Fig. 4, A and
C), we next assessed its effect on the phosphopeptide
distribution. As shown in Fig. 6B, U0126 inhibited the
growth factor-induced phosphorylation of the peptide, which normally
shows an enhanced phosphorylation level, and did not visibly affect the
growth factor-induced dephosphorylation of the other peptides. We
therefore conclude that activation of the Erk MAP kinase pathway
enhances the phosphorylation of that peptide in response to growth
factor stimulation.
We next determined the identity of the two major phosphorylated
peptides, i.e. the one with increased and the one with
decreased phosphorylation in response to growth factor stimulation. We
initially attempted to identify the phosphorylation sites by
radioactive microsequencing, but we were unable to generate sufficient
in vivo phosphorylated TACE for this purpose. We therefore
initiated extensive mutagenesis of the serines following
Met715, first in groups of several serine conversions into
alanines. The G1 mutation of TACE resulted in replacement of
Ser717, Ser718, and Ser723 by
alanines, whereas the G2 mutation replaced Ser747 with an
alanine. These serines are located within the 4-amino acid
Leu716-Met719 and the
Asp720-Met759 CnBr digestion fragments,
respectively. Phosphoamino acid analyses revealed that these mutations
did not affect the ability of serum to enhance or decrease the
phosphorylation level of the two peptides, as detected using wild type
TACE (data not shown). Two other clustered mutations addressed the
phosphorylation states of the serines in the carboxyl-terminal
Asp773-Cys824 segment. The G3 mutation
replaced Ser785, Ser786, and
Ser791, whereas the G4 mutation replaced
Ser803, Ser808, and Ser819 by
alanines. As shown in Fig. 7A
the G3 mutation abolished 32P labeling of the peptide,
which normally shows decreased phosphorylation in response to FGF or
serum stimulation, and did not affect growth factor-induced
phosphorylation of the other peptide. In contrast, the G4 mutation
abolished the phosphorylation of the peptide with increased
phosphorylation in response to growth factor stimulation (Fig.
7B).

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Fig. 7.
Identification of the phosphorylation and
dephosphorylation sites in TACE. A-D, tryptic
peptide mapping of TACE constructs with multiple or single serines
substituted by alanines. The mutations are described under
"Results." Sample preparation and processing were done as in Fig.
6A. WT, wild type. E, the S819A
mutation dramatically reduced TACE and SPRACT phosphorylation in
vivo (left panel). Metabolic labeling with
[35S]cysteine/methionine shows equal expression levels of
wild type and S819A TACE (right panel). We refer to the
legend of Fig. 1 for the detection of 32P- and
35S-labeled TACE (T) and SPRACT
(S).
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Subsequent analyses using single amino acid mutations allowed the
identification of the serines that undergo growth factor-induced phosphorylation or dephosphorylation. In the case of the G3 clustered mutation, single amino acid substitutions identified Ser791
as the phosphorylated residue, which shows decreased phosphorylation in
response to growth factor stimulation. Thus, Ser791 to
alanine mutation abolished phospholabeling of the corresponding peptide
in the absence of growth factor stimulation (Fig. 7C; data
not shown). Complementary single amino acid mutations led to the
identification of Ser819 as the amino acid that shows
growth factor-induced phosphorylation. Accordingly, replacement of
Ser819 by alanine abolished phospholabeling of the peptide
that shows increased phosphorylation in response to growth factor
stimulation (Fig. 7D). These results indicate that growth
factor stimulation induces the phosphorylation of TACE at
Ser819 and dephosphorylation of Ser791.
Accordingly, the S819A mutation of TACE showed a low phosphorylation level, when compared with wild type TACE, in the absence of growth factor stimulation, and largely abolished the growth factor-induced phosphorylation of TACE and SPRACT (Fig. 7E).
Together with the data in Fig. 5B, our results strongly
suggest that activation of the Erk MAP kinase pathway mediates the enhanced Ser819 phosphorylation in response to growth
factor stimulation but had little effect on the dephosphorylation
of Ser791.
TACE Phosphorylation and SPRACT Do Not Affect Growth Factor-induced
TGF-
Ectodomain Cleavage--
The identification of
Ser819 as the major growth factor-induced phosphorylation
site led us to assess its role in growth factor-induced TGF-
shedding. We used the EC2 cell line, which had been derived from
genetically modified mice carrying the
Zn/
Zn TACE and lacked functional TACE expression (31, 42). Cells, transfected with a TGF-
expression plasmid, did not show growth factor-induced TGF-
release,
as measured using our previously established TGF-
ectodomain
shedding assay. In contrast, wild type TACE expression conferred a
basal level of TGF-
release, which was further enhanced in response
to serum stimulation (Fig.
8A). Similarly, the S819A and
the G4 mutants of TACE were also able to confer growth factor-induced TGF-
ectodomain shedding. Furthermore, a TACE mutant with its entire
cytoplasmic domain, except for the proximal two amino acids deleted,
was also able to confer serum-induced TGF-
cleavage (Fig.
8A). We therefore concluded that Ser819
phosphorylation in response to growth factor stimulation or activation of the Erk MAP kinase pathway was not required for growth
factor-induced ectodomain shedding by TACE. Furthermore, ectodomain
shedding by TACE did not require its cytoplasmic domain.

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Fig. 8.
TACE phosphorylation and SPRACT expression do
not detectably affect TGF- shedding by
TACE. A, wild type (WT), the S819A, G4,
and the cytoplasmic domain truncated TACE mutants provide equal
shedding of TGF- . EC2 cells that lack endogenous functional TACE
were cotransfected with expression vectors for transmembrane TGF-
and the TACE mutants, serum-starved overnight, pulsed-labeled with
[35S]cysteine/methionine, and chased with cysteine and
methionine with or without dialyzed FBS as a shedding inducer. Soluble
TGF- released into medium was immunoprecipitated and quantitated.
B, increased SPRACT expression did not affect TGF-
shedding by CHO cells. The cells were transfected with a TGF-
expression plasmid alone ( SPRACT) or together with a
plasmid for the ED/TM mutant (+SPRACT) (see Fig. 3 for
schematic sequence presentation). Pulse-chase analyses were carried out
as described in A. C, expression of TACE and
SPRACT by wild type and mutated TACE forms. EC2 cells transfected with
the indicated expression plasmids were metabolically labeled with
[35S]cysteine/methionine and subjected to
immunoprecipitation with antibody against the TACE cytoplasmic domain.
D, SPRACT expression did not affect TGF- shedding in
EC2 cells. Cells were transfected to express wild type TACE or
M715A/M719A TACE that does not generate SPRACT, with or without the
ED/TM mutant that only expresses SPRACT (see C). Sample
preparation and quantitation of shedding were done as in A
and B.
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To evaluate a possible regulatory role of SPRACT in ectodomain
shedding, we assessed the effect of overexpressed SPRACT on TGF-
ectodomain shedding in transfected CHO cells. In these cells TGF-
ectodomain shedding depended on the endogenous proteases, because an
expression plasmid of TACE was not cotransfected. As shown in Fig.
8B, the basal and serum-induced TGF-
ectodomain shedding
was not affected by SPRACT overexpression.
We also assessed a possible role of SPRACT using transfections of EC2
cells. Cells were transfected with an expression vector for wild type
TACE, thus generating full-size TACE and SPRACT or the
M715A/M719A mutant of TACE, which only expresses TACE but not
SPRACT (Fig. 8C, lanes 2 and 3). As is
apparent from Fig. 8D, both versions of the TACE expression
vectors conferred an equal ability to induce basal and growth
factor-induced ectodomain shedding of TGF-
. We also cotransfected an
expression plasmid for the M715A/M719A mutant of TACE with one
that only expresses SPRACT, i.e. the
ED/TM plasmid (Fig.
3, B and C). As is apparent from the
4th lane in Fig. 8C, this
cotransfection resulted in high expression of SPRACT and a lower level
of the M715A/M719A mutant of TACE, when compared with the
expression of TACE in the absence of overexpressed SPRACT. This lower
level of M715A/M719A mutant TACE expression in the latter
cotransfection experiment may be an artifact, because expression of
SPRACT did not affect endogenous TACE expression in HEK293 cells (data
not shown). As shown in Fig. 8D, coexpression of SPRACT did
not affect the basal and growth factor-induced levels of TGF-
ectodomain shedding, as observed with the M715A/M719A mutant of TACE or
wild type TACE. Together, these data do not allow us to conclude that
coexpression of SPRACT has an effect on ectodomain shedding by
TACE.
 |
DISCUSSION |
We demonstrated the growth factor-induced phosphorylation of the
TACE cytoplasmic domain on serine. We also identified an alternative
translation product, named SPRACT, that corresponds to most of the TACE
cytoplasmic domain. Similarly to TACE, SPRACT undergoes regulated
phosphorylation. Our findings represent a first direct demonstration
that a natural inducer of ectodomain shedding induces phosphorylation
of an ADAM family sheddase. While this manuscript was in preparation,
Diaz-Rodriguez et al. (50) reported that PMA induces
phosphorylation of the TACE cytoplasmic domain and proposed that
PMA- and growth factor-induced phosphorylation occurs through direct
phosphorylation of Thr735 in the TACE cytoplasmic domain by
Erk MAP kinase.
Both the glycosylated pro-TACE as well as mature TACE with its
prosegment removed showed enhanced phosphorylation in response to
growth factor or PMA stimulation. In transfected cells that overexpressed TACE only the pro-TACE form was visibly phosphorylated, but this may have resulted from the high predominance of pro-TACE and
its inefficient maturation (47). It should be noted that phosphorylation of mature TACE was not demonstrated upon PMA
stimulation in previous studies (45, 50). The level of TACE
phosphorylation correlated with its shedding activity following
induction by growth factor or PMA stimulation.
We have shown that enhanced phosphorylation of TACE upon growth factor
stimulation required induction of the Erk MAP kinase signaling pathway.
Accordingly, U0126, an inhibitor of MEK1/2 which prevents the
activation of Erk MAP kinase, inhibits growth factor-induced
phosphorylation of TACE and SPRACT to a level similar to that in the
absence of stimulation. Our previous results demonstrated that
activation of the Erk MAP kinase pathway mediates growth factor-induced
shedding of TGF-
, TNF-
, and L-selectin. Therefore, activation of
MEK1/2 and the downstream Erk MAP kinase is required for growth
factor-induced phosphorylation of the TACE cytoplasmic domain and
ectodomain cleavage (32). In contrast, inhibition of the MEK1/2
activity affected only minimally the PMA-induced phosphorylation of
SPRACT and possibly TACE, suggesting the involvement of an additional
kinase pathway different from the MEK/Erk MAP kinase signaling pathway
in PMA-induced phosphorylation. In addition, constitutively active
forms of either MEK1 or Erk2 induced enhanced TACE phosphorylation and
activated ectodomain shedding.
We demonstrated growth factor-induced phosphorylation of the TACE
cytoplasmic domain on serine only but not on threonine or tyrosine.
Extensive mutagenesis identified Ser819 as the major target
for phosphorylation in response to FGF and serum and Ser791
as the major site for growth factor-induced dephosphorylation. Accordingly, the S819A mutant has lost the ability to increase the
phosphorylation level upon growth factor stimulation. Additionally, the
phosphorylation of Ser791 is markedly reduced upon the
treatment. The dephosphorylation of Ser791 is not inhibited
by U0126, suggesting that it is independent of the Erk MAP kinase
pathway. In contrast, growth factor-induced Ser819
phosphorylation is inhibited by U0126 and thus depends on activation of
the Erk MAP kinase pathway.
The two phosphorylation sites that we identified in the human TACE can
also be found in the mouse, rat, and hamster TACE sequences. Sequence
comparison of the cytoplasmic domains of other adamalysins did not
reveal conservation of a sequence similar to the one flanking the
phosphorylated Ser791 in TACE. Interestingly, the
carboxyl-terminal phosphorylated Ser819 is near a motif
that was identified to interact with PTPH1, which was suggested by
overexpression to down-regulate TACE expression and TNF-
shedding
(49). It is therefore possible that the phosphorylation of
Ser819 plays a role in the regulation of TACE processing.
In addition, we noticed that the Ser-Lys dimer that composes
Ser819 exists with unusually high frequencies in the ADAM
family. At least 13 of the 30 adamalysins with cytoplasmic domains
ranging from 4 (ADAM 26) (62) to 196 amino acids (ADAM 19) (63) have at
least one Ser-Lys sequence, with ADAM 30 containing 6 Ser-Lys repeats
in its 85 amino acid cytoplasmic domain (64). Therefore, whether this
sequence functions as a signaling motif is worth addressing.
While this manuscript was in preparation, Diaz-Rodriguez et
al. (50) demonstrated that TACE shows increased phosphorylation on
serine and threonine in response to PMA, and that Thr735,
located within a favorable MAP kinase consensus site, can be directly
phosphorylated by Erk MAP kinase in response to PMA. We did not find
phosphorylation on threonine in response to growth factor stimulation,
and mutation of Thr735 did not affect growth factor- or
PMA-induced phosphorylation of TACE and SPRACT. The basis for this
discrepancy is unclear but may be related to the use of different cell
types and the use of PMA at a 1 µM concentration by
Diaz-Rodriguez et al. (50), whereas we used 20 nM PMA as inducer.
Our use of TACE and protease inhibitors, as well as defined
experimental conditions and the use of a catalytically inactive TACE,
suggested that SPRACT was not derived from autolytic or proteolytic
degradation of TACE either in the cell or during sample preparation.
Instead, SPRACT appears to be expressed through translational initiation from proximal ATG codons in the TACE cytoplasmic domain. Consequently, mutation of the two proximal ATGs encoding
Met715 and Met719 abolished the expression of
SPRACT. Initiation at internal ATGs within an open reading frame has
been observed previously (65-68)in several cellular mRNAs and,
more commonly, in viral RNAs. The expression of SPRACT in untransfected
human cell lines as well as in transfected cells suggests that SPRACT
is a physiological endogenous protein. The poor retention of SPRACT by
PVDF membrane may explain why it was not recognized previously.
Similarly to TACE, SPRACT underwent regulated phosphorylation in
response to growth factor or PMA stimulation, and CNBr cleavage yielded
the same phosphorylated peptide(s) as TACE. These data suggest that SPRACT may be phosphorylated and dephosphorylated on the same residues
as TACE, and raise the possibility of a regulatory role for SPRACT in
the function of TACE.
We have been unable to define a function for SPRACT and for the growth
factor-induced phosphorylation of TACE and SPRACT. Mutation of
Ser819 into Ala or replacement of Ser803,
Ser808, and Ser819 by alanines (the G4
mutation) did not detectably affect the basal and growth factor-induced
shedding of TGF-
by TACE. Additionally, and consistent with previous
findings using PMA as inducer (42, 69), cytoplasmic truncation still
allowed for basal and growth factor-induced TACE activation.
Furthermore, increased levels of SPRACT did not affect TACE activity,
and mutation in TACE to abolish SPRACT expression did not affect growth
factor-induced shedding either. Together, these data suggest that
growth factor-induced changes in the phosphorylation state of the TACE
cytoplasmic domain do not regulate TACE activation. Nevertheless, more
quantitative assessments and analyses using different ectodomain
cleavage substrates are needed to evaluate further the role of SPRACT
and of growth factor-induced phosphorylation of TACE. In line with this
cautious argument, it has been shown that the disintegrin domain of
TACE is required for shedding of interleukin 1 type II receptor but not
of other proteins tested (42, 69), and TACE displays differential efficiencies in cleaving TGF-
at the distal site and the proximal site (17).
Our analyses are further complicated by the fact that the available
TACE-negative cells have been immortalized with activated Ras and Myc
(31, 42), thereby presumably strongly deregulating endogenous signaling
pathways that normally regulate TACE presentation and function. In
addition, although these cells do not express enzymatically active
TACE, the strategy to inactivate the TACE gene in these cells is
expected to only disrupt the catalytically required structure within
the TACE ectodomain and to allow endogenous expression of an inactive
form of TACE, which nevertheless maintains the regulatory cytoplasmic
sequences. Alternatively, SPRACT and the growth factor-induced changes
in phosphorylation of the TACE cytoplasmic domain may not regulate
activation of TACE per se but rather play a role in
transport, processing, maturation, or presentation of TACE. Future
research will be required to address the role of the cytoplasmic domain
of TACE and its regulated phosphorylation in the elaboration of TACE
function and the regulation of ectodomain shedding.