* Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5332; and Preuss
Laboratory Molecular Neuro-oncology, Brain Tumor Research Center, University of California at San Francisco, San Francisco,
California 94143-0520
The spindle assembly checkpoint prevents
cells whose spindles are defective or chromosomes are
misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the
Erk2 mitogen-activated protein kinase (MAP kinase)
in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic
progression. Here we have investigated whether MAP
kinase function is required for mitotic progression or
the spindle assembly checkpoint in vivo in Xenopus
tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during
prometaphase and metaphase, consistent with the idea
that MAP kinase might regulate or monitor the status
of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase,
into XTC cells in various stages of mitosis to interfere
with MAP kinase activation. We found that mitotic
progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or
metaphase exited mitosis in the presence of nocodazolethe chromosomes decondensed and the nuclear
envelope re-formed
whereas cells injected with buffer
or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes
XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP
kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.
Inhibitors of microtubule polymerization arrest most
cells in a prometaphase-like stage, indicating that a
checkpoint monitors chromosome alignment and/or
the status of the spindle and regulates the metaphase to
anaphase transition. This safeguard helps to ensure accurate transmission of genetic information to the daughter cells. Defects in cell cycle checkpoints increase the frequency of chromosome loss and gene amplification and
may play a role in tumor progression (Hartwell et al., 1994 In the budding yeast Saccharomyces cerevisiae, spindle
disruption arrest depends on the products of three MAD
(mitotic arrest defective) genes (Hardwick and Murray, 1995 In a wide variety of organisms, phosphoprotein dephosphorylation is essential for progression from metaphase to
anaphase. Certain fission yeast and Drosophila mutants
with altered phosphatase activity exhibit metaphase arrest phenotypes (Kinoshita et al., 1991 Minshull et al. (1994) Although MAP kinases are probably best known for their
transient activation when quiescent cells are prompted to
reenter the cell cycle, they are also activated during meiotic maturation in oocytes (Ferrell et al., 1991 MAP kinase may contribute to M phase in other ways as
well. Gotoh et al. (1991b) In this study we set out to evaluate the hypotheses that
p42 or p44 MAP kinase function is important for entry into
mitosis in somatic cells, as suggested by studies of meiotic
maturation; for establishing a mitotic spindle, as suggested
by studies of microtubule dynamics in cytoplasmic Xenopus egg extracts; or for spindle assembly checkpoint control, as suggested by studies of nocodazole-treated, sperm
nucleus-supplemented Xenopus egg extracts. We chose to
carry out our studies in the Xenopus tadpole cell line
(XTC), both because of the availability of good quality homologous reagents for perturbing MAP kinase function,
and because XTC cells remain relatively flat during mitosis, facilitating microscopic examination of mitotic cells.
Here we demonstrate that MAP kinase is present in the
spindle during specific stages of M phase, consistent with
the possibility that MAP kinase regulates the spindle or monitors the status of the spindle. Microinjection of a specific MAP kinase-inactivating enzyme, Xenopus MAP kinase phosphatase-1 or XCL100, has no obvious effect on
normal mitotic progression but renders cells unable to
maintain a mitotically arrested state when mitosis is disrupted by nocodazole treatment. Our results provide in
vivo support for the hypothesis that p42 and/or p44 MAP
kinase function are required for the spindle assembly
checkpoint.
Cell Culture
The Xenopus tadpole cell line (XTC) was obtained from Dr. Tim Stearns
(Stanford University) and grown at room temperature in 70% L-15 medium supplemented with 10% FBS. Cells to be used for microinjection
were plated onto coverslips with photoetched locator grids (Bellco Glass,
Inc., Vineland, NJ) and grown for at least 2 d.
Antibodies
The rabbit MAP kinase antiserum X15 was raised against a Xenopus p42
MAP kinase COOH-terminal peptide (Hsiao et al., 1994 Preparation of Cell Lysates
XTC cells were grown to near confluence. Cells were trypsinized and collected by centrifugation. SDS sample buffer was added to the pelleted
cells.
Lysates from Xenopus oocytes or electrically activated eggs, containing
nonphosphorylated p42 MAP kinase, and from progesterone-treated oocytes or nonactivated eggs, containing phosphorylated p42 MAP kinase,
were prepared as described (Ferrell et al., 1991 Immunoblotting
Oocyte, egg, and XTC cell lysates were separated by SDS 10% polyacrylamide gel electrophoresis, transferred to an Immobilon-P membrane (Millipore Corp., Bedford, MA), and probed with the affinity-purified X15
antibody at 1 µg/ml followed by affinity-purified alkaline phosphatase-conjugated goat anti-rabbit IgG at 1:3,000 (Sigma Chemical Co., St. Louis, MO).
Construction of an XCL100 Expression Plasmid
The wild-type XCL100 cDNA was inserted into pGEX-4T for expression
in bacteria as follows: gel-purified 1.6-kb BamHI-XhoI fragment, which
contained all but the NH2-terminal 130 bp of the XCL100 coding region
plus 0.66 kb of the 3 A cDNA for the catalytically inactive phosphatase XCL100 C260S, which
has a serine residue in place of the essential active site cysteine residue,
was generated by PCR by M.L. Sohaskey. An EcoRI-NheI fragment spanning the mutagenized codon was excised and ligated into pGEX-XCL100
to yield pGEX-XCL100 C260S. The protein products of the wild-type and
mutant forms of pGEX-XCL100 are fused to the COOH terminus of glutathione-S-transferase (GST).
Expression and Purification of XCL100
Overnight cultures of Escherichia coli transformed with pGEX-XCL100
or pGEX-XCL100 C260S were grown and lysed essentially as described
(Wang et al., 1996
We found that the biochemical activity of XCL100 did not survive multiple freeze-thaw cycles or prolonged storage at 4°C. Therefore, for studies
described below, we used XCL100 and XCL100 C260S that had been
stored at 4°C for no more than 48 h, or, in some experiments, used
XCL100 that had been frozen in 10% glycerol, stored at Other Recombinant Proteins
A pRSET-based plasmid for a constitutively active, His6-tagged mutant of
human MKK-1/Mek-1 (with Ser 218 replaced by Glu, Ser 222 replaced by
Asp, and a deletion of amino acids 32-51, hereafter denoted MKK-1*)
was provided by Natalie Ahn (University of Colorado, Boulder). An
NheI-HindIII fragment containing the MKK-1* coding region was subcloned into pFastBac1 for generation of baculoviruses. MKK-1* was expressed in insect cells and purified to homogeneity by nickel-chelate chromatography by Ramesh Bhatt (Stanford University).
Hexahistidine-tagged, kinase-minus (K52R) rat Erk2 was expressed in
E. coli and purified by Chi-Ying Huang (Stanford University; Huang and
Ferrell, 1996a In Vitro Phosphatase Activity Assay
To prepare phosphorylated Erk2 for use as a substrate in an XCL100
phosphatase assay, we incubated recombinant kinase-minus rat Erk2 with
active MKK-1 that had been obtained from Xenopus egg lysates by immunoprecipitation with antibody 662 (Hsiao et al., 1994 MAP Kinase Assay
MAP kinase activity was assessed by a myelin basic protein phosphorylation assay essentially as described (Huang and Ferrell, 1996a Microinjection and Nocodazole Treatment
Micropipettes were pulled on a horizontal pipette puller (Sutter Instrument Co., Novato, CA). Proteins were microinjected with a Narishiga
manual microinjector (Greenvale, NY). The needle concentration of
XCL100 protein (both wild-type and mutant forms) was 1.5 µM, and MKK1* was 10 µM when MKK-1* was injected alone, and 5 µM when MKK-1*
was coinjected with XCL100.
For checkpoint control experiments, we first microinjected the appropriate protein(s), and then aspirated the medium and added fresh F-15
medium containing nocodazole at 50 ng/ml. Cells were viewed with a ×40
phase-contrast objective (NA 0.75) on an inverted microscope (Carl Zeiss,
Inc., Thornwood, NY) equipped with a CCD camera and recorded with
an optical disk recorder (Panasonic, Secaucus, NJ) for up to 4 h after injection or treatment with the drug.
Immunofluorescence
XTC cells were grown on coverslips, fixed with methanol ( Images were obtained using either a ×100, 1.3 NA objective on a Zeiss
microscope equipped with a cooled CCD camera or a ×40, 0.9 NA objective on a Zeiss microscope equipped with a conventional 35-mm camera.
Erk2/p42 MAP Kinase Localizes at the Mitotic
Spindle after Nuclear Envelope Breakdown and
Leaves the Spindle before Anaphase
The localization of some cell cycle regulators provides a
clue to their function. Accordingly, we examined the localization of MAP kinase during M phase in a tissue-culture
cell line, XTC. We determined the most suitable of several
antisera for immunolocalization studies to be X15, an antipeptide antiserum raised against a Xenopus Erk2 (p42)
MAP kinase COOH-terminal peptide (Hsiao et al., 1994
We affinity purified X15 antibody on a peptide column
and characterized the affinity-purified X15 for its specificity in total XTC lysates. Affinity-purified X15 recognized
two bands on immunoblots (Fig. 1), corresponding to Xenopus Erk1 and Erk2.
We then carried out immunofluorescence studies of
XTC cells using affinity purified X15. Fig. 2 shows the results of triple label staining with X15 (fluorescein staining),
antitubulin antiserum (Texas red), and DAPI for DNA
staining. In interphase, MAP kinase was diffusely distributed through the cytoplasm (Fig. 2 A). There was no obvious concentration of MAP kinase in the interphase microtubules.
When cells entered prometaphase, much of the MAP kinase staining remained diffusely cytoplasmic, but some of
the staining became concentrated on the spindle (Fig. 2 B).
MAP kinase was detectable on the spindle during early
prometaphase just after nuclear envelope breakdown, and
the staining increased during metaphase (Fig. 2, B-C). During anaphase, the intensity of the MAP kinase staining of
the spindle decreased to about the level of intensity of
general cytoplasmic staining (Fig. 2 D). Thus, the spindlelike "fingers" of MAP kinase staining seen in Fig. 2 D could
include some spindle-associated MAP kinase, but could also simply represent fingers of cytoplasm between MAP
kinase-free masses of chromosomes. By late anaphase, the
intensity of MAP kinase staining around the spindle was
lower than that in other regions of the cell (Fig. 2 E). MAP
kinase was not detectable in the midbody, the prominent
microtubule-containing structure that forms between the
two daughter cells during cytokinesis (Fig. 2 F). Thus, MAP
kinase was found to be associated with the spindle during
prometaphase and metaphase and to dissociate from the
spindle and midbody, or to become inaccessible to antibody staining during anaphase and cytokinesis.
Preimmune serum yielded no spindle staining (Fig. 2 G),
indicating that the spindle staining observed with affinitypurified X15 was specific. Preincubation of X15 with the
peptide against which it was raised markedly decreased
the intensity of both spindle and cytoplasmic staining (Fig.
3). Finally, single label immunofluorescence studies with
affinity-purified X15 showed spindle staining, confirming that the results seen in the triple label studies were not due to cross-reactivity between secondary antibodies or leakage between the fluorescein and Texas red filters (Fig. 3).
These findings demonstrate that Erk1 or Erk2 is present
in the spindle during specific subphases of mitosis. This
places MAP kinase in an appropriate position to be involved in either regulating the spindle or relaying information about the status of the spindle.
Purified Bacterially Expressed GST-XCL100
Dephosphorylates Erk2 MAP Kinase In Vitro
We set out to determine whether, in intact XTC cells, MAP
kinase activation might be necessary to trigger or maintain
Cdc2 activation, as appears to be the case during meiotic
maturation (Gotoh et al., 1995 We expressed wild-type XCL100 and an engineered catalytically inactive version of XCL100, with Cys 260 mutagenized to Ser (XCL100 C260S), as GST fusion proteins
in bacteria. The proteins were purified to homogeneity by
glutathione affinity chromatography and Mono-Q anion
exchange chromatography (Fig. 4 A). The purified wildtype XCL100 protein potently dephosphorylated p42 MAP
kinase (Fig. 4 B) but not Mos, Mek, or Rsk (Huang, C.Y.-F.,
unpublished data), other enzymes in the MAP kinase pathway. The mutant XCL100 C260S protein did not dephosphorylate MAP kinase (Fig. 4 B).
We then carried out in vitro studies to determine whether
it was likely that we could achieve a high enough concentration of XCL100 by microinjection to interfere with Erk1
and Erk2 activation. By microinjection, we should be able
to increase the cell's XCL100 concentration by ~75 nM
(assuming we can inject 50 fl into a 1 pl cell, and given that
the needle concentration of XCL100 was 1.5 µM). The
abundance of MKK-1 and Erk1/2 in several cell lines has
been estimated to be on the order of 1 µM (Ferrell,
1996b Microinjection of MAP Kinase Phosphatase Does Not
Affect Mitotic Progression
To test whether interfering with MAP kinase activation by
XCL100 microinjection would affect the mitotic progression,
we microinjected XTC cells at different stages of mitosis Table I.
The Effect of Inhibiting MAP Kinase Activation
on the Spindle Assembly Checkpoint: Summary of
Microinjection Data
;
Hartwell and Weinert, 1989
; Murray, 1994
, 1995
).
;
Hardwick et al., 1996
; Li et al., 1994
), three BUB (budding
uninhibited by benzimidazole) genes (Hoyt et al., 1991
;
Roberts et al., 1994
), and MPS1 (multipolar spindle; Lauze
et al., 1995
; Weiss and Winey, 1996
). Vertebrate homologs of
one of these genes, MAD2, have recently been identified and found to be important for the spindle assembly checkpoint, indicating that the mechanisms underlying this
checkpoint are likely to be well conserved evolutionarily
(Chen et al., 1996
; Li and Benezra, 1996
).
; Mayer-Jaekel et al.,
1993
; Ohkura et al., 1989
). Studies with phosphoepitopespecific antibodies have also implicated dephosphorylation in progression to anaphase (Campbell and Gorbsky, 1995
;
Gorbsky and Ricketts, 1993
; Nicklas et al., 1995
). Inactivation of phosphatases that are necessary for anaphase, or
activation of kinases that oppose progression into anaphase,
could be the mechanism through which defective spindles
and misaligned chromosomes bring about mitotic arrest.
implicated the Erk2 or p42 mitogen-activated protein kinase (MAP kinase)1 protein in the
spindle assembly checkpoint through studies of cytoplasmic extracts of Xenopus eggs, a system that offers great biochemical manipulability. Previous work had shown that
even though Xenopus eggs and early embryos lack the
DNA synthesis checkpoint
DNA synthesis inhibitors do
not affect blastomere cleavage
this checkpoint can be reconstituted by the addition of sperm nuclei to a concentration of ~250 per µl (higher than the concentration of nuclei in an early embryo, but lower than the concentration
of ~250,000 nuclei per µl for a 4 pl somatic cell; Dasso and
Newport, 1990
). Minshull et al. (1994)
examined whether
the spindle assembly checkpoint could also be reconstituted by addition of sperm nuclei to the extracts. They found
that it could: extracts supplemented with ~9,000 nuclei
per µl arrested in a mitotic state in the presence of nocodazole, and destruction of cyclins B1 and B2 (but not
cyclin A1) was suppressed. They also noted that p42 MAP
kinase underwent sustained activation in the nocodazoletreated, arrested extracts. Addition of a MAP kinase phosphatase (mouse Mkp-1) that can dephosphorylate and inactivate p42 (Erk2), p44 (Erk1), and p38 (Hog1) MAP
kinases, but is minimally active towards other phosphoproteins (Alessi et al., 1993
; Groom et al., 1996
; Keyse and
Emslie, 1992
; Sun et al., 1993
), resulted in inactivation of
the p42 MAP kinase, destruction of cyclins B1 and B2, and
release of the extracts from mitotic arrest. These studies
showed that MAP kinase, or some closely related protein,
is essential for the spindle assembly checkpoint in this reconstituted in vitro system.
; Haccard et al.,
1990
; Jessus et al., 1991
; Posada et al., 1991
). MAP kinase
activation triggers or facilitates the activation of Cdc2 (Gotoh et al., 1995
; Haccard et al., 1995
; Huang and Ferrell,
1996a
; Kosako et al., 1994), although the mechanism through
which MAP kinase regulates Cdc2 activity remains to be
elucidated. Given the similarities between meiotic and mitotic M-phase regulation, these findings raise the possibility that MAP kinase might participate in the activation of
Cdc2 at the G2-M transition in somatic cells.
observed that MAP kinase is capable of increasing the dynamic instability of microtubules
in Xenopus egg extracts, which raises the possibility that
MAP kinase activity may be necessary for microtubule reorganization at the onset of M phase.
Materials and Methods
) and affinity purified on a peptide column. Affinity-purified X15 was tested for its specificity by immunoblotting XTC cell lysates. An Erk1 antibody (C-16) was
obtained from Santa Cruz Biochemicals (Santa Cruz, CA). MKK-1 antiserum 662, raised against a Xenopus MKK-1/Mek-1 NH2-terminal peptide,
has been described previously (Hsiao et al., 1994
). An antibody that recognizes active, but not inactive, Erks 1 and 2 was purchased from Promega
(Madison, WI). XCL100 antiserum was raised and provided by M.L. Sohaskey (Stanford University).
; Murray, 1991
).
-untranslated region, was ligated into pGEX-4T to
yield pGEX-
N. The NH2-terminal 130-bp fragment was obtained by PCR,
with the 5
primer consisting of a synthetic BamHI site fused to the codon
for the initiator methionine and the 3
primer spanning the natural
XCL100 BamHI site. The fidelity of the PCR product was verified by sequencing. The PCR product was digested with BamHI, gel purified, and
ligated into the BamHI site of pGEX-
N to yield pGEX-XCL100.
). The clarified cell extracts were recycled over a 2.5-ml
column of glutathione agarose (Sigma Chemical Co.) for 5 h at 4°C. The
column was washed with 10 vol of PBS, and fusion proteins were eluted
with 50 mM Tris, pH 8.0, containing 15 mM glutathione. Fractions were
assessed for MAP kinase phosphatase activity, as described below, and
peak fractions were pooled. As has been previously reported, XCL100
and XCL100 C260S manifested substantial proteolytic degradation; much
of the glutathione agarose-purified protein was digested back to the GST
moiety (see Fig. 4 A; see also Sun et al., 1993
). However, we were able to
obtain purified full-length XCL100 or XCL100 C260S proteins by anion
exchange chromatography. Peak fractions from the glutathione agarose
column were applied to a Mono-Q column (Pharmacia Fine Chemicals,
Piscataway, NJ) equilibrated with 20 mM Tris, pH 7.8, 20 mM NaCl, and
2 mM DTT. Proteins were eluted from the column with a linear gradient
of 20 mM-1 M NaCl. Activity was recovered in the fractions containing
640-660 mM NaCl, pooled, and dialyzed against the microinjection buffer
(130 mM KCl, 10 mM NaPipes, pH 7.0, and 1 mM MgCl2 ). Material obtained after the glutathione agarose purification step and after the Mono-Q step exhibited indistinguishable biochemical and biological activities.
Fig. 4.
Purification and characterization of the GST-XCL100 fusion protein. (A) Purification of bacterially expressed GST-XCL100. Full-length GST-XCL100 migrates at ~67 kD. The prominent band at ~27 kD in the glutathione agarose purified sample is GST. After Mono-Q purification, full-length GST-XCL100 was the main band detectable by Coomassie staining, and a single band was detectable by XCL100 immunoblotting. (B) Activity of GST-XCL100. This panel shows a MAP kinase immunoblot of recombinant nonphosphorylated rat Erk2 (MK), and phosphorylated rat Erk2 (MK-P) incubated with no phosphatase, with GST-XCL100, or with the inactive
GST-XCL100 C260S mutant. (C) In vitro titration of GST-XCL100 activity. Various concentrations of GST-XCL100 were mixed with
250 nM MKK-1* and 1 µM Xenopus Erk2, and the system was allowed to reach steady state. MAP kinase activity was determined by a myelin basic protein phosphorylation assay.
[View Larger Version of this Image (22K GIF file)]
70°C, and
thawed just before its use.
) from a plasmid provided by Melanie Cobb and Tom Geppert (University of Texas Southwestern Medical Center) (Robbins et al.,
1993
). A pT7-7-based plasmid for expression of hexahistidine-tagged Xenopus Erk2 was engineered from a Xenopus Erk2 cDNA provided by
Jonathan Cooper and Jim Posada (Fred Hutchinson Cancer Research Center, Seattle, WA), and it was expressed in E. coli and purified by Ramesh
Bhatt.
), and MgATP. The
XCL100 phosphatase assay was performed by incubating phosphorylated
MAP kinase with purified XCL100 fusion proteins in phosphatase reaction buffer (50 mM Hepes, pH 7.5, 1 mg/ml BSA, 2 mM DTT, and 2 mM
EDTA) in a final reaction vol of 60 µl. After incubation for 30 min at
30°C, the reaction was stopped by adding SDS sample buffer, and the sample was boiled. Proteins were separated by SDS 10% polyacrylamide gel
electrophoresis and immunoblotted as described above. Dephosphorylation of Erk2 was detected as a shift of the blotted Erk2 band to a lower apparent molecular weight.
). Reaction
mixtures were analyzed by SDS 12.5% polyacrylamide gel electrophoresis, proteins were transferred to an Immobilon P membrane, and incorporation of 32P into myelin basic protein was quantified using a phosphorimager (Molecular Dynamics, Sunnyvale, CA).
20°C) for 6 min, and then treated with a microtubule stabilizing buffer (25 mM imidazole, pH 7.0, 10 mM KCl, 1 mM MgSO4, 10 mM EGTA, and 20% glycerol) containing 0.1% Triton X-100 (Pierce Chemical Co., Rockford, IL),
followed by washing with PBS. XTC cells were blocked in 3% milk, and
then stained with affinity-purified X15 at 8 µg/ml followed by affinitypurified FITC-conjugated goat anti-rabbit IgG at 1:100 (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD). For triple labeling, the cells
were incubated sequentially with X15 and then mouse monoclonal anti-
-tubulin antibody (Amersham Corp., Arlington Heights, IL) at 1:100.
FITC-conjugated anti-rabbit IgG (1:100) and affinity-purified Texas red-
conjugated goat anti-mouse IgG (1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were mixed and applied. Finally, chromosomes were stained with 4
,6-diamidino-2-phenylindole (DAPI) at 1 µg/
ml for 30 s. Each step was followed by four 10-min rinses with PBS.
Results
). X15 recognizes active and inactive forms of Xenopus Erk2
equally well, both in immunoprecipitations (nondenaturing conditions) and in immunoblots (denaturing conditions; Hsiao et al., 1994
; Fig. 1).
Fig. 1.
The specificity of antibody
X15. Immunoblots of lysates from
XTC cells (lane 1), G2-phase oocytes
(lane 2), and M-phase oocytes (lane 3)
were probed with X15. XTC cells exhibited two bands: a lower band that
comigrated with nonphosphorylated
Erk2, and an upper band that comigrated with both Erk1 and phosphorylated Erk2. The upper band was identified as Erk1, rather than phosphorylated Erk2, based on its cross-reactivities
with other Erk1 and Erk2 antisera, and
on the fact that it did not shift to a
lower apparent molecular weight when
lysates were treated with XCL100 (not
shown).
[View Larger Version of this Image (12K GIF file)]
Fig. 2.
Immunolocalization
of MAP kinase at different
mitotic stages. XTC cells
were fixed, permeabilized,
and subjected to triple label
staining with DAPI, X15, and
a -tubulin antibody (A-F), or
with DAPI, X15 preimmune
serum, and the tubulin antibody (G).
[View Larger Version of this Image (68K GIF file)]
Fig. 3.
Single label immunolocalization of MAP kinase.
XTC cells were fixed, permeabilized, stained with DAPI (not
shown) and X15 or X15 plus
blocking peptide (35 µM), and
examined by fluorescence and
phase microscopy. The cell
shown is in metaphase.
[View Larger Version of this Image (70K GIF file)]
; Haccard et al., 1995
; Huang
and Ferrell, 1996a
; Kosako et al., 1994b
); MAP kinase might
be needed to maintain the dynamic properties of the
M-phase microtubules, as suggested by studies of interphase Xenopus egg extracts (Gotoh et al., 1991b
); or MAP
kinase activity might be required for the spindle assembly checkpoint, as suggested by studies of nocodazole-treated,
nucleus-supplemented cycling Xenopus egg extracts (Minshull et al., 1994
). The approach chosen was to microinject
a Xenopus MAP kinase phosphatase, XCL100, which
could prevent or reverse MAP kinase activation, and determine the consequences by time-lapse video microscopy.
XCL100 and its homologs CL100 (human) and Mkp-1
(mouse) are dual-specificity phosphatases that dephosphorylate the phosphothreonine and phosphotyrosine regulatory sites of MAP kinase, thereby inactivating MAP kinase (Alessi et al., 1993
; Lewis et al., 1995
; Sun et al., 1993
).
CL100/Mkp-1 proteins are highly specific for the p42 Erk2,
p44 Erk1, and p38 Hog1 MAP kinases in vitro (Alessi et
al., 1993
; Groom et al., 1996
; Keyse and Emslie, 1992
; Sun
et al., 1993
). Transient expression of Mkp-1 in COS cells
leads to selective dephosphorylation of MAP kinase from
the spectrum of phosphotyrosine-containing proteins (Sun
et al., 1993
), indicating that XCL100/Mkp-1 proteins are highly specific in vivo as well.
). In Xenopus oocyte extracts, activation of ~25% of
the MKK-1 results in activation of ~50% of the Erk2
(Huang and Ferrell, 1996b
). We added various dilutions of
XCL100 to a mixture of recombinant Xenopus Erk2 (1 µM)
and MKK-1* (250 nM), in the presence of MgATP, and let
the system reach steady state. We found that 38 nM
XCL100 was sufficient to half-maximally inhibit Erk2 activation, 75 nM XCL100 inhibited Erk2 activation by
87%, and 150 nM XCL100 inhibited Erk2 activation completely (Fig. 4 C). Based on these in vitro titrations, it appears reasonable to expect XCL100 microinjection to have
an effect on MAP kinase activation in vivo, provided the microinjected phosphatase is not rapidly degraded or inactivated.
prophase, prometaphase, metaphase, and anaphase
with
purified XCL100 protein, and then followed the injected
cells by time-lapse video microscopy. We found that 20 out
of 20 cells injected with XCL100 (needle concentration 1.5 µM, estimated intracellular concentration 75 nM) progressed to metaphase, initiated anaphase, and cleaved, all
with normal kinetics (Table I). These findings indicate that
either a high level of MAP kinase activity is not required
for normal mitotic progression, or the intracellular concentration of XCL100 achieved by microinjection was not
sufficient to substantially interfere with MAP kinase activation. The fact that some of the same preparations of
XCL100 were found to interfere with checkpoint control,
as described below, argues for the first possibility.
Microinjection of MAP Kinase Phosphatase Causes Mitotically Arrested Cells to Exit Mitosis without Sister Chromatid Segregation, Karyokinesis, or Cytokinesis
Next, we tested the hypothesis that MAP kinase function
is required for the spindle assembly checkpoint, as suggested by experiments in nucleus-supplemented Xenopus egg
extracts (Minshull et al., 1994). Treatment of noninjected,
mitotic XTC cells with nocodazole (0.05 µg/ml) resulted in
rapid disappearance of the spindle (generally within 3 min),
including the kinetochore microtubules (data not shown).
Cells then remained blocked in a pseudo-prometaphase
stage with scattered chromosomes for up to 12 h.
Our initial approach to determining whether XCL100 microinjection might interfere with this arrest was to arrest XTC cells in a pseudo-prometaphase stage by treatment with nocodazole, then inject XCL100 protein, and finally follow the cells to see whether they would exit mitosis. However, the nocodazole-treated XTC cells proved too fragile to microinject; they often swelled and died (as indicated by cessation of cytoplasmic streaming) immediately after injection.
Therefore, we chose to identify and microinject mitotic
cells first, and then immediately thereafter treat them with
nocodazole. We found that cells injected with wild-type
XCL100 protein during prometaphase or metaphase gradually began to exit mitosis ~45 min to 1 h after nocodazole
treatment, without re-forming spindles or carrying out sister chromatid segregation, karyokinesis, or cytokinesis (Table I). Initially, the chromosomes became clustered and
then decondensed, and finally the nuclear envelope reformed and nucleoli appeared (Fig. 5). The resulting cells
resembled typical, flat interphase cells, except that the
cells and their nuclei were unusually large (Fig. 5). When
cells were injected during prophase (before nuclear envelope breakdown) and then treated with nocodazole, they
did not exit mitosis.
We carried out a variety of experiments to assess the specificity of the effects of XCL100 microinjection on metaphase arrest. First, the possibility that cells escaped the checkpoint control because of microinjection trauma was assessed by injection of buffer alone. All cells (10/10) injected with buffer behaved the same as noninjected ones: they remained mitotically arrested for at least 4 h after nocodazole treatment, indicating no detectable effect of microinjection per se (Table I).
Next, we determined whether the effect of XCL100 on mitotic arrest was dependent on its phosphatase activity. We injected cells with purified recombinant XCL100 C260S protein, the inactive mutant form, at a needle concentration of 1.5 µM. All cells (17/17) injected with XCL100 C260S protein maintained normal mitotic arrest for at least 4 h when treated with nocodazole (Fig. 5; Table I). This result indicates that the effect of XCL100 on the spindle assembly checkpoint is dependent on its phosphatase activity.
We also set out to determine whether any active Erk1 or
Erk2 was present in nocodazole-arrested XTC cells, as
should be the case if the effects of XCL100 were due to inactivation of one of these MAP kinases. We prepared lysates from asynchronous XTC cells, from cells incubated
for various lengths of time (15 min through 7 h) with nocodazole, and from cells shaken off of a plate of cells that
had been treated with nocodazole for 16 h. We subjected these samples to immunoblotting with an antiserum that
recognizes active Erk1 and Erk2 (Promega). Small amounts
of active Erk2 were detected in all of the nocodazoletreated samples (~10% of the total Erk2), and no active
Erk1 was detected (data not shown). The presence of
some active Erk2 in the nocodazole-treated cells is compatible with the hypothesis that XCL100's effects on mitotic arrest were due to inactivation of Erk2. However,
similar amounts of active Erk2 were detected in untreated,
asynchronous cells (data not shown). In contrast, Minshull
et al. (1994)
found marked differences in the activity of
Erk2 in interphase vs mitotically arrested cycling extracts.
Thus, mitotic activation of MAP kinase is either more subtle or more difficult to detect in intact XTC cells than it is
in extracts.
XCL100 C260S can bind avidly to phosphorylated Erk2,
and therefore could potentially interfere with MAP kinase
function by preventing MAP kinase from interacting with
its substrates (Sun et al., 1993). However, it seems unlikely
that this type of inhibition would occur to any significant
extent at the concentration of XCL100 C260S we have
injected in our studies. Sun et al. (1993)
found that expression of the C258S mutant of mouse Mkp-1 (the mouse
homolog of XCL100) in COS cells at levels substantially greater than the levels of Erk2 resulted in the coprecipitation of ~30% of the cells' Erk2 with the Mkp-1 C258S.
However, in our case, the concentration of microinjected
XCL100 C260S was ~20-fold lower than the concentration of Erk2 (75 nM vs 1-2 µM). Thus, we would expect
our injected phosphatases to be able to inhibit MAP kinase function only by dephosphorylation, not by sequestration. This is consistent with our finding here that XCL100
interfered with mitotic arrest, and XCL100 C260S did not.
Similarly, we have found that microinjection of wild-type
XCL100 mRNA inhibits progesterone-induced oocyte maturation, whereas XCL100 C260S does not (Sohaskey, M.L., unpublished data).
The Effect of XCL100 on Mitotic Arrest Is Due to Its Effects on p42 or p44 MAP Kinase
We set out to determine whether the effect of XCL100 on
mitotic arrest was due to its effect on p42/p44 MAP kinase,
p38 Hog1, or some other as yet unidentified target. Our
approach was to coinject XCL100 with a constitutively active MAP kinase kinase (MKK-1*) (Mansour et al., 1994).
MKK-1* has no direct effect on XCL100 but exerts an effect on MAP kinase that is opposite to the effect of
XCL100: it phosphorylates the same residues that XCL100
dephosphorylates. MKK-1 is a highly specific protein kinase. It does not phosphorylate p38 Hog1, and biochemical and genetic studies have thus far uncovered no evidence for the existence of substrates of MKK-1 other than
p42 and p44 MAP kinase (Cano and Mahadevan, 1995
;
Ferrell, 1996a
; Lin et al., 1995
; Rouse et al., 1994
). Thus, if
MKK-1* were found to antagonize the effect of XCL100
on mitotic arrest, this would be strong evidence that XCL100's effects on mitotic arrest were due to its effect on
p42 or p44 MAP kinase.
We coinjected XCL100 (needle concentration 1.5 µM) with MKK-1* (needle concentration 5 µM; based on in vitro assays [Fig. 4 C], this concentration should substantially counteract the effects of the coinjected XCL100) into XTC cells at metaphase or prometaphase, and then immediately treated the cells with nocodazole. We found that the injected cells (8/8) did not exit mitosis; they remained blocked at pseudo-prometaphase stage with scattered chromosomes for at least 4 h (Fig. 5; Table I). These findings indicate that MKK-1* inhibited or overcame the effects of XCL100 on mitotic arrest. This supports the hypothesis that the effects of XCL100 on mitotic arrest are due to its effects on p42 or p44 MAP kinase, rather than to some other possible target.
Finally, we assessed whether MAP kinase activation was sufficient to bring about metaphase arrest. We addressed this question by injecting MKK-1* alone (needle concentration 10 µM) into mitotic XTC cells. We found that microinjection of this protein did not bring about a mitotic arrest. This finding suggests either that MAP kinase activation is not sufficient for mitotic arrest, or that we were unable to achieve a high enough level of MAP kinase activation to bring about mitotic arrest.
Spindle Localization of MAP Kinase Provides a Clue to Its Role in Mitosis
Several workers have previously examined the localization
of the Erk1 and Erk2 MAP kinases in interphase tissueculture cells. Erk1/Erk2 have been reported to be distributed in a diffuse (Dikic et al., 1994; Gonzalez et al., 1993
;
Lenormand et al., 1993
; Traverse et al., 1994
) or diffusely
speckled (Chen et al., 1992
) pattern in a variety of quiescent cells and to translocate to the nucleus when the cells
are treated with mitogens. However, recent evidence indicates that, in at least some cell types, MAP kinase may concentrate on or around microtubules (Morishima-Kawashima and Kosik, 1996
; Reszka et al., 1995
). Here we
found that, in interphase XTC cells, the Erk1/Erk2 distribution is diffuse; we saw no examples of microtubule-like staining in these cells.
When XTC cells entered M phase, some MAP kinase immunofluorescence was found to concentrate in the spindle. This could represent MAP kinase translocating to the spindle or, possibly, microtubule-associated MAP kinase undergoing some change in association that made it more accessible to antibody staining. MAP kinase spindle staining was strongest during metaphase and decreased by anaphase, and MAP kinase staining was absent from the cytokinesis midbody.
In some ways, the changes in MAP kinase localization
during mitotic M phase reported here are similar to those
reported by Verlhac et al. (1993) and Choi et al. (1996)
for
meiotic M phase. Verlhac et al. (1993)
found MAP kinase
to concentrate at the spindle poles in mouse oocytes in
meiotic M phase and to become more diffuse in anaphase.
This is similar to our finding, except that we found MAP
kinase to be present throughout the spindle rather than
just at the spindle poles. Choi et al. (1996)
found MAP kinase to concentrate in the miniasters that form near the
chromosomes in mouse oocytes in early meiotic M phase,
again reminiscent of the spindle localization we found.
The presence of MAP kinase in the mitotic spindle puts
it in a position either to monitor the status of the spindle,
or to regulate spindle-associated proteins, or both. It also
places MAP kinase in the proximity of key cell cycle regulators. In particular, cyclin B1/Cdc2 complexes appear to be
largely spindle associated during M phase, and a fraction
of the cyclin B2 is as well (Jackman et al., 1995; Ookata et al.,
1992
, 1993
, 1995
; Pines and Hunter, 1991
; Rattner et al.,
1990
).
Microinjection of MAP Kinase Phosphatase Has No Apparent Effect on Mitotic Progression
Erk2 becomes activated concomitantly with Cdc2 during
oocyte maturation, and manipulations that inhibit the activation of MAP kinase (Gotoh et al., 1995; Huang and Ferrell, 1996a
; Kosako et al., 1994b
) or its upstream activators
(Freeman et al., 1990
; Sagata et al., 1988
) inhibit Cdc2 activation. In addition, microinjection of oocytes with constitutively active MAP kinase (Haccard et al., 1995
), constitutively active Mek-1 (a MAP kinase kinase; Huang et al.,
1995
), constitutively active Raf-1 (a MAP kinase kinase kinase; Muslin et al., 1993
), or Mos (a MAP kinase kinase kinase; Yew et al., 1992
) brings about Cdc2 activation. These
findings place Erk2 upstream of Cdc2 during meiosis and
raise the possibility that MAP kinases may be important
for Cdc2 activation at the onset of mitosis as well. It has
also been shown that purified Erk2 can cause centrosomenucleated microtubules to change from interphase-like stability to M-phase-like instability (Gotoh et al., 1991b
). Thus, MAP kinase could be involved in the changes in microtubule dynamics that occur during both meiosis and mitosis.
However, there is conflicting evidence on whether Erk1
or Erk2 is activated during mitotic M phase (Edelmann et
al., 1996; Heider et al., 1994
; Tamemoto et al., 1992
), and
the question of whether Erk1 or Erk2 function is required
for entry into or progression through mitotic M phase has
not been previously addressed. In the present study we detected small amounts of active Erk2 in M-phase cells but
found no indication that MAP kinase function was important for normal mitotic progression; microinjection of the
MAP kinase phosphatase XCL100 had no obvious effect.
We cannot rule out the possibility that microinjection of a
higher concentration of XCL100, or microinjection of
XCL100 before prophase, might alter mitotic progression.
MAP Kinase Function Is Required for the Spindle Assembly Checkpoint
Previous work demonstrated that Xenopus egg extracts
supplemented with nuclei arrest in a mitotic state when
treated with nocodazole, and that addition of mouse MAP
kinase phosphatase (Mkp-1) to these extracts releases them
from mitotic arrest (Minshull et al., 1994). These findings
suggest a role for MAP kinase in the spindle assembly checkpoint, provided the reconstituted extract system faithfully
recapitulates the checkpoint controls found in intact cells.
Here we present strong evidence that MAP kinase function is required for the spindle assembly checkpoint in intact XTC cells. Microinjection of active XCL100 during prometaphase or metaphase (but not prophase) abrogates the normal spindle assembly checkpoint, whereas microinjection of saline or an engineered inactive XCL100 mutant protein does not. Coinjection of MKK-1*, a constitutively active MAP kinase kinase, abolishes the effect of XCL100 on the spindle assembly checkpoint. This finding argues that the relevant target of XCL100 is p42 or p44 MAP kinase, rather than p38 Hog1 or some adventitious target. It is also possible that the relevant target is some as yet unidentified protein that can be regulated by both XCL100 and MKK-1*.
Previous work has also shown that MAP kinase function
is required for the metaphase arrest that occurs naturally
during the maturation of frog oocytes (Haccard et al., 1993;
Kosako et al., 1994a
; Minshull et al., 1994
); i.e., MAP kinase is an essential mediator of "cytostatic factor" function.
Taken together with Minshull et al.'s studies of the spindle
assembly checkpoint in extracts and the present studies of
the spindle assembly checkpoint in intact cells, it appears
that M-phase arrest in a variety of biological contexts depends upon MAP kinase function. It is therefore possible
that studies of any one of these systems might yield insights into all of them.
At present, the mechanism through which MAP kinase
mediates the spindle assembly checkpoint is unknown. The
possibilities can be organized into three general classes of
mechanism. (a) MAP kinase could regulate the machinery
that detects spindle misassembly or chromosome misalignment. (b) MAP kinase could regulate the cyclin destruction machinery. For example, MAP kinase could regulate whatever specific ubiquitin-conjugating enzymes are responsible for ubiquitinating cyclins B1 and B2. (c) MAP
kinase could convert cyclins B1 and B2 to phosphorylated,
nondestructible forms. This possibility is supported by the
observation that cyclin B1 is a good substrate for p42 MAP
kinase (Izumi and Maller, 1991). Cyclin B2 is not a good
substrate for Erks 1 or 2, but Minshull et al. have demonstrated the existence of one or more cyclin B2 kinase activities that appear to be MAP kinase dependent (Minshull et al., 1994
).
Likewise, it is not understood what lies upstream of MAP kinase in the spindle assembly checkpoint. It is unclear whether the checkpoint makes use of known MAP kinase kinases (MKK-1/Mek-1, MKK-2/Mek-2) and known MAP kinase kinase kinases (Mos, Raf-1, A-Raf, and B-Raf), or whether there are unknown MAP kinase cascade components used specifically for this function. Our hope is that through a combination of in vivo analysis, like that described here, and examination of in vitro systems, as described previously, it may be possible to delineate the signaling pathways used in the spindle assembly checkpoint in detail.
Received for publication 18 October 1996 and in revised form 24 January 1997.
1. Abbreviations used in this paper: DAPI, 4We thank Mike Sohaskey, Chi-Ying Huang, Natalie Ahn, Melanie Cobb, Jon Cooper, Jim Posada, and Tom Geppert for providing plasmids and recombinant proteins, and for helpful discussions; Tim Stearns for XTC cells, helpful discussions, and the use of his immunofluorescence microscope; and Andrew Murray for helpful discussions. We especially thank Tim Mitchison, Claire Walczak, Louise Cramer, and members of the Mitchison laboratory for their hospitality and assistance in the microinjection studies.
This work was supported by a grant from the National Institutes of Health (GM46383) and a Howard Hughes Junior Faculty Scholars award (to J.E. Ferrell, Jr.), and by a postdoctoral fellowship from Cancer Biology training grant CA09302 to X.M. Wang.