From the Department of Biochemistry, University of Leicester,
University Road, Leicester, United Kingdom LE1 7RH, the
§ Department of Physiological Sciences. Medical School,
Framlington Place, Newcastle-upon-Tyne, United Kingdom NE2 4HH, the
¶ Medical Research Council Laboratory of Molecular Cell Biology
and the Department of Pharmacology, University College London, Gower
Street, London, United Kingdom WC1E 6BT, the Department of
Neurobiology, Stanford University School of Medicine, Stanford,
California 94305-5401, and the ** Department of Pharmacology, Nagoya
University School of Medicine, Tsurumai-cho 65, Showa-Ku,
Nagoya 466, Japan
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ABSTRACT |
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The human tyrosine phosphatase
(p54cdc25-c) is activated by phosphorylation at mitosis entry.
The phosphorylated p54cdc25-c in turn activates the p34-cyclin
B protein kinase and triggers mitosis. Although the active p34-cyclin B
protein kinase can itself phosphorylate and activate
p54cdc25-c, we have investigated the possibility that other
kinases may initially trigger the phosphorylation and activation of
p54cdc25-c. We have examined the effects of the
calcium/calmodulin-dependent protein kinase (CaM kinase II)
on p54cdc25-c. Our in vitro experiments show that
CaM kinase II can phosphorylate p54cdc25-c and increase its
phosphatase activity by 2.5-3-fold. Treatment of a synchronous
population of HeLa cells with KN-93 (a water-soluble inhibitor of CaM
kinase II) or the microinjection of AC3-I (a specific peptide inhibitor
of CaM kinase II) results in a cell cycle block in G2
phase. In the KN-93-arrested cells, p54cdc25-c is not
phosphorylated, p34cdc2 remains tyrosine phosphorylated, and
there is no increase in histone H1 kinase activity. Our data suggest
that a calcium-calmodulin-dependent step may be involved in
the initial activation of p54cdc25-c.
Calcium, an intracellular second messenger, is known to be
required for cells to traverse the cell cycle checkpoints at
G1/S, at entry into mitosis (G2/M phase), and
at mitosis exit (reviewed in Refs. 1-3, 4, and 5). A number of studies
have shown that this requirement for calcium during cell division is
mediated by calmodulin
(CaM),1 an intracellular
calcium-binding protein (6, 7). Studies in several species have shown
that the level of calmodulin protein rises 2-fold as cells progress
through the G1/S phase checkpoint (8-10). The results of
several studies also suggest that calcium/CaM may also be involved in
the G2/M phase transition in mammalian cells (reviewed in
Ref. 2). Transient increases in intracellular calcium can be detected
as cells undergo nuclear envelope breakdown and chromatin condensation
(11, 12) and both calcium and CaM are localized to the centrosomal
region of the mitotic apparatus (13-15). The level of the calmodulin
protein is also reported to increase 2-fold when mammalian tsBN2 cells
are induced to undergo premature chromatin condensation upon incubation
at the restrictive temperature (16). In addition, the reduction of
intracellular calmodulin levels using calmodulin antisense RNA (17) or
the use of calmodulin antagonists (9) is reported to block entry of the
cells into mitosis and imply a role for CaM at the G2/M phase checkpoint.
However, the immediate targets of calcium/CaM at the G2/M
phase checkpoint remain largely unknown. Studies in sea urchin eggs and
Xenopus oocytes suggest the target to be a multifunctional calcium/calmodulin-dependent serine/threonine protein
kinase (CaM kinase II). CaM kinase II is a member of a family of
multifunctional calcium/CaM-dependent protein kinases,
which include CaM kinases Ia, Ib, and IV and which have broad substrate
specificity and wide distribution in mammalian tissue (Ref. 18;
reviewed in Refs. 19 and 20). CaM kinases with properties similar to
the mammalian CaM kinase II have been found in a number of species (21-23) including the sea urchin (24). Inhibition of the sea urchin
CaM kinase II using either anti-CaM kinase antibodies or an inhibitory
peptide was found to block nuclear envelope breakdown (24). In
addition, it is reported that microinjection of a cDNA encoding a
calcium/CaM-independent form of CaM kinase II induced maturation in
Xenopus oocytes (25). These observations together with the
finding that mammalian cells contain nuclear isoforms of CaM kinase II
(26) and that the enzyme is localized in the nucleus during interphase
and in the mitotic apparatus of dividing cells (27) suggest that CaM
kinase II may be a positive regulator of the G2/M phase transition.
The targets of the Ca2+/CaM/CaM kinase II second
messenger system must ultimately be the cell cycle control proteins. Of
these, the p34cdc2 protein kinase is generally acknowledged to
be the key mediator of the G2/M phase transition in all
eucaryotic cells (reviewed in Refs. 28 and 29). The active mitotic
kinase (MPF, or mitosis-promoting factor) is a dimer comprising a catalytic subunit,
p34cdc2, and a regulatory subunit, a B-type cyclin (30-32).
Cyclins are a class of proteins that are synthesized during the
interphase of each cell cycle and rapidly degraded at the end of each
mitosis (reviewed in Ref. 33). The activity of the p34cdc2
protein kinase depends not only on its association with cyclin B but
also on its phosphorylation state. Three major sites of phosphorylation
have been identified, corresponding to Thr14,
Tyr15, and Thr161 in the human p34cdc2
protein (34, 35). Phosphorylation of either Thr14 or
Tyr15 inhibits p34cdc2 kinase activity (36, 37),
while phosphorylation of the Thr161 residue is required for
kinase activity (38-40). There is now considerable evidence suggesting
that the dephosphorylation (of Tyr15 and Thr14)
and activation of p34cdc2 at the G2/M phase
checkpoint is initiated by a tyrosine phosphatase, the product of the
fission yeast cdc25 gene or its homologues in other
eucaryotes (41-45). In the species studied so far, the enzymatic
activity of cdc25 has been found to be regulated during the
cell cycle by transcriptional control (46), by translational control
(47), or by a post-translational modification involving phosphorylation
of the Cdc25 protein (48-50). In mammalian cells, the Cdc25-C protein
is known to be phosphorylated at mitosis, and it has been shown that
the p34cdc2-cyclin B protein kinase phosphorylates and
activates the tyrosine phosphatase activity of the Cdc25-C protein
(48). However, this mechanism assumes the presence of some active
p34cdc2-cyclin B protein kinase, the origin of which remains
unknown. Protein kinases other than p34cdc2-cyclin B, such as
Cdk2-cyclin A and Cdk2-cyclin E, can also phosphorylate and activate
the Xenopus Cdc25 protein in vitro (51). However, in Xenopus extracts depleted of both Cdc2 and Cdk2, Cdc25
still undergoes phosphorylation and activation upon the addition of the
protein phosphatase inhibitor microcystin (51). Therefore, an
alternative explanation is that another protein kinase(s)
phosphorylates and activates the enzymatic activity of Cdc25-C. Once
the production of active p34cdc2-cyclin B is initiated, a
positive feedback loop producing more active kinase would ensue, as
Hoffmann and co-workers suggest (48).
In this study, we show that Cdc25-C is phosphorylated during M phase in
mammalian cells and that this phosphorylation can be inhibited by
KN-93, a water-soluble, cell-permeable inhibitor of CaM kinase II.
Treatment of HeLa cells with either KN-93 or AC3-I, a specific peptide
inhibitor of CaM kinase II, inhibits progression of the cells through
the G2/M phase checkpoint. We also show that in
vitro the Cdc25-C protein is a substrate for CaM kinase II and
that the phosphorylated enzyme has a 2.5-3-fold greater tyrosine
phosphatase activity when compared with the nonphosphorylated enzyme.
Cell Culture, Synchronization, and Cell Extracts--
HeLa cells
were cultured in Dulbecco's minimum essential medium (MEM) (Life
Technologies, Inc., Paisley, UK) supplemented with 10% fetal calf
serum (Life Technologies) and antibiotics/antimycotic (penicillin/streptomycin/amphotericin B; Life Technologies). HeLa cells
were synchronized at the G1/S phase boundary using the
thymidine-aphidicolin double block method (52). Mitotic and
G2 phase cells were obtained following 14-18 h of
treatment with nocodazole (50 ng/ml; Sigma) as described previously
(48). HeLa cells blocked at the G1/S phase boundary and S
phase cells (3 h after release from the G1/S phase block;
Ref. 53) were obtained by scraping the cells from the plates with cold
(4 °C) phosphate-buffered saline (PBS) containing 0.4 mM
EDTA. The cells were centrifuged at 1000 rpm for 5 min at 4 °C, and
the cell pellet was lysed in 3 volumes of lysis buffer (50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 0.1% Triton
X-100, 5 mM EDTA, 50 mM NaF, 0.1 mM
Na3VO4, 0.1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml
pepstatin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml
L-1-tosylamido-2-phenylethyl chloromethyl ketone, and 10 µg/ml 1-chloro-3- tosylamido-7-amino-2-heptanone).
Immunoprecipitation and Western Blotting--
For
immunoprecipitation of either the Cdc25-C or the p34cdc2
protein, 200-300 µg of total protein from HeLa cell lysates was
used. The lysates were precleared by incubating with 30 µl of protein A-Sepharose beads (Amersham-Pharmacia Biotech) for 1 h at 4 °C with constant rotation. The lysates were then incubated overnight (15 h) with either a polyclonal antibody to the human Cdc25-C protein
(1:400 dilution) or a polyclonal anti-human Cdc2 antibody (1:200
dilution), at 4 °C. The antibody was recovered by incubating the
lysates with 30 µl of protein A-Sepharose beads for 2 h at 4 °C, and the immunoprecipitates were washed five times in wash buffer (lysis buffer plus 0.05% SDS, 1 mM EGTA, and 100 nM okadaic acid). The washed immunoprecipitates were
dissolved in 60 µl of sample buffer, and the proteins were resolved
using 10% SDS-PAGE. Cdc25-C immunoprecipitates were not boiled before
loading on an SDS gel in order to maintain aggregation of the antibody
chains (48). Western blotting of proteins was performed as described previously (54). For Western blotting, the Cdc25-C antibody was used at
a dilution of 1:200 (1:1000 for the Cdc2 antibody) and detected using 1 µCi of [125I]protein A (Amersham Pharmacia Biotech).
The membranes were subsequently exposed for autoradiography using
Hyperfilm-M (Amersham Pharmacia Biotech) or phosphor image analysis
(Raytek Scientific Ltd., Sheffield, UK). For detection of the Cdc25-C
antibody, using an alkaline phosphatase-linked goat anti-rabbit
secondary antibody (Sigma), the protocols described in Ref. 55 were
used. The tyrosine phosphorylation state of the immunoprecipitated
p34cdc2 was determined by Western blotting with an
anti-phosphotyrosine antibody. The proteins bound to the protein
A-Sepharose beads were resolved using 10% SDS-polyacrylamide gels. A
mouse monoclonal antibody to phosphotyrosine (PY54; Affinity,
Nottingham, UK) was used (1:1000 dilution) for the Western blots as
described previously (54). The level of p34cdc2 in the
immunoprecipitates was determined by Western blotting with the
p34cdc2 antibody (1:1000 dilution).
Expression and Purification of the Recombinant Human Cdc25-C
Protein--
Cdc25-C protein was purified from the inclusion bodies of
a strain of E. coli expressing the full-length protein (56).
Briefly, after isopropyl Protein Kinase and Protein Phosphatase
Assays--
Phosphorylation of Cdc25-C by CaM kinase II was performed
in a final volume of 20 µl containing 50 mM Pipes, pH 7, 10 mM MgCl2, 100 µM EGTA, 200 µM CaCl2, 0.3 µg of calmodulin, 100 µM ATP, and 5 µCi of [ Phosphopeptide and Phosphoamino acid Analysis of
Cdc25-C--
The phosphopeptide and phosphoamino acid analysis of
Cdc25-C following phosphorylation by CaM kinase II was performed
as described previously (57).
Drug Treatment of HeLa Cells--
KN-93 was dissolved as a 10 mM stock in sterile distilled water (KN-92 was dissolved in
Me2SO, 10 mM stock solution) and stored at
Microinjection of HeLa Cells--
HeLa cells were cultured on
sterile 22 × 22-mm coverslips etched with a cross (to aid
relocation of microinjected cells). The cells were blocked and released
from G1/S phase as described above. The peptide was
microinjected into the cells between 4 and 6 h after release from
the G1/S phase arrest. The cells were injected around the
cross essentially as described by Graessman et al. (59). The
AC3-I peptide was dissolved in 0.5× PBS (pH 7.2) and was used at a
needle concentration of 10 µM. To facilitate identification of the microinjected cells, a fluorescent dye, tetramethylrhodamine isothiocyanate-dextran (0.5 mg/ml,
Mr 3000; Molecular Probes, Inc., Eugene, OR) was
incorporated into the peptide solution. The control peptide, AC3-C, was
prepared similarly and used at a needle concentration of 10 mM. Approximately 50-100 cells were injected in five
separate experiments with approximately 0.1-1.0 picoliters of peptide
(1-10% of cell volume as estimated in Ref. 60). Both peptides were
microinjected into the cytoplasm. Using this technique, 85-90% of the
microinjected cells remained viable as assessed by trypan blue
exclusion. After microinjection the cells around the etched cross were
photographed on a Nikon TMS light microscope (× 10 objective) using a
Nikon F-801s camera. Cells were then returned to a 37 °C incubator
for 24 h. After 24 h, the cells were again photographed
around the etched cross and then fixed with 3.7% formaldehyde (in
PBS). Coverslips were mounted in 90% glycerol, 10% PBS, and
tetramethylrhodamine isothiocyanate-dextran-positive cells were counted
under a Zeiss Axiophot epifluorescence microscope.
Reagents--
The protease inhibitors aphidicolin and thymidine
were purchased from Sigma (Poole, UK). All other chemicals were of
"Analar" grade and were purchased from BDH unless indicated otherwise.
CaM Kinase II Phosphorylates and Activates Cdc25-C in
Vitro--
The recombinant human Cdc25-C protein was purified from
bacterial inclusion bodies and renatured. As shown in Fig.
1A (left lane) a major 54-kDa band could be detected on a
Coomassie-stained gel. The 54-kDa protein, when Western blotted,
immunoreacted with a polyclonal antibody to the human Cdc25-C protein
(see Fig. 1A, right lane). To
determine the tyrosine phosphatase activity of the renatured, purified
Cdc25-C protein, we incubated it with the sea urchin
p34cdc2-cyclin B complex (isolated from embryos 40 min
postfertilization) bound to p13suc1-Sepharose beads. We have
shown previously that at 40 min postfertilization the sea urchin
p34cdc2 is phosphorylated on tyrosine and that cyclin B is
present in the complex (54, 61). As shown in Fig. 1, B and
C, Cdc25-C (0.01-1.25 mg/ml) caused a
dose-dependent dephosphorylation of p34cdc2 on
tyrosine, confirming the results of an earlier study (44).
To determine if Cdc25-C was a substrate for CaM kinase II in
vitro, we treated the purified Cdc25-C protein (1 µg) with
varying concentrations of purified, rat brain CaM kinase II (25-200
ng). The samples were subsequently resolved by 10% SDS-PAGE,
electroblotted onto a nylon membrane, and analyzed by autoradiography.
Our results (shown in Fig. 2A,
top) indicate that a 54-kDa protein (Cdc25-C) was strongly
phosphorylated by CaM kinase II in a
calcium/calmodulin-dependent manner to a high stoichiometry
(1.2 mol of phosphate/mol of Cdc25-C). In the absence of Cdc25-C, only
weak phosphorylation was observed, which we presume represents the
known autophosphorylation of the
We performed both phosphopeptide mapping and phosphoamino acid analysis
of Cdc25-C protein after phosphorylation by CaM kinase II (200 ng).
Tryptic phosphopeptide mapping revealed the presence of four
phosphopeptides with negative charge (labeled 1-4, Fig. 2B, left). The minor phosphopeptides present in
the digest were found to be variable and may represent fragments of CaM
kinase II cut out from the polyacrylamide gel together with Cdc25-C. Phosphoamino acid analysis of the four labeled peptides
(numbered 1-4) revealed that Cdc25-C was
phosphorylated on serine residues only (see Fig. 2B,
right).
We also used the recombinant, renatured Cdc25-C protein to determine if
the tyrosine phosphatase activity of Cdc25-C was altered following its
phosphorylation by CaM kinase II. The Cdc25-C protein (0.006-0.024
mg/ml) was phosphorylated with CaM kinase II (200 µg) and then
incubated with its substrate, the inactive, tyrosine-phosphorylated p34cdc2-cyclin B complex, which was isolated from sea urchin
egg extracts using p13suc1-Sepharose beads. We then determined
if there was any increase in the histone H1 kinase activity of the
p34cdc2-cyclin B complex following incubation with the
phosphorylated and nonphosphorylated Cdc25-C protein. As shown in Fig.
2C, we obtained a significant and consistent increase
(approximately 2.5-3-fold) in the histone H1 kinase activity with the
phosphorylated Cdc25-C protein compared with the nonphosphorylated
form. Incubation of the CaMK II-phosphorylated Cdc25-C with vanadate (1 mM) inhibited activation of the p34cdc2-cyclin B
complex as assessed by its histone H1 kinase activity. This result
indicates that in vitro the Cdc25-C protein has enhanced phosphatase activity after phosphorylation by CaM kinase II.
To eliminate the possibility that the phosphorylation of Cdc25-C by CaM
kinase II was fortuitous, possibly the result of incorrect protein
folding during renaturation, we used a soluble form of the human
Cdc25-C protein. We purified the human Cdc25-C glutathione S-transferase fusion protein from bacteria using
glutathione-Sepharose affinity chromatography as described previously
(45). A Western blot of the purified fusion protein with a polyclonal
anti-Cdc25-C antibody (Fig. 2D, left,
lane 1), indicated the presence of two major
polypeptides with molecular masses of 82 and 54 kDa. The 82-kDa band
represents the full-length Cdc25-C fusion protein, while the 54-kDa
band represents a degradation product of Cdc25-C that has also been
described in a previous study (48). To test if the soluble Cdc25-C
fusion protein was a substrate for CaM kinase II, we incubated 2 µg
of the purified protein (a mixture of the 82- and 54-kDa polypeptides)
with 25 ng of CaM kinase II in the presence of
[
We also performed phosphopeptide mapping and phosphoamino acid analysis
of the 82-kDa Cdc25-C fusion protein after phosphorylation by CaM
kinase II (200 ng). The two-dimensional tryptic peptide map of the
Cdc25-C fusion protein indicated that two major acidic peptides
(comparable with peptides 1 and 2 in the 54-kDa protein; see Fig.
2B, inset) were generated, along with two minor
acidic peptides (comparable with peptides 3 and 4 in the 54-kDa
protein; see Fig. 2B and compare with Fig. 2B,
inset). The two minor peptides in the Cdc25-C fusion
protein, however, were not phosphorylated as efficiently as the two
minor peptides (labeled 3 and 4) in the 54-kDa
Cdc25-C protein. Phosphoamino acid analysis of the labeled peptides
indicated that they were phosphorylated exclusively on serine residues
(data not shown). Since both the renatured Cdc25-C protein and the
Cdc25-C fusion protein were both found to be phosphorylated by CaM
kinase II, the remainder of the experiments described in this paper
were performed with the renatured 54-kDa protein.
We also determined whether the Cdc25-C protein could be phosphorylated
by either cAMP-dependent protein kinase or protein kinase
C. Neither of these kinases phosphorylated Cdc25-C (data not shown). In
HeLa cells, the phosphorylation and activation of Cdc25-C is believed
to be mediated by the p34cdc2 kinase present in mitotic cell
extracts (48). We have confirmed and extended this observation and
shown that Cdc25-C is phosphorylated by a pure preparation of starfish
p34cdc2 protein kinase (see Fig. 2D,
right)
KN-93 Blocks the Cell Cycle at the G2/M Phase
Transition--
To investigate a possible role for CaM kinase II in
the regulation of the G2/M phase transition in mammalian
cells, we used KN-93,
(2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]
amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), a specific, water-soluble inhibitor of CaM kinase II (62). We treated a
synchronous population of HeLa cells with KN-93 (10 µM,
added 3 h after removal of the aphidicolin block), and, to determine if the KN-93-treated cells arrest at a specific point in the
cell cycle, we analyzed their cellular DNA content as a function of
time. The result of a representative flow cytometric analysis is shown
in Fig. 3A. Both the control
cells and the KN-93-treated cells had replicated their DNA by 9 h.
By 15 h, there was a reduction in the G2/M population
in the control cells, indicating progression through mitosis and cell
division, whereas in the KN-93-treated cells a gradual accumulation of
cells with a G2/M DNA content was observed.
In a separate experiment, we treated a synchronous population of
cells with either 10 µM KN-92
(2-[N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine),
an inactive analogue of KN-93 (up to 30 µM KN-92 has no
effect on CaM kinase II, cAMP-dependent protein kinase, or
protein kinase C),2 for
comparison with 10 µM KN-93, or with an equivalent volume of Me2SO alone (control cells). Flow cytometric analysis of
the DNA content of the KN-92-treated, KN-93-treated, and control cells (Fig. 3B) indicated that 15 h after the addition of the
drugs, both the control and the KN-92-treated cells had completed one cell division cycle, whereas 59% of the KN-93-treated cells were found
at G2/M with a 4n DNA content. This result provides
additional evidence of the specificity of the KN-93-induced
G2 arrest and demonstrates a role for CaM kinase II in
regulating the G2/M phase transition in HeLa cells.
To determine the tyrosine phosphorylation state of p34cdc2 in
the KN-93-arrested HeLa cells, we immunoprecipitated p34cdc2
and immunoblotted it with either an anti-phosphotyrosine antibody or an
anti-Cdc2 antibody. The results (Fig. 3C) indicate that p34cdc2 was phosphorylated on tyrosine in the KN-93-arrested
cells and at a level comparable with that seen in G2 phase
cells. This result suggests that KN-93 causes G2 phase
arrest by inhibiting activation of the Cdc25-C tyrosine phosphatase.
Since KN-93 was found to arrest HeLa cells specifically at the
G2/M phase transition, we wanted to determine if CaM kinase II was present in this cell line. Its existence in HeLa cells had not
been reported previously. The presence of CaM kinase II in HeLa cells
was determined by assessing the phosphorylation of autocamtide-2 (24),
a specific, synthetic peptide substrate of CaM kinase II. By comparing
the specific activity of the purified enzyme (from rat brain) with that
in HeLa cells, our data (not shown) indicate that CaM kinase II
comprises 0.003% of total cell protein.
KN-93 Inhibits the Phosphorylation of Cdc25-C in HeLa
Cells--
We treated a synchronous population of HeLa cells with 10 µM KN-93 and looked at the phosphorylation of Cdc25-C
using its relative mobility on SDS-polyacrylamide gels as an indication of its phosphorylation state. Using a polyclonal antibody against the
full-length human protein, we immunoprecipitated and then immunoblotted
Cdc25-C from HeLa cell extracts. Fig.
4A (top) shows that
in interphase cells the antibody detected a doublet (54-56 kDa), a
finding consistent with previous reports (41). These bands were not
detected in the control experiment, where duplicate HeLa cell extracts
were immunoprecipitated with a preimmune serum and then immunoblotted
with the Cdc25-C antibody (data not shown). In cells blocked at M phase
for 14 h with nocodazole (50 ng/ml), the Cdc25-C protein is seen
to shift to a higher molecular mass (60 kDa, Fig. 4A,
top). We were unable to detect hyperphosphorylation of
the Cdc25-C protein (as assessed by its mobility on SDS-PAGE) in M
phase cells collected by mitotic shake-off (Fig. 4A,
top). Indeed, hyperphosphorylation has not been reported
under these conditions (48). Since the hyperphosphorylation of Cdc25-C
was only observed in the nocodazole-treated cells, we examined the effect of KN-93 on Cdc25-C phosphorylation in the presence of nocodazole. We added both KN-93 (10 µM) and nocodazole
(50 ng/ml) to a synchronous population of HeLa cells and determined the
state of Cdc25-C phosphorylation 14 h after the addition of the
two inhibitors. As Fig. 4A (top) shows, we did
not observe any change in the mobility of the Cdc25-C protein in cells
treated with both nocodazole and KN-93, while cells treated with
nocodazole alone showed Cdc25-C hyperphosphorylation. This result
indicates that there is a calcium/calmodulin
kinase-dependent step in the phosphorylation of Cdc25-C in
HeLa cells, probably mediated by CaM kinase II or a CaM kinase II-like
enzyme.
We also examined the tyrosine phosphorylation state of p34cdc2
in the KN-93 blocked HeLa cells. Using a polyclonal anti-human cdc2
antibody, we immunoprecipitated p34cdc2 from HeLa cell extracts
prepared at different stages of the cell cycle. One half of the
immunoprecipitate was then immunoblotted with an anti-phosphotyrosine
antibody, and the other half was immunoblotted with the anti-human Cdc2
antibody. Our results show that in the KN-93-arrested cells,
p34cdc2 was present in a tyrosine-phosphorylated state (see
Fig. 4A, center) and that the level of
phosphotyrosine was comparable with that detected in p34cdc2
during the G2 phase of the cell cycle (see Fig. 4,
A, center, and B, left).
Although we have not tested it formally, we presume that the
p34cdc2 kinase also remains inactive in the KN-93-blocked
cells. By way of comparison, in the control cells p34cdc2 was
gradually phosphorylated on tyrosine during interphase (Fig. 4A, center) and dephosphorylated in M phase. The
tyrosine dephosphorylation of p34cdc2 was more apparent in the
nocodazole-arrested cells than in M phase cells collected by mitotic
shake-off (see Fig. 4A, center). The results of
the immunoblot with the p34cdc2 antibody indicated that a
uniform level of p34cdc2 protein was present in the
immunoprecipitates (see Fig. 4, A, bottom, and
B, right).
To confirm the specificity of KN-93 in vivo, we wanted to
eliminate the possibility that the G2/M phase block induced
by KN-93 did not result from a direct inhibition of Cdc25-C phosphatase activity or from inhibition of cyclin B synthesis. To determine if
KN-93 was inhibiting the tyrosine phosphatase activity of Cdc25-C, we
assayed its activity in vitro in the presence and absence of KN-93 (10 µM). The Cdc25-C protein was incubated with its
substrate, the inactive, tyrosine-phosphorylated sea urchin
p34cdc2-cyclin B complex (isolated on p13suc1-Sepharose
beads). Following the incubation, the phosphotyrosine content of
p34cdc2 was determined by immunoblotting with an
anti-phosphotyrosine antibody. The results of this experiment, shown in
Fig. 4C (top and bottom), indicate
that KN-93 had no effect on the tyrosine phosphatase activity of
Cdc25-C (compare lanes 2 and 3).
However, incubation of Cdc25-C in the presence of 1 mM
vanadate, an inhibitor of tyrosine phosphatases (63), completely
inhibited its enzyme activity (lane 4). In the
presence of vanadate, the level of tyrosine phosphorylation of
p34cdc2 was comparable with that of control samples
(lanes 1 and 5), which were not
treated with Cdc25-C. To test the effect of KN-93 on the synthesis of
cyclin B, we used a monoclonal antibody to human cyclin B1 to
immunoprecipitate and then immunoblot cell extracts from KN-93-blocked
cells and from G2 and G1 phase cells. The
results, shown in Fig. 4D (top and
bottom), indicate that the cyclin antibody detected a 54-kDa
protein and that the cyclin B1 protein was present in the
KN-93-arrested cells (lane 3) at a level
comparable with that detected in the G2 phase cells
(lane 1). G1/S cyclin B levels are
shown for comparison (lane 2). This result led us
to conclude that KN-93 does not inhibit cell cycle progression by
inhibiting the synthesis of cyclin B1.
KN-93 Does Not Inhibit Okadaic Acid-induced Phosphorylation of
Cdc25-C in HeLa Cells--
In mammalian BHK1 cells, okadaic acid (OA)
is known to cause transient activation of the p34cdc2 kinase,
causing cells to enter into a premature mitotic state by inducing
nuclear envelope breakdown and chromatin condensation (64). We
determined if KN-93 was able to inhibit the okadaic acid-induced
activation of the p34cdc2 protein kinase in HeLa cells. We
treated a synchronous population of HeLa cells, in early S or
G2 phase, either with OA alone (0.5 µM) or
with OA (0.5 µM) and KN-93 (10 µM).
p34cdc2 was immunoprecipitated from cell extracts and prepared
at intervals after the addition of OA, and its histone H1 kinase
activity and its tyrosine phosphorylation state were measured.
Treatment of S phase or early G2 phase HeLa cells with 0.5 µM OA caused the flattened cells to round up within 30 min of the addition of OA (not shown). Fig.
5 (top) shows that we also
observed an increase in the histone H1 kinase activity of
p34cdc2 at 30 min and at 90 min after the addition of OA when
compared with the control, untreated cells. The increase in the histone H1 kinase activity was associated with the dephosphorylation of p34cdc2 on the tyrosine residue (see Fig. 5, bottom,
lane 2, 30 min and lane 3, 90 min). We also
found that 10 µM KN-93 did not inhibit either the
OA-induced increase in the histone H1 kinase activity of
p34cdc2 (Fig. 5, top) or the OA-induced tyrosine
dephosphorylation of p34cdc2 (see Fig. 5, bottom,
lane 5). These data demonstrate that that KN-93
has no direct effect on histone H1 kinase activity in
vitro.
A Peptide Inhibitor of CaM Kinase II (AC3-I) Arrests HeLa Cells in
G2--
The finding that KN-93 caused a G2/M
phase block in HeLa cells prompted us to test whether a specific
peptide inhibitor of CaM kinase II would also arrest HeLa cells in
G2. HeLa cells were arrested at G1/S phase
using an aphidicolin block. After release from the block (4-6 h later)
the cells were microinjected with AC3-I, a selective peptide inhibitor
of CaM kinase II (65), or a control, inactive peptide (AC3-C) in which
a single alanine residue had been substituted for an arginine. In each
experiment, typically between 50 and 100 cells were microinjected with
either AC3-I or AC3-C to a final cytoplasmic concentration of 1 µM. To aid detection of the microinjected cells, both
peptides were dissolved in PBS containing tetramethylrhodamine
isothiocyanate-dextran as described under "Materials and Methods."
After 24 h, the cells were fixed, and the number of fluorescent
cells was scored. The results (mean ± S.E.) from five independent
experiments are shown in Fig.
6A. The AC3-I peptide
inhibited the proliferation of HeLa cells. These cells remained
arrested in G2 to 24 h after microinjection, their
chromatin remained decondensed as visualized by staining with Hoechst
33258 (data not shown), and they failed to divide. We infer that the
cells are arrested in G2, since they were released into S
phase by the removal of aphidicolin. Trypan blue exclusion indicated
that the majority of the microinjected cells (>95%) remained viable
24 h postinjection. In contrast, the cells microinjected with the
control peptide (AC3-C) continued to replicate, as seen by a 73%
increase in cell number 24 h after microinjection.
We also determined the selectivity of AC3-I for CaM kinase II over
protein kinase C, CaM kinase I, and CaM kinase IV. For CaM kinase II,
the IC50 (using 10 µM autocamtide-2 as
substrate) was found to be approximately 3 µM. For
protein kinase C (using myelin basic protein as substrate) the
IC50 was approximately 500 µM, or 100-fold
less effective. We have found that AC3-I had no effect on the activity
of either CaM kinase IV (at concentrations of up to 100 µM using GS-10 as substrate) or CaM kinase I (at concentrations of up to 200 µM using 50 µM
site 1 peptide as substrate (66)), whereas it inhibited CaM kinase II
activity with an IC50 of 1 µM (using 3 µM autocamtide-3 as substrate). Thus, AC3-I has a
100-fold selectivity for CaM kinase II over either protein kinase C or
CaM kinase IV and at least 200-fold over CaM kinase 1.
The results we have obtained in this study suggest that CaM kinase
II or a CaM kinase II-like activity is required at the G2/M
phase transition in HeLa cells. This result is consistent with previous
studies, which also indicate a requirement for CaM kinase II at mitosis
entry in sea urchin embryos (24) and Xenopus oocytes (25).
The target(s) of CaM kinase II at the G2/M phase transition, however, have not been identified. We show here that one
possible target may be the Cdc25-C protein. A current model for the
activation of the p34cdc2-cyclin B mitotic kinase (Ref. 48 and
Fig. 7) requires the presence of a small
amount of phosphorylated, active Cdc25-C enzyme to trigger the
activation of the p34cdc2-cyclin B protein kinase. Once
activated, the p34cdc2-cyclin B protein kinase engages a
positive feedback loop in which it phosphorylates and further activates
Cdc25-C, which, in turn, increases activation of the
p34cdc2-cyclin B protein kinase (48). It has been shown in HeLa
cells that the Cdc25-C enzyme is phosphorylated and activated by the p34cdc2 protein kinase (45). While we do not disagree with this
result and indeed have reproduced it, our data suggest that CaM kinase II may function as a trigger to initiate the phosphorylation and activation of Cdc25-C.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
MATERIALS AND METHODS
-D-thiogalactopyranoside
induction, the bacterial pellet was resuspended in 10 ml of cold
(4 °C) TEN (50 mM Tris-HCl, pH 8, 0.5 mM
EDTA, 0.3 M NaCl), and the bacteria was lysed by the
addition of 10 ml of lysozyme (5 mg/ml) and incubation for 30 min at
4 °C. 1 ml of 10% Nonidet P-40 was then added, and the incubation
continued for another 10 min. The lysate was diluted with 75 ml of 1.5 M NaCl, 12 mM MgCl2 and incubated
with DNase I (0.2 mg/ml) for 60 min at 4 °C. The lysate was then
sonicated for 30 s at 4 °C and centrifuged at 12,500 × g for 30 min. The pellet was resuspended in 40 ml of cold
(4 °C) TEN, mixed with 1 ml of lysozyme (10 mg/ml), and incubated on
ice for 10 min. 1 ml of 10% Nonidet P-40 was added to the lysate
before centrifugation at 15,000 × g for 15 min. The pellet,
containing mostly inclusion bodies, was resuspended in 10 ml of 6 M urea, 25 mM dithiothreitol, 50 mM
Tris-HCl, pH 8. The suspension was boiled for 5 min at 100 °C,
cooled to 37 °C, and centrifuged for 10 min at 10,000 × g. The supernatant was recovered, warmed to 37 °C, and
diluted into 100 ml of warm (37 °C) 50 mM Tris-HCl, pH
8, 10% glycerol, 5 mM glutathione (reduced), 0.5 mM glutathione (oxidized), 0.4 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin. After overnight
incubation at 37 °C, the solution was centrifuged for 30 min at
14,000 × g. The supernatant was recovered and dialyzed
against 3 × 500 ml of 50 mM Tris-HCl, pH 8, 1 mM dithiothreitol, 10% glycerol. The 82-kDa Cdc25-C fusion
protein was recovered from Escherichia coli and purified
using glutathione-Sepharose chromatography, as described in Ref.
48.
-32P]ATP (3000 Ci/mmol, Amersham Pharmacia Biotech), 2.5 µg of Cdc25-C, and 50-200
ng of CaM kinase II. The reaction was incubated at 30 °C for 5 min
before termination of the reaction by the addition of 20 µl of 2×
sample buffer. Phosphorylation of Cdc25-C by the purified
p34cdc2 protein kinase was performed in a final volume of 25 µl containing 50 mM Tris-HCl, pH 7.4, 10 mM
MgCl2, 80 mM
-glycerophosphate, 6 mM EGTA, 10 µM ATP, 10 µM
[
-32P]ATP, 2.5 µg of Cdc25-C, and 1 µl of Cdc2
kinase. The reaction was incubated at 30 °C for 10 min before being
terminated by the addition of 25 µl of 2× sample buffer.
Phosphorylation of Cdc25-C by cAMP-dependent protein kinase
and protein kinase C was performed as described previously (57). The
tyrosine phosphatase activity of Cdc25-C or Cdc25-C following
phosphorylation by CaM kinase II was assessed using a
p34cdc2-cyclin B complex isolated from sea urchin embryos (40 min after fertilization) using p13suc1-Sepharose beads as
described previously (54). The p13 precipitates were washed three times
with assay buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 2 mM dithiothreitol) before the
addition of Cdc25-C. The tubes were incubated for 20 min at 25 °C,
washed three times with buffer C, and assayed for H1 kinase activity as
described (58). H1 kinase activity was quantitated by excising the
histone H1 bands from the dried gels, dissolving in NCS tissue
solubilizer (Amersham Pharmacia Biotech) and scintillation counting, or
by phosphor image analysis. Alternatively, the p13 beads were washed three times in wash buffer, resuspended in 2 volumes of 2× sample buffer, and used for Western blotting to determine the tyrosine phosphorylation state of p34cdc2. The H1 kinase activity of the
p34cdc2 immunoprecipitates from HeLa cells was assayed as
described by Gu et al. (53).
20 °C. KN-93 (10 µM) and KN-92 (10 µM)
were added to HeLa cells (approximately 50% confluent cultures) 3 h after release from the aphidicolin-thymidine block. Samples were
collected at 2-3-h intervals over an 18-22-h period. After washing
the cells in cold (4 °C) PBS containing 0.4 mM EDTA, the
cells were either lysed in lysis buffer or fixed in 1 ml of cold
(
20 °C) methanol (70%). The fixed cells were washed two times in
PBS and incubated in the dark for 60 min with PBS containing propidium
iodide (Sigma) and RNase I (type 1-AS; Sigma). Analysis of the DNA
content was performed using an EPICS Elite cell sorter (Coulter
Electronics, Luton, UK). Okadaic acid (potassium salt; Affinity) was
dissolved in Me2SO at a concentration of 2 mM.
Okadaic acid (0.5 µM) was added to HeLa cells 3 h
after release from an aphidicolin-thymidine block. The cells were
scraped from the plates and washed in cold (4 °C) PBS containing 0.4 mM EDTA and 0.5 µM okadaic acid. The cell
pellet was lysed in 3 volumes of lysis buffer.
RESULTS
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Fig. 1.
The recombinant Cdc25-C protein causes
tyrosine dephosphorylation of the sea urchin p34cdc2-cyclin B
complex. A, left lane, Coomassie
Blue-stained gel showing the bacterially expressed Cdc25-C protein (54 kDa) after its solubilization from inclusion bodies. Right
lane, immunoblot of the Cdc25-C protein using a polyclonal
antibody to the human Cdc25-C protein. Molecular mass standards are
shown on the right (kDa). B, the inactive,
tyrosine-phosphorylated, sea urchin p34cdc2-cyclin B complex
was isolated using p13suc1-Sepharose beads and incubated with
varying concentrations of the recombinant Cdc25-C protein and then
analyzed by immunoblotting with a monoclonal anti-phosphotyrosine
antibody (PY54). The result of the Western blot with the
anti-phosphotyrosine antibody is shown. C, quantitation of
the data shown in B.
- (51 kDa) and
- (66 kDa)
subunits of CaM kinase II (19). The nylon membrane from the preceding
experiment was then immunoprobed with an anti-cdc25-C antibody and
visualized using a colorimetric detection assay as described under
"Materials and Methods." The result of the Western blot (shown in
Fig. 2A, bottom) indicates that the mobility of
the Cdc25-C protein in the SDS gel was retarded following
phosphorylation by CaM kinase II. A slight upshift of Cdc25-C was seen
(see Fig. 2A, bottom, lane
3) following phosphorylation by 100 ng of CaM kinase II. The
upshift was readily apparent (see Fig. 2A,
bottom, lane 4) when 200 ng of CaM
kinase II was used; the mobility of Cdc25-C was increased by
approximately 9 kDa, from 54 to 63 kDa.
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Fig. 2.
A, phosphorylation of Cdc25-C by CaM
kinase II in vitro. Top, recombinant Cdc25-C protein (1 µg) was incubated with Ca2+ (100 µM),
calmodulin (100 µg/ml), CaM kinase II (25-200 ng), and 30 µM [ -32P]ATP (lanes
1-4), without Ca2+ (lane
5) or without the Cdc25-C protein (lane
6). The samples were electrophoresed on a 10%
polyacrylamide gel and blotted onto nylon membrane. The autoradiogram
of the blotted proteins is shown. Bottom, the blot from
A was then immunoprobed with the Cdc25-C antibody and
developed using an alkaline phosphatase-conjugated goat anti-rabbit
secondary antibody. Molecular mass standards (kDa) are shown shown on
the right. B, phosphopeptide and phosphoamino acid analysis
of Cdc25-C phosphorylated by CaM kinase II in vitro. The
recombinant Cdc25-C protein (1-2 µg) was phosphorylated by CaM
kinase II (100 ng) as described under "Materials and Methods."
Left, two-dimensional phosphopeptide analysis of
phosphorylated, recombinant, renatured Cdc25-C. Phosphopeptides were
electrophoresed (pH 3.5) in the first dimension (horizontal)
followed by chromatography in the second dimension
(vertical). Left inset,
two-dimensional tryptic phosphopeptide map of the Cdc25-C glutathione
S-transferase fusion protein phosphorylated with CaM kinase
II (100 ng). Right, phosphoamino acid analysis of
recombinant, renatured Cdc25-C phosphorylated by CaM kinase II. The
phosphorylated Cdc25-C band was excised from the dried gel, digested
with trypsin, and hydrolyzed with HCl. The products were then resolved
by electrophoresis by on cellulose TLC plates followed by
autoradiography. The mobility of unlabeled phosphoserine,
phosphothreonine, and phosphotyrosine standards is shown. C,
CaM kinase II phosphorylates and activates Cdc25-C. The recombinant
Cdc25-C protein was phosphorylated by CaM kinase II (100 ng) and then
incubated with the sea urchin p34cdc2-cyclin B complex bound to
p13suc1-Sepharose beads, either in the absence or presence of
vanadate (1 mM). The activity of the p34cdc2-cyclin
B complex was assayed using histone H1 as substrate. Each
bar shows the mean ± S.E. of four experiments.
Left, phosphorylation of the Cdc25-C fusion protein by CaM
kinase II. Lane 1, a Western blot of the Cdc25-C
fusion protein using an antibody to the human protein; Lane
2, phosphorylation of the Cdc25-C fusion protein (2 µg) with 25 ng of CaM kinase II; lane 3, phosphorylation of
the Cdc25-C fusion protein (2 µg) with 25 ng of CaM kinase II in the
absence of Ca2+ or CaM. Right, phosphorylation
of the 54-kDa, recombinant Cdc25-C protein by the purified starfish
p34cdc2-cyclin B protein kinase. Lane 1,
Cdc25-C plus p34cdc2 kinase; lane 2, histone H1 plus
p34cdc2 kinase; lane 3, Cdc25-C plus CaM kinase
II.
-32P]ATP, resolved the proteins by 10% SDS-PAGE, and
analyzed protein phosphorylation by autoradiography. Both the 54- and
the 82-kDa polypeptides were phosphorylated to a high level by CaM
kinase II (Fig. 2D, left, lane
2). Phosphorylation of the polypeptides was
Ca2+/CaM-dependent, since no significant
phosphorylation was observed when both were excluded from the
incubation mixture (Fig. 2D, left,
lane 3).
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Fig. 3.
KN-93 arrests HeLa cells with a
G2 DNA content. A, flow cytometry DNA
profiles of control HeLa cells (left) or HeLa cells treated
with 10 µM KN-93 (right). B, flow
cytometry DNA profiles of HeLa cells (18 h after release from the
aphidicoloi/thymidine cell cycle block) treated with Me2SO
alone (left), 10 µM KN-92 (middle),
or 10 µM KN-93 (right). C,
p34cdc2 is phosphorylated on tyrosine in KN-93-arrested HeLa
cells. p34cdc2 was immunoprecipitated from either a synchronous
culture of HeLa cells at various intervals after release from an
aphidicolin/thymidine block or from cells arrested in G2
phase with KN-93 (10 µM). One-half of each sample was
immunoblotted with an anti-phosphotyrosine antibody (top)
and the other half with the p34cdc2 antibody
(bottom). Samples from left to right
are as follows: G1/S phase blocked cells, S phase cells,
G2 phase cells, nocodazole-arrested cells in M phase, and
KN-93 (10 µM)-arrested cells.
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Fig. 4.
KN-93 inhibits phosphorylation of Cdc25-C at
mitosis. A (top), immunoprecipitation and
immunoblot analysis of Cdc25-C from a synchronous culture of HeLa
cells. Samples from left to right are as follow:
cells arrested at the G1/S phase boundary, S phase cells,
G2 phase cells, M phase cells, nocodazole-blocked (50 ng/ml) cells at M phase, and in cells treated with both KN-93- (10 µM) and nocodazole- (50 ng/ml) treated cells.
Center, KN-93 inhibits dephosphorylation of p34cdc2
on tyrosine. p34cdc2 was isolated from a synchronous culture of
HeLa cells using an antibody to the human protein. One half of each
sample was immunoblotted with an anti-phosphotyrosine antibody, and the
other half was immunoblotted with the p34cdc2 antibody.
Bottom, a Western blot of the immunoprecipitated
p34cdc2 protein. B, quantitation of the data shown
in A. C (top), KN-93 does
not inhibit the tyrosine phosphatase activity of Cdc25-C in
vitro. The recombinant Cdc25-C protein (0.12 mg/ml) was incubated
with the inactive, tyrosine-phosphorylated, sea urchin
p34cdc2-cyclin B complex on p13suc1-Sepharose beads
(lane 2) or with Cdc25-C in the presence of 10 µM KN-93 (lane 3), 10 µM vanadate (lane 4), or buffer
alone (lanes 1 and 5).
Bottom, Quantitation of the data shown above. D
(top), immunoprecipitation and immunoblot analysis of cyclin
B from HeLa cells (G2 phase) using a monoclonal antibody to
the human cyclin B1 protein. Lane 1,
G2 phase HeLa cells. Lane 2, HeLa
cells blocked at the G1/S phase boundary. Lane
3, KN-93-arrested HeLa cells. Bottom,
quantitation of the data shown above.
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Fig. 5.
Okadaic acid induces activation of the
p34cdc2 kinase in HeLa cells. Top, the
p34cdc2 kinase was immunoprecipitated, using an
anti-p34cdc2 antibody, from a synchronous population of HeLa
cells following the addition of okadaic acid (0.5 µM).
The histone H1 kinase activity associated with the immunoprecipitated
p34cdc2 was assayed as described under "Materials and
Methods." Each bar represents the mean ± S.E. of
three experiments. Bottom, immunoblot analysis of
p34cdc2, immunoprecipitated from OA-treated HeLa cells, using
an anti-phosphotyrosine antibody. Lane 1, control
cells without OA at 0 min; lane 2, 30 min after
the addition of OA; lane 3, 90 min after the
addition of OA; lane 4, cells at 90 min without
OA; lane 5, cells with OA plus KN-93 at 90 min.
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Fig. 6.
AC3-I, a peptide inhibitor of CaM kinase II
blocks the HeLa cell cycle in G2. A, the
effect of microinjecting the CaM kinase II inhibitory peptide (AC3-I)
or the control peptide (AC3-C) on the HeLa cell cycle. For each
experiment, 50-100 cells were injected 4-6 h after release from the
G1/S phase block with either AC3-I (1 µM
cytoplasmic concentration) or AC3-C (1 µM cytoplasmic
concentration). 24 h after microinjection, the cells were fixed
and processed for immunofluorescence as described under "Materials
and Methods." Each column represents the mean ± S.E.
of five independent experiments. The total number of cells injected
with either AC3-I or AC3-C was 311. 24 h after microinjection, the
number of cells microinjected with AC3-I was 294 and that with AC3-C
was 527. B, light microscopy photographs of HeLa cells
microinjected with either AC3-I (1 µM) or the control
peptide AC3-C (1 µM). The cells were photographed just
after microinjection (0 h), and the same field of cells was
photographed 24 h after microinjection. All the cells shown in
each field were microinjected. Scale bar, 10 µm.
DISCUSSION
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Fig. 7.
A model for the multifunctional CaM
kinase-dependent activation of the p34cdc2-cyclin B
protein kinase (modified from Ref. 45). Phosphorylation of Cdc25-C
by a multifunctional CaM kinase initiates activation of the tyrosine
phosphatase and the formation of a small amount of active
p34cdc2-cyclin B protein kinase. Once formed, the active
p34cdc2-cyclin B protein kinase increases its own production
via a positive feedback loop.
We have studied the regulation of Cdc25-C enzyme activity by CaM kinase II using both in vitro and in vivo methods. Our in vitro results indicate that the 54-kDa Cdc25-C recombinant protein, isolated from the bacterial inclusion bodies, is a substrate for CaM kinase II. A possible criticism regarding the use of the renatured, recombinant Cdc25-C protein is that its phosphorylation by CaM kinase II may result from the exposure of nonspecific phosphorylation sites in an incompletely or incorrectly folded protein. To address this criticism, we have shown that the soluble Cdc25-C fusion protein can also be phosphorylated by CaM kinase II. The similarity of the phosphopeptide maps, of the renatured Cdc25-C protein and the Cdc25-C fusion protein, suggest that CaM kinase II phosphorylates the same sites in both proteins, although our data indicate that CaM kinase II phosphorylates two sites in the renatured protein more efficiently. That Cdc25-C is a substrate for CaM kinase II is also consistent with the finding that the homologue of Cdc25-C in the fungus Aspergillus nidulans, a product of the nimTcdc25 gene, can also be phosphorylated by CaM kinase II in vitro (2). The human Cdc25-C protein contains four CaM kinase II substrate consensus sequences (RXX(S/T); Ref. 67), the potential phosphorylation sites being serine 38, serine 216, serine 449, and serine 451 (56). Our phosphoamino acid analysis data shows that the Cdc25-C protein is specifically phosphorylated on serine residues only and is consistent with the phosphorylations being within the four putative CaM kinase II consensus sequences present within the human Cdc25-C protein. This result provides further evidence for the phosphorylation of CaM kinase II-specific sites in the Cdc25-C protein.
Both the human and the Xenopus Cdc25-C enzymes are known to be phosphorylated at multiple sites (48, 68), although the phosphorylation sites that regulate Cdc25-C activity in vivo have not all been identified, perhaps one reason being the reported low abundance of the Cdc25-C protein in mammalian cells (48). More is known about the kinase(s) that may regulate the activity of the Cdc25-C enzyme in vivo. Protein kinases able to phosphorylate the Xenopus Cdc25-C protein include p34cdc2-cyclin A, p34cdc2-cyclin B, Cdk2-cyclin A, Cdk2-cyclin E (51, 68), and the serine/threonine protein kinase, Plx1 (69), whereas only p34cdc2-cyclin B (and not p34cdc2-cyclin A) is able to phosphorylate the human Cdc25-C enzyme (48). Phosphorylation of both the human and the Xenopus Cdc25-C enzyme enhances its tyrosine phosphatase activity and also retards its mobility on SDS-PAGE, by between 13 kDa (human Cdc25-C; Ref. 48) and 30 kDa (Xenopus Cdc25-C; Ref. 49). A negative regulator of Cdc25 activation has also recently been identified. Chk1, a serine/threonine protein kinase (70, 71), is activated in response to DNA damage and has been shown to phosphorylate and inactivate both the fission yeast and human Cdc25 enzyme. It is unclear why multiple protein kinases phosphorylate the Cdc25 enzyme. One possibility is that phosphorylation by a specific protein kinase which enhances binding of Cdc25 to proteins such as Pin1 (72) and 14-3-3 (73) may serve to regulate its activity by altering its subcellular localization (72).
Both p34cdc2 and Plx1 can phosphorylate and activate the Xenopus Cdc25 enzyme (69). However, it is clear from phosphopeptide mapping data (69) that other, unidentified protein kinase(s) are also able to phosphorylate the Cdc25 enzyme. In this study, we have demonstrated that the phosphorylation of Cdc25-C by CaM kinase II results in a 2.5-3-fold greater tyrosine phosphatase activity when compared with the nonphosphorylated enzyme. The relatively modest increase in the activity of the CaM kinase II-phosphorylated Cdc25-C in our assay may be a reflection of the fact that we have used a substrate bound to p13suc1-Sepharose beads instead of soluble p34cdc2 (where a 10-fold increase in the activity of the phosphorylated Cdc25-C enzyme has been reported (49)) or an artificial substrate such as p-nitrophenyl phosphate (where a 4-5-fold increase in the activity of the phosphorylated cdc25-C enzyme has been reported (48)). Our data are consistent with a previous study that also reported a 3-4-fold activation of the phosphorylated Cdc25-C enzyme when assayed using immobilized p34cdc2-cyclin B as substrate (68) and are consistent with the reported modest increase in HeLa cells between interphase and mitosis (84). We have also shown that the phosphorylation of Cdc25-C by CaM kinase II, as by p34cdc2 (48, 68), retards the electrophoretic mobility of the protein on SDS-polyacrylamide gels.
Similar phosphorylation and activation of the Xenopus Cdc25 protein by CaM kinase II in vitro has also been reported (51). Izumi and Maller (51) concluded that Cdc25 was unlikely to be a physiological substrate for CaMK II, since high concentrations of the calcium chelator EGTA, present in the Xenopus extracts, would apparently inhibit any Ca2+/CaM-dependent enzymatic activity. However, we note that in Xenopus extracts prepared in the presence of high concentrations of EGTA, a calcium-dependent activation and inactivation of the p34cdc2-cyclin B protein kinase can be demonstrated (74). The existence of local transient increases in Xenopus egg extracts (74) is consistent with the finding that p34cdc2 insensitive phosphorylation site mutants of the Xenopus Cdc25 protein show small but detectable shifts in electrophoretic mobility in both mitotic and microcystin-treated extracts (51). Our data are therefore in accord with the observation that another kinase (in addition, that is, to the p34cdc2-cyclin B, p34cdc2-cyclin A, Cdk2-cyclin A, and Cdk2-cyclin E protein kinases) is able to phosphorylate and activate the Cdc25-C tyrosine phosphatase (51, 68).
Our in vivo experiments have been aimed at determining the physiological role of CaM kinase II in the activation of the human Cdc25-C enzyme. Using a functional assay of CaM kinase II activity (the phosphorylation of a synthetic peptide substrate, autocamtide-2) we have demonstrated the presence of this enzyme in HeLa cells. Autocamtide-2 is a specific substrate for CaM kinase II, and, unlike syntide-2, the autocamtide series of peptide substrates is not phosphorylated by either cAMP-dependent protein kinase or protein kinase C (75). There is a possibility that other multifunctional CaM kinases (types I and IV), which may be present in HeLa cells, also contribute toward the phosphorylation of autocamtide-2. However, this is unlikely, since CaM kinase IV phosphorylates autocamtide-2 poorly (76), while CaM kinase I has a substrate specificity that is similar to cAMP-dependent protein kinase rather than CaM kinase II (77, 78). These observations suggest that, in HeLa cells, it is CaM kinase II that is primarily responsible for the phosphorylation of autocamtide-2.
To inhibit the HeLa cell CaM kinase II, we used KN-93, a specific, water-soluble inhibitor of CaM kinase II that is not prone to nonspecific, hydrophobic interactions (IC50 0.37 µM; Ref. 62). The addition of KN-93 to a synchronous culture of HeLa cells caused the cells to arrest with a G2 content of DNA, while cells treated with KN-92, an inactive analogue of KN-93 lacking only the hydroxyethyl group, did not affect cell division in HeLa cells. This result is consistent with previous reports of a KN-93-induced G2 phase arrest in HeLa cells (79). Our data show that in the KN-93-arrested cells the Cdc25-C enzyme did not become hyperphosphorylated and p34cdc2 remained in an inactive, tyrosine-phosphorylated form. However, we note the possibility that the KN-93-induced G2 phase arrest may result from the inhibition of other unidentified CaM kinase II-regulated proteins involved in regulating entry into mitosis or, indeed, other unidentified kinases that may be affected by KN-93, a possibility we discuss below.
At first glance, these results, which involve the inhibition of CaM kinase II, seem to contradict the finding that expression of a constitutive form of CaM kinase II also leads to cell cycle arrest at the G2/M phase checkpoint (80). The constitutive expression of CaM kinase II, however, causes cells to arrest at the G2/M phase checkpoint with a high level of p34cdc2-associated histone H1 kinase activity and, therefore, is not inconsistent with our results, which suggest a role for CaM kinase II in the activation of the Cdc25-C tyrosine phosphatase and subsequent activation of the p34cdc2 protein kinase.
Previous studies using calmodulin inhibitors have also reported the inhibition of cell cycle progression at G2 (9), although because of the hydrophobic nature of the drugs used, other CaM-dependent enzymes, as well as CaM kinase II, were inhibited (81). KN-93 is a water-soluble inhibitor of CaM kinase II, and at the concentrations used in this study it is reported to inhibit cAMP-dependent protein kinase, protein kinase C, myosin light chain kinase, and a calcium-dependent phosphodiesterase by less than 10% (62). KN-93 is known to inhibit both CaM kinase I and IV (IC50 values of 2.7 and 50 µM, respectively).2 Therefore, it is possible that the KN-93-induced G2 phase arrest of HeLa cells may also result from the inhibition of CaM kinase I activity. To exclude a role for either CaM kinase I or IV at the G2/M phase transition, we microinjected AC3-I, a specific peptide inhibitor of CaM kinase II (65), into a synchronized population of HeLa cells. Upon microinjection in G2 phase cells, AC3-I, but not AC3-C, blocked entry into M phase. This result demonstrates that the AC3-I-induced cell cycle cycle arrest is most likely to be mediated by the inhibition of CaM kinase II, although our microinjection study does not allow us to monitor the phosphorylation state of Cdc25-C in these cells. The inference is that Cdc25-C is not hyperphosphorylated and hence not activated in the AC3-I-arrested cells. Since our peptide inhibitor data complement the KN-93 data, it seems clear that a multifunctional Ca2+/CaM-dependent protein kinase, almost certainly CaM kinase II, is required for Cdc25-C activation.
We have additionally demonstrated the specificity of the KN-93-induced G2/M phase block by showing that it does not inhibit either the synthesis of cyclin B, the regulatory subunit of the p34cdc2 protein kinase, or the tyrosine phosphatase activity of the Cdc25-C enzyme. The inability of KN-93 to inhibit the OA-induced activation of p34cdc2 indicates that OA can bypass the calcium-dependent regulatory step for mitosis entry in mammalian cells as it does in early sea urchin embryos, where OA-induced mitosis is insensitive to calcium chelators and a peptide inhibitor of CaM kinase II (82). OA is a phosphatase inhibitor. The identity of the phosphatase whose inhibition bypasses upstream controls and leads to mitosis entry is not known. It may inhibit the protein phosphatase 2A-like INH phosphatase activity (83). Inhibition of Cdc25-C-directed phosphatase activity may lead to activation of Cdc25 and thus p34cdc2 protein kinase activity via the positive feedback mechanism postulated by Hoffmann and co-workers (48), eliminating the need for CaM kinase-dependent phosphorylation of Cdc25. OA treatment might also lead directly to enhanced activating phosphorylation of p34cdc2 itself.
We propose a hypothetical model (see Fig. 7) for the activation of the
p34cdc2-cyclin B protein kinase at mitosis entry that is
consistent with our data in this study. The first step involves
activation of a multifunctional
Ca2+/CaM-dependent protein kinase (CaM kinase
II) as a result of an increase in Cai, which initiates the
phosphorylation and activation of Cdc25-C. In the second step, the
phosphorylated Cdc25-C enzyme triggers activation of the
p34cdc2 protein kinase, which, in accordance with the published
data (48, 68), phosphorylates and activates additional Cdc25-C enzyme
in an autoamplification loop. An autocatalytic model of this sort
predicts that CaMK II-dependent Cdc25-C phosphorylation will be difficult to test by measurement of Cdc25-C phosphorylation in vivo, since the major part of Cdc25-C phosphorylation
will be due to p34cdc2. We note that recent studies have used
cell lines overexpressing Cdc25-C (presumably because Cdc25-C is a low
abundance protein) for phosphopeptide map analysis of Cdc25-C (73),
although it is unclear whether all of the sites phosphorylated on the
overexpressed protein are the same as those phosphorylated on the
native protein. Our data, therefore, indicate that CaMK II may be the
enzyme that triggers the phosphorylation and activation of Cdc25-c at
mitosis entry in mammalian cells.
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ACKNOWLEDGEMENTS |
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We thank Marcel Doree for the purified starfish p34cdc2-cyclin B protein kinase, Paul Russell for the Cdc25-C antibody and the Cdc25-C-producing E. coli, Arnold Pizzey and Michael Aitchison for help with flow cytometry, Chris Norbury for the human p34cdc2 antibody, Jacqueline Hayles for the p13-producing E. coli, Ingrid Hoffmann for the Cdc25-C fusion protein clone, and Nora Sotelo-Kury for the AC3-I inhibitor studies.
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FOOTNOTES |
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* This work was supported by a Wellcome Trust Program grant (to M. J. W.) and by a Wellcome Trust fellowship (to R. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Physiology, University College London,
Gower St., London, United Kingdom WC1E 6BT.
To whom correspondence should be addressed.
2 R. Patel, M. Holt, R. Philipova, S. Moss, H. Schulman, H. Hidaka, and M. Whitaker, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: CaM, calmodulin; CaM kinase II, calcium/calmodulin-dependent protein kinase II; PAGE, polyacrylamide gel electrophoresis; OA, okadaic acid; PBS, phosphate-buffered saline; Pipes, 1,4-piperazinediethanesulfonic acid.
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REFERENCES |
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