Mechanism of Mitosis-specific Activation of MEK1*

Angus HardingDagger §, Nichole GilesDagger , Andrew BurgessDagger , John F. Hancock§, and Brian G. GabrielliDagger

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha -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), MEK1Delta 32-51, and MEK1(T286A) were a generous gift from Dr. Andy Catling (16). GFP- and HA-tagged MEK1Delta 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).

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 -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).

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 [gamma -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).

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 -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-alpha -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


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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 (black-square), S (black-diamond ), and G2/M (open circle ) 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 (black-square) and S100 (diamond ) fractions. F, level of pERK in the P100 (black-square) and S100 (diamond ) 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).

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).


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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.

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.


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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.

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).


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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.

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.


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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.

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 (MEK1Delta 32-51). HeLa cells were transfected with HA-tagged versions of wild-type MEK1 and MEK1Delta 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 MEK1Delta 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-MEK1Delta 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-MEK1Delta N51(S218D/S222D). The cultures were either untreated or arrested in mitosis with nocodazole. The cells were fractionated and immunoblotted with the indicated antibodies.

The insensitivity of the MEK1Delta 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-MEK1Delta N51) or GFP (GFP-MEK1Delta N51). Immunoblotting of the P100 fraction prepared from mitotic cells expressing GFP-MEK1Delta 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 MEK1Delta 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-MEK1Delta N51 with anti-HA antibody revealed a similar loss of HA tag in the P100 fraction (data not shown).

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-MEK1Delta 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-MEK1Delta N51(S218D/S222D) mutant activated ERK in the P100 fraction of asynchronously growing cells (Fig. 6C). Interestingly, GFP-MEK1Delta 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).

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.


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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

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-MEK1Delta 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 MEK1Delta 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 MEK1Delta N51 (31). Nevertheless, the ability of truncated MEK1Delta N51 to activate membrane-bound ERK is lost in mitosis, demonstrating that additional mitosis-specific mechanisms exist to inhibit MEK activity for ERK.

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.

    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.

    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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DISCUSSION
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