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INTRODUCTION |
SET was first identified as a gene that was fused to the
CAN gene in a patient with acute undifferentiated leukemia,
apparently as a result of a translocation (1). SET is a 39-kDa
phosphoprotein widely expressed in human and mouse tissues and found
predominantly in the cell nuclei, although it has been recently found
associated to the endoplasmic reticulum (1-4). SET belongs to a family
of proteins that also includes nucleosome assembly protein 1, testis-specific protein Y-encoded, the suppressor of presenilin 2 protein, the brain protein MB20, the differentially expressed nucleolar
transforming growth factor
1 target, and the cell division
autoantigen 1 (5-8).
Not much is known about the role of SET in the regulation of cell
activities, although several possible functions of this protein have
been identified. SET has been found to be identical to the
template-activating factor I, a host protein necessary for DNA
replication of the adenovirus genome (9). A long acidic domain in the
C-terminal region of SET is essential for template-activating factor I
activity (9). SET has been also identified as a potent inhibitor of the
protein phosphatase 2A (10), a phosphatase involved in the regulation
of cell cycle progression (11). Moreover, SET has been shown to bind to
nucleosomal histones and in that way protects histones from acetylation
by a variety of histone acetyl transferases (11). Because histone
acetylation has been involved in the regulation of chromatin
condensation and transcription (12), SET might be involved in the
regulation of these processes (13). Recently, it has been described
that SET binds to the cyclin-dependent kinase
(CDK)1 inhibitor
p21Cip1 in vivo and in vitro and that
SET might revert the inhibition produced by p21Cip1 on
cyclin E-CDK2 complexes but not on cyclin A-CDK2 (14). SET also binds
to p35nck5a (15), an activator of the neuronal CDK5 kinase
functioning as a cyclin in neurons to support CDK5 activity (16). SET
interacts specifically with B-type cyclins, although the functional
significance of this interaction has not been elucidated (17). In
general, the known functions of SET clearly relate it with the control of cell cycle progression. However, the cell cycle functional steps
regulated by SET still remain unclear.
In the work reported here, we analyzed in detail the domains of
p21Cip1 and SET involved in their association, and we have
identified that SET might play a role in mitosis by regulating cyclin
B-CDK1 activity.
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EXPERIMENTAL PROCEDURES |
Plasmids--
SET human cDNA in a pET plasmid was a gift
from Dr. Damuni (Pennsylvania State University). The open
reading frame of SET cDNA was amplified by PCR using the
following specific primers: sense,
5'-gggggaattctaatgtcggcgcaggcggcc-3', and antisense,
5'-ggggggaagcttagtcatcttctccttcat-3'. The PCR product was
purified, digested by EcoRI and HindII, and cloned in-frame into pGEX-KG.
cDNAs corresponding to different SET fragments (SET1, aa 1-80;
SET2, aa 81-180; SET3, aa 181-277) were generated by PCR using the
full-length SET cDNA as a template. The primers used to generate the different constructs were the following: SET1 sense,
5'-gggggaatctaatgtcggcgcaggcggcc-3', and SET1 antisense,
5'-ggggaagcttatttgggattttggcgatca-3'; SET2 sense,
5'-ggggcatatgttttgggtaacaacatttgt-3', and SET2 antisense, 5'-gggggggaagcttcctgctggctttattct-3'; SET3 sense,
5'-ggggcatatgaggcagcatgaggaaccaga-3', and SET3 antisense,
5'-ggggggaagcttagtcatcttctccttcat-3'. After amplification, SET1
cDNA was digested with EcoRI and HindIII, whereas SET2 and SET3 were digested with NdeI and
HindII. All fragments were cloned into pGEX-4T-2 vector.
cDNA fragments of p21Cip1 (N terminus, aa 1-90; C
terminus, aa 91-164) were obtained by PCR using specific primers and
human p21Cip1 cDNA as a template. They were inserted
into pGEX-4T-2 by BamHI-HindIII sites.
Green Fluorescent Protein (GFP) and DsRed
Constructs--
The GFP and DsRed expression vectors were gifts from
Dr. H. P. Rahn (Munich). All SET constructs were cloned into
BamHI-HindIII sites, and p21Cip1 was
cloned into EcoRI-HindIII sites.
Protein Expression and Purification--
Glutathione
S-transferase (GST) fusion proteins were expressed in
Escherichia coli and subsequently purified by absorption to
glutathione-Sepharose beads (Amersham Biosciences) as previously described (18). In several cases GST was separated from the GST fusion
proteins by digestion with thrombin protease according to the
manufacturer (Sigma).
Cell Cultures and Transfections--
The colon carcinoma HCT116
cell line and the monkey COS-7 cell line were cultured in Dulbecco's
modified Eagle's medium (Biological Industries) containing 10%
heat-inactivated fetal calf serum. Both cell types were maintained at
37 °C in a humidified atmosphere containing 5% CO2.
Transfections of COS and HCT166 cells were performed with Effectene
(Qiagen) as directed by the manufacturer. Cells were incubated
overnight at 37 °C, and fresh medium was added after 12-18 h. Then
cells were harvested at different times after transfection. To measure
the effect of GFP-SET overexpression on CDK1 activity, transfected
cultures were subjected to cell sorting to separate GFP- or
GFP-SET-expressing cells. Cell sorting gave a 90% of purity. Then
separated cells were subjected to immunoprecipitation and kinase assay
as described below.
Antibodies--
Antibodies against cyclin A were from Santa Cruz
(H-432). All the other antibodies, including anti-cyclin B1 (05-158),
anti-cyclin E (06-459), anti-phospho histone H1 (06-597), and
anti-phospho histone H3 (06-570), were from Upstate Biotechnology.
Immunofluorescence--
Transfected cells were seeded in culture
dishes containing glass coverslips and allowed to grow for at least
24 h. Cells were fixed with ethanol:acetic acid (95:5) and
incubated with primary antibodies for 1 h at 37 °C. After
washing three times (5 min each) in phosphate-buffered saline, cells
were incubated with secondary antibodies conjugated with cyanine 3 (Jackson) for 45 min at 37 °C. After washing, coverslips were
mounted on slides with Mowiol (Calbiochem). Immunofluorescence was
recorded using a confocal laser fluorescence microscope.
To analyze the intracellular localization of DsRed-SET and
GFP-p21Cip1, cells transfected with these plasmids were
fixed with 4% paraformaldehyde for 10 min at room temperature. After
washing, coverslips were mounted on slides with Mowiol, and
immunofluorescence was recorded using a confocal laser fluorescence microscope.
Immunoprecipitation and CDK1 Kinase Assays--
Cells were lysed
in buffer A (50 mM Tris-HCl, pH 7.4, 250 mM
NaCl, 5 mM EDTA, 50 mM NaF, 0.1% Triton X-100,
0.5 µg/µl aprotinin, 10 µg/µl leupeptin, 1 mM
phenylmethylsulfonyl fluoride, and 0.1 mM
Na3VO4) for 30 min on ice. Lysates (0.1-0.5 mg
of protein) were incubated with 2 µg of anti-cyclin B1 antibodies
overnight at 4 °C. Then, protein G-agarose beads (Sigma) were added,
and samples were incubated for 1 h at 4 °C. After 3 washes in
buffer A and 2 in kinase buffer (50 mM Tri-HCl, pH 7.6, 20 mM MgCl2), immunoprecipitates were resuspended
in a final volume of 30 µl of kinase buffer containing 4 µg of
histone H1 (Roche Molecular Biochemicals) and 2 mM
dithiothreitol. Then they were incubated with different amounts of
recombinant SET or/and GST-p21Cip1 for 10 min. A kinase
assay reaction was initiated by the addition of 30 µM ATP
including 10 µCi of [
-32P]ATP (Amersham
Biosciences). After incubation for 30 min at 30 °C, reactions were
stopped by the addition of Laemmli sample buffer. Histone H1 was
separated on 12% SDS-polyacrylamide gels, which were then stained with
Coomassie Blue and dried. Phosphorylation was detected by autoradiography.
Pull-downs with Purified Proteins--
Pull-down experiments
were performed as previously described (19). SET protein, the different
fragments of SET, and the different peptides and fragments of
p21Cip1 were coupled to BrCN-activated Sepharose 4B, as
indicated by the manufacturer. Then, samples (1-4 µg of protein)
were incubated with 2 µg of immobilized protein or peptide in the
binding buffer (50 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 1 mM dithiothreitol, 1% Triton X-100) for 2 h
at room temperature. The beads were then extensively washed with
binding buffer. The unbound fraction was the supernatant of the first
wash. The bound fraction was extracted from the beads with Laemmli
sample buffer. Unbound and bound proteins were analyzed by
SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining.
Laser-scanning Confocal Analysis--
Cell cycle analysis was
studied by laser-scanning cytometry to determine the cellular DNA
content in transfected cells. After transfection, cells were fixed with
70% methanol for 2 h at room temperature and washed with
phosphate-buffered saline. Then, they were incubated with 50 µg/ml
propidium iodide (Sigma) and 200 µg/ml RNase for 10 min. The
coverslips were seeded onto a slide using a mounting medium containing
25% propidium iodide (100 µg/ml in phosphate-buffered saline) and
75% glycerol. Slides were scanned using the 20× objective and 5 mW of Argon laser power. A red fluorescence threshold and a
minimum cell size of 100 pixels identified cells for measurement. At
least 10,000 fluorescent-positive cells were analyzed.
Binding Experiments--
To analyze the binding of cyclin B to
SET or to the mutated form SET1 + 2, 15 mg of Molt 4 extracts (lysed in
buffer A) were loaded onto a SET-Sepharose 4B or SET1 + 2-Sepharose 4B
columns (3 mg, each column). After washing in 50 volumes of the same
buffer, proteins bound to the column were eluted with the same buffer containing 1 M NaCl. The eluted proteins were subjected to
Western blotting using anti-cyclin B1 antibodies.
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RESULTS |
Identification of the p21Cip1 Domains That Interact
with SET--
We previously reported that SET binds to a peptide of
p21Cip1 containing aa 145-164 (14). However, the binding
analysis performed in this previous study did not cover all the
p21Cip1 sequence. To define more precisely the SET binding
domains of p21Cip1, this protein was fractionated in two
parts we named NT domain (aa 1-90) and CT domain (aa 91-164). Then,
the binding of SET to these fragments was analyzed by pull-down
experiments. Results showed that SET only bound to the CT domain of
p21Cip1 (data not shown).
We subsequently analyzed the binding of SET to four synthetic peptides
from this CT domain of p21Cip1, 1 (aa 84-98), 2 (aa
99-121), 3 (aa 122-139), and 4 (aa 140-164). Peptides were coupled
to Sepharose 4B and pull down experiments were performed. Results
showed that SET only significantly bound to peptide 4 (Fig.
1A). These results together
with those previously reported (14) indicate that the
p21Cip1 region, including aa 140-164, contains the SET
binding domain of p21Cip1. To analyze in detail the
putative SET-binding sites in this p21Cip1 region, we
studied the binding of SET to 4 overlapping peptides from this domain
named 4A (aa 140-150), 4B (aa 145-155), 4C (aa 150-160), and 4D (aa
155-164). Pull-down experiments showed that SET bound to peptides 4A,
4C, and 4D but not to 4B (Fig. 1B), indicating that
p21Cip1 binds to SET by two separated domains (aa 140-144
and aa 156-160). To further confirm the relevance of the region
including aa 155-160 in the binding to SET, we generated mutated
p21Cip1 fragments by changing aa 157 (Leu) and aa 159 (Phe)
to Asp. Specifically, we generated the mutated peptides 4C-DID
(aa 150-160 with the mutations), 4-DID (aa 140-164 with the
mutations), and also the fragment CT-DID (aa 84-164 with the
mutations). The binding analysis clearly indicated that all these
fragments containing the mutation did not associate with SET (Fig.
2). Thus, the
157LIF159 domain of the C terminus of
p21Cip1 is essential for the SET-p21Cip1
interaction.

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Fig. 1.
Binding of SET to synthetic peptides from the
p21CT domain. A, pull-down experiments using purified
SET and peptide 1 (aa 84-98), 2 (aa 99-121), 3 (aa 122-139), or 4 (aa 140-164) from the p21CT domain coupled to Sepharose 4B. The amount
of SET bound (B) or not bound (NB) to the beads
was analyzed by gel electrophoresis. B, purified SET was
incubated with p21Cip1 peptides 4A (aa 140-150), 4B (aa
145-155), 4C (aa 150-160), and 4D (aa 155-164) coupled to Sepharose
4B. The amount of SET bound (B) or not bound (NB)
to the different peptides was analyzed by gel electrophoresis.
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Fig. 2.
Change of the p21Cip1
157LIF159 domain by
157DID159 inhibits the binding of SET.
p21Cip1-mutated fragments were generated by changing aa 157 (Leu) and aa 159 (Phe) to Asp. Peptides 4C-DID, 4-DID, and
fragment CT-DID (all of them containing the mutations) were coupled to
Sepharose 4B beads. The amount of SET bound (B) or not bound
(NB) to p21-4C- DID (A) or p21-4-DID
(B) beads was analyzed by gel electrophoresis.
C, the binding of p21CT or p21CT-DID to SET-Sepharose
beads was analyzed by gel electrophoresis.
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Identification of the SET Domains That Interact with
p21Cip1--
To identify the SET domains that interact
with p21Cip1, we generated three SET fragments, SET1 (aa
1-80), SET2 (aa 81-180), and SET3 (aa 181-277) (Fig.
3A). Then the binding of these
three fragments of SET to p21Cip1 peptides 1-4 coupled to
Sepharose 4B was analyzed by pull-down experiments. Results showed that
SET2 and SET3 but not SET1 associated with p21Cip1 peptide
4 (Fig. 3B). Neither SET2 nor SET3 bound to the mutated peptide 4-DID (Fig. 3B). Then, the binding of SET fragments
to p21Cip1 peptides 4A (aa 140-150), 4B (aa 145-155), 4C
(aa 150-160), and 4D (aa 155-164) was studied. Results indicated that
fragments SET2 and SET3 bound to peptides 4C and 4D, although SET3 in
addition bound also to peptide 4A (Fig. 3C). These results
revealed that p21Cip1 putative binding sites are present in
SET2 and SET3 fragments. The binding site from SET2 only binds to aa
156-160 of p21Cip1, whereas the binding site(s) in SET3
might associate with aa 140-144 and/or aa 156-160 of
p21Cip1.

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Fig. 3.
Binding of SET fragments to
p21Cip1 peptides. A, SET was fragmented in
three parts named SET1 (aa 1-80), SET2 (aa 81-180), and SET3 (aa
181-277). B, the amount of these fragments bound
(B) or not bound (NB) to p21Cip1
peptides 1-4 or 4-DID coupled to Sepharose 4B was analyzed by gel
electrophoresis. C, the amount of SET fragments bound
(B) or not bound (NB) to p21Cip1
peptides 4A (aa 140-150), 4B (aa 145-155), 4C (aa 150-160), and 4D
(aa 155-164) or 4C-DID coupled to Sepharose 4B was analyzed by gel
electrophoresis.
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Nuclear Co-localization of SET and p21Cip1--
COS
cells were transfected with GFP-p21Cip1 constructs alone or
together with DsRed-SET, and the cellular localization of both proteins
was subsequently analyzed by confocal microscopy. In some cells a clear
co-localization between both proteins was observed (Fig.
4A). In many cases a clear
co-localization was observed in perinucleolar areas (Fig.
4A). In contrast, in other cells p21Cip1 and SET
were separated (Fig. 4, B and C). These results
indicate a temporal better than a constitutive association of SET with p21Cip1 in the cells. Immunocytochemical studies to detect
endogenous proteins using anti-SET and anti- p21Cip1
antibodies also showed the co-localization of both proteins in a
significant number of cells (Fig. 4D). Whether this temporal association is related to specific phases of the cell cycle is under
study in our laboratory.

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Fig. 4.
Intracellular distribution of
p21Cip1 and SET in COS cells. Cells were
transfected with DsRed-SET and GFP-p21Cip1 as described
under "Experimental Procedures," and the intracellular distribution
of both proteins was analyzed by confocal microscopy. A, a
diffused pattern of both proteins, showing that a clear co-localization
in perinucleolar areas was observed in a significant number of cells.
B, in other cells a punctuated pattern of SET and a diffused
pattern of p21Cip1 without co-localization was observed.
C, other cells showed a punctuated pattern of both
proteins without co-localization. D,
immunofluorescence experiments using anti-SET and
anti-p21Cip1 antibodies.
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Overexpression of SET Blocks Cell Cycle at
G2/M--
To analyze the possible role of SET
in cell cycle regulation we studied the effect of SET overexpression on
cell cycle progression. By using laser-scanning confocal analysis, we
determined the amount of DNA in COS and HCT116 cells transfected with
GFP-SET or GFP. As shown in Fig. 5, SET
overexpression induced a clear increase in the number of cells at
G2/M and a decrease in the number of cells in
G1 in both cell types. These results suggest that
overexpression of SET blocks cell cycle at G2/M. To further
confirm this possibility, experiments analyzing the effect of SET
overexpression on histone H3 phosphorylation (this phosphorylation only
occurs in mitotic cells) were performed on COS and HCT116 cells.
Results revealed that in both cell types transfected with SET, no
phosphorylated histone H3 was observed (Fig.
6, A and B). In
contrast, cells transfected only with GFP showed this phosphorylation
(Fig. 6, A and B). Overexpression of SET did not
block the cell cycle at S phase since transfected cells showed
bromodeoxyuridine incorporation (data not shown). These results support
the hypothesis that SET might be involved in the regulation of
G2/M transition.

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Fig. 5.
Effect of SET overexpression on cell
cycle. By using laser-scanning confocal analysis, the amount of
cellular DNA in COS (upper panel) and HCT116 (lower
panel) cells transfected with GFP-SET (empty bars) or
GFP (filled bars) was determined. Results are represented as
the mean values of the percentage of cells in the different phases of
the cell cycle ± S.D. of eight independent experiments carried
out in duplicate. Statistically significant differences were evaluated
by Student's t test. **, p < 0.001.
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Fig. 6.
Effect of SET overexpression on
phosphorylation of histones H3 and H1. HCT116 (A and
C) and COS (B and D) cells were
transfected with GFP-SET or GFP as indicated under "Experimental
Procedures." Then cells were subjected to immunofluorescence analysis
using specific antibodies against phosphorylated H3 (A and
B) or H1 (C and D) histones.
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Role of SET on Cyclin B-CDK1 Activity--
Evidence presented here
showing that overexpression of SET blocked cell cycle progression at
G2/M and the fact that SET binds to cyclin B (17) suggests
that SET alone or together with p21Cip1 might be involved
in the regulation of cyclin B-CDK1 activity. Thus, we first studied the
effect of SET overexpression on histone H1 phosphorylation (histone H1
phosphorylation depends on cyclin B-CDK1 activity). These experiments
were performed on COS and HCT116 cells transfected with GFP-SET or GFP
as a control. Results revealed that cells overexpressing SET did not
show H1 phosphorylation, whereas those transfected only with GFP did
(Fig. 6, C and D). We subsequently studied the
effect of SET overexpression on cyclin B-CDK1 activity. At 48 h
after transfection with GFP-SET or GFP alone, COS and HCT116 cells were
subjected to immunoprecipitation using anti-cyclin B antibodies. Then
immunoprecipitates were analyzed for CDK1 activity. Results indicated
that after SET transfection cyclin B-CDK1 activity was clearly
inhibited in both cell types (Fig.
7A). To analyze whether SET
might directly regulate cyclin B-CDK1 activity, HCT116 cell extracts
were immunoprecipitated with specific anti-cyclin B antibodies. Then
CDK1 activity was determined in the immunoprecipitates in the presence
or absence of exogenous SET. As shown in Fig. 7B, CDK1
activity was inhibited by the addition of 1-2 µM
purified SET. Inhibition was dose-dependent, and the effect
was similar to that observed by recombinant GST-p21Cip1
(Fig. 7B). Interestingly, this effect was specific for
cyclin B-CDK1 because cyclin A-CDK2 and cyclin E-CDK2 activities were not affected by the addition of purified SET (Fig.
8A). SET and p21Cip1 generate an additive inhibitory effect. The
addition of 0.5 µM SET and 0.5 µM
p21Cip1 inhibited cyclin B-CDK1 activity, whereas at these
doses SET and p21Cip1 added alone only produced a limited
inhibition (Fig. 8B). To determine which domain of SET was
involved in the inhibition of cyclin B-CDK1 activity, we studied the
effect of SET fragments (SET1, SET2, and SET3) on this activity.
Results indicate that SET3 fragment contains the cyclin B-CDK1
inhibitory domain (Fig. 8C). To further confirm this
possibility, we analyzed the binding of cyclin B to a mutated form of
SET lacking the SET3 fragment (SET1 + 2). Affinity columns of SET or
SET1 + 2 were prepared and loaded with cell extracts. After extensive
washing, bound proteins were eluted, and the presence of cyclin B in
the eluates was analyzed by Western blot. As shown in Fig.
9A, cyclin B bound to SET but
not to SET1 + 2, indicating that SET3 was responsible for cyclin B
binding. Overexpression of SET1 + 2 in COS cells showed an
intracellular localization both nuclear and cytoplasmic, similar
to the distribution of GFP but different to SET-GFP that was mainly
nuclear (Fig. 9B). The overexpression of this mutated form
of SET was not able to block cell cycle at G2/M (Fig.
9C). These results support the SET3 domain as responsible
for the regulation of G2/M transition.

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Fig. 7.
Role of SET on cyclin B-CDK1 activity.
A, COS and HCT116 cells were transfected with SET-GFP or GFP
alone. At 48 h after transfection, cells were separated using a
cell sorter. Cell extracts (0.1 mg) were subjected to
immunoprecipitation using anti-cyclin B antibodies. Immunoprecipitates
(IP) were then analyzed for CDK1 activity as indicated under
"Experimental Procedures." B, HCT116 cell extracts were
immunoprecipitated with specific anti-cyclin B antibodies. Then CDK1
activity was determined in the immunoprecipitates in the absence
(C+) or in the presence of different concentrations of
exogenous SET, GST-SET, or GST-p21Cip1.
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Fig. 8.
SET specifically inhibits cyclin B-CDK1
activity, and its effect is additive to that of p21Cip1.
A, HCT116 cell extracts were immunoprecipitated with
specific antibodies against cyclins B, A, or E. Then CDK activities
associated with these cyclins were determined in the immunoprecipitates
(IP) in the absence (C+) or in the presence of
different concentrations of exogenous SET. B, cyclin B-CDK1
activity was determined in immunoprecipitates from HCT116 cell extracts
in the absence (C+) or in the presence of 0.5 µM SET or p21Cip1 alone or in combination.
C, cyclin B-CDK1 activity was determined in
immunoprecipitates from HCT116 cell extracts in the absence
(C+) or in the presence of 2 µM concentrations
of the different SET fragments (SET1, SET2, and SET3).
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Fig. 9.
Overexpression of a mutant form of SET,
lacking aa 181-277, did not block cell cycle progression.
A, Molt-4 extracts were loaded in affinity columns of SET or
SET lacking aa 181-277 (SET1 + 2), and the binding of cyclin B1 was
analyzed by Western blot. B, intracellular localization of
GFP, GFP-SET1 + 2, or GFP-SET in transfected COS cells. C,
by using laser-scanning confocal analysis, the amount of cellular DNA
in COS cells transfected with GFP (black bars), GFP-SET
(gray bars), and GFP-SET1 + 2 (empty bars) was
determined. Results are represented as the mean values of the
percentage of cells in the different phases of the cell cycle ± S.D. of three independent experiments carried out in duplicate.
Statistically significant differences were evaluated by Student's
t test. (* p < 0.01).
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DISCUSSION |
SET is a cyclin B-interacting protein that recently has been shown
to associate with the CDK inhibitor p21Cip1 (14, 17). We
report here that SET inhibits cyclin B-CDK1 activity both in
vivo and in vitro and that this inhibitory capacity is
additive to that of p21Cip1. Thus, SET might be involved in
the regulation of G2/M transition.
Our results revealed that SET binds to two specific domains of
p21Cip1 located at the C terminus (aa 140-144 and aa
156-164). The later domain is essential for the binding of SET to
p21Cip1 since specific mutations in this region block the
association between both proteins. Thus, domain 140-144 might help the
binding, but it is not enough for maintaining a stable association with SET when domain 156-164 is mutated. The C terminus of
p21Cip1, which includes the 156-164 domain, also binds a
number of proteins including proliferating cell nuclear antigen, the E7
oncoprotein of the human papilloma virus, Gadd45, c-Myc, and calmodulin
among others (19-23). This fact suggests an important role for this
p21Cip1 region in the control of cellular functions.
Studies to analyze the ability of SET to compete with these other
p21Cip1-binding proteins for the association with
p21Cip1 are still lacking. Thus, the possible involvement
of SET on the modulation of the binding of p21Cip1 to these
important cell cycle regulators is still unknown.
SET binds to p21Cip1 also by two domains, one located at
the central region of SET, whereas the other one is placed at the
C-terminal region. These results suggest that SET might associate with
two p21Cip1 molecules at the same time or, alternatively,
that the two SET-binding sites of p21Cip1 (aa 140-145 and
156-164) might bind to two different domains of SET. Experimental work
to answer this question is under way in our laboratory.
A previous report demonstrated by immunoprecipitation experiments that
SET and p21Cip1 might be associated in vivo
(14). Here we demonstrate the in vivo co-localization of
both proteins by ectopically expressing p21Cip1 and SET
followed by confocal microscopy analysis but also by immunofluorescence
studies using specific antibodies. Results reported here indicate that
p21Cip1 and SET clearly co-localize in the nucleus;
however, patterns showing that both proteins might also be separated
inside the nucleus were also observed in a significant number of cells.
These results suggest that both proteins might interact at specific moments of the cell cycle but not in others. A possibility is that both
proteins might interact during mid-late S phase. The perinucleolar
pattern of co-localization (Fig. 4A) suggests that both
proteins might interact during the mid-S phase since a perinucleolar pattern of replication factories has been observed at this stage of S
phase (24). In addition, preliminary results obtained in our laboratory
indicate that SET co-localizes with bromodeoxyuridine in the
replication factories. Studies to define the kinetics of p21Cip1-SET interactions during cell cycle are under way in
our laboratory.
Overexpression of SET in two different cellular types, COS and HCT116,
induced the inhibition of cell cycle progression at G2/M
transition. This block is associated with the inhibition of cyclin
B-CDK1 activity, indicating that SET might be involved in the control
of mitosis entry by regulating the activity of cyclin B-CDK1 complexes.
Results from in vitro experiments revealing that SET
inhibits cyclin B-CDK1 activity at concentrations similar to those of
p21Cip1 strongly support this hypothesis. Interestingly,
the inhibitory effect of SET and p21Cip1 are additive,
suggesting that they might cooperate in regulating cyclin B-CDK1
activity under specific circumstances, as for instance after DNA
damage. This cooperation in the inhibition of cyclin B-CDK1 might be
similar to that occurring between Gadd45 and p21Cip1 after
DNA damage (25, 26). The specific domain of SET involved in cyclin
B-CDK1 inhibition is located at the C-terminal region (SET3) that
contains a long very acidic domain (aa 225-277). Recently, it has been
shown that Gadd45 binds to CDK1 and inhibits the activity of cyclin
B-CDK1 complexes (27, 28). Interestingly, the Gadd45 domain responsible
for the inhibition is an acidic patch including the sequence DEDDDR
(28). Moreover, Ran, a small nuclear GTPase implicated in both cell
cycle progression and nuclear export (29, 30), also contains an acidic
motif with similar amino acid composition in its C-terminal domain
(DEDDDL) (31). It has been shown that overexpression of Ran induces
G2/M arrest, whereas deletion of the DEDDDL motif abolishes
this activity (29). Interestingly, the C-terminal region of SET
contains an acidic sequence 260DEDDDE265 that
is very similar to those of Gadd45 and Ran, involved in the regulation
of cyclin B-CDK1 activity and G2/M transition. All these
results suggest that this sequence included in the C terminus of SET is
the one involved in the inhibition of cyclin B-CDK1 activity and,
consequently, in G2/M block. However, this still remains to
be proved.
Results reported here together with recent reports indicate that
SET has a relevant role in the regulation of CDK activities. A previous
report revealed that SET blocks the inhibitory effect of
p21Cip1 on cyclin E-CDK2, thus allowing a cyclin E-CDK2
activity in the presence of p21Cip1 (14). In this case, SET
plays a role as an "activator" of these complexes in
G1. Recently, it has been shown that SET binds to p35nck5a and p35nck5ai (15). Both proteins are
activators of the neuronal CDK5 (16). SET and p35nck5a
co-localize in the nucleus of cultured cortical neurons, and in
vitro experiments indicated that SET enhanced the activity of
p35nck5a-CDK5. Interestingly, the acidic tail of SET is
required for the stimulatory effect of p35nck5a-CDK5 (15).
Results reported here indicate that in addition to the positive
regulation of CDK2 and CDK5 activities, SET might also play a role as a
negative regulator of CDK1. This information reveals that SET plays a
dual role in the cell cycle as a positive regulator of G1/S
and as a negative regulator of mitosis entry. A possibility that has to
be investigated is that SET might be involved in blocking cyclin B-CDK1
during S phase and G2, avoiding a premature activation of
these complexes before cells would be ready to enter mitosis.
SET is also a potent inhibitor of protein phosphatase 2A, which is
involved in different cellular functions, including cell proliferation
(10). This fact suggest a concerted regulation by SET of
phosphorylation/dephosphorylation of nuclear proteins during cell
proliferation but also during other cellular processes requiring the
activity of CDKs.
Results of this study are the first to demonstrate that SET is a
negative regulator of cyclin B-CDK1 in vivo and in
vitro and as a consequence of mitosis entry. The fact that SET is
also involved in the regulation of chromatin structure by modulating histone acetylation (11) places this protein in a key position in the
regulation of cell cycle. On the one hand, SET might be involved in the
control the phosphorylation status of nuclear proteins by regulating
CDK and protein phosphatase 2A activities. On the other hand, SET might
control transcriptional activity of chromatin by regulating histone
acetylation and chromatin condensation. The protein p21Cip1
might participate in these processes, although its role still remains unclear.