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INTRODUCTION |
Sp1 is a ubiquitous transcription factor that is particularly
important for the regulation of TATA-less genes that encode housekeeping proteins (1-4). Although Sp1 generally has been considered constitutively to regulate gene expression (5), its activity
and cellular content have been shown to be regulated during development
(6-9), cellular proliferation (10, 11), apoptosis (12), and other
cellular processes (13-19). Most of this regulation occurs through
either post-translational modifications of Sp1 or by alterations in the
abundance of Sp1 protein. The principal known post-translational
modifications of Sp1 are phosphorylation and glycosylation through the
O-linkage of the monosaccharide, N-acetylglucosamine
(O-GlcNAc)1
(20-22). Phosphorylation of Sp1 occurs under a variety of
circumstances. It has been shown that altered levels of phosphorylation
of Sp1 result in changed DNA binding activity (6, 23). With regard to
O-glycosylation of Sp1, our laboratory has proposed two
potential roles for O-GlcNAc in Sp1 function. By using a
model peptide based on the Sp1 transcriptional activation domain, we
previously showed that the hydrophobic interactions into which this
peptide entered were blocked by O-GlcNAc modification of the
peptide (24). The other potential role of O-GlcNAc in Sp1
function is in the control of Sp1 stability (25). We found that under
the experimental conditions of glucose starvation and adenylate cyclase
activation, the O-GlcNAc modification of most of the
cellular proteins vanished. Sp1 was among the proteins so modified;
however, Sp1 was the only transcription factor examined that was
degraded by a protease that could be inhibited by specific inhibitors
of the 26 S proteasome. Conversely, treatment of cells with glucose or
glucosamine resulted in increased protein modification by
O-GlcNAc and stabilization of Sp1 to proteolytic
degradation. These observations have established a correlation between
the glycosylation state of Sp1 and its ability to be proteolytically
degraded. Furthermore, these results suggested that the degradation of
Sp1 under these circumstances was relatively specifically targeted at
Sp1. However, Sp1 is probably not the only protein whose stability is
associated with the O-GlcNAc state. It has been shown that
an O-glycosylated protein, p67, controls the phosphorylation
and activity of elongation factor 2 in protein synthesis (26, 27).
Under conditions of serum starvation, p67 is first deglycosylated and
then degraded (28). Loss of p67 results in the phosphorylation of
elongation factor 2 and the inhibition of protein synthesis. We have
proposed that the O-GlcNAc state of Sp1 or perhaps other
proteins may be involved in a mechanism that coordinates cellular
nutritional status with growth and general protein synthesis.
That the proteasome-dependent degradation of Sp1 appears to
be relatively selective is in concordance with evidence that
transcription factor abundance or activity can be selectively regulated
by proteolytic mechanisms (29-32). This selective protein degradation
is usually accomplished by the 26 S proteasome (32, 33). How the
proteasome recognizes its target is quite variable. In general, those
proteins that are destined for degradation are first modified
covalently by the attachment of multiple ubiquitin peptides, which, in
turn, are recognized by the proteasome (31, 33-35). Furthermore,
proteasome substrate proteins appear to be recognized through a variety
of sequence motifs (31). The examples include the
proline-glutamate/aspartate-serine-threonine (PEST) sequence in short
lived proteins (36-41), the N-end rule for unstable proteins (42-44),
the glycine-rich region (GRR) in NF-
B precursor p105 (45), the
destruction box in mitotic cyclins (46, 47), and other motifs in
various labile proteins (48-50). But how Sp1 is targeted to the
proteasome-dependent degradation is still unknown.
In this study, we established an in vitro system to analyze
Sp1 degradation. This system allowed the identification of the N-terminal 54 amino acids of Sp1 as the major domain that targets Sp1
for proteasome-dependent degradation. We also showed that Sp1 degradation is a two-step process. An endoproteolytic cleavage occurs downstream of this target region, followed by the degradation of
the C-terminal portion. This target domain in Sp1 is both necessary and
sufficient for the targeting of this transcription factor for the
endocleavage, and this domain does not need to be ubiquitinated for
this function. The elucidation of this Sp1 target domain will facilitate studies that connect protein O-glycosylation with
the control of Sp1 abundance.
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EXPERIMENTAL PROCEDURES |
Materials--
LLnL
(N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal)
and forskolin were purchased from Sigma. Lactacystin was purchased from Dr. E. J. Corey (Harvard University, Boston), and
clasto-lactacystin
-lactone was purchased from
Calbiochem. The above drugs were dissolved in dimethyl sulfoxide
(Me2SO) and stored at
20 °C for later use.
Glutathione-Sepharose and reduced free glutathione were purchased from
Amersham Pharmacia Biotech. Glucosamine, ATP, AMP-PNP,
N-ethylmaleimide, hemin, 2,4-dinitrophenol, 2-deoxyglucose, phosphocreatine, creatine phosphokinase, and thrombin were purchased from Sigma. Monoclonal anti-GST antibody was purchased from Sigma. Polyclonal anti-Gal4 DNA binding domain (amino acids 1-147) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antiserum to Sp1 (3517) was raised against the C-terminal portion of Sp1 as described previously (11).
Cell Culture--
Normal rat kidney (NRK) cells were grown in
Dulbecco's modified Eagle's medium (DMEM) with 10% newborn calf
serum (NCS), nonessential amino acids (Life Technologies, Inc.), 100 µg/ml penicillin (Sigma), and 50 µg/ml gentamicin (Sigma) at
37 °C in a humidified incubator with 7.0% CO2 (51). For
stimulation of the cells, exponentially growing cells were seeded at
2.5 × 106 of cells per 100-mm diameter dish at
approximately 30% confluency. After overnight incubation, the cell
culture medium was changed to glucose-free DMEM containing 10% NCS,
and the cells were incubated for additional 20 h. The cells were
then treated with 5 mM glucosamine or 100 µM
forskolin as indicated in glucose-free DMEM containing 10% NCS, and
the incubation was continued for an additional 24 h. Lactacystin
and LLnL were added to the cells at a concentration of 20 and 50 µM, respectively, 24 h prior to harvest. BSC40 cells were grown in DMEM with 10% NCS, 100 µg/ml penicillin, and 50 µg/ml gentamicin at 37 °C in a humidified incubator with 7.0% CO2.
Vaccinia Virus Expression and Purification of Recombinant GST
Fusion Proteins--
The full-length human Sp1 cDNA (kindly
provided by Dr. James Kadonaga) was cloned into the pTM3GST vector, and
a recombinant vaccinia virus was generated as described previously (25,
52). The cDNAs encoding various fragments of Sp1 were obtained by
restriction enzyme digestion or PCR amplification using high fidelity
Pfu DNA polymerase (Stratagene, La Jolla, CA) and cloned
into the pTM3GST vector. The resulting plasmids (pTM3GST-Sp1 fragment) were transfected into BSC40 cells by electroporation at 250 V and 500 microfarads. After overnight recovery, the cells were infected with
recombinant vaccinia virus VTF7-3 containing the T7 RNA polymerase
coding sequence (53). At 24 h after infection, whole cell extract
was prepared by freezing and thawing the cells three times in the
extraction buffer (20 mM Tris (pH 7.5), 0.5 M
NaCl, 0.5% Nonidet P-40, 1 mM MgCl2, 0.5 mM EDTA, 20% glycerol, 1 mM dithiothreitol
(DTT), 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 0.5 mM
phenylmethylsulfonyl fluoride (PMSF)). Glutathione-Sepharose beads were
incubated with the whole cell extract for 30 min at 4 °C. The
glutathione beads were then collected and washed three times in the
extraction buffer. The fusion proteins bound to the glutathione beads
were either eluted with 20 mM reduced free glutathione or
cleaved from GST with 4 units of thrombin per mg of fusion protein.
Nuclear Extract Preparation and the Reconstituted Degradation
Assay--
Nuclear extract was prepared as described (54).
Approximately 2 × 107 cells were washed by ice-cold
phosphate-buffered saline twice and scraped into 6 ml of cold
phosphate-buffered saline. The cells were collected by centrifugation
for 3 min at 2,000 × g at 4 °C. The cells were
resuspended in 1 ml of ice-cold buffer A (10 mM HEPES-KOH
(pH 7.9 at 4 °C), 3 mM MgCl2, 10 mM KCl, 0.5% Nonidet P-40, 1 mM DTT, and 1 mM PMSF) and allowed to stand on ice for 10 min. The cells
were gently vortexed for 10 s and centrifuged for 15 s at
16,000 × g to pellet nuclei. The supernatant was
thoroughly decanted, and the nuclei were resuspended in 80 µl of
buffer C (20 mM HEPES (pH 7.9 at 4 °C), 20% glycerol,
1.5 mM MgCl2, 300 mM NaCl, 0.2 mM EDTA, 1 mM DTT, and 1 mM PMSF).
The suspension was intermittently homogenized by pipetting over a
period of 30 min incubation on ice and then centrifuged for 15 min at
16,000 × g at 4 °C. The resulting supernatant was
collected and designated as nuclear extract (the protein concentration
is approximately 5 µg/µl). The ATP-depleted nuclear extract was
prepared as described (55). For the reconstituted degradation assay,
the glutathione-eluted GSTSp1 or other GST fusion protein
(approximately 10 ng) was mixed with nuclear extract (containing 50 µg of protein) in the presence or absence of 330 µM
LLnL. The mixing reaction was performed in a volume of 12 µl in 20 mM HEPES (pH 7.9), 250 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 9%
glycerol, 1 mM DTT, and 1 mM PMSF and was incubated on ice or room temperature for 2 h or as indicated. The
proteins in the reaction mixture were then separated by
SDS-polyacrylamide gel electrophoresis (PAGE) and analyzed by Western
blot analysis using anti-GST antibody. In the experiment involved
clasto-lactacystin
-lactone, nuclear extract was
preincubated with
-lactone for 25 min at room temperature prior to
the addition of GSTSp1 protein. To test the ATP dependence of the
degradation assay, the indicated reagents were added at the following
final concentrations: 2 mM ATP, 5 or 20 mM
AMP-PNP, 5 mM MgCl2, 5 mM NEM, and 100 µM hemin. The
ATP-regenerating system consisted of 10 mM creatine phosphate and 100 µg/ml creatine phosphokinase (55, 56). These reagents were added to the nuclear extract, and the mixture was incubated at 30 °C for 20 min. Purified GSTSp1 protein was added to
the preincubated mixture, and the incubation was continued at 30 °C
for another 60 min. The reaction mixture was then subjected to Western
blot analysis using anti-GST antibody.
Western Blot Analysis--
The samples were resolved by SDS-PAGE
on 8 or 10% gels, transferred to ECL-Hybond nitrocellulose membranes
(Amersham Pharmacia Biotech), blotted with the primary antibody
overnight at 4 °C and with the secondary antibody (anti-rabbit or
mouse immunoglobulin, horseradish peroxidase-linked whole antibody from
donkeys, Amersham Pharmacia Biotech) for 1 h at room temperature,
and then subjected to enhanced chemiluminescence detection (Amersham
Pharmacia Biotech).
Mass Spectroscopy--
The vaccinia virus-expressed GST-SpV or
GST-Sp1 was purified and immobilized on glutathione-Sepharose beads.
The beads were mixed with differently treated NRK nuclear extracts for
1 h at room temperature. The beads were then extensively washed
and cleaved with thrombin to release the Sp1 fragment from GST; 5 M guanidine was applied to elute all the components into
the solution. The resulting solution was subjected to mass spectroscopy
analysis. Matrix-assisted laser desorption ionization-time-of-flight
(MALDI-TOF) mass spectroscopy was carried out on a Perspective
Biosystems (Framingham, MA) Voyager Elite MALDI-TOF mass spectrometer.
Samples were mixed with a saturated solution of
-cyano-4-hydroxycinnamic acid in a water/acetonitrile (50:50)
mixture acidified with 0.1% trifluoroacetic acid. A 1-µl aliquot of
the sample was spotted onto the gold plate target. Ionization of the
sample was accomplished with a nitrogen laser operated at 337 nm. A
delayed extraction method was used in the determination of molecular
mass. Measurement of ion flight times through the drift region of the
mass spectrometer was carried out with a Tektronix (Beaverton, OR)
model TDS784A oscilloscope. The instrument was calibrated with external
molecular weight standards.
 |
RESULTS |
In Vitro Reconstituted Degradation System--
To map the domain
of Sp1 protein that targets its degradation, we established an in
vitro reconstituted system for Sp1 degradation. Our previous
studies had shown that when NRK cells were glucose-starved and
stimulated with forskolin, Sp1 protein was rapidly degraded in intact
cells, and the degradation was inhibited by the proteasome inhibitors,
lactacystin and LLnL, but not by a cysteine protease inhibitor, E64-D
(25). Sp1 protein was not degraded when the cells were glucose-starved
alone, or treated with glucosamine, or cultured in normal
glucose-containing medium (5 mM). Furthermore, we showed,
by using mixing experiments, that intact Sp1 in nuclear extract from
glucosamine-treated cells could be degraded by nuclear extract from
cells previously treated with glucose starvation and forskolin.
Based on these observations, we established an Sp1 degradation assay
using purified GSTSp1 that had been expressed recombinantly in a
vaccinia virus-based protein expression system. This GSTSp1 protein was
mixed with nuclear extract from differently treated NRK cells. The
mixture was incubated on ice for 2 h before protein separation on
SDS-PAGE. Western blot analysis using anti-GST monoclonal antibody was
performed to detect the degradation of GSTSp1. As shown in Fig.
1A, GSTSp1 was not degraded by
nuclear extract from cells that had been glucosamine-treated (Fig.
1A, lane 1), whereas GSTSp1 was degraded by extract from
glucose-starved and forskolin-stimulated cells (Fig. 1A,
lane 2). A degradation product, termed GSTSpX (Fig.
1A, lane 2), was detected with the GST antibody and is
discussed later. The degradation of GSTSp1 was inhibited by the
addition to the reaction mixture of the proteasome inhibitor, LLnL,
suggesting that this degradation was proteasome-dependent (Fig. 1A, lane 3). As negative controls, vaccinia
virus-expressed GST, GST-epidermal growth factor receptor extracellular
domain, and GST-SpE (SpE contains domain B, a glutamine-rich activation domain of Sp1 (24)) were not degraded under the same conditions (Fig.
1A, lanes 4-12). Similarly, we also observed that GST-Sp1 was not degraded by nuclear extract from the cells that had been glucose-starved alone or cultured in normal glucose-containing medium
(data not shown). The degradation of GSTSp1 in vitro has been correlated with the degradation of Sp1 in vivo.

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Fig. 1.
In vitro reconstituted degradation
system. A, substrate specificity. Vaccinia
virus-expressed GST-Sp1, GST, GST-epidermal growth factor receptor, and
GST-SpE (10 ng) were exposed to nuclear extract (50 µg of protein)
from glucosamine-treated (labeled as GlcN) or
glucose-starved and forskolin-treated (labeled as For.) NRK
cells in a total volume of 12 µl; 330 µM LLnL was added
to the reaction as indicated. The reaction mixture was incubated on ice
for 2 h, then separated by SDS-PAGE, and subjected to Western blot
analysis with anti-GST antibody. B, the degradation of
GST-Sp1 has the characteristics of the proteasome. Left
panel, the reagents, ATP, AMP-PNP, MgCl2, NEM, hemin,
or combinations of them, were preincubated with nuclear extract
(NE) from differently treated NRK cells
(Forskolin-ATP indicates that the nuclear extract was
derived from cells that had been treated with forskolin and glucose
starvation and had been depleted of ATP). Recombinant GST-Sp1 protein
was added to the preincubated mixture, and the reaction continued to incubate at 30 °C for
another 60 min, followed by Western blot analysis with anti-GST
antibody. Right panel, the state of the endogenous Sp1 in
the NRK nuclear extracts used for the experiments shown in the
left panel was determined by Western blot analysis with
anti-Sp1 antibody. C, proteasome-specific inhibitors inhibit
the degradation of GSTSp1. Left panel, GSTSp1 was mixed with
nuclear extract from glucosamine-treated (lane 1),
glucose-starved, and forskolin-treated (lane 2), forskolin
plus 20 µM lactacystin (lane 3) or 50 µM LLnL-treated (lane 4) cells. The reaction
mixture was incubated for 30 min at room temperature, followed by
Western blot analysis with anti-GST antibody. Right panel,
the nuclear extract (50 µg) from glucose-starved and
forskolin-treated cells was preincubated with Me2SO
(lane 2), 100 µM -lactone (lane
3), or 330 µM -lactone (lane 4) in a
total volume of 11 µl for 25 min at room temperature. GSTSp1 (10 ng)
was then added to the preincubation mixture, and the incubation was
continued for additional 30 min at room temperature, followed by
Western blot analysis with anti-GST antibody.
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Given that LLnL is also a calpain inhibitor (57, 58), we tested whether
the degradation of GSTSp1 in this reconstituted system had other
features characteristic of the 26 S proteasome-dependent degradation (56). As shown in the left panel of Fig.
1B, the addition of the nonhydrolyzable ATP analog, AMP-PNP,
to the reaction mixture at 5 and 20 mM concentrations
blocked the degradation of GSTSp1 (Fig. 1B, lanes
2-4). Furthermore, we also prepared ATP-depleted nuclear
extract. NRK cells were glucose-starved and stimulated with forskolin.
At 2 h before cell harvest, 2,4-dinitrophenol and 2-deoxyglucose
were added to the culture medium to deplete the cellular ATP. As shown
in Fig. 1B, lane 5, GSTSp1 was not degraded by this nuclear
extract. However, when 2 mM ATP and Mg2+ were
added to the extract, degradation of GSTSp1 was restored (lane
8). The addition of Mg2+ alone or Mg2+
with AMP-PNP did not stimulate the degradation (lanes 7 and
9). Together, these data demonstrate that the degradation of
Sp1 in this reconstituted system is ATP-dependent. As shown
in lanes 10 and 11, the addition of 5 mM NEM or 100 µM hemin to the reaction inhibited the degradation of GSTSp1, showing that the degradation of
GSTSp1 in this system was sensitive to NEM and hemin. The ATP dependence and the sensitivity to hemin and NEM are all features of the
26 S proteasome and suggest that the in vitro degradation of GSTSp1 is proteasome-dependent. The right
panel shows a Western blot analysis using anti-Sp1 antibody to
determine the content of endogenous Sp1 in the nuclear extract used in
the reconstituted assay. The endogenous Sp1 protein level in the
nuclear extract confirms our previous observation that the treatments
of the cells resulted in the respective preservation or loss of Sp1
in vivo.
To confirm further the degradation of GSTSp1 in this reconstituted
system is proteasome-dependent, we made use of the
proteasome-specific inhibitor, lactacystin. Lactacystin has been known
to inhibit specifically the 20 S and 26 S proteasome activity without
inhibiting any other protease yet tested, including the serine
proteases trypsin and chymotrypsin and the cysteine proteases papain,
calpain I and II, and cathepsin B (59-62). In aqueous solution,
lactacystin undergoes spontaneous hydrolysis to form an intermediate
clasto-lactacystin
-lactone that is the sole species to
interact with the proteasome and inhibit its activity (63-65). As
shown in the left panel of Fig. 1C, GSTSp1 was
degraded by the nuclear extract prepared from glucose-starved and
forskolin-treated cells (lane 2). However, GSTSp1 was not
degraded by the nuclear extract from the cells that had been treated
with forskolin plus 20 µM lactacystin or 50 µM LLnL (Fig. 1C, left panel, lanes 3 and
4). Furthermore, we tested if the active lactacystin
derivative
-lactone can directly inhibit GSTSp1 degradation in
vitro. As shown in the right panel of Fig.
1C, the addition of increasing amounts of
-lactone to the
in vitro reaction mixture blocked the degradation of GSTSp1 in a dose-dependent manner (lanes 3 and
4). However, the in vitro treatment of nuclear
extract with lactacystin at a similar concentration failed to block
significantly GSTSp1 degradation (data not shown). In our reaction
system, the transient accumulation of
-lactone could be blocked by
the large amounts of glutathione from GSTSp1 preparation (59, 64, 65).
Nevertheless, our results strongly suggest that GSTSp1 degradation in
the in vitro reconstituted system is
proteasome-dependent, and this system can mimic what happens in the living cells.
The N-terminal Region of Sp1 Contains a Target Domain for Its
Degradation--
The reconstituted degradation system allowed us to
map the domain of Sp1 protein that targets its degradation. The domain structure of Sp1 has been well characterized. As shown in Fig. 2A, the glutamine-rich domains
in the N-terminal half of the protein confer transactivation. The
C-terminal zinc finger domain binds DNA (66). We made a series of N-
and C-terminal deletion fragments of Sp1 that are termed SpN (amino
acids 81-621 of Sp1), SpBS (amino acids 245-621), SpBC (amino acids
345-621), SpZD (amino acids 621-778), SpBCD (amino acids 245-778),
SpV (amino acids 1-81), and SpK (amino acids 1-54), as shown in Fig.
2A. These fragments of Sp1 were expressed as GST fusion
protein using the vaccinia virus expression system and were subjected
to the reconstituted degradation assay with nuclear extract prepared
either from cells treated with glucosamine (inactive extract) or cell
treated with glucose deprivation and forskolin (active extract). LLnL
was added to the active extract to block proteasome activity. Fig.
2B, lanes 1, 4, 7, 10, 13, and 16, shows the
effect of the inactive extract (labeled GlcN) on the various
GST fusion proteins and therefore indicates the amount of the input
GST-Sp1 fusion protein. Similarly, the lanes labeled LLnL
show the effect of proteasome blockade on the active extract and also
indicate the input GST-Sp1 fusion protein level. As shown in Fig.
2B, lanes 1-3, full-length GST-Sp1 was almost
completely degraded by active nuclear extract, producing a degradation
product, GSTSpX, of about 35 kDa. All the other Sp1 domains (SpN, SpBS,
SpBC, SpZD, and SpBCD) were degraded by the active extract to a
considerably lesser degree than the full-length Sp1, although some
degradation fragments were produced (lanes 4-18). These
data suggest that there may be multiple structural elements that are
responsible for Sp1 degradation, but the major one does not lie in the
five domains shown in Fig. 2B. By elimination, the major
targeting domain appears to reside in the N-terminal 81 amino acids of
Sp1. As shown in Fig. 2A, this N-terminal region of Sp1
contains a segment from amino acid 12 to 46 that is glycine-rich, with
14 glycine residues in this 34-amino acid region. This glycine-rich feature is reminiscent of the glycine-rich region (GRR, it contains 15 glycines from amino acids 372-394 of p105) in NF-
B precursor p105
that serves as a proteasome processing signal for this transcription factor (45). To determine whether this N-terminal region of Sp1 can
also serve as a proteasome-dependent processing signal, we
made GST fusion proteins that contain only the first 81 amino acids
(GSTSpV) or the first 54 amino acids (GSTSpK). As shown in Fig.
2C, GSTSpV was degraded by the forskolin-treated nuclear extract, and from it was generated the same 35-kDa GSTSpX degradation product as from full-length Sp1 (Fig. 2C, lane 8). Because
GSTSpK is so close in size to the degradation product, GSTSpX, it was impossible to determine whether GSTSpK was degraded. These data are
compatible with the notion that SpV contains a target for Sp1
degradation, perhaps the GRR-like domain.

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Fig. 2.
The N-terminal 81 amino acids of Sp1 contain
the major target domain for Sp1 degradation. A, the
structural domains of Sp1 protein, the deletion Sp1 fragments that were used in the
following studies, and the amino acid sequence of the N-terminal 81 amino acids of Sp1. B, vaccinia virus-expressed GST fusion
proteins containing full-length Sp1 or Sp1 deletion fragments were
applied to the in vitro reconstituted degradation system as
described in Fig. 1. The reaction mixture was subjected to Western blot
analysis with anti-GST antibody. C, the N-terminal 81 amino
acids of Sp1 (SpV) contain the target domain for its degradation.
GSTSpV (amino acids 1-81 of Sp1) and GSTSpK (amino acids 1-54 of Sp1)
were applied to the reconstituted degradation system. GST-Sp1 and
GST-SpN were used as controls. The reaction mixture was subjected to
Western blot analysis with anti-GST antibody.
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The Target Domain of Sp1 Directs the Degradation of a Heterologous
Protein--
To determine whether the N-terminal 81 amino acids of Sp1
(SpV) can act as an independent signal for Sp1 processing, we
constructed a fusion protein, GST-SpV-Gal4, in which SpV was fused to
the C-terminal of GST and the N-terminal of yeast Gal4 DNA-binding domain (amino acids 1-147) (Fig.
3A). We also constructed
GST-SpK-Gal4 to determine whether the N-terminal 54 amino acids of Sp1
contain the degradation signal. These fusion proteins were then
subjected to the in vitro degradation assay. As shown in
Fig. 3B, GSTSpVGal4 was degraded by active extract and
produced a similarly sized degradation product, GSTSpX, as GSTSp1 (Fig.
3B, lane 5). GSTSpKGal4 was also degraded and produced the
same size degradation product as GSTSp1 (Fig. 3B, lane 8).
The two heterologous proteins were expressed as a doublet that may be
due to posttranslational modifications of Gal4 (67). As specificity
controls, GSTGal4 or GSTSpEGal4 (SpE contains amino acids 424-521 of
Sp1) were not degraded under the same condition, although the Gal4
portion of the molecule appears to have been inefficiently cleaved to
produce a larger GST fusion product. These data indicate that the first
54 amino acids of Sp1 contain a sequence that can confer processing to a heterologous protein, in this case a segment of Gal4. This N-terminal element of Sp1 therefore appears both necessary as the main target domain for Sp1 degradation and sufficient as an independent processing signal for an unrelated protein.

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Fig. 3.
The N-terminal 54 amino acids of Sp1 direct
the degradation of a heterologous protein. A, the
structure of the heterologous proteins. The Sp1 fragment was fused to
the C terminus of GST and to the N terminus of Gal4 DNA binding domain
(amino acids 1-147). B, the degradation of the heterologous
proteins. The fusion proteins, GSTSpVGal4, GSTSpKGal4, GSTGal4, and
GSTSpEGal4 were applied to the reconstituted degradation system as
described in Fig. 1. GSTSp1 was used as a control. Shown is the Western
blot analysis of the fusion proteins with an anti-GST antibody.
For., forskolin.
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Exogenous Target Domain Competitively Blocks Sp1
Degradation--
To determine whether the processing of Sp1 is a
saturable process, we loaded the reconstituted degradation assay
with excess SpV to determine the effect on holo-Sp1 degradation. As
shown in Fig. 4 lanes 3-5,
the addition of increasing amounts of GSTSpV inhibited the degradation
of GSTSp1. This inhibition was specific in that the addition of large
amounts of GST alone did not alter GSTSp1 degradation (lanes
6-8). These data show that exogenous target domain SpV can
competitively block the degradation of holo-Sp1. Our results suggest
that the factors required for Sp1 degradation are saturable by the
target domain.

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Fig. 4.
Exogenous target domain competitively blocks
Sp1 degradation. Increasing amounts of vaccinia virus-expressed
GSTSpV or GST were mixed with forskolin-treated nuclear extract
(NE) as indicated for 15 min at room temperature. After
preincubation, GSTSp1 substrate was added into the reaction for
additional 30 min at room temperature. The reaction mixture was
subjected to Western blot analysis with anti-GST monoclonal
antibody.
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Ubiquitination of the Target Domain Is Not Required for Sp1
Processing--
To determine whether ubiquitination is required for
recognition of the N-terminal target domain of Sp1, we mutated the only two potential ubiquitination sites in this domain (the first 81 amino
acids). The lysine residues at positions 9 and 12 were mutated to
alanine residues by site-directed mutagenesis. The resulting mutant,
GST-Sp1mKK, was expressed, purified, and subjected to the in
vitro degradation assay described previously. As shown in Fig.
5, GSTSp1mKK was degraded (lanes
3-5) as efficiently by the active extract as did wild-type Sp1,
and the processing generated the same size product as wild-type GSTSp1.
The proteasome inhibitor, LLnL, also inhibited the degradation of this
mutant Sp1 (lane 5). This result suggests that
ubiquitination of the first 81 amino acids of Sp1 is not required for
its recognition by the proteasome-dependent system. Our
data do not exclude the possibility that another region of Sp1 is
modified by ubiquitination in the process of Sp1 degradation.

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Fig. 5.
Ubiquitination of the N-terminal 81 amino
acids of Sp1 is not required for Sp1 processing. GSTSp1mKK, in
which the lysine residues at the positions 9 and 12 of Sp1 were mutated
to alanine residues, was applied to the reconstituted degradation assay
as described in Fig. 1. Wild-type GSTSp1 was used as a control. Western
blot analysis with anti-GST antibody was performed. For.
forskolin.
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The Degradation of Sp1 Is a Two-step Process--
To understand
better the process of Sp1 degradation, we performed a time course
experiment using GSTSp1 as the substrate in the reconstituted system.
GSTSp1 protein was exposed to the activated nuclear extract for
different incubation times, and the degradation products were analyzed
by Western blotting both with anti-GST and anti-Sp1 sera. Fig.
6A shows the Western blot
using the GST antiserum. By 90 min, the GSTSp1 had vanished, while the
GSTSpX product had accumulated to a maximal degree (Fig. 6A,
lanes 2-7). That GSTSpX is about 6 kDa larger than GST alone
suggests that this cleavage product results from an endoproteolytic
cleavage within the Sp1 portion of the fusion protein, resulting in a
C-terminal extension of GST. This notion was supported by the finding
that when the same protein blot was stripped and reblotted with an anti-Sp1 antibody, a new band, termed Sp1a, was detected at about the
89-kDa position (Fig. 6B, lanes 2-7). Sp1a is smaller than Sp1 by about 6 kDa, suggesting that the GSTSp1 is cleaved at a position
corresponding to about 6 kDa from the N terminus of the Sp1 portion of
the fusion protein.

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Fig. 6.
The degradation of Sp1 is a two-step
process. A, a time course experiment of GSTSp1
degradation. GSTSp1 was mixed with activated nuclear extract
(NE) at room temperature for the indicated periods. The
reaction mixture was separated on SDS-polyacrylamide gels and subjected
to Western blot analysis with anti-GST antibody. B, the
membrane in A was stripped and reblotted with anti-Sp1
antibody. C, a time course experiment of GSTSpVGal4
degradation. GSTSpVGal4 was incubated with activated NRK nuclear
extract at room temperature for the indicated periods. The reaction
mixture was analyzed by Western blot with anti-GST antibody.
D, the membrane in C was stripped and reblotted
with anti-Gal4 DNA binding domain (DBD) (Gal4 amino acids
1-147) antibody.
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During this time course experiment, Sp1a first accumulated, reaching a
maximum level between 5 and 25 min of incubation in the activated
extract (Fig. 6B, lanes 2 and 4). The level of
Sp1a then decreased at longer incubation times, suggesting that the Sp1a band was first produced and then degraded (Fig. 6B, lanes 5-7). In contrast, the GST-SpX remnant peptide is not degraded. The addition of proteasome inhibitor, LLnL, inhibited the initial endocleavage step (Fig. 6, A and B, lane
8).
To determine further the role of the first 81 amino acids of Sp1 in
this degradation process, we performed the same time course experiment
using the heterologous protein substrate, GSTSpVGal4. As shown in Fig.
6C, the GSTSpVGal4 substrate was also endocleaved, generating the N-terminal peptide GSTSpX, which accumulated with increasing incubation time (lanes 2-6). We reblotted the
same membrane with anti-Gal4 DNA-binding domain polyclonal antibody to
detect the C-terminal Gal4 peptide after the endocleavage. As shown in
Fig. 6D, the level of Gal4 peptide also accumulated with
increasing incubation time (lanes 2-6), suggesting that the Gal4 peptide was not subsequently degraded like Sp1a. The proteasome inhibitor, LLnL, still blocked the initial endocleavage step (Fig. 6,
C and D, lane 7). These results suggest that Sp1
degradation is a two-step process as follows: an endoproteolytic
cleavage followed by the degradation of the C-terminal portion and that the N-terminal 81 amino acids of Sp1 only direct the endocleavage step,
and additional signals may be necessary for the further degradation of
the C-terminal portion of Sp1.
Mapping of the Endocleavage Site of Sp1 Degradation--
To
confirm the notion that Sp1 is endoproteolytically cleaved and to
determine the endocleavage site of Sp1, we used mass spectroscopy to
determine the molecular mass of the degradation product SpX. Since the
amino acid sequence of Sp1 is known, it was possible to determine the
last amino acid in SpX from a precision determination of its molecular
mass. Because GSTSpV degradation produces the same size product as
GSTSp1, and because the protein expression system yields higher
quantities of GSTSpV, GSTSpV was used as the starting substrate for the
generation of GSTSpX. The GSTSpX was generated by exposure of GSTSpV to
activated extract. The GSTSpX was repurified and cleaved with thrombin
at the thrombin cleavage site encoded in the expression vector at the C
terminus of GST. The upper panel of Fig.
7A shows a mass spectroscopic analysis of GSTSpV that had been exposed to inactive extract made from
glucosamine-stimulated cells. Three peaks were detected. The
9,661.97-Da peak has a mass corresponding to SpV; the 26,126-Da peak
corresponds to GST; and the 35,791.8-Da peak corresponds to GSTSpV that
failed to be cleaved by thrombin. The lower panel of Fig.
7A shows the analysis of GSTSpV that had been exposed to
activated extract. Again, three major peaks were detected, indicating
that the GSTSpV was processed to completion, producing the degradation
product GSTSpX. The SpX corresponds to the 5,961.92-Da peak. The other
peaks represent GST and GSTSpX uncleaved by thrombin.

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Fig. 7.
The endocleavage occurs between
Leu56 and Leu57 of Sp1. A,
mapping of the endocleavage site by mass spectroscopy. GSTSpV
immobilized on glutathione-Sepharose beads was mixed with
glucosamine-treated NRK nuclear extract (NE) (upper
panel) or glucose-starved and forskolin-stimulated NRK nuclear
extract (lower panel) at room temperature for 1 h. The
glutathione beads were washed extensively with thrombin cleavage
buffer. The proteins bound on glutathione beads were digested with
thrombin to cleave the GST moiety from the fusion protein and eluted
from the beads with 5 M guanidine. The elute was subjected
to mass spectroscopy analysis. The upper panel shows the
spectrum resulting from exposure of the fusion protein to inactive
extract; the lower panel shows the spectrum resulting from
exposure of the fusion protein to active extract. B, the
amino acid sequence of SpV peptide indicating the endocleavage
site.
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From the molecular mass determination of SpX, it can be deduced that
the endocleavage occurred between Leu56 and
Leu57 of Sp1 (Fig. 7B). Subsequently, the GSTSpX
remnant from GSTSp1 was similarly analyzed, and the endocleavage site
was mapped to the same leucine residues (data not shown). These results
confirm the notion that Sp1 is endoproteolytically cleaved at a site
just downstream of the GRR-like sequence.
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DISCUSSION |
The transcription factor Sp1 is particularly important in the
transcriptional regulation of TATA-less genes, including growth factors, receptors, enzyme involved in DNA synthesis, critical regulators of the cell cycle, and other regulatory proteins (4, 5).
Previously, we provided evidence that Sp1 is degraded in vivo by the proteasome system in cells exposed to the stress of glucose deprivation and cAMP accumulation (25). The cAMP signal is
often associated with nutrient deprivation in lower eukaryotes, whereas
in mammalian cells, cAMP accumulation results in cell cycle arrest at
G1 phase (68-70). Thus, the Sp1 degradation under the
experimental conditions may reflect the extremes of Sp1 regulation during physiological processes. In this study, we show that the degradation of recombinant Sp1 can be accomplished in
vitro by using nuclear extract from cells treated by the same
nutritional stress. This reconstituted system also appears to degrade
Sp1 through an ATP-dependent system that has features
characteristic of the proteasome. The use of this reconstituted system
allowed us to define the principal structural determinant in Sp1 that directs its degradation.
We identified the N-terminal 54 amino acids as the major determinant
that directs Sp1 to a two-step processing through a
proteasome-dependent mechanism. The endocleavage step
appears to be an integral part of proteasome processing. For the
degradation of Sp1, this first step is blocked by LLnL, hemin, and NEM
and is ATP-dependent, all features of the 26 S proteasome
(71, 72). More directly, the endocleavage step is also blocked in
nuclear extract from cells treated in vivo with
proteasome-specific inhibitor lactacystin and is blocked in
vitro by treatment of nuclear extract with the active
-lactone
derivative of lactacystin. Blockade of the first endocleavage step
results in blockade of the subsequent degradation of the protein, and
Sp1 fragments lacking the N-terminal 81 residues containing the target
region and endocleavage site are relatively poor substrates for the
proteasome-dependent system. Thus, the N-terminal region of
Sp1 appears necessary for efficient proteasome-dependent processing. This N-terminal sequence also appears to be sufficient for
the endocleavage, although it is not sufficient for degradation of the
C-terminal fragment that is generated by the cleavage. That is, this
element, when fused to a heterologous protein, can confer
endoproteolytic processing to the fusion protein. However, the
fragment of Gal4 liberated by the endocleavage was not subsequently degraded. This result suggests that the segment of protein C-terminal to the target region must have properties that allow its further degradation by the proteasome system. Of note, Sp1 fragments lacking the N-terminal target region could be degraded, albeit much less efficiently than proteins containing that target domain. A PEST sequence has been identified between amino acids 487 and 508 of Sp1
(10). Whether this PEST motif plays a role in the further degradation
of the C-terminal portion of Sp1 remains to be elucidated. Furthermore,
the endocleavage of Sp1 is a saturable process, in that excessive
amounts of the N-terminal Sp1 element result in the blockade of
holo-Sp1 processing and degradation. This saturability suggests that
the Sp1 N terminus is specifically recognized by the
proteasome-dependent system. Finally, mutation of the only two lysine residues in the N-terminal domain of Sp1 did not affect the
degradation event, implying that the N-terminal target sequence in Sp1
need not be ubiquitinated. However, we have not entirely ruled out the
possibility that the remaining C-terminal portion of Sp1 is
ubiquitinated. Although ubiquitination is usually a requirement for
proteasome degradation, the degradation of ornithine decarboxylase and
c-Jun has been shown to be proteasome-dependent but
ubiquitin-independent (73, 74). We have identified this N-terminal
region of Sp1 as a target for proteasome-dependent degradation; however, the mechanism by which it targets to the proteasome remains unclear.
In this N-terminal target domain of Sp1, there is a glycine-rich-like
region that has several similarities to the glycine-rich region (GRR)
in NF-
B p105 that directs the proteasome processing of this NF-
B
precursor (45). The GRR in p105 directs a similar two-step processing
to this protein as follows: a GRR-dependent endoproteolytic
cleavage, followed by the degradation of the C-terminal portion that is
the inhibitory subunit of p105. The N-terminal portion liberated by the
cleavage remains intact and is the active form of NF-
B subunit p50.
However, for p105, the degradation event occurs in the cytosol, and the
net result is an activation of this transcription factor. In contrast,
the two-step processing of Sp1 appears to occur in the nucleus and
results in the disposal of Sp1. Interestingly, the short segment of Sp1
that is at the N-terminal of the GRR does not appear to be degraded.
Whether this residual fragment plays a role in the cell is unclear, but there is some prior evidence that this N-terminal region of Sp1 can be
bound by cellular factors that have an effect on Sp1 function (75). For
both Sp1 and p105, the endoproteolytic cleavage occurs at a position
C-terminal to the GRR, and the amino acid residues at the site of
cleavage are not important determinants of the cleavage. Rather, it
appears that the spacing between the GRR and the cleavage site is the
major determinant of the cleavage site. As evidence for this notion, we
mapped the exact site of cleavage in Sp1 and found it immediately
C-terminal to Leu56, 10 residues downstream of the GRR. We
also showed that the Sp1-derived fusion protein GSTSpKGal4, which
contains only the first 54 amino acids of Sp1 and, therefore, lacks the
leucine residues found at the cleavage site in the native protein, is
efficiently cleaved at a similar position C-terminal to the GRR that
was observed for GSTSp1. Similarly, for p105, the endoproteolytic
cleavage occurs downstream of the GRR at a fixed position relative to
the GRR that does not depend on specific downstream sequences.
Taken together, these results suggest that the N-terminal region of Sp1
is a primary recognition site for proteasome-dependent degradation (Fig. 8). This recognition
results first in an endocleavage that generates two fragments. For both
Sp1 and p105, the C-terminal fragment is further degraded, leaving the
N-terminal fragment intact. The further degradation of the C-terminal
fragment by the proteasome system appears to require properties in
addition to the GRR. This two-step processing provides an additional
level of control of protein fate. It allows for the partial degradation of target proteins and the generation of remnants that have altered biological functions.

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Fig. 8.
A model for Sp1 degradation. Sp1 protein
is first cleaved between Leu56 and Leu57
residues and produces two peptides. The C-terminal peptide is
subsequently degraded, whereas the small N-terminal peptide remains
intact. This two-step processing is analogous to the processing of
NF- B precursor p105.
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