School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK
* Author for correspondence (e-mail: stephen.taylor{at}man.ac.uk)
Accepted 8 June 2002
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Summary |
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Key words: Mitosis, Kinetochore, Cenp-F, Protein farnesylation, Farnesyl transferase inhibitor
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Introduction |
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Chromosome segregation in mitosis depends on kinetochores, complex protein
structures that assemble at the centromeres of chromosomes
(Rieder and Salmon, 1998).
Kinetochores not only tether and move chromosomes along microtubule fibres but
they also play a central role in the checkpoint mechanism that delays anaphase
onset until all the chromosomes achieve correct, bipolar attachments
(Amon, 1999
). Although a number
of kinetochore components have been identified, their precise roles are only
partially defined (Maney et al.,
2000
; Pidoux and Allshire,
2000
). Cenp-F was first identified as a human autoantigen that
localises to kinetochores during mitosis
(Rattner et al., 1993
). During
interphase Cenp-F is part of the nuclear matrix but upon entry into mitosis
Cenp-F is released into the cytosol and a sub-pool becomes detectable at
kinetochores (Casiano et al.,
1993
; Liao et al.,
1995
; Zhu et al.,
1995a
). Cenp-F remains kinetochore associated until anaphase onset
when it re-localises to the spindle midzone. Thus, Cenp-F belongs to the
family of `chromosome passenger' proteins
(Earnshaw and Bernat, 1991
).
Cenp-F is a cell-cycle-regulated protein, reaching maximum levels in G2/M
followed by rapid degradation after mitosis
(Liao et al., 1995
;
Zhu et al., 1995a
). The 3210
amino acid protein is mainly coiled coil sequence, containing two internal
repeats, several leucine heptad repeats and a bipartite nuclear localisation
sequence. Expression of epitope-tagged deletion mutants indicates that the
kinetochore localisation domain resides in the C-terminal region of the
protein (Zhu et al., 1995b
).
Yeast two hybrid screens show that the C-terminal domain of Cenp-F is also
capable of interacting with itself, the kinetochore-associated kinesin-related
motor protein Cenp-E, and the spindle checkpoint component Bub1
(Zhu et al., 1995b
;
Chan et al., 1998
;
Jablonski et al., 1998
).
Although these observations suggest that Cenp-F may play a role in kinetochore
assembly and/or the spindle checkpoint, the function of Cenp-F remains
obscure. Significantly, Cenp-F and Cenp-E end with CAAX farnesylation
sequences and recently both have been shown to be farnesylated
(Ashar et al., 2000
). These
observations raise the possibility that the G2/M effects induced by FTIs are
due to inhibition of Cenp-E and Cenp-F.
To determine whether Cenp-F plays a role in the spindle checkpoint we generated cell lines expressing a C-terminal Cenp-F mutant. Rather than compromising spindle checkpoint function, expression of this mutant delays progression through G2/M. Significantly, this effect requires an intact CAAX farnesylation motif. We also show that the CAAX motif and farnesyl transferase activity are required for Cenp-F localisation to kinetochores, the nuclear envelope at the G2/M transition and for its degradation after mitosis. Not only do these observations highlight new roles for farnesylation as a signal for cell-cycle-regulated protein localisation and degradation but they also suggest that FTI-induced G2/M delays may be due, at least in part, to Cenp-F dysfunction.
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Materials and Methods |
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Retroviral plasmids
The retroviral plasmid pLPCX (Clontech) was modified to include the
5' untranslated sequence from the human lamin A cDNA and an N-terminal
myc tag (Taylor and McKeon,
1997) to create pLPCX-Myc. This plasmid was used as the empty
vector control. cDNA fragments encoding C630 and the CAAX mutants were
generated by PCR amplification of a Cenp-F cDNA, obtained from Don Cleveland
(University of California at San Diego), cloned into pLPCX-Myc and sequenced.
The GFP open reading frame was generated by PCR amplification of pGFPemd-N1[F]
(Packard) and N-mBub1 was as previously described
(Taylor and McKeon, 1997
). All
plasmids were purified by ion exchange chromatography (Qiagen).
Antibody production
To create the sheep polyclonal anti-Cenp-F antibody, SCF.1, a central
portion of Cenp-F encoding amino acids 1363-1640 was cloned as a
BamHI/NotI fragment into pGex-4T-3 (Pharmacia) and
transformed into the E. coli strain BL21. Expression and purification
of the GST-fusion protein, sheep immunisation and affinity purification were
all carried out as described (Taylor et
al., 2001).
Transfections and retroviral infections
Transient transfections of BHK and HeLa cells were carried out as described
(Taylor et al., 2001). For
retroviral infections, approximately 1x107 Phoenix-Ampho
cells were transfected in 10 cm dishes with 27 µg of the pLPCX-Myc-based
vectors using the ProFection calcium phosphate transfection kit (Promega). DNA
precipitates were washed away after 24 hours then, 48 hours post transfection,
the tissue culture supernatant was harvested, filtered through a 0.22 µm
filter (Millipore) and diluted 2:3. Approximately 1x106
TA-HeLa cells in 10 cm dishes were then infected for 24 hours in the presence
of 8 µg/ml polybrene (Sigma). Virus containing media was then removed and,
48 hours after infection, the cells were expanded into three 10 cm dishes and
cultured in the presence of 0.5 µg/ml puromycin (Clontech). After 4 days of
selection this procedure typically resulted in 3x107
puromycin-resistant cells when using pLPCX-Myc. These cells were then expanded
for immediate analysis or frozen. To determine the expression efficiency,
puromycin resistant HeLa cells infected with control and GFP viruses were
analysed by flow cytometry on a FACScan (Becton Dickinson).
Cell cycle analysis
To determine relative cell number, 1.25x104 cells were
plated in 12-well dishes and then fixed in 4% formaldehyde at the times
indicated. Following washes in PBS, cells were stained with 0.1% crystal
violet for 30 minutes and then washed with several volumes of water. Bound dye
was then extracted with 10% acetic acid and the optical density at 590 nm was
determined. Within an experiment, each point was determined in triplicate and
each growth curve was performed at least twice. Synchronisations, DNA content
and mitotic index measurements were all carried out as described
(Taylor et al., 2001).
Immunofluorescence and western blotting
Cell fixation, labelling and fluorescence microscopy were all carried out
as described (Taylor et al.,
2001) using the following primary antibodies: mouse anti-Myc tag
(9E10, 1:300); mouse anti-tubulin (TAT-1, 1:100); sheep anti-Cenp-F (SCF.1,
1:2500); mouse anti-lamin B2 (Zymed, 1:50); rabbit anti-phospho-histone H3
(Upstate Biotech, 1:700). Secondary antibodies used were either Cy2- or Cy3-
conjugated donkey anti-rabbit, anti-mouse or anti-sheep antibodies (Jackson
Immunoresearch) diluted 1:1000. Preparation of cell extracts and western
blotting were all carried out exactly as described
(Taylor et al., 2001
) using
the following primary antibodies: sheep anti-Cenp-F (SCF.1, 1:2500); mouse
anti-Myc tag (9E10, 1:300); mouse anti-tubulin (TAT-1, 1:100).
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Results |
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Expression of C630 delays entry into mitosis but does not prevent
chromosome segregation
The observations described above show that expression of C630 delays but
does not block G2/M progression. Because the mitotic index of the control and
C630 cultures was not significantly different (not shown) we reasoned that
C630 was inducing a G2 rather than a mitotic delay. To test this we measured
the mitotic index of control and C630 cultures following exposure to the
microtubule toxin nocodazole. We reasoned that if C630 delayed progression
through G2, the accumulation of mitotic cells would be slower compared with
that observed in a control population. In contrast, if expression of C360 had
no effect on mitotic entry but delayed progression through mitosis, mitotic
cells would accumulate at a similar rate in both control and C630 cultures.
Fig. 3 shows that the mitotic
index of either asynchronous or synchronised populations of cells expressing
C630 is reduced relative to control cells in the presence of nocodazole
(Fig. 3A,B), suggesting that
expression of C630 does indeed delay progression through G2 rather than
mitosis. To determine whether expression of C630 had an effect on spindle
assembly or chromosome segregation we compared mitotic cells in both control
and C630 cultures. The number of metaphases and anaphases did not appear
different (Fig. 3C) and the
spindle morphology did not appear different
(Fig. 3D). While we cannot rule
out the possibility that expression of C630 has a subtle effect on mitotic
chromosome alignment and segregation, these observations are more consistent
with the notion that C630 delays progression through G2 and/or the onset of
mitosis.
|
The CAAX motif of C630 is required for delaying progression through
G2/M
Recently, Cenp-F has been shown to be farnesylated
(Ashar et al., 2000). In
addition FTIs inhibit proliferation and can delay G2/M progression in some
cell lines (Vogt et al., 1997
;
Ashar et al., 2001
). Based on
these and our observations described above, we formulated the following
hypothesis. We reasoned that perhaps Cenp-F binds to some unknown protein(s)
and/or membrane structure in a farnesylation-dependent manner and that this is
required for timely progression through G2/M. Thus, when farnesylation of
Cenp-F is inhibited by FTIs this interaction is less efficient resulting in a
G2/M delay. Likewise expression of C630, which contains the CAAX motif and
should therefore be an efficient farnesylation substrate, should compete for
this interaction, thus also delaying G2/M progression. This hypothesis
predicts that if the CAAX motif in C630 is mutated to prevent farnesylation it
should be a less efficient competitor and thus not delay G2/M.
Therefore, based on mutations that inhibit farnesylation of Ras
(Kato et al., 1992) we mutated
the CAAX motif in C630, replacing the cysteine and valine with a serine and
aspartic acid, creating C630 C:S and C630 V:D, respectively. We also deleted
the CAAX motif (C630
4) and the C-terminal 150 amino acids (C630
150). HeLa cells were then infected in parallel with viruses encoding
C630, the four CAAX mutants and a control virus. Puromycin-resistant cells
were selected, expanded and all six lines analysed in parallel. Significantly,
cells expressing the CAAX mutants grew better than cells expressing C630
(Fig. 4A). Whereas cells
expressing C630 doubled every 3 days, cells expressing the CAAX mutants
doubled every 1.5 days and cells infected with the control virus doubled
approximately every day. DNA content histograms of asynchronous cultures
showed that cells expressing the CAAX mutants did not accumulate in G2/M to
the same extent as those expressing C630
(Fig. 4B). To investigate cell
cycle timing in more detail, cells were released from a G1/S block, harvested
every 3 hours and then analysed by flow cytometry to determine DNA content. To
facilitate comparison of all six lines we calculated the 4n/2n DNA content
ratio at each time point. In all cases the 4n/2n ratio increased following
release as the cells progressed into G2/M, and then decreased after 9-12 hours
as the cells completed mitosis and returned to G1
(Fig. 4C). At 9 and 12 hours,
the 4n/2n ratio of the C630 culture was markedly higher than that of the
control cells, consistent with G2/M delay. Significantly, however, the CAAX
cultures exhibited 4n/2n ratios more typical of the control cells, indicating
that expression of the CAAX mutants does not delay G2/M progression as
markedly as C630. In addition, cells expressing the CAAX mutants behaved more
like control cells rather than C630-expressing cells when exposed to
nocodazole over a 30-hour period (Fig.
3A).
|
While these observations suggest that the phenotype induced by expression of C630 does indeed require an intact CAAX sequence, mutation of the CAAX motif does not appear to completely alleviate the C630 phenotype. This raises the possibility that mutation of the CAAX motif simply leads to lower expression levels and/or protein misfolding. However, immunoblotting of whole cell lysates prepared from all six lines shows that mutation of the CAAX motif does not result in lower expression levels (Fig. 4D). Furthermore, immunofluorescence of late G2 cells shows that the CAAX mutants localise to the nuclei in the vast majority of cells (Fig. 4E), demonstrating that mutation of the CAAX motif does not result in cytoplasmic aggregation of C630. Thus, as predicted by the hypothesis outlined above, these observations suggest that the phenotype induced by expression of C630 does indeed depend to a large extent on an intact CAAX motif.
Cenp-F localises to the nuclear envelope at the G2/M transition
To determine whether C630 disrupts the localisation of endogenous Cenp-F we
first generated a polyclonal antibody, SCF.1, against a central portion of
Cenp-F (Fig. 1A). Consistent
with previous observations indicating that Cenp-F is part of the nuclear
matrix in interphase but mainly cytosolic in mitosis
(Casiano et al., 1993;
Liao et al., 1995
), SCF.1
detects a single protein significantly greater than 200 kDa that remains in
the cell pellet following mild detergent extraction of interphase cells
(Fig. 1C). Following similar
extraction of mitotic cells this protein is present in the supernatant. SCF.1
stains the nucleus of some but not all interphase cells
(Fig. 5A,B) consistent with
cell cycle regulation of Cenp-F (Liao et
al., 1995
). In prometaphase, SCF.1 stains the cytoplasm and
kinetochores (Fig. 5A,C). Taken
together, these observations suggest that SCF.1 specifically recognises
Cenp-F.
|
When detectable, Cenp-F localised exclusively to the nucleus in most
interphase cells. However, in interphase cells that showed early signs of
phospho-histone H3 staining, Cenp-F also localised to the nuclear periphery
(Fig. 5B). In prophase cells
this peripheral localisation was more pronounced
(Fig. 5D). Whereas previous
reports have suggested that this nuclear rim staining is because soluble
Cenp-F is excluded from the chromatin upon mitotic entry
(Rattner et al., 1993;
Liao et al., 1995
), our
observations are more consistent with the notion that Cenp-F localises to the
nuclear envelope in late G2 and early mitosis. Indeed, in very early prophase
cells where chromatin condensation is only just visible and only a few Bub1
foci are apparent, Cenp-F clearly localises to what appears to be the nuclear
envelope (Fig. 5E). To confirm
this we co-stained HeLa cells for Cenp-F and lamin B2.
Fig. 6A shows that Cenp-F
localises not only to the nuclear envelope around the nuclear periphery but
also to invaginations within the middle of the nucleus. However, note that
Cenp-F and lamin B2 do not appear to colocalise but rather localise to
different regions of the nuclear envelope resulting in alternating red and
green spots in the merged image (Fig.
6A,B). Because there is no sign of chromosome condensation and the
nucleoli are still intact, the cell shown in
Fig. 6A must be in late G2.
Therefore it seems unlikely that localisation of Cenp-F to the nuclear
envelope is simply due to exclusion from the chromatin following breakdown of
the nuclear matrix at the onset of chromosome condensation. Rather, a subpool
of Cenp-F appears to concentrate at the nuclear envelope in late G2, through
the G2/M transition and into early mitosis until envelope breakdown.
|
The CAAX motif of Cenp-F is required for nuclear envelope and
kinetochore localisation
To determine whether C630 disrupts the localisation of endogenous Cenp-F,
the six lines were stained to detect myc-tagged proteins and Cenp-F
(Fig. 7A and data not shown).
Cenp-F was readily detectable at nuclear envelopes and kinetochores in control
cells but not in cells expressing C630. Significantly however, Cenp-F was
frequently observed at nuclear envelopes and kinetochores in cells expressing
the CAAX mutants (Fig. 7A,B).
Furthermore, whereas C630 clearly localises to kinetochores, the C:S mutant
appears not to (Fig. 7A). To
confirm this, prometaphase cells were stained to detect myc-tagged proteins
and Bub1 then analysed by deconvolution microscopy
(Fig. 7C). C630 and Bub1
clearly co-localise to kinetochores resulting in yellow foci in the merged
image. In contrast, the C:S and V:D mutants do not co-localise with Bub1
resulting in separate red and green foci in the merged image. Thus, this
analysis suggests not only that an intact CAAX motif is required for
kinetochore localisation of C630 but also that the ability of C630 to disrupt
the localisation of endogenous Cenp-F depends on the CAAX sequence.
|
Farnesyl transferase activity is required for efficient localisation
of Cenp-F to the nuclear envelope and kinetochores
The simplest explanation for the observations described above is that the
CAAX farnesylation motif of Cenp-F is required for both nuclear envelope and
kinetochore localisation. To test whether farnesyl transferase activity is
required for correct localisation, we analysed the effect of the FTI SCH 66336
(Ashar et al., 2000) on Cenp-F
localisation. For these experiments we chose to use HeLa cells for the
following reason. Consistent with previous observations
(Ashar et al., 2001
), we
observed that culturing HeLa cells in SCH 66336 for more than 72 hours results
in a G2/M cell cycle delay (not shown). However, the cell cycle effects of SCH
66336 following a 24 hour exposure are minimal (not shown). Therefore, by
analysing HeLa cells that have been exposed to SCH 66336 for less than 24
hours we reasoned that we could analyse the effect of the FTI on Cenp-F
localisation independently of any effects on cell cycle progression.
While Cenp-F was detectable at both nuclear envelopes and kinetochores in the presence of the FTI, the frequency and intensity of Cenp-F staining appeared diminished (not shown). To investigate this further, we analysed HeLa cells cultured with and without the FTI following release from G1/S block (Fig. 8A). As cells progressed into G2 in the absence of the FTI, nuclear envelope staining became more frequent; after 10-12 hours, as the cells completed mitosis and returned to G1, nuclear envelope staining became less frequent. However, in the presence of FTIs the number of cells with Cenp-F detectable at the nuclear envelope was reduced by approximately 50%. To illustrate this, Fig. 8B shows two early prophase cells: the chromatin shows signs of condensation but the nucleoli are still distinct and the nuclear envelopes are intact as judged by the lamin B2 staining. However, while Cenp-F is detectable at the nuclear envelope in untreated cells, it is not apparent in the presence of the FTI (Fig. 8B). To investigate the effect on kinetochore localisation, FTI-treated HeLa cells were arrested in mitosis and then stained to detect Cenp-F and phospho-histone H3. In control cells, Cenp-F is detectable both in the cytoplasm and on the chromatin as discrete foci (Fig. 8C). In contrast, in the presence of the FTI, Cenp-F is only present in the cytoplasm and chromatin-associated foci are not apparent. Thus, these observations indicate that farnesyl transferase activity is required for efficient localisation of Cenp-F to both nuclear envelopes and kinetochores.
|
Farnesyl transferase activity is required for degradation of Cenp-F
after mitosis
Strikingly, after 24 hours in the presence of FTIs the vast majority of
HeLa cells stained positive for Cenp-F (not shown) consistent with them being
in G2 (Ashar et al., 2001).
However, because a 24 hour exposure to SCH 66336 has only minimal effect on
cell cycle progression in HeLa cells (see above), this observation raised the
possibility that inhibition of farnesyl transferase activity may disrupt
Cenp-F turnover. To test this, HeLa cells were released from a G1/S block in
the presence and absence of SCH 66336. After 15 hours, most cells had
completed mitosis and returned to G1 (Fig.
9A). In the absence of the FTI, Cenp-F levels were low in the vast
majority of cells (Fig. 9B),
consistent with Cenp-F being degraded after mitosis
(Liao et al., 1995
). The few
Cenp-F-positive cells were typically phospho-histone-H3-positive, indicating
that they were still in mitosis. In contrast, in the presence of the FTI the
vast majority of interphase, phospho-histone-H3-negative nuclei contained
Cenp-F (Fig. 9B). Furthermore,
immunoblotting of total cell lysates prepared from HeLa cells synchronised at
G1/S by a double thymidine block in the presence of the FTI contained high
levels of Cenp-F protein (Fig.
9C). In contrast, in the absence of the FTI, Cenp-F was not
detectable in G1/S suggesting that farnesyl transferase activity is indeed
required for Cenp-F degradation after mitosis.
|
To determine whether the CAAX motif is required for degradation we examined the expression levels of C630 and the CAAX mutants following release from a G1/S block. Both Cenp-F and C630 were not detectable immediately after release from a G1/S block but were clearly present in G2 cells 12 hours after release (Fig. 9D). In contrast, the C630 C:S mutant was present both in G1/S and G2 suggesting that the CAAX motif is required for the cell-cycle-regulated turnover of C630. Whether the C630 C:S protein present in G1/S cells is due to post-mitotic persistence of protein synthesised in the previous cell cycle or the failure to degrade newly synthesised protein remains to be seen. However, taken together, these results suggest that farnesylation of Cenp-F is required for its degradation after mitosis.
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Discussion |
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Farnesylation of Cenp-F is required for proliferation and progression
through G2/M
Expression of C630 inhibits both cellular proliferation and progression
through G2/M (Fig. 2).
Previously, transient transfection of CV-1 cells with plasmids expressing
C-terminal Cenp-F fragments resulted in the accumulation of cells in G2/M
(Zhu et al., 1995a). However,
because transient transfections are refractory to detailed cell cycle
analysis, the significance of this observation was unclear. In addition,
generation of stable cell lines was uninformative
(Zhu et al., 1997
), possibly
because the anti-proliferative effects resulted in clones with very low
expression levels. Here, we have used retroviral infections to overcome both
of these limitations. Importantly, the strategy employed typically results in
85-95% of the cells stably expressing the cDNA of interest at high levels
during the period in which the cell cycle analysis was performed (Figs
2,3,4).
Using this strategy, we have shown that both the proliferation defect and the
G2/M delay induced by expression of C630 depends on an intact CAAX
farnesylation motif (Fig. 4).
What can these observations tell us about the endogenous Cenp-F protein?
Clearly, any interpretations must take into account our experimental strategy,
namely the expression of truncation mutants. First, in the absence of a
clearly defined function for Cenp-F it is not clear whether expression of C630
exerts a dominant negative or a dominant active effect. In addition, owing to
technical constraints we have been unable to determine the expression levels
of C630 relative to endogenous Cenp-F. Furthermore, we cannot rule out the
possibility that expression of C630 interferes with proteins other than
Cenp-F, for example, by simply competing for farnesyl transferase activity.
Second, although mutation of the CAAX motif alleviates the phenotype induced
by expression of C630, we cannot rule out the possibility that these mutations
simply result in misfolding of the C630 protein, effectively producing a null
mutant. However, despite these caveats, the simplest explanation for our
observations is that C630 competes with Cenp-F for one or more CAAX-dependent
interactions, thus interfering with the function of the endogenous Cenp-F
protein. The observations based on expression of C630 and the CAAX mutants
are, however, only consistent with the notion that farnesylation of Cenp-F is
required for timely progression through G2/M, its localisation and its
degradation after mitosis. Nevertheless, the observations showing that FTIs
interfere with G2/M progression (Vogt et
al., 1997
; Ashar et al.,
2001
), the localisation and degradation of Cenp-F (Figs
8,
9) lend further support to this
hypothesis. Clearly, however, a more definitive test will require analysing
Cenp-F in a more genetically tractable system.
Mutation of the CAAX motif in C630 does not completely abolish the
proliferation and cell cycle effects suggesting that expression of C630 may
exert its effects via two different mechanisms. While one mechanism is clearly
CAAX dependent, the second mechanism appears to be CAAX independent. One
possible explanation for the CAAX-independent mechanism comes from the
observation that the C-terminal domain of Cenp-F has a large number of
potential phosphorylation sites (Liao et
al., 1995; Zhu et al.,
1995a
). In addition, Cenp-F has been shown to be phosphorylated in
mitosis (Zhu et al., 1995a
).
Therefore, overexpression of C630, and the CAAX mutants that still have many
potential phosphorylation sites, might compete for Cdk1 and/or other kinase
activities, and thus reduce the efficiency of certain phosphorylation events
required for mitotic entry. However, taking into account the observation that
C630 is expressed at much lower levels relative to the CAAX mutants
(Fig. 4D), the predominant
mechanism by which C630 inhibits proliferation and cell cycle progression
clearly depends on the CAAX motif.
FTIs can suppress tumour cell growth, both in cell culture and in animal
models, and consequently several FTIs are undergoing phase I clinical trials
(reviewed by Sinensky, 2000;
Crul et al., 2001
). However,
the molecular mechanisms underlying the anti-proliferative effects of FTIs
remain obscure. In particular, the cell cycle effects of FTIs are multiphasic:
while cells with Ha-Ras mutations accumulate in G1, other FTI-sensitive cells
accumulate in G2/M (Vogt et al.,
1997
; Ashar et al.,
2001
). The observation that both the anti-proliferative and G2/M
effects of C630 expression are CAAX dependent
(Fig. 4) suggests that
FTI-induced effects may be mediated, at least in part, by inhibiting
farnesylation of Cenp-F. In some cells that exhibit G2/M delays in response to
FTIs, chromosome alignment defects have been observed
(Crespo et al., 2001
). Thus,
the G2/M delay may be due to spindle checkpoint activation resulting in a
prolonged mitosis. In addition, taxol and FTIs can act synergistically to
induce mitotic arrest (Moasser et al.,
1998
), suggesting that FTIs may inhibit proteins required for the
correct attachment of kinetochores to microtubules. A key FTI target in these
cells is likely to be the kinesin-related motor protein, Cenp-E, which has
recently been shown to be farnesylated, and has previously been shown to be
required for both tethering chromosomes to microtubules and spindle checkpoint
signalling (Schaar et al.,
1997
; Wood et al.,
1997
; Chan et al.,
1999
; Abrieu et al.,
2000
; Ashar et al.,
2000
; Yao et al.,
2000
). Although inhibition of farnesyl transferase activity does
not appear to alter kinetochore localisation of Cenp-E, it does diminish the
affinity of Cenp-E for microtubules (Ashar
et al., 2000
; Crespo et al.,
2001
).
It is possible that FTI-induced chromosome alignment defects may be due to
inhibition of Cenp-F. Indeed, Cenp-F localises to kinetochores and can
interact with the spindle checkpoint protein Bub1. Furthermore, two Cenp-F
related proteins, Okp1 and HCP-1, are required for chromosome segregation in
S. cerevisiae and C. elegans, respectively
(Moore et al., 1999;
Ortiz et al., 1999
). However,
there is no evidence at present that Cenp-F plays a role in chromosome
alignment in human cells. Indeed, we have not observed any chromosome
segregation defects or mitotic delays in cells expressing C630. In contrast,
when treated with nocodazole, lines expressing C630 accumulate mitotic cells
slower than controls (Fig. 3)
which, taken together with our cell cycle analysis
(Fig. 2), is consistent with
either a delay in G2 progression or in the G2/M transition. Further evidence
that Cenp-F may play a role in G2 or the G2/M transition comes from our
observations that Cenp-F localises to the nuclear envelope in late G2
(Fig. 5). This localisation
appears to be dependent on an intact CAAX motif and farnesyl transferase
activity (Figs 6,
7). Thus, in some lines
FTI-induced G2/M delays may be due to inhibition of Cenp-F function in late G2
or at the G2/M transition.
Cenp-F, a link between kinetochores and the nuclear envelope
The CAAX motif of C630 is required for its ability to efficiently localise
to kinetochores and its ability to efficiently displace endogenous Cenp-F from
kinetochores (Fig. 7). In
addition, inhibition of farnesyl transferase activity reduces the levels of
Cenp-F at kinetochores (Fig.
8). However, it has previously been reported that Cenp-F remains
present at kinetochores following exposure to FTIs
(Ashar et al., 2000;
Crespo et al., 2001
). Indeed,
at high antibody concentrations we can detect Cenp-F at kinetochores in
FTI-treated cells (not shown). Furthermore, FTI treatment does not completely
abolish nuclear envelope localisation of Cenp-F
(Fig. 8). One reason why FTIs
might have only partial effects is that Cenp-F could be alternatively
prenylated in the presence of FTIs. However, this is unlikely because SCH
66336 appears to block prenylation of Cenp-F completely under conditions where
K-sRas is alternatively prenylated (Ashar
et al., 2000
). Farnesylated proteins are typically associated with
membranes and although Cenp-F localises to the nuclear envelope in late G2,
there is no evidence for membranes at kinetochores
(McEwen et al., 1998
).
Although we cannot rule out the possibility that Cenp-F needs to associate
first with the nuclear envelope in order to subsequently target the
kinetochore, we have no evidence that C630 localises to the nuclear envelope.
Yet, C630 does localise to kinetochores
(Fig. 7), suggesting that
nuclear envelope localisation is not a prerequisite for kinetochore
localisation. Thus, taking these observations together, we suggest that
farnesylation of Cenp-F is not absolutely required for kinetochore
localisation but rather increases the affinity of the protein-protein
interaction(s) responsible for Cenp-F localisation.
Interestingly, two nucleoporins, hNup133 and hNup107, localise to
kinetochores in mitosis (Belgareh et al.,
2001). In addition, the kinetochore attachment checkpoint
components Mad1 and Mad2 localise to nuclear pores in interphase
(Campbell et al., 2001
). While
the significance of these observations is unclear it is possible that, by
first localising to the nuclear envelope, Cenp-F mediates the recruitment of
these proteins to kinetochores upon entry into mitosis. Therefore, a key
question is how is the localisation of Cenp-F coupled with cell cycle
progression? Perhaps the tight binding of Cenp-F to the nuclear matrix may be
a sequestration mechanism that prevents it from interacting with the nuclear
envelope in interphase. Alternatively, Cenp-F is phosphorylated in G2/M
(Zhu et al., 1995a
) and
whereas the C-terminal 215 amino acids contain nine SP/TP motifs, the
remainder of the protein (2995 amino acids) has only four. Therefore,
hyperphosphorylation may expose the C-terminus, thus promoting
farnesylation-mediated interactions. In addition, the rapid degradation of
Cenp-F after mitosis may then liberate binding partners thus allowing them to
reassociate with the nuclear envelope in G1.
Farnesylation as a signal for cell-cycle-regulated proteolysis
Ubiquitin-mediated proteolysis is required to drive cell cycle progression.
In mitosis, the anaphase-promoting complex or cyclosome (APC/C), an E3
ubiquitin ligase that targets anaphase inhibitors and mitotic cyclins for
degradation, is activated by two additional subunits, Cdc20 and Hct1/Cdh1,
which regulate early and late mitotic events, respectively (reviewed by
Morgan, 1999). Previous
studies show that Cenp-F is rapidly degraded shortly after mitosis
(Liao et al., 1995
). While the
mechanism by which Cenp-F is degraded is unknown, induction of a Cdh1
transgene in human cells leads to loss of Cenp-F protein
(Sorensen et al., 2000
),
suggesting that Cdh1-APC/C may target Cenp-F for ubiquitin-mediated
proteolysis. Consistently, Cenp-F contains several KEN sequences that target
proteins for Cdh1-APC/C-mediated degradation
(Pfleger and Kirschner,
2000
).
Our observations also suggest that farnesyl transferase activity is
required for degradation of Cenp-F after mitosis
(Fig. 9). While it is possible
that this is an indirect effect due to inhibition of the proteolysis
machinery, we feel this is unlikely as cell cycle progression is largely
unaffected following treatment of HeLa cells with SCH 66336 for up to 24 hours
(D.H. and S.S.T., unpublished). Our analysis of C630 suggests that
farnesylation of Cenp-F may be directly required for its degradation. The
expression profile of C630, which importantly contains two KEN sequences,
mirrors that of endogenous Cenp-F. C630 levels are low during G1/S, speak in
G2/M and then fall after mitosis (Fig.
2). Significantly, mutation of the CAAX motif renders C630
insensitive to this cell cycle regulation
(Fig. 9). Farnesylation alone
is unlikely to be a direct signal for degradation as other farnesylated
proteins are relatively stable (Crul et
al., 2001). Therefore, perhaps only in concert with other
degradation signals, such as KEN motifs, does farnesylation contribute to
targeting proteins for proteolysis.
Interestingly, the cell cycle effects induced by FTIs are often not
immediate. Rather there is a gradual decrease in proliferation over 5-7 days
and significant cell cycle effects are typically apparent only after 2-3 days
(Ashar et al., 2001;
Crul et al., 2001
). Thus, it
is possible that the proliferation defects caused by FTIs are not due to
immediate dysfunction of farnesylated proteins but rather due to the failure
to degrade proteins such as Cenp-F. Finally, Cenp-F has been used as a
proliferation marker both in hematopoietic and solid tumours
(Landberg et al., 1996
). Our
observations now open up a new opportunity for using Cenp-F accumulation as a
marker to determine whether FTIs effectively inhibit farnesyl transferase
activity in animal models and patients.
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Acknowledgments |
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