From the Cancer Biology Program, Centre for
Immunology and Cancer Research, and the § Institute for
Molecular Bioscience, University of Queensland,
Queensland 4102, Australia
Received for publication, January 30, 2003
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ABSTRACT |
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Activation of cyclin B-Cdc2 is an absolute
requirement for entry into mitosis, but other protein kinase pathways
that also have mitotic functions are activated during
G2/M progression. The MAPK cascade has well
established roles in entry and exit from mitosis in
Xenopus, but relatively little is known about the
regulation and function of this pathway in mammalian mitosis. Here we
report a detailed analysis of the activity of all components of the
Ras/Raf/MEK/ERK pathway in HeLa cells during normal G2/M. The focus of this pathway is the dramatic activation of an
endomembrane-associated MEK1 without the corresponding activation of
the MEK substrate ERK. This is because of the uncoupling of MEK1
activation from ERK activation. The mechanism of this uncoupling
involves the cyclin B-Cdc2-dependent proteolytic cleavage
of the N-terminal ERK-binding domain of MEK1 and the phosphorylation of
Thr286. These results demonstrate that cyclin B-Cdc2
activity regulates signaling through the MAPK pathway in mitosis.
Growth factors signal cells to proliferate via a complex mechanism
that links receptor activation to the cell cycle machinery. Receptor
activation induces cells to progress from quiescence (G0)
into G1 phase. Constant receptor stimulation is required until the restriction point is reached in mid-to-late G1.
Once the restriction point is passed, cell cycle progression is
regulated autonomously by the cell cycle machinery (1). The primary
components of this cell cycle machinery are a family of
serine/threonine kinases, the cyclin-dependent kinases.
Cyclin-dependent kinases are regulated by association with
regulatory cyclin subunits and by phosphorylation (2). Passage through
the eukaryotic cell cycle requires successive activation of different
complexes between cyclin and cyclin-dependent kinase
complexes. Progression from G2 to M phase is controlled by
cyclin B-Cdc2.
The MAPK1 cascade, controlled
by the small GTPase Ras, is critical for progression from
G1 to S phase (3). Growth factor receptors recruit Ras
guanine nucleotide exchange factors to the plasma membrane to activate
Ras, which then recruits and activates Raf kinases. In turn, activated
Raf phosphorylates and activates the dual-specificity MAPK kinases
(MEK1 and MEK2) that phosphorylate and activate the MAPKs ERK1 and
ERK2. Activated ERK phosphorylates a wide range of effector proteins,
including transcription factors that increase expression of proteins
required for cell cycle progression into S phase (3). The MAPK pathway
has a clearly defined role in G1 phase progression, but may
also have a role in the G2/M phase of the cell cycle. In
cycling Xenopus egg extracts, addition of activated MEK
during interphase blocks cyclin B-Cdc2 activation by maintaining Wee1
(4, 5). ERK activity is dispensable for normal mitotic progression in
Xenopus, although ERK activity is required for the proper
functioning of the mitotic spindle (6).
The role of the MAPK pathway in mammalian mitosis is less clear. The
biochemical activities of endogenous ERK1 and ERK2 have been shown to
decrease as cells enter mitosis (7, 8), yet other studies conclude that
activation of the MEK/ERK cascade is required for normal progression
into mitosis (9, 10). Components of the MAPK pathway have been shown to
associate with the mitotic apparatus of mammalian cells. Activated MEK
and ERK localize to the mitotic spindle from prophase to anaphase and to the midbody during cytokinesis (11). Activated ERK also colocalizes with the kinetochore motor protein CENP-E, raising the possibility that
CENP-E is a downstream effector for ERK during mitosis (12). Blocking
MEK activity in cycling somatic cells does not significantly affect
mitotic entry, but it does slow progression through mitosis, probably
by slowing the CENP-E-dependent chromosome movement
coordinated by the mitotic spindle (13).
A number of groups have investigated the regulation of Raf-1 in mitosis
using ectopic expression of Raf-1 and nocodazole treatment to produce a
mitotic checkpoint-arrested cell population. However, nocodazole
treatment itself causes a rapid activation of Raf-1 independent of
mitosis (10), probably due to disruption of the microtubule network
(14). Thus, little is actually known about the activity of the
endogenous Ras/Raf/MEK/ERK pathway in normal G2/M progression.
In this study, we examined the expression and activation of all
components of the Ras/Raf/MEK/ERK pathway during G2/M
progression. To avoid the use of microtubule-disrupting drugs like
nocodazole that directly influence Raf activity independent of the cell
cycle, HeLa cells were synchronized at G1/S and allowed to
progress through mitosis in the absence of drugs. Endogenous proteins
of the MAPK cascade were then analyzed biochemically. This approach
excludes potentially confounding results arising from nocodazole
treatment and ectopic protein expression. We show that membrane-bound
MEK1 is strongly activated as cells transit into mitosis, but that this
activation is uncoupled from ERK, which is inactive in mitosis. The
uncoupling of MEK activation from ERK is mediated by direct modifications to MEK1 by the mitotic cell cycle machinery and requires
active cyclin B-Cdc2.
Reagents and Plasmids--
Roscovitine was purchased from
BIOMOL Research Labs Inc. Compound 5 was a kind gift from Professor
John Lazo (University of Pittsburgh). 4,6-Diamidino-2-phenylindole
(dihydrochloride, hydrate) was purchased from Sigma. Rabbit anti-N-Ras,
anti-K-Ras, anti-H-Ras, anti-B-Raf, anti-MEK1C, anti-MEK1N, anti-MEK2C,
anti-MEK2N, anti-ERK1, and anti-ERK2 polyclonal antibodies were
purchased from Santa Cruz Biotechnology Inc. Anti-phospho-MEK,
anti-MEK1/2, and anti-phosphothreonine/proline polyclonal antibodies
and anti-HA, anti-Raf-1, and anti-phospho-ERK antibodies were purchased
from Cell Signaling Technology. Rabbit anti-cyclin B1 and anti-Cdc2
polyclonal antibodies were as previously described (15). Mouse
anti- Cell Lines and Culture Conditions--
HeLa and 293H
(Invitrogen) cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% bovine donor serum (Serum Supreme, BioWhittaker,
Inc.). Assays for Mycoplasma were carried out monthly to
ensure that the cultured cells were free of contamination. HeLa cells
were synchronized using a double thymidine block release protocol as
described (17). Mitotic shake-off was used to obtain a highly enriched
mitotic cell population from double thymidine-synchronized cells
passing through mitosis. 293H cells were transfected using
LipofectAMINE 2000 (Invitrogen) as directed by the manufacturer. HeLa
cells were transfected by electroporation. Cell cycle status of
cultures was assessed by flow cytometry as described previously
(17).
Cell Fractionation and Immunoblotting--
Floating and attached
cells were harvested in ice-cold PBS and pelleted by low speed
centrifugation (1000 rpm) at 4 °C. PBS was removed, and cell pellets
were resuspended in 0.5 ml of buffer A (10 mM Tris-HCl (pH
7.5), 25 mM NaF, 5 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, and 100 µM NaVO4) and homogenized by passage through
a 23-gauge needle. The post-nuclear supernatants were spun at
100,000 × g; the supernatants (S100) were removed; and the sedimented fractions (P100) were rinsed and sonicated for 5 min in
100 µl of ice-cold buffer A. Protein content was measured by the
Bradford reaction. P100 and S100 aliquots were snap-frozen and
stored at Two-dimensional Gel Electrophoresis--
Membrane-bound MEK1 was
solubilized for two-dimensional analysis by addition of 100 µl of
0.5% SDS in PBS to a 1% Nonidet P-40 membrane pellet (from a 10-cm
dish of mitotic HeLa cells) and incubation at 37 °C for 10 min with
vigorous shaking and then addition of 150 µl of 5% Nonidet P-40 and
sonication for 10 min at 4 °C in a sonicating water bath. The
solubilized protein was acetone-precipitated and resuspended in 250 µl of rehydration buffer (5 M urea, 2 M
thiourea, 2% CHAPS, 2% SB310, 4 mM Tris-Cl (pH 9.5), and
bromphenol blue to color) with freshly added dithiothreitol and pH
3-10 carrier ampholytes (two-dimensional) to final concentrations of
50 mM and 1%, respectively. The first dimension was
resolved on a pH 3-10 linear ImmobilineTM DryStrip
(Amersham Biosciences) for 100 kV-h after 18 h of rehydration. The
second dimension was resolved on 12% SDS-polyacrylamide gel. Western
transfer and immunoblotting were performed as described above.
Kinase Assays--
Raf kinase assays were performed essentially
as described (19). Briefly, equivalent P100 aliquots (20 µg of total
protein) were incubated in kinase buffer (buffer A containing 0.5 mM ATP and 5 mM MgCl2) at 30 °C
for 30 min with recombinant MEK and ERK (tube A; to measure total Raf,
MEK, ERK, and nonspecific kinase activities), ERK alone (tube B; to
measure MEK, ERK, and nonspecific kinase activities), or buffer alone
(tube C; to measure ERK and nonspecific kinase activities). Primary
reactions were diluted in buffer A, and an aliquot was incubated in a
second reaction containing [
To measure cytosolic Raf activity, volumes of the S100 fraction
proportional to the P100 aliquots assayed above were diluted into 1 ml
(final volume) of buffer B (50 mM Tris-HCl (pH 7.5), 75 mM NaCl, 5 mM MgCl2, 25 mM NaF, 5 mM EGTA, 100 µM
NaVO4, 1% Nonidet P-40, 1 mM dithiothreitol, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 5 µg/ml pepstatin A).
Samples were rotated for 3 h at 4 °C either with 2 µg of
anti-Raf-1 antibody plus 10 µl of protein G beads or with 2 µg of
anti-B-Raf antibody plus 10 µl of protein A beads. The beads were
washed three times with buffer B and three times with buffer A and then
resuspended in kinase buffer for assaying. Kinase assays were performed
on the washed beads using the coupled MEK/ERK assay as described above. To ensure comparable results, P100 and S100 fractions were assayed at
the same time using the same reaction buffers and then quantified simultaneously by phosphorimaging. As the P100 and S100 fractions analyzed were equivalent, total measurable Raf kinase activity was
determined by adding total P100, S100 Raf-1, and S100 B-Raf kinase
activities together. This value is referred to as total Raf kinase
activity. Cyclin B1 kinase assays were performed as described (17).
Immunofluorescent Staining--
For immunostaining, cells were
grown on poly-L-lysine-coated coverslips. Coverslips were
washed with PBS, and then the cells fixed with 3% paraformaldehyde or
ice-cold methanol and stored at Analysis of the Ras/Raf/MEK/ERK
Pathway during the Cell Cycle--
To examine the expression and
activity of the components of the Ras/Raf/MEK/ERK pathway during cell
cycle progression, we synchronized HeLa cell cultures with a double
thymidine block. Using this protocol, >80% of the cells progressed
synchronously through the S, G2, and M phases and into the
subsequent G1 phase. By 6 h after release from the
thymidine block, >85% of the cells progressed to G2 phase
and, after 9 h, entered mitosis (Fig.
1A). Entry and exit from
mitosis were determined by measuring cyclin B1-Cdc2 kinase activity.
Cyclin B1-Cdc2 activity increased at 8 h after release from the
thymidine block, peaked at 9-10 h, and returned to basal levels by
12 h (Fig. 1B).
These synchronized cells were used for a detailed biochemical analysis
of the expression levels and activities of the endogenous components of
the Ras/Raf/MEK/ERK pathway throughout the cell cycle. In HeLa cells,
K-Ras and N-Ras were strongly expressed at constant levels throughout
the cell cycle (Fig. 1G). H-Ras was also expressed, but at
very low levels (data not shown). Ras-GTP loading was measured in a
sensitive GST-Raf-RBD binding assay that permits GTP loading of each
Ras isoform to be assessed simultaneously (18). N-Ras displayed two
peaks of GTP loading. The first, smaller peak occurred 12 h after
release from the thymidine block and correlated with exit from mitosis.
The major peak of N-Ras-GTP loading occurred in late G1
phase (18 h), consistent with a role in G1/S transition
(Fig. 1, C and G). In contrast, K-Ras-GTP levels remained uniformly low throughout the cell cycle, and H-Ras-GTP loading
was undetectable (data not shown).
Membrane-associated and cytosolic pools of Raf, MEK, and ERK have
different specific activities (19) and possibly access to different
substrates. We therefore measured the activities of Raf, MEK, and ERK
in membrane (P100) and cytosolic (S100) fractions. The activities of
B-Raf and Raf-1 were measured in a coupled MEK/ERK kinase assay (19).
Total P100-associated Raf kinase activity and S100-associated B-Raf and
Raf-1 kinase activities were assayed. The P100 Raf kinase activity
profile had two peaks (Fig. 1, D and H). The
first peak occurred 8-9 h after release from the thymidine block in
late G2/early mitosis. Interestingly, this early peak of
Raf kinase activity did not correlate with the small peak of Ras-GTP
loading that occurred in late mitosis (Fig. 1, C and
D). The second peak of P100 Raf kinase activity occurred in
G1 phase (14-18 h) and correlated with the major peak of
N-Ras-GTP loading (Fig. 1, C and D). Cytosolic
B-Raf activity essentially mirrored P100-associated Raf activity,
whereas cytosolic Raf-1 was not activated during mitosis, but was
activated in G1 (Fig. 1H). The levels of P100
Raf-1 and B-Raf proteins were constant throughout S and G2
(up to 8 h), fell during mitosis (at 9-12 h), and then increased
again during G1 (at 15-18 h). The levels of cytosolic Raf-1 and B-Raf were constant (Fig. 1H).
MEK activation was assayed with phospho-specific antisera that
recognize activated MEK1 and MEK2 (pMEK). Two peaks of pMEK were
observed. The first peak occurred in late G2/early mitosis and was entirely confined to the P100 membrane fraction (Fig. 1,
E and I). The second, smaller peak occurred in
G1 phase (14-18 h), but was entirely confined to the S100
fraction. Both peaks of MEK activation corresponded with peaks of Raf
activity (Fig. 1, D and E). The levels of
membrane-associated MEK1 and, to a lesser extent, MEK2 decreased during
mitosis (10-12 h), but cytosolic MEK levels remained constant (Fig.
1I). The activation of ERK was assayed in the same fractions
using activation- and phospho-specific antisera (pERK). The
level of pERK dramatically decreased in both P100 and S100 fractions
during mitosis, but rapidly recovered during G1 progression
(Fig. 1, F and J). The levels of ERK1 and ERK2
did not change throughout the cell cycle (Fig. 1J).
Immunofluorescent staining for pMEK revealed strong staining of mitotic
cells (Fig. 2). This mitotic pMEK
staining pattern was observed in both asynchronously growing and
synchronized cultures, thus was not an artifact of the synchrony
protocol. The staining was dispersed throughout the cells, although
weak accumulation at the spindle poles was observed in some cells (data
not shown), as reported previously (11). Interphase cells had low
levels of phospho-MEK staining. Therefore, these results support the results from pMEK immunoblot analysis (Fig. 1, E and
I).
Cyclin B-Cdc2 Activity Is Required to Uncouple Mitotic MEK and ERK
Activation--
The very high level of membrane-bound pMEK in mitotic
cells surprisingly correlated with a loss of active ERK, whereas by contrast, ERK was strongly activated in G1 in response to
the more modest peak of MEK activity (Fig. 1, E and
F). This observation strongly suggested that the coupling
between MEK and ERK activation was lost in mitosis. To establish
whether this loss of coupling was a direct consequence of entry into
mitosis, synchronized HeLa cells were arrested in late G2
phase by treatment with Compound 5, an inhibitor of the Cdc2 activator
Cdc25 (20), or with roscovitine, a direct inhibitor of Cdc2 (21).
Membrane fractions of these G2-arrested cells were compared
with untreated mitotic shake-off HeLa populations by immunoblotting for
pMEK and pERK using ERK2 as a protein input control. Both Cdc2
inhibitor-treated samples had reduced pMEK levels relative to normal
mitotic cells, but pERK levels in the drug-treated cells were
substantially higher than those in normal mitotic cells, which had
barely detectable levels of pERK (Fig.
3A). This result suggests that
a mitotic factor either blocks access of activated MEK to ERK or
directly modifies activated MEK so that it is unable to activate ERK.
To differentiate between these possibilities, the activity of
membrane-associated MEK was assayed in an in vitro coupled
ERK1 kinase assay. Membrane-associated pMEK from roscovitine-treated
G2-arrested cells had a 10-fold higher specific activity
than pMEK from control mitotic cell populations (Fig. 3B).
These results strongly suggest that activated MEK is directly modified
as cells enter mitosis to inhibit its ability to activate ERK.
MEK1 (but Not MEK2) Is Cleaved and Activated during
Mitosis--
Earlier studies that examined MEK activity in mitosis
(10, 22) did not detect the dramatic MEK activation shown in Fig. 1I, but these studies analyzed only detergent lysates of
mitotic cells. We therefore tested the detergent solubility of
P100-associated pMEK. P100 fractions prepared from mitotic HeLa cells
were solubilized in 1% Nonidet P-40, and the soluble and insoluble
fractions were immunoblotted for pMEK, MEK1, and MEK2. pMEK was
insoluble in 1% Nonidet P-40, whereas MEK1 was highly soluble, and
MEK2 had low solubility (Fig.
4A).
To determine which MEK isoform was activated during mitosis, the
Nonidet P-40-insoluble P100 fraction of mitotic HeLa cells was
electrophoresed on high resolving SDS-polyacrylamide gel and then
immunoblotted for pMEK with antibodies raised against the N and C
termini of MEK1 (anti-MEK1N and anti-MEK1C antibodies) and MEK2
(anti-MEK2N and anti-MEK2C antibodies). The phosphorylated form of MEK
migrated with an apparent molecular mass of ~37 kDa. Anti-MEK1C
antibody detected three bands, a 44-kDa band corresponding to
unmodified MEK1 and cytosolic pMEK and two more rapidly migrating bands, the most abundant of which comigrated with the 37-kDa band detected by anti-pMEK antibody (Fig. 4B). Anti-MEK1N,
anti-MEK2N, and anti-MEK2C antibodies did not detect a 37-kDa band. To
confirm that the 37-kDa band detected by anti-pMEK antibody was indeed MEK1, the mitotic P100 fraction was resolved by two-dimensional isoelectric focusing/SDS-PAGE and then immunoblotted for pMEK and for
MEK1 and MEK2 with an antibody that detects both MEK isoforms (anti-MEK1/2 antibody). pMEK exactly comigrated with the minor species
detected by anti-MEK1/2 antibody, confirming the identity of the
species as a truncated form of pMEK1 (Fig. 4C).
MEK1 Cleavage Is Dependent on Cyclin B-Cdc2 Activity--
To
determine the timing of MEK1 cleavage, the Nonidet P-40-insoluble P100
fraction from the experiment shown in Fig. 1I was run on
high resolving SDS-polyacrylamide gel and immunoblotted with anti-MEK1C
antibody. Full-length 44-kDa MEK1 was the major species in S and
G2, but 37-kDa MEK1 increased in late G2 to
become the major form in mitosis (Fig.
5A). As cells exited mitosis
and entered G1, the shorter form was lost, and the
full-length 44-kDa form of MEK1 reappeared. This suggested that MEK1
was cleaved during mitosis and that the cleaved protein was stable only
during mitosis. To confirm that MEK1 cleavage was a mitosis-specific event, synchronized HeLa cells were allowed to progress into mitosis or
were arrested in G2 by addition of the Cdc2 inhibitor
roscovitine. P100 fractions from these cells were then immunoblotted
with anti-pMEK, anti-MEK1C, and anti-MEK1N antibodies. Blocking cyclin
B-Cdc2 activity and entry into mitosis reduced MEK activation and MEK1 cleavage (Fig. 5B), confirming that MEK1 cleavage is a
mitosis-specific event that is dependent on cyclin B-Cdc2 kinase
activity.
Mitosis-specific Cleavage of the N-terminal ERK-binding Domain of
MEK1--
The detection of mitotic pMEK1 with only anti-MEK1C and
anti-MEK1/2 antibodies suggested that the faster migrating species was
the result of proteolytic cleavage of the N-terminal region of MEK1.
The apparent size difference between full-length 44-kDa MEK1 and
mitotic pMEK1 was 5-7 kDa, indicating that the cleavage occurred in
the region of residues 40-60. This was investigated using a MEK1
internal deletion mutant with residues 32-51 removed (MEK1
The insensitivity of the MEK1
The N terminus of MEK1 contains the ERK-binding domain (23). The
removal of this region could explain the uncoupling of MEK1 activation
from ERK activation in mitosis. To test this hypothesis, the level of
endogenous ERK activation was examined in the P100 and S100 fractions
from mitotic cultures overexpressing a constitutively activated form of
MEK1 (HA-MEK1(S218D/S222D)) or GFP-MEK1 Phosphorylation of MEK1 Thr286 Uncouples MEK1
Activity from ERK Activation--
The preceding experiments suggested
that cleavage of the N-terminal ERK-binding domain was not sufficient
to fully uncouple mitotic P100 MEK1 activation from ERK phosphorylation
in vivo, except in mitotic cells. MEK1 contains consensus
sequences for phosphorylation by cyclin B-Cdc2 at Thr286
and Thr292, and both of these residues are phosphorylated
by cyclin B-Cdc2 in vitro, resulting in down-regulation of
MEK1 activity (24). However, phosphorylation of Thr292 does
not appear to affect MEK1 activity (16); therefore, we examined the
phosphorylation of Thr286 in mitosis and its role in
regulating mitotic MEK1 activity by substituting Thr286
with Ala in HA-tagged wild-type MEK1 (HA-MEK1(T286A)). Cells transfected with empty vector, MEK1, or MEK1(T286A) were arrested in
mitosis by 18 h of nocodazole treatment and then fractionated for
analysis. Ectopic expression of MEK1 or MEK1(T286A) did not affect
mitotic MEK1 activation, as pMEK levels remained constant in all
transfected cell populations. Immunoblotting of the S100 and P100
fractions with antibody specific for phosphothreonine/proline consensus
sites for cyclin B-Cdc2-dependent phosphorylation revealed a strong band corresponding to ectopic MEK1, which was greatly reduced
with mutation of Thr286 (Fig.
7). Examination of the level of ERK
activation in these transfected cultures showed that the T286A
substitution was sufficient to allow robust ERK activation during
mitosis in both the membrane and cytosolic fractions. We conclude that
MEK1 phosphorylation by cyclin B-Cdc2 at Thr286 is
necessary to uncouple MEK and ERK activation during mitosis.
There has been evidence from a number of eukaryotic models that
components of the Ras/Raf/MEK/ERK signaling pathway are involved in
mitosis, but their precise role and regulation have remained elusive.
In this work, we present a comprehensive study of the activity of
individual components of the Ras/Raf/MEK/ERK pathway throughout the
cell cycle.
Our study has provided some insights into the role of Ras in cell cycle
progression. We found that, in HeLa cells, N-Ras and K-Ras were both
strongly expressed, but only N-Ras was significantly activated during
the cell cycle. It is currently unclear why K-Ras was not activated,
although Ras isoforms do have different biological functions in
vivo (25). The Ras isoforms vary in their ability to activate
downstream effectors such as Raf and phosphatidylinositol 3-kinase (26)
and in their sensitivity to activation by specific guanine nucleotide
exchange factors (18). N-Ras has also been shown to have a role in
suppressing apoptosis (27), and it is possible that the selective
activation of N-Ras during normal cell cycle progression is related in
some way to an anti-apoptotic role. The small peak of N-Ras activation
in late mitosis/early G1 that was not correlated with Raf
activation could be especially relevant in this context. Alternatively,
this N-Ras activity may be involved in activation of the Rho family
GTPases that function in exit from mitosis and cytokinesis. In
contrast, N-Ras-GTP loading in G1 appears to be directly
linked with activation of Raf, MEK, and ERK. The peak of mitotic Raf
activation did not correlate with an increase Ras activation;
nevertheless, some activated N-Ras-GTP was present when Raf was active.
Thus, Raf activation during normal mitosis may be
Ras-dependent, but other factors or activation mechanisms
specific to mitosis probably augment Raf activity in an as yet
undefined manner. This may account for the Ras-independent mitotic Raf
activity reported by others (10, 28). The mitotic peak of Raf activity
correlates well with mitotic MEK1 activation, suggesting that Raf
activates MEK1 during G2/M progression.
The major finding of this study is the dramatic activation of MEK1
during mitosis and the uncoupling of this activation from the
activation of its substrate ERK. Other groups have reported MEK
activation in G2/M (10, 11, 13, 22), but the extent of
activation has been overlooked, probably due to mitotically activated
MEK1 being restricted to a detergent-insoluble membrane fraction.
Although MEK1 is strongly activated in mitosis, we observed very little
concomitant ERK activation, in agreement with previous studies (8, 10,
13). This uncoupling of MEK from ERK activation in mitosis is
particularly apparent when compared with the strong ERK activation in
the subsequent G1 phase, which correlates with a much more
modest level of MEK activation. Thus, it appears that the focus of the
MAPK pathway in mitosis is the activation of MEK1, in contrast to its
G1 role of activating ERK. The observation that there is
considerably more active MEK present during G2/M progression than during any other stage of the cell cycle poses several
intriguing questions concerning MEK activation during mitosis. How is
MEK1 specifically activated during mitosis? How is such a robust
activation of MEK so efficiently uncoupled from its downstream effector
ERK? And finally, what function does MEK have in somatic cell
G2/M progression?
Mitotic MEK1 activation and its uncoupling from ERK activation are
dependent on cyclin B-Cdc2 activity. A number of mechanisms contribute
to this uncoupling. We have shown that MEK1 is cleaved during mitosis,
removing a 5-7-kDa N-terminal segment of the protein, and this is
dependent on cyclin B-Cdc2 activity. The mitotic proteolytic cleavage
site in MEK1 appears to be located after residue 51 because GFP-MEK1 Cyclin B-Cdc2 can phosphorylate MEK1 at Thr286 and
Thr292 in vitro, and this phosphorylation
reduces MEK1 activity for ERK (24). We have demonstrated that
phosphorylation of Thr286 occurs in vivo in
mitosis and is necessary to uncouple active MEK1 from ERK during
mitosis. Mutation of Thr286 did not affect the
mitosis-specific N-terminal cleavage of MEK1, suggesting that the
cyclin B-Cdc2 dependence of this event is not through the direct
modification of MEK1, but is an indirect consequence of its activity.
The mitotic activation of MEK1 during mitosis suggests a novel function
for this MEK isoform during mitotic progression. MEK1 and MEK2 are
highly homologous, although their sequences are divergent in the
N-terminal 30-40 residues and the
Thr286/Thr292 phosphorylation sites, which are
not conserved in MEK2 (24). The almost complete sequence identity in
the region of the putative mitotic cleavage site makes it difficult to
propose a model for the isoform-specific cleavage and activation of
MEK1 during mitosis, although tertiary structural considerations
involving the nonconserved N-terminal regions may be involved. We have
provided evidence that Raf is active in G2/M; however, the
mechanism specifically targeting MEK1 for activation or alternately
blocking MEK2 activation requires further investigation.
The down-regulation of ERK activation by two distinct mechanisms may be
essential in facilitating proper G2/M progression, as ERK
is able to up-regulate the cyclin B-Cdc2 inhibitor Wee1 kinase and
thereby delay entry into mitosis (4, 5). It is also tempting to
speculate that membrane-associated mitotically activated MEK1
may have substrates other than ERK during mitosis. In this context, it
has been suggested that the MEK contribution to Golgi fragmentation in
mitosis involves MEK phosphorylation of non-ERK substrates (22).
However, immunofluorescent staining performed during this study showed
that pMEK was widely dispersed in mitosis, and co-staining with a Golgi
marker revealed that the majority of mitotically activated MEK1 was not
Golgi-associated.2 Further
ultrastructural and fractionation studies will be required to
definitively localize mitotically activated MEK1 as a first step in
unraveling the mechanisms involved in the isoform-specific activation
and cleavage of MEK1 as well as identifying potential novel MEK substrates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-tubulin monoclonal antibody was purchased from Amersham
Biosciences. Horseradish peroxidase-conjugated anti-rabbit and
anti-mouse and fluorescein isothiocyanate-conjugated anti-mouse goat
polyclonal antibodies were purchased from Zymed Laboratories
Inc. Plasmid pLNCAL-HA-tagged wild-type MEK1,
MEK1(S218D/S222D), MEK1
32-51, and MEK1(T286A) were a
generous gift from Dr. Andy Catling (16). GFP- and HA-tagged MEK1
N51
was produced by PCR of the open reading frame of wild-type MEK1 or
MEK1(S218D/S222D) from residue 51 to the termination codon, such that
it was cloned in-frame into the pEGFP vector (Clontech).
70 °C. Protein expression levels were determined by
quantitative immunoblotting. 20 µg of P100 and an equivalent volume
of S100 were resolved on SDS-polyacrylamide gels and transferred to
polyvinylidene difluoride membranes by semidry transfer. Proteins were
detected using the appropriate primary antibodies followed by
horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary
antibody. Bands were visualized by enhanced chemiluminescence (ECL),
and relative luminescence was quantified by phosphorimaging (Bio-Rad).
Levels of Ras-GTP were assayed using bacterially expressed GST-Raf-RBD(K85A) to bind Ras-GTP, which was detected by immunoblotting with Ras isoform-specific antibodies (18).
-32P]ATP and myelin basic
protein. The radioactivity incorporated into myelin basic protein was
quantified by phosphorimaging. Total P100 Raf kinase activity was the
difference in the activities in tubes A and B, as P100-associated Raf-1
and B-Raf are insoluble in 1% Nonidet P-40 (19). Total P100 MEK
activity was the difference in activities in tubes B and C. MEK-specific activity was estimated by dividing the total P100 MEK
kinase activity by the amount of P100 phospho-MEK present (determined
by quantitative Western analysis as described above).
20 °C until required. Coverslips
were washed three times with PBS and then blocked in blocking buffer
(PBS containing 0.1% Tween 20 and 3% bovine serum albumin) for 1 h at room temperature. The cells were stained with anti-
-tubulin
antibody (1:1000 dilution in blocking buffer), washed five times for 5 min, stained with Texas Red-conjugated anti-mouse antibody (1:300
dilution in blocking buffer containing 10 µg/ml
4,6-diamidino-2-phenylindole for DNA counterstaining) for 30 min,
washed five times with PBS, and mounted on slides. Photomicroscopy was
performed as described previously (17).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Analysis of the MAPK pathway during the cell
cycle. HeLa cells were synchronized by double thymidine
block/release, and the MAPK pathway was analyzed as cells progressed
through mitosis into the subsequent G1 phase. This
experiment was repeated three times with similar results. A,
fluorescence-activated cell sorter analysis of the DNA content of cells
harvested after release. The percentage of cells in G1
( ), S (
), and G2/M (
) for each time point is
shown. Entry into G2 phase occurred 6 h after release,
and entry into mitosis occurred 9-10 h after release. B,
cyclin B1-Cdc2 kinase activity assayed as a marker for mitosis. Peak
mitosis is indicated by the dashed line. C,
Ras-GTP loading (only N-Ras is shown). D, total Raf kinase
activity. E, level of pMEK in the P100 (
) and S100 (
)
fractions. F, level of pERK in the P100 (
) and S100 (
)
fractions. G-J, the levels and activities of Ras
(G), B-Raf and c-Raf-1 (H), MEK1 and MEK2
(I), and ERK1 and ERK2 (J).
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Fig. 2.
MEK is activated in mitosis. HeLa cells
were fixed in 3% paraformaldehyde and immunostained for pMEK, and DNA
was stained with 4,6-diamidino-2-phenylindole. Mitotic cells
(Mt) were identified and photographed using confocal
microscopy. Shown are mitotic cells displaying typical strong pMEK
staining. The surrounding interphase cells displayed little staining. A
similar pattern was observed with methanol fixation.
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Fig. 3.
Cyclin B-Cdc2 activity uncouples MEK
phosphorylation from ERK activation during mitosis. A,
synchronized HeLa cells were treated with 20 µM Compound
5 or 50 µM roscovitine 7 h after release and
harvested at 10 h, when controls were at peak mitosis. Cells were
fractionated, and the P100 membrane fractions were then immunoblotted
for pMEK and pERK. ERK2 was used as an input control. B,
synchronized HeLa cells were treated with roscovitine as described for
A; equivalent untreated mitotic HeLa cells were
fractionated; and the P100 membrane fraction was assayed for MEK kinase
activity. Identical membrane fractions were immunoblotted for pMEK.
MEK-specific activity was calculated as described under "Materials
and Methods." ERK2 was immunoblotted as an input control.
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Fig. 4.
MEK1 (but not MEK2) is cleaved and activated
in mitosis. A, mitotic HeLa cells prepared from
synchronized cultures by mechanical shake-off were fractionated, and
the membrane fraction was solubilized in buffer A with or without 1%
Nonidet P-40 (NP40). Insoluble (I) and soluble
(S) fractions were run in triplicate on SDS-polyacrylamide
gel and probed for pMEK, MEK1, and MEK2. B, the Nonidet
P-40-insoluble fraction from mitotic HeLa cells was run on high
resolving SDS-polyacrylamide gel and transferred to a membrane. The
membrane was cut into strips and probed for pMEK, MEK1 with an N
terminus-specific antibody (anti-MEK1N) and a C terminus-specific
antibody (anti-MEK1C), and MEK2 with an N terminus-specific antibody
(anti-MEK2N) and a C terminus-specific antibody (anti-MEK2C). The
strips were carefully realigned upon development so that mobility
shifts between the isoforms could be observed. C, the
Nonidet P-40-insoluble fraction from a 10-cm dish of HeLa cells
arrested in mitosis was resolved by two-dimensional gel electrophoresis
and transferred to a membrane. The membrane was marked with three
orientation spots and then probed for pMEK, stripped, and reprobed for
MEK1 and MEK2 with anti-pan MEK1/2 antibody. Two exposures
(exp) of the MEK1/2 blot are shown. The arrow
indicates the minor form of MEK detected by anti-MEK1/2 antibody, which
overlaid the strong pMEK spot.
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Fig. 5.
MEK1 cleavage is dependent on cyclin B-Cdc2
activity. A, samples from the experiment shown in Fig.
1 were assayed for cyclin B1-Cdc2 activity as a marker for mitosis. The
Nonidet P-40-insoluble P100 fraction was immunoblotted with anti-MEK1C
antibody to indicate the time point at which MEK1 cleavage occurred.
B, synchronized HeLa cells were allowed to progress into
mitosis or arrested at G2 by addition of 50 µM roscovitine added 7 h after release. The Nonidet
P-40-insoluble membrane fractions were run on high resolving
SDS-polyacrylamide gel and immunoblotted with anti-pMEK, anti-MEK1N,
and anti-MEK1C antibodies. A G1/S cell population at time 0 is included for comparison.
32-51).
HeLa cells were transfected with HA-tagged versions of wild-type MEK1
and MEK1
32-51 and either untreated or arrested in mitosis with
nocodazole, which produced essentially identical accumulation of the
faster migrating pMEK species observed in normal mitotic samples. S100
and P100 fractions were prepared and immunoblotted for the HA-tagged
proteins. The level of HA-tagged wild-type MEK1 in the P100 fraction
from mitotic cells was reduced compared with the untreated
asynchronously growing culture (Fig. 6A). This paralleled the
reduction in MEK1 detected with anti-MEK1N antibody in normal mitotic
P100 fractions (Fig. 5B). There was little change in the
level of HA-tagged MEK1
32-51 in the P100 fractions from
asynchronous and nocodazole-arrested mitotic cells (Fig.
6A). The levels of these proteins were constant in the S100 fractions from asynchronous and mitotic cells.
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Fig. 6.
The N-terminal ERK-binding domain of MEK1 is
proteolytically cleaved in mitosis. A, 293H cells were
transfected with empty vector or HA-tagged MEK1 constructs and
harvested as asynchronous (without nocodazole) or mitotic (with 0.5 µg/ml nocodazole for 18 h) populations. Cells were fractionated
into S100 and Nonidet P-40-insoluble P100 fractions, normalized for
protein content, and then immunoblotted for the HA epitope.
B, HeLa cells were transfected with either GFP or
GFP-MEK1 N51; then after 8 h, the cells were treated with
nocodazole for 18 h to arrest them in mitosis. Cells were
fractionated as described for A and immunoblotted with the
indicated antibodies. C, HeLa cells were transfected with
empty vector (mock), HA-MEK1(S218D/S222D) (DD),
or GFP-MEK1
N51(S218D/S222D). The cultures were either untreated or
arrested in mitosis with nocodazole. The cells were fractionated and
immunoblotted with the indicated antibodies.
32-51 constructs to N-terminal
cleavage suggested either that the deleted residues 32-51 contain the
proteolytic cleavage site or that the tertiary structure of the deleted
protein masks the cleavage site. To discriminate between these
possibilities, the N-terminal 51 residues of MEK1 were replaced with
either an HA tag (HA-MEK1
N51) or GFP (GFP-MEK1
N51).
Immunoblotting of the P100 fraction prepared from mitotic cells
expressing GFP-MEK1
N51 revealed the presence of the full-length
chimera, detected by both anti-MEK1C and anti-GFP antibodies, and an
abundant 37-kDa MEK1 protein, detected only by anti-MEK1C antibody and
corresponding to MEK1
N51 cleaved of the GFP moiety. This band
comigrated with 37-kDa pMEK (Fig. 6B). The full-length
chimera was the major form of the protein detected in the corresponding
S100 fractions (Fig. 6B). Immunoblotting of HA-MEK1
N51
with anti-HA antibody revealed a similar loss of HA tag in the P100
fraction (data not shown).
N51(S218D/S222D). The N-terminal deletion mutant failed to activate ERK in the S100 fraction compared with constitutively activated full-length
MEK1, but the GFP-MEK1
N51(S218D/S222D) mutant activated ERK in the P100 fraction of asynchronously growing cells (Fig. 6C).
Interestingly, GFP-MEK1
N51(S218D/S222D) failed to activate ERK in
the P100 fraction of mitotic cells, although ERK activation by
MEK1(S218D/S222D) was unaffected (Fig. 6C).
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Fig. 7.
Mitotic phosphorylation of MEK1
Thr286 uncouples MEK and ERK activation. HeLa cell
were transfected with either empty vector or the indicated HA-tagged
MEK1 constructs and then arrested in mitosis with nocodazole.
Cells were fractionated and immunoblotted with
anti-phosphothreonine/proline antibody (p-TP), anti-HA tag
antibody, or anti-endogenous pMEK, pERK, or ERK2 antibody. The
anti-phosphothreonine/proline band in the wild-type (wt)
MEK1-expressing cells was confirmed as transfected HA-MEK1 by
immunoprecipitation of the HA-tagged protein and
anti-phosphothreonine/proline immunoblotting (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
N51 remained sensitive to mitotic cleavage. The MEK1 N-terminal region contains an ERK-binding domain, and deletion or
proteolytic removal of this domain by anthrax toxin lethal factor or
caspases renders MEK1 incapable of activating ERK (29, 30). Here we
show that, in asynchronous cell populations, the loss of the
ERK-binding domain is sufficient to completely inhibit cytosolic ERK
activation, but that the ERK-binding domain is not required for
MEK1
N51 to activate membrane-bound ERK. One possible mechanism that
may account for this is the presence of scaffold proteins such as MP-1
that can bind both MEK and ERK and thereby compensate for the loss of
the ERK-binding domain from MEK1
N51 (31). Nevertheless, the ability
of truncated MEK1
N51 to activate membrane-bound ERK is lost in
mitosis, demonstrating that additional mitosis-specific mechanisms
exist to inhibit MEK activity for ERK.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Andy Catling for MEK1 expression plasmids and Professor John Lazo for Compound 5.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Queensland Cancer Fund and the National Health and Medical Research Council of Australia (to B. G. G. and J. F. H.). The Institute for Molecular Bioscience is a special research center for the Australian Research Council.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.
¶ Australian Research Council Research Fellow. To whom correspondence should be addressed: Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland 4102, Australia. Tel.: 61-7-3240-7129; Fax: 61-7-3240-5946; E-mail: bgabrielli@cicr.uq.edu.au.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M301015200
2 A. Harding and B. G. Gabrielli, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; pMEK, phospho-MEK; ERK, extracellular signal-regulated kinase; pERK, phospho-ERK; HA, hemagglutinin; GFP, green fluorescent protein; PBS, phosphate-buffered saline; GST, glutathione S-transferase; RBD, Ras-binding domain; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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