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Molecular and Cellular Biology, June 1999, p. 4465-4479, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Cytoplasmic Localization of Human cdc25C during
Interphase Requires an Intact 14-3-3 Binding Site
Sorab N.
Dalal,
Colleen M.
Schweitzer,
Jianmin
Gan, and
James A.
DeCaprio*
Dana-Farber Cancer Institute and Harvard
Medical School, Boston, Massachusetts 02115
Received 30 July 1998/Returned for modification 8 September
1998/Accepted 1 March 1999
 |
ABSTRACT |
cdc25C induces mitosis by activating the cdc2-cyclin B complex. The
intracellular localization of cyclin B1 is regulated in a cell
cycle-specific manner, and its entry into the nucleus may be required
for the initiation of mitosis. To determine the cellular localization
of cdc25C, monoclonal antibodies specific for cdc25C were developed and
used to demonstrate that in human cells, cdc25C is retained in the
cytoplasm during interphase. A deletion analysis identified a
58-amino-acid region (amino acids 201 to 258) in cdc25C that was
required for the cytoplasmic localization of cdc25C. This region
contained a specific binding site for 14-3-3 proteins, and mutations in
cdc25C that disrupted 14-3-3 binding also disrupted the cytoplasmic
localization of cdc25C during interphase. cdc25C proteins that do not
contain a binding site for 14-3-3 proteins showed a pancellular
localization and an increased ability to induce premature chromosome
condensation. The cytoplasmic localization of cdc25C was not altered by
irradiation or treatment with the nuclear export inhibitor
leptomycin B. These results suggest that 14-3-3 proteins may negatively
regulate cdc25C function by sequestering cdc25C in the cytoplasm.
| |
INTRODUCTION |
In eukaryotic cells, an active
cyclin-dependent kinase complex, cdc2-cyclin B1, promotes entry into
mitosis. Prior to mitosis, kinase activity is inhibited by
phosphorylation of the cdc2 catalytic subunit on two residues,
threonine 14 (T14) and tyrosine 15 (Y15) (reviewed in reference
46). Several kinases, including wee1 (3, 21,
48), mik1 (32), and myt1 (36, 42),
phosphorylate cdc2 at residues T14 and Y15 and inhibit mitotic
progression. Entry into mitosis is dependent on dephosphorylation of
the T14 and Y15 residues, resulting in the formation of an active
cdc2-cyclin B complex (reviewed in reference 46).
Inhibition of cdc2 dephosphorylation is a target of the DNA replication
and DNA damage checkpoints in both yeast and mammalian cells (reviewed
in references 45 and 46).
Dephosphorylation of cdc2 and subsequent entry into mitosis are
catalyzed by the dual-specificity phosphatase, cdc25C (11, 27, 33,
40, 57), which in turn is regulated by cell cycle-dependent phosphorylation events (15, 24). Hyperphosphorylation of
cdc25C during mitosis is thought to stimulate its phosphatase activity (19, 22, 24, 29). Although the specific kinase that
phosphorylates cdc25C during mitosis is unknown, several candidate
kinases activate cdc25C in vitro. cdc25C may be phosphorylated by its
own substrate, an active cdc2-cyclin B complex, to create an
autoactivation loop (19, 22, 56). Other studies have
reported that the mitosis-specific hyperphosphorylation of
cdc25C can occur in the absence of both cdc2 and cdk2 (23),
suggesting that other kinases may activate cdc25C during mitosis. A
candidate for the cdc25C M-phase kinase is the product of the
Xenopus Plx1 gene, a polo-like kinase. Plx1 can
phosphorylate cdc25C in vitro and stimulate cdc25C phosphatase activity
in vitro (28); however, it is not yet clear whether Plx1
stimulates the mitotic activation of cdc25C in vivo.
Premature activation of cdc25C is prevented by phosphorylation of
specific residues in cdc25C during interphase that are distinct from
the sites phosphorylated in M phase. Piwnica-Worms and colleagues have
demonstrated that during interphase, the major phosphorylation site in
human cdc25C is a serine residue at position 216 (S216) (47). Conversely, S216 is not phosphorylated during mitosis, suggesting that phosphorylation of this residue may contribute to the
negative regulation of cdc25C activity (49, 52). Consistent with the above hypothesis, expression of a cdc25C mutant that substituted alanine for serine 216 (S216A) induced premature entry into
mitosis by override of a DNA replication checkpoint and a -radiation-induced DNA damage checkpoint (49). Several
kinases that promote the phosphorylation of the S216 residue in cdc25C have recently been identified. Piwnica-Worms and colleagues purified from HeLa cells a kinase, C-TAK1, that specifically phosphorylates residue S216 in vitro (47, 50). cdc25C can also be
phosphorylated by chk1, a DNA damage checkpoint kinase that was first
identified in fission yeast (9, 49, 52, 60, 61). chk1 is
activated by phosphorylation in response to -radiation-induced DNA
damage, leading to a cell cycle arrest in G2 (9,
52). Recent work with the fission yeast Schizosaccharomyces
pombe has shown that cdc25 can also be phosphorylated by another
kinase activated by DNA damage, cds1 (65). This pathway is
replicated in other eukaryotes, as Kumagai et al. have demonstrated
that cdc25C is phosphorylated and capable of responding to checkpoint
control in Xenopus laevis extracts that have been depleted
of chk1 (30). A human homolog of cds1, chk2, has recently
been cloned and found to phosphorylate cdc25C at S216 in vitro
(38). Therefore, the S216 residue in cdc25C may be a
substrate for multiple kinases that specifically inhibit its activity
in response to the S-phase or DNA damage checkpoints.
Phosphorylation of cdc25C at S216 by chk1 or C-TAK1 in vitro
results in the generation of a binding site for the 14-3-3 family of
proteins (44, 49, 50, 52). A consensus binding site for
14-3-3 was first identified by Muslin et al. as RSXpSXP (X being any
amino acid and pS indicating phosphoserine) (44). Recent
work by Yaffe and colleagues has led to refinement of the 14-3-3 binding consensus to R[S/Ar][+/S]pS[L/E/A/M]P as well as identification of a second consensus, RX[Ar/S][+]pS[LEAM]P (where Ar is an amino acid with an aromatic side chain and + is an amino acid with a basic side chain) (62). A peptide corresponding to the first sequence is present in residues 213 to 218 of cdc25C. 14-3-3 proteins have been shown to bind to cdc25C during interphase, at
a time when S216 is phosphorylated (49). Similarly, cdc25 has been shown to form a complex with the 14-3-3
and 14-3-3
proteins in X. laevis egg extracts (31). Given
that S216 is critical for regulation of cdc25C activity and can be
phosphorylated by chk1, 14-3-3 proteins may inhibit cdc25C function in
response to DNA damage (49). Consistent with this model, two
14-3-3 homologues in fission yeast, rad24 and rad25, are required for
the response to radiation induced DNA damage and G2
checkpoint control (1, 8). The rad24 and rad25 proteins
associate with the S. pombe cdc25 protein in vitro when
cdc25 is phosphorylated by either chk1 or cds1 (65).
Notably, expression of 14-3-3
can be induced by
-radiation-induced DNA damage in human cells (18).
The activity of a number of cellular proteins is regulated by their
cell cycle-dependent intracellular localization. For example, cyclin B1
is located in the cytoplasm during interphase and then enters the
nucleus just prior to mitosis (51). Several reports have
demonstrated that the cytoplasmic retention of cyclin B1 is maintained
due to the presence of a strong nuclear export signal (NES) (13,
59, 63). Toyoshima and colleagues demonstrated that inhibition of
cyclin B1 nuclear export led to an override of the DNA damage
checkpoint upon treatment with caffeine (59). Similarly, Jin
et al. demonstrated that a constitutively nuclear cyclin B1 (cyclin B1
was fused to the simian virus 40 [SV40] nuclear localization signal
[NLS]) protein could override a DNA damage-induced checkpoint arrest
in cooperation with a cdc2 mutant that could not be phosphorylated at
T14 and Y15 (cdc2AF) (25). Notably, in both studies, the
nuclear localization of cyclin B1 was necessary but not sufficient to
override the DNA damage checkpoint and promote entry into mitosis.
Similarly, Hagting et al. observed premature mitosis when an
export-defective cyclin B1 mutant was coexpressed with either cdc2AF,
cdc25C, or a dominantly active mutant of cdc25C, S216G (13).
These results suggest that the DNA replication and DNA damage
checkpoints affect mitotic progression by regulating cyclin B1
localization as well as by modulating cdc25C function.
Although cdc25C activity can be regulated by complex formation with
14-3-3 proteins, association of 14-3-3 proteins with cdc25C has not
been shown to decrease cdc25C phosphatase activity in vitro (31,
49). This leads to the question of how 14-3-3 proteins may affect
cdc25C function. cdc25C has been reported to contain a bipartite NLS in
its N terminus (47), and at least three reports suggested
that human cdc25C was a predominantly nuclear protein (10, 12,
39). However, an exogenously expressed Myc epitope-tagged human
cdc25C protein was reported to be localized primarily in the cytoplasm
during interphase and then translocated to the nucleus just prior to
entry into M phase (16). To address this issue, a panel of
monoclonal antibodies (MAbs) specific for human cdc25C was developed
and used to demonstrate that the endogenous human cdc25C protein is
localized in the cytoplasm during interphase in multiple cell types.
The region in cdc25C required for the cytoplasmic localization was
mapped to a 14-3-3 binding site in cdc25C. Thus, cytoplasmic
localization of cdc25C may depend on its ability to associate with
14-3-3 proteins, suggesting that 14-3-3 proteins prevent mitotic
progression by sequestering cdc25C in the cytoplasm.
| |
MATERIALS AND METHODS |
Cell strains and transfections.
The human osteosarcoma cell
line U-2OS and the primary diploid human fibroblast strains MRC-5 and
WI-38 were obtained from the American Type Culture Collection and
cultured in Dulbecco's modified Eagle's medium (Cellgro) supplemented
with 10% Fetal Clone-I serum (Hyclone), 100 U of penicillin per ml,
and 100 µg of streptomycin per ml. Cells were transfected by calcium
phosphate precipitation as described previously (58).
Briefly, 15 µg of the cdc25C plasmid was used per 100-mm-diameter
dish transfected, and the total amount of DNA was adjusted to 25 µg
with pBluescript (pBSK
). To perform the localization and
premature chromosome condensation (PCC) assays, cells were transfected
on a coverslip in a 35-mm-diameter dish; 3 µg of each cdc25C plasmid
was cotransfected with either 2 µg of pBSK or 2 µg of
the hemagglutinin (HA) epitope-tagged cyclin B1 plasmid per
35-mm-diameter dish. To perform the cell cycle synchrony experiments, 15 µg of cdc25C plasmid, 8 µg of pBSK, and 2 µg of
a CD19 cDNA (64) were transfected per 100-mm-diameter dish.
To perform the 14-3-3 binding assays, 15 µg of cdc25C plasmid was
cotransfected with 7.5 µg of pBSK and 2.5 µg of
HA-14-3-3 per 100-mm-diameter dish.
Plasmids.
The N-terminal Myc-tagged cdc25C cDNA has been
described previously (16). All of the cdc25C constructs were
tagged with the Myc epitope (EQKLISEEDL [5, 43]) and
expressed from the SV40 promoter in plasmid pSG5-L (54). The
SV40 NLS (PKKKRKVEDPAV) was fused to the C terminus of cdc25C by PCR
using the primers (5' ggg aag ctt gga tcc ATG GAG CAG AAG CTC 3'
and 5' ggg gtc gac TCA CAC CGC CCG GAT CTT CTA CTT TCC GTT
TCT TTT TGG GTG GGC TCA TGT CCT TCA CC 3' for all
oligonucleotides, coding sequences are in uppercase). The mutation of
the active-site residue of the phosphatase domain, cysteine 377 to
serine (C377S) (4, 11, 40), was generated by PCR using the
oligonucleotides 5' TGA GGA GAA TTC AGA GTG GAA CAC GAT 3'
and 5' ggg tct aga gga tcc TCA TGG GCT CAT GTC CTT CAC CAG
3' and inserted into the wild-type (WT) cdc25C constructs as a
BamHI-EcoRI fragment. The deletion mutants
1-258, 1-200, and 1-150 were amplified from WT cdc25C by using the
oligonucleotides 5' ggg aga tct TCA TAA ACA TAA GCC CTT CCT 3', 5'
ggg aga tct TCA TTT CAG GGA AAA CTC CAT 3', and 5' ggg aga
tct TCA ATT TGC AGA TGA ACT ACA 3', respectively, with the
oligonucleotide 5' ggg aag ctt gga tcc ATG GAG CAG AAG CTC 3'
in PCRs. The mutant 259-473 was constructed by performing a PCR
with the oligonucleotides 5' aaa aag ctt ATG GAG CAG AAG CTC ATC
TCA GAG GAG GAC CTG GGA TCC AAG AAG ACA GTC TCT CTG TGT 3' and
5' ggg tct aga gga tcc TCA TGG GCT CAT GTC CTT CAC CAG 3'. The deletion mutants 
201-258 and 201-258 C377S were
cloned by PCR amplifying either WT cdc25C or the C377S mutant with the oligonucleotides 5' ttt gga tcc AAG AAG ACA GTC TCT CTG TGT GAC 3'
and 5' aaa ctc gag TTA AGA TCT TTA TCA TGG GCT CAT GTC CTT CAC CAG AAG GGC 3' and inserted into pSG5-L as a
BamHI-XhoI fragment. Subsequently, another PCR
fragment generated with the oligonucleotides 5' aaa aag ctt ATG
GAG CAG AAG CTC ATC TCA GAG GAG GAC CTG 3' and 5' ggg gga
tcc TTT CAG GGA AAA CTC CAT TAA TTC ATG TG 3' was inserted into
the above construct as a HindIII-BamHI
fragment. The point mutant of the serine residue at position 216 to
alanine (S216A) was generated by PCR with the oligonucleotides 5'
AAC AGG CCT AGA CTG AAG and 5' ggg tct aga gga tcc TCA TGG
GCT CAT GTC CTT CAC CAG 3' and inserted in a three-way ligation
into pSG5-L. The clones NEScdc25C and NESS216A were generated by
cloning cdc25C or S216A as BamHI fragments downstream of the
influenza virus HA epitope (YPYDVPDYA [6]) and the
human immunodeficiency virus type (HIV-1) Rev NES (LQLPPLERLTLD
[7]) in pSG5-L (55). Cyclin B1 was PCR
amplified by using the primers 5' ccc gga tcc ATG GCG CTC CGA GTC
ACC AGG AAC TCG and 5' ggg ctc gag TTA CAC CTT TGC CAC AGC
CTT GGC TAA ATC 3' and fused to the HA epitope in pcDNA3 (Invitrogen). Cyclin B1 was cloned as a
BamHI-XhoI fragment downstream of the SV40 NLS
and the HA epitope in pSG5-L (55) to generate HA-NLSB1.
14-3-3 (62) was cloned as a
BamHI-XhoI fragment into pcDNA3 downstream of the
HA epitope to generate HA-14-3-3. All clones were verified by DNA sequencing.
Antibodies.
To generate specific MAbs to cdc25C, a
glutathione S-transferase (GST) fusion to the first 258 amino acids of human cdc25C (GST 1-258) was constructed. The protein
was purified from Escherichia coli on glutathione-Sepharose
beads as described by the manufacturer (Pharmacia). The protein was
eluted off the beads overnight at 4°C in 20 mM glutathione-100 mM
Tris (pH 8.0)-120 mM NaCl, and dialyzed against phosphate-buffered
saline (PBS). RBF/DnJ mice were immunized and fusions to NS1 myeloma
cells were performed as described elsewhere (14). Four
specific MAbs to cdc25C, TC14, TC15, TC19, and TC113, were identified.
The antibodies were subtyped with an isotype kit (IsoStrip; Boehringer
Mannheim) and belong to the immunoglobulin G1 (IgG1) subclass. The
phosphorylated histone (phospho-histone) H3-specific antiserum (Upstate
Biotechnology) was used at a dilution of 1:200 for immunofluorescence.
Tissue culture supernatants of the mouse monoclonal hybridomas anti-HA (12CA5) and anti-Myc (9E-10) were used at a dilution of 1:50 for immunofluorescence analysis and Western blotting. Tissue culture supernatants of the hybridomas DG122 (anti-GST), TC14, TC15, and TC113
were used at a dilution of 1:50, while the TC19 supernatant was used
without dilution for Western blotting of GST fusion proteins. The
anti-cdc25C rabbit polyclonal antibody (C-20; Santa Cruz Biotechnology) was used at a dilution of 1:500 for Western blotting. For
immunofluorescence analysis, ascites fluid was generated for TC14,
TC15, TC19, and TC113 and used at a dilution of 1:50. A mixture of
TC14, TC15, and TC19 ascites fluid (each at a 1:500 dilution) was used
to perform Western blotting in the immunoprecipitation Western blotting (IP/Western) experiments. TC14 (1:10), TC15 (1:10), and TC113 (1:100)
high-titer tissue culture supernatants were used at the dilutions
indicated for direct Western blotting. The anti-Myc rabbit polyclonal
antibody (A-14; Santa Cruz) was diluted 1:1,000 for immunofluorescence
and 1:500 for Western blotting. Tissue culture supernatants of the
cyclin B1 antibody CB169 (Upstate Biotechnology) was used at a dilution
of 1:50 for immunostaining. The anti-Rb (retinoblastoma protein) MAb
(MAb 245; Pharmingen) was used at a dilution of 1:500 for Western
blotting, and 2 µl of antibody was used to immunoprecipitate Rb from
cellular extracts. The secondary antibodies goat anti-mouse
IgG-rhodamine and goat anti-rabbit IgG-fluorescein isothiocyanate
(FITC) (Boehringer Mannheim) were diluted 1:1,000 for
immunofluorescence assays. The MPM2 antibody was used at a dilution of
1:1,000 for immunofluorescence.
Immunoprecipitation and Western blotting.
To determine the
specificity of the anti-cdc25C MAbs, E. coli strains
expressing GST fusion to full-length human cdc25A (GST-cdc25A) and
human cdc25B (GST-cdc25B) or the first 258 amino acids of human cdc25C
(GST-1-258) were induced to synthesize the fusion protein as described
by the supplier (Pharmacia). The bacterial pellets were boiled in
sodium dodecyl sulfate (SDS) buffer (58) and separated in
SDS-polyacrylamide gels. Proteins were transferred to polyvinylidene
difluoride membranes, and the immune complexes were detected as
described previously (58) with an alkaline phosphate-conjugated goat anti-rabbit IgG (Boehringer Mannheim). To map
the epitopes of the different antibodies, various cdc25C constructs
were translated in vitro in the presence of
[35S]methionine in the Promega TNT coupled transcription
translation system. The in vitro translation products were incubated
with the various antibodies and protein A-Sepharose in 500 µl of
NET-N (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40 [NP-40]) for 2 h at 4°C. The immune complexes were washed three times with NET-N, boiled in SDS buffer, separated in
SDS-polyacrylamide gels, and analyzed by autoradiography.
Whole-cell extracts of U-2OS cells were prepared in EBC buffer (50 mM
Tris-HCl [pH 8.0], 120 mM NaCl, 0.5% NP-40, 10 µg of aprotinin per
ml, 10 µg of leupeptin per ml, 0.1 mM phenylmethylsulfonyl fluoride,
50 mM NaF, 1 mM sodium orthovanadate, 1 mM EDTA). Extracts were cleared
by centrifugation at 12,000 × g for 15 min at 4°C. Two micrograms of EBC extract was rocked overnight at 4°C with protein A-Sepharose beads and either 200 µl of the antibody
supernatants (9E-10, TC14, TC15, and TC113) or 2 µl of ascites fluid
(TC19). The immune complexes were washed three times with NET-N and
resolved in an SDS-7.5% polyacrylamide gel, followed by Western
blotting with a mixture of TC14, TC15, and TC19 or the anti-cdc25C
rabbit polyclonal antibody (Santa Cruz). Whole-cell extracts for direct Western blots were prepared by harvesting cells by trypsinization and
boiling the cell pellet in 50 mM Tris (pH 8.0)-2% SDS for 10 min. The
extracts were cleared by centrifugation, and 25 to 50 µg of extract
was loaded onto an SDS-10% polyacrylamide gel. The antibody antigen
complexes were detected by enhanced chemiluminescence as instructed by
the manufacturer (Pierce).
To detect transiently transfected Myc-tagged cdc25C, 40 to 44 h
posttransfection, cells were extracted in 1 ml of EBC buffer
and the
extracts were rocked overnight at 4°C with protein A-Sepharose
beads
and 100 µl of 9E-10 tissue culture supernatant. Immune complexes
were
washed thrice with NET-N and detected by Western blotting
as described
above. In the experiments where HA-14-3-3
was cotransfected
with
cdc25C, the extracts were lysed in EBC as described above
and then
immunoprecipitations were performed with antibodies to
either the Myc
(9E-10) or HA (12CA5) epitope overnight as described
above.
Cellular fractionation.
Fractionation of U-2OS cells was
performed by using a modified version of a protocol described by
Schreiber et al. (53). Briefly, U-2OS cells at ~50%
confluence were harvested by trypsinization. Whole-cell extracts were
made with EBC. Cytoplasmic extracts were prepared by harvesting cells
in buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 1 mM EDTA, 0.1 mM EGTA,
1 mM dithiothreitol, 10 µg of aprotinin per ml, 10 µg of leupeptin
per ml, 0.1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 1 mM sodium
orthovanadate) on ice for 10 min. Additional phosphatase inhibitors (10 µM cypermethrin, 200 µM dephostatin, 200 nM okadaic acid, 25 nM
tautomycin) were added to buffer A when indicated. The extracts were
spun for 30 s in a microcentrifuge at 4°C. The cytoplasmic
extract was removed, and the nuclear pellets from the two different
preparations were pooled and extracted with EBC to generate nuclear
extracts. Two milligrams of each extract was immunoprecipitated with a
MAb to cdc25C (TC113) or to Rb (MAb 245). The immune complexes were
resolved in an SDS-7.5% polyacrylamide gel and Western blotted for
the presence of either cdc25C or Rb. For direct Western blotting of the
extracts, 100 µg of each fraction was separated in an SDS-10% polyacrylamide gel and then Western blotted with a mixture of the
protein A-purified cdc25C MAbs.
Localization and PCC assays.
To detect endogenous cdc25C
protein by immunofluorescence, cells were fixed in a solution of 4%
paraformaldehyde in PBS for 20 min at room temperature (RT) and
subsequently washed with PBS and permeabilized with 0.3% Triton X-100
in PBS for 10 min at RT. The fixed permeabilized cells were incubated
with primary antibody, diluted 1:50 in PBS containing 3% bovine serum
albumin and 0.1% NP-40, overnight at 4°C. The coverslips were washed
alternately with PBS containing 0.1% NP-40 and PBS six times and then
incubated with the appropriate secondary antibody in PBS containing 3%
bovine serum albumin and 0.1% NP-40 for 30 minutes at RT. The cells
were washed again as described above and counterstained with the DNA dye 4',6-diamidino-2-phenylindone (DAPI) at a concentration of 5 µg/ml. Confocal images were obtained by using a Bio-Rad MRC-1024/2P instrument interfaced with a Zeiss Axiovert S100 microscope. A krypton-argon laser with emission lines at 488 and 568 nm was used for
conventional excitation of fluorescein and rhodamine with bandpass
emission filters at 522 and 598 nm. A Spectra-Physics Tsunami
femtosecond pulsed laser tuned to 770 nm was used for the multiphoton
excitation of DAPI.
To determine the localization of cdc25C in
-irradiated cells, U-2OS
cells were plated in a 100-mm-diameter dish containing
a coverslip. The
next day the medium was changed to medium containing
200 µM mimosine
to induce a G
1 arrest (
26); 20 h later, the
mimosine was removed, and the cells were washed twice and refed
with
fresh medium; 6 h after mimosine removal, cells were irradiated
in
a Gamma-cell 40 irradiator (MDS Nordion) at a dosage of 6 Gy.
After
irradiation, one set of plates was incubated with leptomycin
B at a
concentration of 2 ng/ml for 18 h (
13,
59); 18 h
postirradiation,
the cells were washed with PBS, and the coverslips
were removed
and fixed with paraformaldehyde as described above. After
fixation,
the coverslips were stored in PBS and then stained with a MAb
to either cdc25C (TC113) or cyclin B1 (CB169). The remainder of
the
cells were harvested by trypsinization, fixed in 70% ethanol,
stained
with propidium iodide, and processed for fluorescence-activated
cell
sorting (FACS) as previously described (
64).
Localization of the transfected cdc25C and cyclin B1 constructs was
determined by performing indirect immunofluorescence with
antibodies to
the HA or Myc epitope. To perform the PCC assays,
asynchronously
growing U-2OS cells were transfected with the various
cdc25C and cyclin
B1 expression plasmids. At 40 to 44 h after
transfection, the
cells were fixed and stained with antibodies
to the appropriate epitope
tags. In each experiment, at least
100 transfected cells were counted.
The results presented are
the averages of at least three independent
experiments. To perform
the cell cycle synchrony experiments, U-2OS
cells were plated
in a 100-mm-diameter dish containing a coverslip. The
next day
the medium was changed with either regular medium or medium
containing
200 µM mimosine to induce a G
1 arrest
(
26). Approximately 2
h later, the cells were
transfected as described above; 6 h posttransfection,
the DNA
precipitate was removed, and the cells were fed twice
with medium
containing mimosine; 20 h after the mimosine was first
added, the
mimosine was removed, and the cells washed twice and
fed with fresh
medium. At the indicated time after mimosine removal,
the cells were
washed with PBS, and the coverslip was removed
and fixed with
paraformaldehyde as described above. After fixation,
the cells on
coverslips were stained with an anti-Myc rabbit polyclonal
antibody
(Santa Cruz) and a MAb to cyclin B1 (CB169). The remainder
of the cells
were harvested by trypsinization and stained with
antibody to CD19
conjugated to FITC (Caltag) and then processed
for FACS as previously
described (
64). An S-phase arrest was
induced in certain
transfections by the addition of 100 µM hydroxyurea
(HU).
| |
RESULTS |
Generation of specific MAbs to cdc25C.
A diagram of the cdc25C
constructs used in this study is shown in Fig.
1. A panel of MAbs to cdc25C, TC14, TC15,
TC19, and TC113, was generated by immunizing mice with a GST fusion
protein containing the first 258 residues of human cdc25C. The
specificity of these antibodies for cdc25C was tested by Western blot
analysis of bacterial lysates expressing GST-cdc25A (Fig.
2A, lane 1), GST-cdc25B (lanes 2 and 3),
or the N-terminal GST-cdc25C fusion protein (GST 1-258; lane 4). All
three fusion proteins were recognized by the anti-GST MAb DG122. In
contrast, the anti-cdc25C antibodies failed to react with either
GST-cdc25A or GST-cdc25B but did react with cdc25C. These results
suggest that each of these antibodies could specifically detect cdc25C
and not cdc25A or cdc25B.
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FIG. 1.
Diagram of the human cdc25C constructs used. All cdc25C
constructs contained an N-terminal Myc epitope tag (black box). A
14-3-3 binding motif of cdc25C (RSPSMP) is phosphorylated at the second
serine residue (S216) in the consensus (44, 47, 49), and
mutation of the serine residue to alanine abolishes binding to 14-3-3 proteins (49). The phosphatase domain (hatched box) is
located in the C terminus with the active site (HCEFSSER) shown.
Substitution of the cysteine residue at position 377 with serine
disrupts phosphatase activity (4, 11, 40). The N-terminal
box (vertical lines) represents the HA epitope and the HIV-1 Rev NES,
and the C-terminal stippled box represents the SV40 large-T-antigen
NLS.
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FIG. 2.
Specificity of MAbs to cdc25C. (A) MAbs were raised to a
GST fusion protein that contained the first 258 amino acids of human
cdc25C (GST 1-258). Bacterial lysates expressing either GST-cdc25A (10 µl; lane 1), GST-cdc25B (20 and 30 µl; lanes 2 and 3, respectively), or GST 1-258 (10 µl; lane 4) were separated in an
SDS-7.5% polyacrylamide gel and Western (W.) blotted with DG122, a
mouse MAb specific for GST, and the four cdc25C antibodies, TC14, TC15,
TC19, and TC113. (B) Indicated Myc-tagged cdc25C constructs were
translated in vitro with [35S]methionine; 15 µl of in
vitro translate was immunoprecipitated with the indicated antibodies,
and the immunoprecipitated proteins were visualized by autoradiography.
The 201-258 construct contains an in-frame deletion of amino acids
201 to 258 (lane 2). 201-258 (211-221) and 201-258
(222-246) (lanes 3 and 4) are mutant proteins that contain insertions
of the indicated amino acids into the 201-258 mutant. 1-258,
1-150, and 259-473 (lanes 5 to 7) are shown in Fig. 1. (C)
cdc25c diagram showing where the MAbs bind. The N-terminal myc tag is
recognized by MAb 9E-10.
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To map the epitopes in cdc25C recognized by the MAbs, in vitro
translates of various cdc25C deletion mutants tagged with the
Myc
epitope (Fig.
1) were used in immunoprecipitation reactions.
The
anti-Myc antibody 9E-10 immunoprecipitated each of the cdc25C
constructs as expected (Fig.
2B). The cdc25C MAbs could
immunoprecipitate
WT cdc25C as well as an N-terminal fragment
expressing the first
258 residues (1-258) (Fig.
2B, lanes 1 and 5) but
not a C-terminal
fragment of cdc25C (259-473) that was not part of the
immunizing
peptide (lane 7). Of the four antibodies generated, only
TC14
could immunoprecipitate 1-150 (lane 6), suggesting that it
recognized
an epitope contained within the first 150 amino acids of the
protein.
The other three MAbs failed to immunoprecipitate the
201-258
or 1-150 protein (lanes 2 and 6), suggesting that they
recognize
epitopes between residues 201 and 258. TC15 and TC113
recognized
an epitope located between amino acids 222 and 246, as
determined
by the observation that they could immunoprecipitate the
201-258
(222-246) mutant (lane 4). TC19 recognized an epitope between
amino acids 211 and 246 that was distinct from the epitope recognized
by TC15 and TC113, based on its inability to immunoprecipitate
either
201-258 (211-221) or
201-258 (222-246) (lanes
3 and
4) and its ability to immunoprecipitate both the
201-210 and
247-258 proteins (data not shown). Therefore the four MAbs
recognize
at least three different epitopes in
cdc25C.
The cdc25C antibodies were tested for the ability to immunoprecipitate
endogenous cdc25C in lysates prepared from U-2OS cells.
All four cdc25C
antibodies specifically immunoprecipitated a doublet
that migrated at
approximately 55 kDa in U-2OS cells upon Western
blotting with either a
mixture of the cdc25C MAbs or an anti-cdc25C
rabbit polyclonal
antiserum (C-20; Santa Cruz) raised against
the C terminus of cdc25C
(Fig.
3A, lanes 2 to 5 and lanes 7 to
10, respectively). A similar doublet was observed in the normal
diploid
human fibroblast line, MRC-5 (data not shown). The anti-Myc
MAb 9E-10,
which belongs to the same subclass as the anti-cdc25C
antibodies,
failed to immunoprecipitate the doublet (lanes 1 and
6). It has been
reported previously that the slower-migrating
band in this doublet is
due to phosphorylation (
47,
49). The
immunoprecipitates were
treated with lambda phosphatase, resulting
in a loss of the upper band
and an enhancement of the lower band,
confirming that the upper band in
the doublet was a phosphorylated
version of the lower band (data not
shown). These results suggest
that all four MAbs could specifically
immunoprecipitate both the
phosphorylated and unphosphorylated forms of
cdc25C.
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FIG. 3.
cdc25C is present in the cytoplasm of asynchronously
growing human cells. (A) Two-milligram aliquots of EBC extracts
prepared from U-2OS cells were immunoprecipitated with cdc25C-specific
antibodies TC14 (lanes 2 and 7), TC15 (lanes 3 and 8), TC113 (lanes 4 and 9), and TC19 (lanes 5 and 10) or the Myc antibody 9E-10 (lanes 1 and 6). The immune complexes resolved in SDS-7.5% polyacrylamide gels
followed by Western blotting with either a mixture of TC14, TC15, and
TC19 (lanes 1 to 5) or an anti-cdc25C rabbit polyclonal antibody (Santa
Cruz) (lanes 6 to 10). The position of cdc25C, which migrated as a
doublet, is indicated by the bracket. The arrow indicates the
immunoglobulin heavy chain. The positions of the molecular weight
markers are indicated in kilodaltons to the left of each gel. B. U-2OS
(lanes 1, 4, and 7), MRC-5 (lanes 2, 5, and 8), and WI-38 (lanes 3, 6, and 9) cells were harvested by trypsinization, and the cell pellets
were boiled in 2% SDS; 50 (lanes 1 to 6) or 25 (lanes 7 to 9) µg of
protein extract was separated in an SDS-10% polyacrylamide gel, and
then Western blotting was performed with the indicated antibodies. The
immune complexes were detected by using the Pierce Supersignal system.
TC14, TC15, and TC113 all recognized a specific doublet at ~55 kDa in
each of the different cell types. In addition to this band, they
specifically recognized a band at ~47 kDa (open arrow) in U-2OS
cells. On longer exposures, this band was also detected in MRC-5 and
WI-38 cells. No other specific signal was detected with these
antibodies. (C) Two-milligram aliquots of whole-cell (lanes 1 and 4),
cytoplasmic (lanes 2 and 5), or nuclear (lanes 3 and 6) extracts
prepared from U-2OS cells were immunoprecipitated with either an
anti-Rb antibody (MAb 245; Pharmingen) (lanes 1 to 3) or an anti-cdc25C
antibody (TC113) (lanes 4 to 6). The immune complexes were resolved in
an SDS-7.5% polyacrylamide gel and Western blotted with MAb 245 (lanes 1 to 3) or a mixture of TC14, TC15, and TC19 (lanes 4 to 6). The
cdc25C doublet is indicated by the bracket. The thick arrow indicates
the immunoglobulin heavy chains; the position of Rb on the gel is
indicated by the thin arrow. (D) Two-milligram aliquots of whole-cell
(lane 1), cytoplasmic (lanes 2 and 3), or nuclear (lane 4) extracts
prepared from U-2OS cells were immunoprecipitated with the cdc25C MAb
(TC113). The cytoplasmic extracts in lane 3 were prepared in the
presence of a cocktail of phosphatase inhibitors (10 µM cypermethrin,
200 µM dephostatin, 200 nM okadaic acid, and 25 nM tautomycin).
cdc25C is indicated by a bracket; the arrow indicates the position of
the immunoglobulin heavy chain.
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To further demonstrate the specificity of these antibodies, we
tested their abilities to recognize cdc25C in direct Western
blot analyses. Whole-cell extracts prepared from U-2OS, MRC-5,
and
WI-38 cells were resolved on an SDS-10% polyacrylamide gel
and
Western blotted with three different cdc25C MAbs (Fig.
3B).
The three
cdc25C MAbs recognize a doublet at ~55 kDa in U-2OS
(lanes 1, 4, and
7), MRC-5 (lanes 2, 5, and 8), and WI-38 (lanes
3, 6, and 9) cells. A
band at ~47 kDa is seen predominantly in
U-2OS cells, though it can
be seen in the other cell types at
longer exposures. To determine if
this band is related to cdc25C,
we performed an immunoprecipitation
with each of the MAbs followed
by Western blotting with C-20. C-20
recognizes both the 55-kDa
doublet and the 47-kDa band (data not
shown). Similar results
were obtained when the Western blotting was
performed with a pool
of the cdc25C MAbs (data not shown). This result
suggests that
the 47-kDa band is likely to be a truncated product of
cdc25C
and not a cross-reactive
species.
To determine the intracellular localization of cdc25C, biochemical
fractionation experiments were performed in asynchronously
growing
U-2OS cells. Whole-cell, cytoplasmic, or nuclear extracts
were prepared
as described in Materials and Methods. cdc25C was
detected in the whole
cell as well as the cytoplasmic extracts
but not in the nuclear
fraction (Fig.
3C lanes 4 to 6). As a control,
IP/Western experiments
were also performed to determine the localization
of Rb, previously
demonstrated to be localized to the nucleus
(
34,
41). As
shown in Fig.
3B, Rb was present in the nuclear
extracts and no Rb was
detectable in the cytoplasmic fraction
(lanes 1 to 3). These results
suggest that the cytoplasmic extracts
were not contaminated by nuclear
proteins. A significant increase
in amount of the faster-migrating band
of the cdc25C doublet was
observed when cytoplasmic fractions were
prepared from U-2OS cells
compared to the whole-cell extracts (Fig.
3D;
compare lanes 1
and 2), suggesting that cdc25C may have been undergoing
dephosphorylation
while the extract was prepared. To test this
hypothesis, cytoplasmic
extracts were prepared in the presence of
additional phosphatase
inhibitors. Under these conditions, only the top
band of the doublet
can be seen (lane 3); cdc25C is not present in the
nuclear fraction
(lane 4). This latter result suggests that all of the
endogenous
cdc25C protein exists in the phosphorylated
form.
The localization of cdc25C changes prior to mitosis in human
cells.
To confirm the cytoplasmic localization of endogenous
cdc25C in human cells, indirect immunofluorescence analysis was
performed on normal diploid human fibroblasts as well as immortal human cell lines. As shown in Fig. 4A, a
specific cytoplasmic signal for cdc25C was obtained in U-2OS cells when
they were immunostained with either TC14 or TC113. A similar staining
pattern in U-2OS was obtained with each of the other cdc25C MAbs, TC15
and TC19 (data not shown). The normal diploid human fibroblast strains, MRC-5 and WI-38, also showed a cytoplasmic staining pattern for cdc25C
with MAb TC113 (Fig. 4A) as well as with each of the other MAbs (data
not shown).
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FIG. 4.
Cellular localization of cdc25C in human cells. (A)
Indirect immunofluorescence with the cdc25C antibodies was performed in
multiple cell types. U-2OS cells immunostained with either TC14 (left)
or TC113 (right) are shown in the top panels. The primary human
fibroblasts, MRC-5 (bottom left) and WI-38 (bottom right), were
immunostained with antibody TC113. In each pair, the left panel shows
antibody staining while the right panel shows the DAPI stain of the
same field (original magnification, ×100). (B) MRC-5 cells were
immunostained with polyclonal sera specific for phospho-histone H3 ( pH3) (17), a MAb to cdc25C (TC113), and DAPI, as indicated,
and visualized by confocal microscopy. The merge of the cdc25C and the
pH3 staining is shown in the bottom left panel. A cell that stains
positively for histone H3 and shows partial chromatin condensation
(DAPI) is indicated by the thick arrow. A cell that does not stain with
the phospho-histone H3 antibody in which the chromatin is not condensed
is indicated by the thin arrow (original magnification, ×63). (C)
U-2OS cells were treated with mimosine to induce a G1
arrest. Six hours after mimosine release, cells were irradiated
(-IR) with a dose of 6 Gy and then incubated in either the presence
or absence of the crm1 inhibitor leptomycin B (LMB) for an additional
18 h. Cells were immunostained with either an anti-cdc25C antibody
(TC113) or an anti-cyclin B1 antibody (CB169). In each pair, the left
panel shows immunofluorescence while the right panel shows the DAPI
stain of the same field (original magnification, ×100). The cell cycle
profiles of the cells in mimosine (mimosine), cells 6 h after
release from mimosine (6 hours), and the -irradiated cells (-IR)
and irradiated cells treated with leptomycin B (-IR + LMB) are
shown.
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To determine whether the cellular localization of cdc25C was altered
over the cell cycle, MRC-5 cells were stained with a
MAb to cdc25C
(TC113) and a polyclonal serum specific for phospho-histone
H3 followed
by confocal microscopy. Histone H3 is not phosphorylated
in
G
1 and S phases. Phosphorylation of histone H3 is initiated
during G
2, and the intensity and pattern of the signal
phospho-histone
H3 signal changes as cells progress from G
2
to M phase, with a
steady accumulation of the phosphorylated form of
the protein
until anaphase (
17). Cells that show no
phospho-histone H3 in
the nucleus are in G
1 or S phase of
the cell cycle, while cells
that show a punctate pattern of
phospho-histone H3 in the nucleus
are in early G
2 phase
(
17). Cells that did not stain for phospho-histone
H3 showed
a cytoplasmic staining pattern for cdc25C; in contrast,
cells that
stain positively with the antibodies to phospho-histone
H3 contain
cdc25C both in the nucleus and the cytoplasm, as evidenced
by the
yellow staining in the merged image (Fig.
4B). The timing
of the change
in cellular localization just prior to mitosis is
similar to that
reported for cyclin B1 (
13,
51,
59) and
suggests that, like
cyclin B1, cdc25C enters the nucleus just
prior to mitosis in human
cells.
The human cyclin B1 protein enters the nucleus just prior to mitosis
due to the inactivation of a NES in cyclin B1 (
13,
59).
Notably, DNA damage prevents inactivation of the NES, thus
forcing
cyclin B1 to be retained in the cytoplasm (
13,
51,
59). To
determine whether DNA damage affects the localization
of cdc25C, U-2OS
cells were initially synchronized in G
1 phase
with mimosine
(Fig.
4C). Six hours after mimosine was removed,
the cells had entered
S phase (Fig.
4C). cdc25C and cyclin B1
could both be detected in the
cytoplasm by immunostaining during
S phase, while only cdc25C was
expressed in cells arrested in
G
1 with mimosine (data not
shown). At this point, the cells were
irradiated and then incubated
in the absence or presence of
leptomycin B for 18 h. The
-irradiated U-2OS cells arrested almost
exclusively in the
G
2 phase of the cell cycle (Fig.
4C). Immunostaining
of
-irradiated U-2OS cells with a MAb to cdc25C (TC113) or cyclin
B1
(CB169) demonstrated that both proteins remained in the cytoplasm
(Fig.
4C). The leptomycin B-treated cells also showed a G
2 arrest
similar to that for the cells that were
irradiated but not treated
with leptomycin B (Fig.
4C). The localization of cdc25C was not
altered
when the cells were treated with leptomycin B (Fig.
4C).
In contrast,
in cells treated with leptomycin B, the localization
of cyclin B1 was
dramatically altered, with a marked increase
in the nuclear signal
being observed (Fig.
4C), in agreement with
results published by other
groups (
13,
59). These results
suggest that although both
cyclin B1 and cdc25C are maintained
in the cytoplasm in
-irradiated
cells, the cytoplasmic localization
and subsequent nuclear transport of
these two proteins are regulated
by different mechanisms in human
cells.
A 58-amino-acid domain in cdc25C controls its
cellular localization.
To determine the regions of cdc25C that
participate in its normal cytoplasmic localization during interphase,
various Myc epitope-tagged cdc25C deletion constructs were transiently
expressed in U-2OS cells. The cells were immunostained with the
anti-Myc MAb 9E-10 and imaged by confocal microscopy. Wild-type cdc25C was found to be localized in the cytoplasm (Fig.
5A), similar to results for the
endogenous protein and to previously published results (10,
16). A full-length cdc25C protein with an inactive phosphatase
domain, C377S (4, 11, 40), as well as an N-terminal cdc25C
construct containing residues 1 to 258, lacking the catalytic domain,
also localized to the cytoplasm (Fig. 5A and data not shown),
suggesting that the phosphatase activity of cdc25C was not required for
its cytoplasmic retention. Further deletion into the N terminus
(construct 1-200) resulted in a pancellular distribution, with the
protein being present in both the cytoplasmic and nuclear compartments
(Fig. 5A). An N-terminal deletion mutant, 259-473, that contained the
entire catalytic domain was also pancellular (data not shown). These
results suggest that residues 201 to 258 contributed to the cytoplasmic
localization of cdc25C.
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FIG. 5.
Cytoplasmic localization of cdc25C is dependent on a
14-3-3 binding motif. (A) U-2OS cells were transiently transfected with
the indicated cdc25C constructs and visualized by confocal microscopy
with the anti-Myc antibody 9E-10 ( myc) and DAPI staining. The arrow
highlights cells showing condensed fractured chromatin indicative of
PCC (original magnification, ×63). (B) Extracts of U-2OS cells
cotransfected with plasmids expressing an HA-tagged 14-3-3 cDNA and
the indicated Myc-tagged cdc25C constructs were immunoprecipitated with
an anti-Myc antibody (9E-10) or an anti-HA antibody (12CA5). The immune
complexes were washed and then resolved in either an SDS-7.5%
polyacrylamide gel (lanes 1 to 4, top panel) or an SDS-10%
polyacrylamide gel (lanes 5 and 6, top panel; lanes 7 to 18, middle and
bottom panels). The cdc25C constructs were detected by Western blotting
(WB) with a polyclonal Myc antibody (Santa Cruz), and the HA 14-3-3
was detected by Western blotting with the anti-HA MAb (12CA5).
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To determine if residues 201 to 258 were necessary for the cytoplasmic
localization of cdc25C, a mutant with an in-frame deletion
of residues
201 to 258 (
201-258) was constructed. As shown in
Fig.
5A,
201-258 showed a pancellular localization similar to
that observed for
1-200. A consensus binding site for 14-3-3 proteins
is contained in
residues 201 to 258 (213-218). Phosphorylation
of the serine residue
at position 216 is required for binding
to 14-3-3 proteins, and
mutation of S216 to alanine has been shown
to prevent phosphorylation
at this site, resulting in a loss of
14-3-3 binding (
49). To
determine whether S216 was required
for cytoplasmic retention of
cdc25C, we generated a substitution
mutant that altered the serine
residue at position 216 to alanine
(S216A). As shown in Fig.
5A, S216A
showed a pancellular localization
similar to that
of the mutant
201-258, suggesting that S216
may be required for
retention of cdc25C in the cytoplasm. A mutant
that substituted an
aspartic acid for serine, S216D, also showed
a pancellular localization
(data not shown). Mutation of another
serine residue in the same
region, S214, did not alter the cytoplasmic
localization of cdc25C
(data not shown).
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FIG. 6.
Effect of localization on cdc25C function. (A) U-2OS
cells transfected with Myc-tagged cdc25C and cyclin B1 were
immunostained with antibodies to the Myc epitope (-myc) the
mitosis-specific antibody MPM2 (-MPM2) and DAPI. A cell
immunostained with both the Myc and MPM2 antibodies showed condensed
fractured chromatin when stained with DAPI (thin arrow). An
untransfected cell undergoing mitosis is stained by the MPM2 antibody
(thick arrow). (B) U-2OS cells transfected with the indicated
Myc-tagged cdc25C constructs were immunostained with the anti-Myc
antibody (9E-10) and DAPI. More than a 100 Myc-positive,
cdc25C-expressing cells were counted, and the percentage of cells
containing condensed fragmented chromatin was determined in three
independent experiments. (C) U-2OS cells were treated with mimosine to
induce a G1 arrest. Cells were transfected with cdc25C and
a CD19 cDNA in the presence of mimosine. Six hours after transfection,
the DNA was removed and the cells were refed twice with
mimosine-containing medium; 20 h after mimosine was first added to
the cells, cells were refed with fresh medium; cells were then
harvested at the indicated time points for FACS analysis or were
immunostained for Myc-tagged cdc25C and the endogenous cyclin B1 as
described in the text and with DAPI to determine PCC. Six hours after
mimosine was removed, 100 µM HU was added to one set of plates for
6 h. Open bars indicate the percentage of cells undergoing PCC;
filled bars indicate the percentage of transfected cells staining
positively for cyclin B1. A, asynchronous. The percentages of
CD19-positive cells in G1, S, and G2 phases at
the indicated timepoints are tabulated at the bottom. (D) Cells from
the experiment described above were stained with a polyclonal antibody
to the Myc epitope (-myc) a MAb to cyclin B1 (-B1), or DAPI.
Cells arrested in mimosine (0) show little staining for cyclin B1.
After mimosine release, the percentage of cells staining for cyclin B1
increases until all cells stain for cyclin B1 (12 and 12+HU). All cells
undergoing PCC (thin arrow) show staining for cyclin B1 (original
magnification, ×60).
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The ability of these cdc25C mutant proteins to form a complex with
14-3-3 proteins was tested. It has been reported that the
Xenopus cdc25 protein forms a complex with 14-3-3
(
31). To
determine if human cdc25C associated with
14-3-3
, wild-type cdc25C
or the different mutants were transfected
into U-2OS cells with
an expression construct for a HA epitope-tagged
14-3-3
cDNA (HA-14-3-3
).
Extracts from the transfected cells were
prepared, and immunoprecipitations
were performed with antibodies to
either the HA or Myc epitope.
As shown in Fig.
5B, each of the cdc25C
proteins (Fig.
5B, lanes
1 to 6) and the HA-14-3-3
protein (lanes 13 to 18) was expressed
at equivalent levels in each transfection. Both WT
cdc25C and
the 1-258 protein formed a complex with 14-3-3
in vivo,
as demonstrated
by their ability to coprecipitate the HA-14-3-3
protein (lanes
8 and 11). In contrast, S216A,
201-258, and 1-200
were unable
to form a complex with the HA-14-3-3
protein above
background
levels (lanes 9, 10, and 12) (
49). In cells
transfected with
the vector and HA-14-3-3
, an immunoprecipitation
performed with
the Myc antibody did not result in the coprecipitation
of the
HA-14-3-3
protein (lane 7). These results demonstrate that
the
proteins that show a pancellular distribution by immunostaining,
such as S216A,
201-258, and 1-200 also failed to form a complex
with 14-3-3
, while the proteins that show a cytoplasmic staining
pattern, such as wild-type cdc25C and 1-258, could bind to 14-3-3
.
Pancellular localization of cdc25C results in an increase in its
ability to induce PCC.
To determine whether the alteration of
cdc25C localization affected its function, each of these cdc25C mutants
was tested for its ability to induce PCC in U-2OS cells. A minimum
amount of cdc25C expression plasmid was used in all experiments to
yield equivalent levels of expression detectable by both Western
blotting and immunofluorescence analysis (Fig. 5). Some of the cells
expressing Myc-tagged cdc25C (Fig. 5A) or cdc25C and an HA-tagged
cyclin B1 (Fig. 6A) contained condensed
fractured chromatin by DAPI staining as previously reported (13,
16, 49). Cells that contain condensed fractured chromatin by DAPI
staining also stained positively with the mitosis-specific antibody
MPM2 (2) (Fig. 6A). The morphology of the condensed
chromatin in cells undergoing PCC was readily distinguishable from a
normal mitosis (Fig. 6A).
To determine whether the transient expression of cdc25C was more likely
to induce PCC effectively during S and G
2 phases when
cells
expressed cyclin B1, a cell cycle synchrony experiment was
performed.
U-2OS cells were transfected with a wild-type cdc25C
plasmid and an
expression plasmid for the cell surface marker
CD19 to monitor the cell
cycle profile of the transfected cells.
The cell cycle profile was
monitored by staining cells with anti-CD19
antibodies cross-linked to
FITC and propidium iodide followed
by FACS analysis. During
transfection, cells were initially arrested
in G
1 phase by
mimosine treatment (
26) and then released from
the
G
1 block by removing mimosine and monitored over a period
of 27 h. A transfection in which the cells were not treated with
any drugs was also performed (asynchronous). Cyclin B1 expression
was
monitored by immunostaining with a MAb to cyclin B1 (CB169),
and the
transfected cells were identified by immunostaining with
an anti-Myc
antiserum (Santa Cruz). All Myc-tagged cdc25C-expressing
cells that
contained condensed fractured chromatin also stained
positively for
endogenous cyclin B1 (Fig.
6D). Cells in mimosine
were mostly in
G
1 phase of the cell cycle (Fig.
6C, 0) and showed
low
levels of PCC (9%) and cyclin B1 (22%) staining (Fig.
6C and
D).
Cells maintained in mimosine for the duration of the experiment
remained arrested in G
1 phase. Neither the levels of PCC
nor the
number of cells staining positively for cyclin B1 increased
significantly
in those transfections (data not shown). After release
from mimosine,
cells progressed through the cell cycle and entered S
phase (6
h, >60% S phase) and then G
2 (12 h) (Fig.
6C),
with a concomitant
increase in the number of cells undergoing PCC (14 and 27% at
6 and 12 h, respectively) and staining for cyclin B1
(66 and 94%,
respectively) (Fig.
6C and D). HU was added to one set of
transfected
plates after release from mimosine to induce an S-phase
arrest.
As can be seen in the FACS analysis, HU was effective at
preventing
cells from entering G
2 between 6 and 12 h
after mimosine removal.
However, HU did not slow the increase in
percentage of cells expressing
cyclin B1, as the number of cells
staining for cyclin B1 in cells
treated with HU was similar to the
number staining positively
in the absence of HU at the same time point
(Fig.
6C and D; compare
12 to 12+ HU). Furthermore, the percentage of
cells showing condensed
fractured chromatin increased in the presence
of HU (Fig.
6C).
The percentage of cells undergoing PCC at 27 h
after release from
mimosine was similar to that observed in the
untreated asynchronously
growing cells not treated with either mimosine
or HU (asynchronous).
These results suggest that the appearance of
condensed fractured
chromatin reflected an abnormal mitotic event that
is dependent
on the presence of cyclin B1 and transfected cdc25C.
Furthermore,
this most likely represents a premature mitotic event,
given that
transient expression of cdc25C could induce a similar
phenotype
in the presence of
HU.
PCC assays were performed to compare the activity of the wild-type
cdc25C construct to those of the cdc25C mutants. These
assays were
performed in asynchronously growing cells, and levels
of PCC were
determined at a time similar to the asynchronous time
point in the cell
cycle synchrony experiment. Approximately 30%
of the cells that
expressed the wild-type Myc epitope-tagged cdc25C
construct showed PCC
(Fig.
6B). A cdc25C mutant with an inactive
phosphatase domain, C377S,
was unable to induce PCC above background
levels (Fig.
6B, left panel),
consistent with the notion that
cdc25C phosphatase activity was
required for entry into mitosis.
Similarly, the 1-258 and 1-200
mutants (lacking the C-terminal
catalytic domain) were unable to induce
PCC at levels above background
(data not shown). Consistent with
previous results (
49), the
14-3-3 binding-defective mutant
S216A induced PCC at levels greater
than WT (Fig.
6B, left panel).
Similarly,
201-258, which also
does not bind 14-3-3
, was able to
induce PCC in about 50% of
the transfected cells (Fig.
6A, right
panel). The specificity
of this effect could be demonstrated by the
inability of the corresponding
active site mutant,
201-258 C377S,
to induce PCC at significant
levels. In cells that did not undergo PCC,
these proteins demonstrated
a pancellular localization (Fig.
5A).
Furthermore, when WT, S216A,
or
201-258 was cotransfected into
cells with an expression construct
for cyclin B1, more than 90% of the
transfected cells showed evidence
of PCC (Fig.
7B and C and data not shown). However,
the active-site
mutants C377S and
201-258 C377S were unable to
cooperate with
cyclin B1 to induce PCC, with the levels of PCC
remaining at background
levels (data not shown). These data are
consistent with the data
from the cell cycle synchrony experiment that
suggest that the
induction of PCC by cdc25C requires the presence of
cyclin B1.
They also provide support for the hypothesis that an intact
14-3-3
binding motif contributes to regulation of cdc25C activity.
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FIG. 7.
Effect of a heterologous NES or NLS on cdc25C
localization and function. (A) U-2OS cells were transfected with
constructs expressing either the Myc-tagged NLS or HA-tagged NES fusion
of cdc25C. The cells were fixed and visualized with indirect
immunofluorescence with antibody to the HA or Myc epitope (left panel
of each pair) and DAPI (right panel of each pair). The original
magnification on all the panels is ×60 except for the S216ANLS panel
(×40). (B) WT cdc25C and the NES fusions were transfected into U-2OS
cells either alone (left) or in the presence of ectopically expressed
cyclin B1 (right). (C) WT and the NLS fusions were transfected into
U-2OS cells alone (left), cotransfected with cyclin B1 (middle), or
cotransfected with an NLS-tagged version of cyclin B1 (right).
|
|
Targeting cdc25C expression to either the cytoplasm or the nucleus
results in a decrease in its ability to induce PCC.
The results
described above suggest that the cytoplasmic retention of cdc25C by
14-3-3 proteins may prevent premature mitosis. Furthermore, they
suggest the possibility that the entry of cdc25C into the nucleus is
required to initiate mitosis. A prediction of this model would be that
a cdc25C protein that cannot accumulate in the nucleus would not induce
PCC as efficiently as the WT protein, while a cdc25C protein that is
nuclear in interphase cells would induce PCC at levels greater than the
WT protein. To test this hypothesis, cdc25C was fused to either the
HIV-1 Rev NES (7) or the SV40 NLS. As shown in Fig. 7A, the
NEScdc25C fusion localized to the cytoplasm in interphase cells as
expected. Significantly, the NESS216A fusion, containing a substitution
mutation in the 14-3-3 binding domain, was also excluded from the
nucleus (Fig. 7A), a phenotype distinct from that observed for the
S216A mutant. This result suggests that the heterologous NES can induce
the cytoplasmic localization of cdc25C independent of its potential to
bind to 14-3-3 proteins. Conversely, the cdc25CNLS fusion localized exclusively to the nucleus in interphase cells, as did the
corresponding 14-3-3 binding site mutant, S216ANLS (Fig. 7A),
suggesting that the SV40 NLS could act dominantly over the cdc25C
cytoplasmic retention signal. Each of these cdc25C constructs was
expressed at equivalent levels as determined by IP/Western analysis
(data not shown).
The cdc25C constructs with heterologous localization signals were
tested for the ability to induce PCC. As shown in the left
panel of
Fig.
7B, the NEScdc25C and NESS216A fusions induced PCC
at similar
levels despite the loss of the 14-3-3 binding site
in NESS216A (Fig.
6B, left panel). Notably, these NES fusions
did not induce PCC as well
as WT. To test whether the NEScdc25C
fusions could cooperate with
cyclin B1 to induce PCC, they were
transfected into U-2OS cells with an
expression vector for an
HA epitope-tagged cyclin B1. Upon coexpression
of WT cdc25C and
cyclin B1 in U-2OS cells, more than 90% of the
transfected cells
showed evidence of PCC (Fig.
7B, right panel). This
effect requires
cdc25C phosphatase activity, as when cyclin B1 was
cotransfected
with a catalytically inactive cdc25C mutant, C377S, the
levels
of PCC were at background levels (data not shown). Similarly,
when either the NEScdc25C or NESS216A fusion was cotransfected
with
cyclin B1, more than 90% of the transfected cells showed
PCC (Fig.
7B,
right panel). These results suggest that the NES
fusion proteins had no
intrinsic phosphatase defect and were fully
capable of inducing PCC in
the presence of sufficient cyclin B1.
To test if the NEScdc25C fusions
could cooperate with a nuclear
version of cyclin B1, they were
cotransfected with a nuclear version
of cyclin B1, HA-NLSB1 (B1 was
fused to the T-antigen NLS), into
U-2OS cells. HA-NLSB1 was
predominately a nuclear protein, although
there was a significant
signal observed in the cytoplasm as well
(data not shown). Notably,
when the HA-NLSB1 construct was coexpressed
with the NEScdc25C fusions,
more than 90% of the cells expressing
both proteins underwent
PCC (data not shown). This high degree
of cooperativity may be
due to the presence of readily detectable
amounts of the HA-NLSB1
protein in the cytoplasm of U-2OS cells
(data not
shown).
The ability of the nuclear versions of cdc25C to induce PCC was also
compared to that of WT cdc25C. Both the cdc25CNLS and
S216ANLS
fusion proteins were not as effective as the WT cdc25C
protein at
inducing PCC in U-2OS cells (Fig.
7C, left panel).
Furthermore,
coexpression of these constructs with cyclin B1 resulted
in only 70%
of the cells showing PCC(Fig.
7C, middle panel). In
cells not
showing PCC, cdc25CNLS and S216ANLS were nuclear whereas
the
transfected cyclin B1 remained cytoplasmic (data not shown),
suggesting
that the NLS fusion proteins did not alter the localization
of cyclin
B1 and that the reduced level of PCC exhibited by these
nuclear cdc25C
proteins may be due to limiting amounts of cyclin
B1 in the nucleus
(
59). To test whether an NLS-tagged version
of cyclin B1
could cooperate with the NLS fusion proteins to induce
PCC, cdc25CNLS
and S216ANLS were cotransfected with HA-NLSB1 into
U-2OS cells. When
transfected alone, HA-NLSB1 did not induce PCC
above background levels
(data not shown). However, when HA-NLSB1
was cotransfected with either
cdc25CNLS or S216ANLS, more than
90% of the transfected cells showed
evidence of PCC (Fig.
7B,
right panel). The specificity of this effect
was demonstrated
by the failure of the inactive mutant C377SNLS to
induce PCC in
cooperation with HA-NLSB1 (data not shown). This result
suggests
that the nuclear versions of cdc25C were not compromised in
the
ability to function as phosphatases but failed to induce PCC at
WT
levels due to limiting amounts of cyclin B1 in the nucleus
due to the
rapid nuclear export of cyclin B1 (
13,
51,
59).
| |
DISCUSSION |
The results presented herein suggest that the ability of 14-3-3 proteins to regulate cdc25C function is mediated, at least in part, by
promoting the retention of cdc25C in the cytoplasmic compartment during
interphase. Cytoplasmic retention of cdc25C by 14-3-3 required an
intact 14-3-3 binding motif. cdc25C mutants that do not contain a
14-3-3 binding motif, either by deletion or mutation of the S216
residue, showed a pancellular distribution when transiently expressed
in U-2OS cells, in contrast to the predominantly cytoplasmic appearance
of a WT construct. It was observed that transient expression of cdc25C
with a disrupted 14-3-3 binding site led to a significantly increased
tendency to induce PCC compared to WT cdc25C expressed under similar
conditions. The same 14-3-3 binding site in cdc25C has been reported to
represent an important regulatory motif affecting the activity of
cdc25C during the DNA replication and under DNA damage checkpoint
(49). Therefore, 14-3-3 proteins may serve to regulate
cdc25C activity by restricting its cellular localization to the
cytoplasm under various conditions.
Cytoplasmic localization of human cdc25C.
There has been some
controversy in the field regarding the cellular localization of cdc25C.
Previously, three studies reported that cdc25C was a nuclear protein in
human cells (10, 12, 39). However, each of these studies
used a polyclonal serum that had the potential to recognize both cdc25A
and cdc25B, in addition to cdc25C. For example, Girard et al. used a
polyclonal rabbit antiserum raised against a peptide derived from the
catalytic domain of starfish cdc25 (12). Every residue
within this peptide was perfectly conserved in the catalytic domains of
all three human cdc25 proteins. Therefore, this antiserum may have
recognized cdc25A and cdc25B in addition to cdc25C. Two other groups
generated polyclonal antibodies by immunizing rabbits with bacterially
produced, full-length cdc25C protein followed by affinity purification
with the immunogen (10, 39). The purified antisera may have
recognized highly conserved elements within cdc25A, cdc25B, and cdc25C.
Notably, cdc25A was reported to be a nuclear protein with a predicted
molecular mass (58 kDa) similar to that of cdc25C (53 kDa)
(20). In contrast to the cdc25C antiserum, the cdc25A
antibody generated in this latter study was raised against an
N-terminal peptide in cdc25A not present in cdc25C and therefore was
unlikely to recognize cdc25C (20).
In this report, four different MAbs were generated to the N-terminal
258 residues of cdc25C, a region not highly conserved
with either
cdc25A or cdc25B. These MAbs specifically recognized
at least three
distinct epitopes within cdc25C and could not detect
cdc25A or cdc25B
by Western blotting. Immunostaining of a number
of primary human cell
strains and immortal cell lines with each
of these antibodies revealed
a specific cytoplasmic signal. The
cytoplasmic localization of cdc25C
was confirmed by the appearance
of a specific Western blot signal of
the appropriate molecular
weight in the cytoplasmic but not the nuclear
extracts prepared
from U-2OS cells. The cytoplasmic localization of the
endogenous
cdc25C protein was consistent with earlier reports for
transiently
expressed, epitope-tagged cdc25C protein. These
observations were
extended in this report to demonstrate that the
cytoplasmic localization
of transiently expressed cdc25C was dependent
on an intact 14-3-3
binding
motif.
Effect of 14-3-3 on cdc25C function and subcellular
localization.
S216 has been identified as the major
phosphorylation site in cdc25C during interphase (47, 49),
suggesting that its phosphorylation may contribute to the negative
regulation of cdc25C activity during interphase. It was subsequently
demonstrated that phosphorylation of S216 results in the generation of
a binding site for 14-3-3 proteins that may serve to inhibit cdc25C
function (44, 49, 62). However, the precise manner in which
14-3-3 proteins inhibit cdc25C function was not known. The results
presented here suggest that an intact 14-3-3 binding site in cdc25C may
be required for the cytoplasmic retention of cdc25C during interphase.
Binding to 14-3-3 was disrupted by substitution of residue 216 with
alanine (Fig. 5B). Loss of the 14-3-3 binding site in the transiently expressed construct, S216A or 201-258, resulted in pancellular localization of cdc25C (Fig. 5A) and an increased ability to induce PCC
compared to WT cdc25C (Fig. 6B). These results suggest that at least
one mechanism by which 14-3-3 proteins regulate cdc25C function is by
inducing its cytoplasmic localization.
At least seven different 14-3-3 proteins have been identified in
mammalian cells (
62). This leads to the question of which
14-3-3 protein modulates cdc25C function in response to the DNA
replication or DNA damage checkpoints. 14-3-3
and 14-3-3
have
been shown to complex with cdc25 in
Xenopus egg extracts
(
31).
Another human homologue, 14-3-3
, may have a
specific role in
a G
2/M checkpoint, given that its
expression was stimulated by
DNA damage induced by
irradiation in a
p53-dependent manner
(
18). Furthermore, 14-3-3
overexpression induced a G
2 arrest,
leading to the
speculation that at least part of this effect may
be dependent on its
interaction with cdc25C. It is possible that
cdc25C is regulated by any
one of several 14-3-3 proteins in response
to distinct DNA replication
and damage checkpoints. In this report,
14-3-3
was shown to bind
specifically to cdc25C and required
an intact S216 residue. Whether
other 14-3-3 proteins can bind
specifically to cdc25C remains to be
tested.
Phosphorylation of the S216 residue may also occur in response to a
variety of situations. Three kinases (chk1, chk2, and
C-TAK1) have been
shown to be capable of specifically phosphorylating
the S216 residue in
cdc25C, resulting in the generation of a consensus
binding site for
14-3-3 proteins (
38,
49,
50,
52). Notably,
C-TAK1 was found
to be cytoplasmic in human cells and could potentially
promote the
phosphorylation of cdc25C in the cytoplasm, resulting
in association
with 14-3-3 proteins and cytoplasmic retention
(
50). In
contrast, chk1 and chk2 have been reported to be nuclear
proteins
(
38,
52). chk1 and chk2 may phosphorylate the S216
residue
when cdc25C enters the nucleus. Whether this phosphorylation
of S216
can promote the nuclear export of cdc25C is not known.
It has been
recently reported that
S. pombe cdc25 may be exported
from
the nucleus in complex with rad24, a 14-3-3 homolog. This
export is
abolished in a strain lacking chk1 (
37). Therefore,
in
response to DNA damage, chk1 may promote the nuclear export
of a cdc25
protein that has entered the nucleus
prematurely.
The ability of 14-3-3 proteins to sequester important regulatory
proteins was previously demonstrated for the apoptosis inducer
BAD
(
66). Phosphorylation at serine residues within two 14-3-3
binding consensus sites in BAD promotes the formation of a complex
with
14-3-3 proteins, leading to its retention in the cytoplasm.
Upon
dephosphorylation of these residues, BAD becomes capable
of forming a
complex with BCL-X
L within the mitochondrial membrane
fraction. Association with BAD inhibits the antiapoptotic activity
of
BCL-X
L, resulting in apoptosis and cell death
(
66). Therefore,
it appears that 14-3-3 proteins may serve
an important role in
cell survival and cell division by sequestering
regulatory proteins
in the
cytoplasm.
Cellular localization and PCC.
In the PCC assays described
here, WT cdc25C was capable of inducing fractured condensed chromatin
in 25 to 30% of the transfected cells. This percentage was much higher
than that reported previously (13, 49). The difference in
frequency of PCC observed in these assays compared to previous reports
(13, 49) may be attributable to the differences in cell type
(U-2OS cells versus HeLa) or methods used for expression (transient
transfection in this study versus inducible stable cell lines or
microinjection of cDNA in others). Furthermore, the cell cycle
synchrony experiments shown here suggest that PCC was initiated only in
the presence of cyclin B1. In asynchronously growing U-2OS cells, more
than 60% of the cells were in S and G2 phases of the cell
cycle, with a similar percentage containing cyclin B1, as determined by
immunostaining (Fig. 6). Therefore, the high numbers observed in the
PCC assay may reflect the highly proliferative nature of these cells.
Consistent with this finding was the observation that cotransfection of
cdc25C and cyclin B1 resulted in nearly all of the cells undergoing PCC
(Fig. 7) (13, 16). The higher numbers of PCC observed in
this assay system permitted the analysis of cdc25C mutant proteins with
lower activity than the WT construct.
The timely translocation of cdc25C from the cytoplasm to the nucleus
may be an important component of the entry into mitosis.
The increased
tendency of the 14-3-3 binding site mutant S216A
to induce PCC may
indicate that cdc25C should be excluded from
the nucleus until the
appropriate time in the cell cycle. The
requirement for nuclear entry
for initiation of mitosis by cdc25C
was supported by the reduced
tendency of the cdc25C constructs
containing the heterologous NES.
Paradoxically, a cdc25C construct
containing a heterologous NLS was not
more active than the WT
construct. Since the cytoplasmic localization
of cyclin B1 in
interphase cells was unaltered in the presence of
NLS-tagged cdc25C,
it may be argued that cdc25CNLS was unable to
activate a cytoplasmic
cdc2-cyclin B complex. Consistent with this
possibility cdc25C-NLS
was as active as WT cdc25C when coexpressed with
an NLS-tagged
variant of cyclin B1. The reduction in activity of the
NLS and
NES constructs may imply that there may be a dynamic shuttling
of cdc25C into various cellular compartments during the cell cycle
that
was altered by the presence of the heterologous NES or NLS
tag.
Cyclin B1 is maintained in the cytoplasm in interphase cells due to the
presence of an N-terminal NES (
13,
59,
63).
Several recent
reports have suggested that the entry of cyclin
B1 into the nucleus is
necessary but not sufficient for M-phase
progression (
13,
25,
59). The cytoplasmic localization signal
in cdc25C shows no
apparent homology to the NES of cyclin B1 (
13,
51,
59),
suggesting that the intracellular localization of
cdc25C and cyclin B1
may be regulated by different mechanisms.
This is further supported by
the observation that treatment of
U-2OS cells with leptomycin B led to
the nuclear accumulation
of cyclin B1 (
13,
59,
63), but the
cytoplasmic localization
of cdc25C was not altered, and there was no
indication of an increased
tendency for cells to undergo PCC or advance
past G
2 phase and
enter mitosis. These results are
consistent with the hypothesis
that cdc25C may not be excluded from the
nucleus due to the presence
of an NES but maybe retained in the
cytoplasm by complex formation
with 14-3-3 proteins in U-2OS cells.
However, it has been reported
that the rad24 gene product of
S. pombe, a 14-3-3 homologue, promotes
the nuclear export of cdc25 in
a crm1-dependent manner (
37).
It is possible that the
association of cdc25C with 14-3-3 proteins
results in the nuclear
export of cdc25C via a mechanism that is
not sensitive to leptomycin B
in U-2OS cells. The differential
regulation of the intracellular
localization of cdc25C and cyclin
B1 by the checkpoint apparatus in
human cells may provide an independent
regulation of the entry into
mitosis whereby, under certain conditions,
cdc25C would be restricted
to the cytoplasm and be unable to activate
a cdc2-cyclin B complex that
had already translocated to the nucleus.
It has been shown in
Xenopus that phosphorylation of consensus
cdc2 sites in
cyclin B1 are required to inhibit its nuclear export
(
35,
63). Given that cdc25C also undergoes hyperphosphorylation
during
mitosis, it is possible that the cytoplasmic retention
of cdc25C is
disrupted by phosphorylation of consensus cdc2 sites.
Thus, activation
of cdc25C in the cytoplasm could promote the
nuclear transport of
cyclin B1-cdc2 complexes as well as its own
transport, resulting in
further activation of cyclin B-cdc2 complexes
in the
nucleus.
We propose the following model for cdc25C regulation. cdc25C is
retained in the cytoplasm by association with 14-3-3 proteins
during
interphase. This effect is dependent on an intact residue
S216 that is
most likely constitutively phosphorylated by any
one of several kinases
during interphase. In late G
2, S216 is
dephosphorylated,
resulting in the loss of 14-3-3 binding and
free diffusion or active
transport of cdc25C into the nucleus.
Independently, but perhaps at a
similar time, the NES of cyclin
B1 is inactivated, leading to
accumulation of cyclin B1-cdc2 complexes
in the nucleus (
13,
35,
51,
59,
63). Subsequently, the
activity of the cdc25C phosphatase
is stimulated by hyperphosphorylation,
resulting in the activation of
nuclear cdc2-cyclin B complexes.
This model does not exclude the
possibility that the regulation
of cdc25C by 14-3-3 proteins occurs
through other mechanisms in
addition to regulating the subcellular
localization of
cdc25C.
| |
ACKNOWLEDGMENTS |
We thank R. Scully for plasmid pSG5-L, Y. Sanchez, S. Elledge, P. Saha, and A. Datta for GST-cdc25A and GST-cdc25B, Michael Yaffe for GST
14-3-3, and W. R. Sellers for the HA-NLS and HA-NES constructs.
We thank Peter Marks and William Connors at the Brigham and Womens
Hospital confocal facility for help with generating the confocal images
and Steven R. Grossman and Michael Rokas for their help with antibody
purification. We thank D. M. Livingston, P. Adams, J. Ayté,
H. Chao, S. Patankar, and H. Stubdal for critically reading the
manuscript. We also thank the other members of the DeCaprio laboratory
for their help and encouragement.
S.N.D. was supported by a fellowship from the Leukemia Society of
America. J.A.D. is a Scholar of the Leukemia Society of America. This
work was supported in part by Public Health Service grants CA-63113 and
CA-50661.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dana-Farber
Cancer Institute and Harvard Medical School, 44 Binney St., Boston, MA 02115. Phone: (617) 632-3825. Fax: (617) 632-4760. E-mail:
james_decaprio{at}dfci.harvard.edu.
| |
REFERENCES |
| 1.
|
Al-Khodairy, F., and A. M. Carr.
1992.
DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe.
EMBO J.
11:1343-1350[Medline].
|
| 2.
|
Davis, F. M.,
T. Y. Tsao,
S. K. Fowler, and P. N. Rao.
1983.
Monoclonal antibodies to mitotic cells.
Proc. Natl. Acad. Sci. USA
80:2926-2930[Abstract/Free Full Text].
|
| 3.
|
Den Haese, G. J.,
N. Walworth,
A. M. Carr, and K. L. Gould.
1995.
The wee1 protein kinase regulates T14 phosphorylation of fission yeast cdc2.
Mol. Biol. Cell
6:371-385[Abstract].
|
| 4.
|
Dunphy, W. G., and A. Kumagai.
1991.
The cdc25 protein contains an intrinsic phosphatase activity.
Cell
67:189-196[Medline].
|
| 5.
|
Evan, G. I.,
G. K. Lewis,
G. Ramsay, and J. M. Bishop.
1985.
Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.
Mol. Cell. Biol.
5:3610-3616[Abstract/Free Full Text].
|
| 6.
|
Field, J.,
J. I. Nikawa,
D. Broek,
B. MacDonald,
L. Rodgers,
I. A. Wilson,
R. A. Lerner, and M. Wigler.
1988.
Purification of a RAS-responsive adenyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method.
Mol. Cell. Biol.
8:2159-2165[Abstract/Free Full Text].
|
| 7.
|
Fischer, U.,
J. Huber,
W. C. Boelens,
I. W. Mattaj, and R. Luhrmann.
1995.
The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs.
Cell
90:1051-1060.
|
| 8.
|
Ford, J. C.,
F. Al-Khodairy,
E. Fotou,
K. S. Sheldrick,
D. J. F. Griffiths, and A. M. Carr.
1994.
14-3-3 protein homologs required for the DNA damage checkpoint in fission yeast.
Science
265:533-535[Abstract/Free Full Text].
|
| 9.
|
Furnari, B.,
N. Rhind, and P. Russell.
1997.
Cdc25 mitotic inducer targeted by Chk1 DNA damage checkpoint kinase.
Science
277:1495-1497[Abstract/Free Full Text].
|
| 10.
|
Gabrielli, B. G.,
C. P. C. DeSouza,
I. D. Tonks,
J. M. Clark,
N. K. Hayward, and K. A. O. Ellem.
1996.
Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells.
J. Cell Sci.
109:1081-1093[Abstract].
|
| 11.
|
Gautier, J.,
M. J. Solomon,
R. N. Booher,
J. F. Bazan, and M. W. Kirschner.
1991.
cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2.
Cell
67:197-211[Medline].
|
| 12.
|
Girard, F.,
U. Strausfeld,
J.-C. Cavadore,
P. Russell,
A. Fernandez, and N. J. C. Lamb.
1992.
cdc25 is a nuclear protein expressed constitutively throughout the cell cycle in nontransformed mammalian cells.
J. Cell Biol.
118:785-794[Abstract/Free Full Text].
|
| 13.
|
Hagting, A.,
C. Karlsson,
P. Clute,
M. Jackman, and J. Pines.
1998.
MPF localization is controlled by nuclear export.
EMBO J.
17:4127-4138[Medline].
|
| 14.
|
Harlow, E., and D. Lane.
1988.
Antibodies: a laboratory manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 15.
|
Hartley, R. S.,
R. E. Rempel, and J. L. Maller.
1996.
In vivo regulation of the early embryonic cell cycle in Xenopus.
Dev. Biol.
173:408-419[Medline].
|
| 16.
|
Heald, R.,
M. McLoughlin, and F. McKeon.
1993.
Human Wee1 maintains mitotic timing by protecting the nucleus from cytoplasmically activated cdc2 kinase.
Cell
74:463-474[Medline].
|
| 17.
|
Hendzel, M. J.,
Y. Wei,
M. A. Mancini,
A. V. Hooser,
T. Ranalli,
B. R. Brinkley,
D. P. Bazett-Jones, and C. D. Allis.
1997.
Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation.
Chromosoma
106:348-360[Medline].
|
| 18.
|
Hermeking, H.,
C. Lengauer,
K. Polyak,
T.-C. He,
L. Zhang,
S. Thiagalingam,
K. W. Kinzler, and B. Vogelstein.
1997.
14-3-3 is a p53-regulated inhibitor of G2/M progression.
Mol. Cell
1:3-11[Medline].
|
| 19.
|
Hoffmann, I.,
P. R. Clarke,
M. J. Marcote,
E. Karsenti, and G. Draetta.
1993.
Phosphorylation and activation of human cdc25C by cdc2-cyclin B and its involvement in the self amplification of MPF at mitosis.
EMBO J.
12:53-63[Medline].
|
| 20.
|
Hoffmann, I.,
G. Draetta, and E. Karsenti.
1994.
Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition.
EMBO J.
13:4302-4310[Medline].
|
| 21.
|
Igarashi, M.,
A. Nagat,
S. Jinno,
K. Suto, and H. Okayama.
1991.
Wee1+ like gene in human cells.
Nature
353:80-83[Medline].
|
| 22.
|
Izumi, T., and J. L. Maller.
1993.
Elimination of cdc2 phosphorylation sites in the cdc25 phosphatase blocks initiation of M-phase.
Mol. Biol. Cell
4:1337-1350[Abstract].
|
| 23.
|
Izumi, T., and J. L. Maller.
1995.
Phosphorylation and activation of the Xenopus cdc25 phosphatase in the absence of cdc2 and cdk2 kinase activity.
Mol. Biol. Cell
6:215-226[Abstract].
|
| 24.
|
Izumi, T.,
D. H. Walker, and J. L. Maller.
1992.
Periodic changes in phosphorylation of the Xenopus cdc25 phosphatase regulate its activity.
Mol. Biol. Cell
3:927-939[Abstract].
|
| 25.
|
Jin, P.,
S. Hardy, and D. O. Morgan.
1998.
Nuclear localization of cyclin B1 controls mitotic entry after DNA damage.
J. Cell Biol.
141:875-885[Abstract/Free Full Text].
|
| 26.
|
Krek, W., and J. A. DeCaprio.
1995.
Cell synchronization.
Methods Enzymol.
254:114-124[Medline].
|
| 27.
|
Kumagai, A., and W. G. Dunphy.
1991.
The cdc25 protein controls tyrosine dephosphorylation of the cdc2 protein in a cell-free system.
Cell
64:903-914[Medline].
|
| 28.
|
Kumagai, A., and W. G. Dunphy.
1996.
Purification and molecular cloning of Plx1, a cdc25-regulatory kinase from Xenopus egg extracts.
Science
273:1377-1380[Abstract].
|
| 29.
|
Kumagai, A., and W. G. Dunphy.
1992.
Regulation of the cdc25 protein during the cell cycle in xenopus extracts.
Cell
70:139-151[Medline].
|
| 30.
|
Kumagai, A.,
Z. Guo,
K. H. Emami,
S. X. Wang, and W. G. Dunphy.
1998.
The Xenopus Chk1 protein kinase mediates a caffeine-sensitive pathway of checkpoint control in cell-free extracts.
J. Cell Biol.
142:1559-1569[Abstract/Free Full Text].
|
| 31.
|
Kumagai, A.,
P. S. Yakowec, and W. G. Dunphy.
1998.
14-3-3 proteins act as negative regulators of the mitotic inducer cdc25 in Xenopus egg extracts.
Mol. Biol. Cell
9:345-354[Abstract/Free Full Text].
|
| 32.
|
Lee, M. S.,
T. Enoch, and H. Piwnica-Worms.
1994.
mik1+ encodes a tyrosine kinase that phosphorylates p34cdc2 on tyrosine 15.
J. Biol. Chem.
269:30530-30537[Abstract/Free Full Text].
|
| 33.
|
Lee, M. S.,
S. Ogg,
M. Xu,
L. L. Parker,
D. J. Donoghue,
J. L. Maller, and H. Piwnica-Worms.
1992.
cdc25+ encodes a protein phosphatase that dephosphorylates p34cdc2.
Mol. Biol. Cell
3:73-84[Abstract].
|
| 34.
|
Lee, W.-H.,
J.-Y. Shew,
F. D. Hong,
T. W. Sery,
L. A. Donoso,
L.-J. Young,
R. Bookstein, and E. Y.-H. P. Lee.
1987.
The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity.
Nature
329:642-645[Medline].
|
| 35.
|
Li, J.,
A. N. Meyer, and D. J. Donoghue.
1997.
Nuclear localization of cyclin B1 mediates its biological activity and is regulated by phosphorylation.
Proc. Natl. Acad. Sci. USA
94:502-507[Abstract/Free Full Text].
|
| 36.
|
Liu, F.,
J. J. Stanton,
Z. Wu, and H. Piwnica-Worms.
1997.
The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the endoplasmic reticulum and Golgi complex.
Mol. Cell. Biol.
17:571-583[Abstract].
|
| 37.
|
Lopez-Girona, A.,
B. Furnari,
O. Mondesert, and P. Russell.
1999.
Nuclear localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein.
Nature
397:172-175[Medline].
|
| 38.
|
Matsuoka, S.,
M. Huang, and S. J. Elledge.
1998.
Linkage of ATM to cell cycle regulation by the chk2 protein kinase.
Science
282:1893-1897[Abstract/Free Full Text].
|
| 39.
|
Millar, J. B. A.,
J. Blevitt,
L. Gerace,
K. Sadhu,
C. Featherstone, and P. Russell.
1991.
p55CDC25 is a nuclear protein required for the initiation of mitosis in human cells.
Proc. Natl. Acad. Sci. USA
88:10500-10504[Abstract/Free Full Text].
|
| 40.
|
Millar, J. B. A.,
C. H. McGowan,
G. Lenaers,
R. Jones, and P. Russell.
1991.
p80cdc25 mitotic inducer is the tyrosine phosphatase that activates p34cdc2 kinase in fission yeast.
EMBO J.
10:4301-4309[Medline].
|
| 41.
|
Mittnacht, S., and R. A. Weinberg.
1991.
G1/S phosphorylation of the retinoblastoma protein is associated with an altered affinity for the nuclear compartment.
Cell
65:381-393[Medline].
|
| 42.
|
Mueller, P. A.,
T. R. Coleman,
A. Kumagai, and W. G. Dunphy.
1995.
Myt1: a membrane-associated inhibitory kinase that phosphorylates cdc2 on both threonine-14 and tyrosine-15.
Science
270:86-90[Abstract/Free Full Text].
|
| 43.
|
Munro, S., and H. R. B. Pelham.
1984.
Use of peptide tagging to detect proteins expressed from cloned genes: deletion mapping functions of Drosophila hsp70.
EMBO J.
3:3087-3093[Medline].
|
| 44.
|
Muslin, A. J.,
J. W. Tanner,
P. M. Allen, and A. S. Shaw.
1996.
Interaction of 14-3-3 with signaling proteins is mediated by recognition of phosphoserine.
Cell
84:889-897[Medline].
|
| 45.
|
Nurse, P.
1998.
Checkpoint pathways come of age.
Cell
91:865-867.
|
| 46.
|
Nurse, P.
1994.
Ordering S phase and M phase in the cell cycle.
Cell
79:547-550[Medline].
|
| 47.
|
Ogg, S.,
B. Gabrielli, and H. Piwnica-Worms.
1994.
Purification of a serine kinase that associates with and phosphorylates human cdc25C on serine 216.
J. Biol. Chem.
269:30461-30469[Abstract/Free Full Text].
|
| 48.
|
Parker, L. L., and H. Piwnica-Worms.
1992.
Inactivation of the p34cdc2-cyclin B complex by the human wee1 tyrosine kinase.
Science
257:1955-1957[Abstract/Free Full Text].
|
| 49.
|
Peng, C.-Y.,
P. R. Graves,
R. S. Thoma,
Z. Wu,
A. S. Shaw, and H. Piwnica-Worms.
1997.
Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of cdc25C on serine-216.
Science
277:1501-1505[Abstract/Free Full Text].
|
| 50.
|
Peng, C. Y.,
P. R. Graves,
S. Ogg,
R. S. Thoma,
M. J. Byrnes,
Z. Wu,
M. T. Stephenson, and H. Piwnica-Worms.
1998.
C-TAK1 protein kinase phosphorylates human cdc25C on serine 216 and promotes 14-3-3 protein binding.
Cell Growth Differ.
9:197-208[Abstract].
|
| 51.
|
Pines, J., and T. Hunter.
1994.
The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B.
EMBO J.
13:3772-3781[Medline].
|
| 52.
|
Sanchez, Y.,
C. Wong,
R. S. Thoma,
R. Richman,
Z. Wu,
H. Piwnica-Worms, and S. J. Elledge.
1997.
Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to cdk regulation through cdc25.
Science
277:1497-1501[Abstract/Free Full Text].
|
| 53.
|
Schreiber, E.,
P. Matthias,
M. M. Muller, and W. Schaffner.
1989.
Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells.
Nucleic Acids Res.
17:6419[Free Full Text].
|
| 54.
| Scully, R., and D. M. Livingston. 1997. Unpublished data.
|
| 55.
| Sellers, W. R. 1998. Unpublished data.
|
| 56.
|
Strausfeld, U.,
A. Fernandez,
J.-P. Capony,
F. Girard,
N. Lautredou,
J. Derancourt,
J.-C. Labbe, and N. J. C. Lamb.
1994.
Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis.
J. Biol. Chem.
269:5989-6000[Abstract/Free Full Text].
|
| 57.
|
Strausfeld, U.,
J. C. Labbe,
D. Fesquet,
J. C. Cavadore,
A. Picard,
K. Sadhu,
P. Russell, and M. Doree.
1991.
Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human cdc25 protein.
Nature
351:242-245[Medline].
|
| 58.
|
Stubdal, H.,
J. Zalvide, and J. A. DeCaprio.
1996.
Simian virus 40 large T antigen alters the phosphorylation state of the RB-related proteins p130 and p107.
J. Virol.
70:2781-2788[Abstract].
|
| 59.
|
Toyoshima, F.,
T. Moriguchi,
A. Wada,
M. Fukuda, and E. Nishida.
1998.
Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint.
EMBO J.
17:2728-2735[Medline].
|
| 60.
|
Walworth, N.,
S. Davey, and D. Beach.
1993.
Fission yeast chk1 protein kinase links the rad checkpoint pathway to cdc2.
Nature
363:368-371[Medline].
|
| 61.
|
Walworth, N. C., and R. Bernards.
1996.
Rad-dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint.
Science
271:353-356[Abstract].
|
| 62.
|
Yaffe, M. B.,
K. Rittinger,
S. Volinia,
P. R. Caron,
A. Aitken,
H. Leffers,
S. J. Gamblin,
S. J. Smerdon, and L. C. Cantley.
1998.
The structural basis for 14-3-3:phosphopeptide binding specificity.
Cell
91:961-971.
|
| 63.
|
Yang, J.,
E. S. G. Bardes,
J. D. Moore,
J. Brennan,
M. Powers, and S. Kornbluth.
1998.
Control of cyclin B1 localization through regulated binding of the nuclear export factor, CRM1.
Genes Dev.
12:2131-2143[Abstract/Free Full Text].
|
| 64.
|
Zalvide, J.,
H. Stubdal, and J. A. DeCaprio.
1998.
The J domain of simian virus 40 large T antigen is required to functionally inactivate RB family proteins.
Mol. Cell. Biol.
18:1408-1415[Abstract/Free Full Text].
|
| 65.
|
Zeng, Y.,
K. C. Forbes,
Z. Wu,
S. Moreno,
H. Piwnica-Worms, and T. Enoch.
1998.
Replication checkpoint requires phosphorylation of the phosphatase cdc25 by Cds1 or Chk1.
Nature
395:507-510[Medline].
|
| 66.
|
Zha, J.,
H. Harada,
E. Yang,
J. Jockel, and S. J. Korsmeyer.
1996.
Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-XL.
Cell
87:619-628[Medline].
|
Molecular and Cellular Biology, June 1999, p. 4465-4479, Vol. 19, No. 6
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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-
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-
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-
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[Full Text]
-
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[Abstract]
[Full Text]
-
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[Abstract]
[Full Text]
-
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(2001). LIP1, a cytoplasmic protein functionally linked to the Peutz-Jeghers syndrome kinase LKB1. Hum Mol Genet
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[Abstract]
[Full Text]
-
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[Abstract]
[Full Text]
-
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276: 42971-42977
[Abstract]
[Full Text]
-
Aytac, U., Claret, F.-X., Ho, L., Sato, K., Ohnuma, K., Mills, G. B., Cabanillas, F., Morimoto, C., Dang, N. H.
(2001). Expression of CD26 and Its Associated Dipeptidyl Peptidase IV Enzyme Activity Enhances Sensitivity to Doxorubicin-induced Cell Cycle Arrest at the G2/M Checkpoint. Cancer Res.
61: 7204-7210
[Abstract]
[Full Text]
-
Zhao, H., Piwnica-Worms, H.
(2001). ATR-Mediated Checkpoint Pathways Regulate Phosphorylation and Activation of Human Chk1. Mol. Cell. Biol.
21: 4129-4139
[Abstract]
[Full Text]
-
Chen, M.-S., Hurov, J., White, L. S., Woodford-Thomas, T., Piwnica-Worms, H.
(2001). Absence of Apparent Phenotype in Mice Lacking Cdc25C Protein Phosphatase. Mol. Cell. Biol.
21: 3853-3861
[Abstract]
[Full Text]
-
Zilliacus, J., Holter, E., Wakui, H., Tazawa, H., Treuter, E., Gustafsson, J.-A.
(2001). Regulation of Glucocorticoid Receptor Activity by 14-3-3-Dependent Intracellular Relocalization of the Corepressor RIP140. Mol. Endocrinol.
15: 501-511
[Abstract]
[Full Text]
-
Bunney, T. D., van Walraven, H. S., de Boer, A. H.
(2001). 14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase. Proc. Natl. Acad. Sci. USA
10.1073/pnas.061437498v1
[Abstract]
[Full Text]
-
Rhind, N., Russell, P.
(2001). Roles of the Mitotic Inhibitors Wee1 and Mik1 in the G2 DNA Damage and Replication Checkpoints. Mol. Cell. Biol.
21: 1499-1508
[Abstract]
[Full Text]
-
Lee, J., Kumagai, A., Dunphy, W. G.
(2001). Positive Regulation of Wee1 by Chk1 and 14-3-3 Proteins. Mol. Biol. Cell
12: 551-563
[Abstract]
[Full Text]
-
Vielhaber, E., Eide, E., Rivers, A., Gao, Z.-H., Virshup, D. M.
(2000). Nuclear Entry of the Circadian Regulator mPER1 Is Controlled by Mammalian Casein Kinase I varepsilon. Mol. Cell. Biol.
20: 4888-4899
[Abstract]
[Full Text]
-
Takai, H., Tominaga, K., Motoyama, N., Minamishima, Y. A., Nagahama, H., Tsukiyama, T., Ikeda, K., Nakayama, K., Nakanishi, M., Nakayama, K.-i.
(2000). Aberrant cell cycle checkpoint function and early embryonic death in Chk1-/- mice. Genes Dev.
14: 1439-1447
[Abstract]
[Full Text]
-
Kurz, E. U., Leader, K. B., Kroll, D. J., Clark, M., Gieseler, F.
(2000). Modulation of Human DNA Topoisomerase IIalpha Function by Interaction with 14-3-3epsilon. J. Biol. Chem.
275: 13948-13954
[Abstract]
[Full Text]
-
Busby, E. C., Leistritz, D. F., Abraham, R. T., Karnitz, L. M., Sarkaria, J. N.
(2000). The Radiosensitizing Agent 7-Hydroxystaurosporine (UCN-01) Inhibits the DNA Damage Checkpoint Kinase hChk1. Cancer Res.
60: 2108-2112
[Abstract]
[Full Text]
-
Zhou, B.-B. S., Chaturvedi, P., Spring, K., Scott, S. P., Johanson, R. A., Mishra, R., Mattern, M. R., Winkler, J. D., Khanna, K. K.
(2000). Caffeine Abolishes the Mammalian G2/M DNA Damage Checkpoint by Inhibiting Ataxia-Telangiectasia-mutated Kinase Activity. J. Biol. Chem.
275: 10342-10348
[Abstract]
[Full Text]
-
Graves, P. R., Yu, L., Schwarz, J. K., Gales, J., Sausville, E. A., O'Connor, P. M., Piwnica-Worms, H.
(2000). The Chk1 Protein Kinase and the Cdc25C Regulatory Pathways Are Targets of the Anticancer Agent UCN-01. J. Biol. Chem.
275: 5600-5605
[Abstract]
[Full Text]
-
Raleigh, J., O'Connell, M.
(2000). The G(2) DNA damage checkpoint targets both Wee1 and Cdc25. J. Cell Sci.
113: 1727-1736
[Abstract]
-
RHIND, N., BABER-FURNARI, B.A., LOPEZ-GIRONA, A., BODDY, M.N., BRONDELLO, J.-M., MOSER, B., SHANAHAN, P., BLASINA, A., MCGOWAN, C., RUSSELL, P.
(2000). DNA Damage Checkpoint Control of Mitosis in Fission Yeast. Cold Spring Harb Symp Quant Biol
65: 353-360
[Abstract]
-
Baber-Furnari, B. A., Rhind, N., Boddy, M. N., Shanahan, P., Lopez-Girona, A., Russell, P.
(2000). Regulation of Mitotic Inhibitor Mik1 Helps to Enforce the DNA Damage Checkpoint. Mol. Biol. Cell
11: 1-11
[Abstract]
[Full Text]
-
Morris, M. C., Heitz, A., Mery, J., Heitz, F., Divita, G.
(2000). An Essential Phosphorylation-site Domain of Human cdc25C Interacts with Both 14-3-3 and Cyclins. J. Biol. Chem.
275: 28849-28857
[Abstract]
[Full Text]
-
Hu, B., Zhou, X.-Y., Wang, X., Zeng, Z.-C., Iliakis, G., Wang, Y.
(2001). The Radioresistance to Killing of A1-5 Cells Derives from Activation of the Chk1 Pathway. J. Biol. Chem.
276: 17693-17698
[Abstract]
[Full Text]
-
Vogt, A., Adachi, T., Ducruet, A. P., Chesebrough, J., Nemoto, K., Carr, B. I., Lazo, J. S.
(2001). Spatial Analysis of Key Signaling Proteins by High-content Solid-phase Cytometry in Hep3B Cells Treated with an Inhibitor of Cdc25 Dual-specificity Phosphatases. J. Biol. Chem.
276: 20544-20550
[Abstract]
[Full Text]
-
Curman, D., Cinel, B., Williams, D. E., Rundle, N., Block, W. D., Goodarzi, A. A., Hutchins, J. R., Clarke, P. R., Zhou, B.-B., Lees-Miller, S. P., Andersen, R. J., Roberge, M.
(2001). Inhibition of the G2 DNA Damage Checkpoint and of Protein Kinases Chk1 and Chk2 by the Marine Sponge Alkaloid Debromohymenialdisine. J. Biol. Chem.
276: 17914-17919
[Abstract]
[Full Text]
-
Crawford, D. F., Piwnica-Worms, H.
(2001). The G2 DNA Damage Checkpoint Delays Expression of Genes Encoding Mitotic Regulators. J. Biol. Chem.
276: 37166-37177
[Abstract]
[Full Text]
-
Bunney, T. D., van Walraven, H. S., de Boer, A. H.
(2001). 14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase. Proc. Natl. Acad. Sci. USA
98: 4249-4254
[Abstract]
[Full Text]
-
Brunet, A., Kanai, F., Stehn, J., Xu, J., Sarbassova, D., Frangioni, J. V., Dalal, S. N., DeCaprio, J. A., Greenberg, M. E., Yaffe, M. B.
(2002). 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport. JCB
156: 817-828
[Abstract]
[Full Text]
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Abstract
Introduction
