1 Department of Molecular Biology, Umeå University, Sweden
2 Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA
* Author for correspondence (e-mail: Martin.Gullberg{at}molbiol.umu.se)
Accepted 2 October 2002
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Summary |
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Key words: Microtubule, Phosphoprotein, Oncoprotein 18, GTP Phosphohydrolase
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Introduction |
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Op18 is a cytosolic protein that promotes transition from a growing to a
shrinking MT; this transition is referred to as a catastrophe. Op18 appears to
lack a defined tertiary structure in solution but can be divided into three
distinct regions, namely a largely unstructured N-terminus and a tandem repeat
of two weakly homologous -helical regions (see
Fig. 1A)
(Gigant et al., 2000
;
Wallon et al., 2000
). Op18
binds soluble tubulin, and the overall shape of the complex has been revealed
by transmission electron microscopy and a low-resolution X-ray structure
(Gigant et al., 2000
;
Steinmetz et al., 2000
). The
Op18-tubulin complex can be described as two tubulin heterodimers in a
slightly curved head-to-tail alignment (i.e. tandem-tubulin dimers) with each
one of the tandem Op18 helical repeats contacting one heterodimer. These
tandem tubulin dimers are also stabilized by longitudinal
and ß
tubulin subunit interactions. In accordance with these cooperative
interactions within the ternary complex, Op18 binds two tubulins according to
a two-site positive cooperative model, which minimizes complexes with single
tubulin heterodimers (Larsson et al.,
1999
). The 4 Å resolution X-ray structure allowed
unambiguous alignment of the relative locations of the subunits and thus
rejection of alternative models of the complex
(Segerman et al., 2000
;
Wallon et al., 2000
). The
N-terminus was not visible in the 4 Å structure, and the resolution was
insufficient to resolve the orientation of the extended Op18 helix relative to
the tandem tubulin dimers. However, it was recently shown that a peptide
corresponding to the N-terminal 10 residues of Op18 can be cross-linked close
to helix 10 of
-tubulin, which is involved in both longitudinal and
lateral contacts within the MT lattice
(Muller et al., 2001
). This
suggests that the N-terminus of Op18 is located at the
-tubulin subunit
end of the tandem tubulin dimers.
|
The ß-tubulin subunit of the heterodimer contains an exchangeable GTP
site, termed the E site, and hydrolysis at the E site provides the energy that
drives the dynamic properties of MTs. Within the MT polymer, hydrolysis of the
E-site-bound GTP of ß-tubulin is believed to be triggered by a catalytic
Glu residue in a loop on the preceding -tubulin subunit
(Nogales, 2001
). We have shown
that Op18-tubulin complex formation inhibits nucleotide exchange and
stimulates GTP hydrolysis (Larsson et al.,
1999
). On the basis of these data and the observed longitudinal
arrangement of the two tubulin heterodimers, it has been proposed that Op18
stimulates tubulin GTPase activity by promoting interactions between the E
site of ß-tubulin and the catalytic loop on
-tubulin within the
tandem tubulin dimer complex (Steinmetz et
al., 2000
).
Three major models have been proposed for how Op18 destabilizes MTs, namely
that (i) Op18 acts as a pure tubulin-sequestering protein
(Curmi et al., 1997;
Jourdain et al., 1997
), (ii)
Op18 is a specific catastrophe promotor
(Belmont and Mitchison, 1996
)
and (iii) Op18 mediates at least two distinct activities, namely catastrophe
promotion, which requires the N-terminus of Op18, and a tubulin-sequestering
activity observed in vitro, which requires both of the helical repeats but not
the N-terminus (Howell et al.,
1999b
; Larsson et al.,
1999
). Recent structural data readily explain how the two helical
repeats of Op18 form a tubulin-sequestering complex
(Gigant et al., 2000
). However,
the role of the unstructured N-terminus is still elusive but, given that three
out of four Ser phosphorylation sites are located in this region, it has been
speculated that the N-terminus is of regulatory importance (for a review, see
Lawler, 1998
). Consistently,
Op18 activity is switched off by either multi-site phosphorylation during
mitosis (Larsson et al.,
1997
), which is essential to allow cell division
(Marklund et al., 1996
), or in
response to single or dual phosphorylation by two distinct kinase systems
during interphase of the cell cycle (Gradin
et al., 1998
; Melander Gradin
et al., 1997
).
Here we demonstrate phosphorylation-regulated autonomous tubulin-directed activities by the N-terminus of Op18. The data suggest that autonomous N-terminal interactions with tubulin have a different functional outcome from formation of a sequestering ternary Op18-tubulin complex by the first and second helical repeats of Op18. Thus, the results provide a framework for better understanding of the reported functional dichotomy of Op18.
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Materials and Methods |
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Assays of tubulin GTPase activity
Analysis of tubulin GTPase activity was performed in PEM buffer adjusted to
pH 6.8 with NaOH (80 mM piperazine-N,N'-bis[2-ethanesulfonic acid], 1 mM
EGTA, 4 mM Mg2+) containing 5 mM adenyl-5'-yl
imidodiphosphate (AMP-PNP; to inhibit non-specific ATPase activity) as
described previously (Segerman et al.,
2000). In brief, tubulin (TL238, Cytoskeleton, Inc) was incubated
with [
-32P]GTP, the resulting
tubulin-
-[32P]GTP complexes recovered by centrifugation
through a desalting column (P-30 Micro Bio-Spin, Bio-Rad) and single-turnover
GTP hydrolysis followed at 37°C for up to 40 minutes. Control experiments
showed that the Op18 preparations used neither bind nor hydrolyze
[
-32P]GTP or cause dissociation of tubulin bound
[
-32P]GTP. Nucleotide hydrolysis was quantified by ascending
chromatography as described previously
(Segerman et al., 2000
), which
allows reproducible analysis of less than 0.2% hydrolysis of
[
-32P]GTP.
Transfection of the KA8 subclone derived from human K562
erythroleukemia cells
The KA8 cell line, a transfected derivative of K562 cells, expresses an
ectopic integrin 8 subunit (provided by Louis Reichardt)
(Muller et al., 1995
). By
plating this transfected K562 derivative on a plastic surface coated with the
bacterial Yersinia pseudotuberculosis invasin protein, which
activates ß1-intergins (Arencibia et
al., 1997
), the otherwise spherical KA8 cells spread out on the
surface, which facilitated morphological analysis of mitotic cells. For
expression of Op18 deletion mutants in human cell lines, coding regions were
cloned as HindIII to BamHI fragments into the corresponding
sites of the EBV-based shuttle vector pMEP4 (Invitrogen)
(Groger et al., 1989
).
Transfection of the pMEP4 derivatives into KA8 cells was performed as
previously described for the original K562 cell line
(Marklund et al., 1994
).
Conditional expression was achieved by employing the hMTIIa promoter, which
can be suppressed by low concentrations of EDTA (20 µM) and induced by Cd
(Marklund et al., 1994
). For
transfection, 12 µg DNA was used, and expression was induced with 0.5 µM
Cd.
Quantification of ectopic Op18 and analysis of Op18
phosphoisomers
To analyze Op18-NR-helix phosphoisomers, we employed a native PAGE system
that separates Op18 according to the charge differences introduced by each of
the four identified phosphorylations
(Marklund et al., 1993). To
determine ectopic expression levels, cell extracts were separated by SDS-PAGE
together with graded amounts of recombinant Op18 and Op18-NR-helix proteins as
described previously (Marklund et al.,
1994
). The ratio of ectopic proteins versus endogenous Op18 was
determined by probing western blots with affinity-purified rabbit antibodies
and comparing them with a standard of recombinant proteins with known protein
content (Brattsand et al.,
1993
).
Analysis of MT polymerization status, flow cytometric analysis and
immunofluorescence
The cellular content of MT polymers was determined essentially as described
previously (Holmfeldt et al.,
2001). In brief, cells were resuspended in an MT-stabilizing
buffer containing 0.05% saponin, to extract soluble tubulin, and subsequently
fixed in 4% paraformaldehyde/0.05% glutaraldehyde. The remaining polymerized
tubulin was stained with anti-
-tubulin (clone B-5-1-2, Sigma) and
fluorescein-conjugated rabbit anti-mouse immunoglobulin. Fluorescence was
quantified by flow cytometry using a FACS-caliber together with the Cell Quest
software (Becton-Dickinson). To allow calculation of the percentage of
polymerized tubulin in relation to the total amount of cellular tubulin, the
polymerization status in vector-control-transfected cells was determined by
quantitative western blotting (mean of three independent determinations:
58±12%). Relative fluorescence intensities of extracted cells were
normalized in each experiment assuming that the level in vector-control cells
corresponds to 58% polymerized tubulin. This procedure faithfully reproduced
the results obtained by quantification of soluble and particulate tubulin by
western blot analysis (Marklund et al.,
1996
) but with increased reproducibility. Within the time limits
of the experiments, ectopic Op18 does not alter the cellular levels of total
tubulin in K562 or KA8 cells. Analysis of DNA content and quantification of
mitotic cells, using the MPM-2 antibody, was performed by flow cytometric
analysis as described previously (Marklund
et al., 1996
). Immunofluorescence analysis was performed as
described elsewhere (Holmfeldt et al.,
2001
).
MT assembly
The assembly of individual MTs seeded from axoneme fragments was visualized
using video-enhanced differential interference contrast (DIC) microscopy as
described previously (Howell et al.,
1999b; Vasquez et al.,
1994
). Assembly conditions were such that MTs assembled only from
axonemes, and the total amount of tubulin incorporated into MT polymer was
insignificant compared with the total tubulin concentration. A PEM buffer
adjusted to pH 7.5 was used in the present study. To prevent any possible
adherence of the NR-helix to the slide or coverslip, glass surfaces were
blocked with 5 mg/ml casein for 5 minutes
(Vasquez et al., 1994
). We
estimate that the detection limit for MTs assembled from axonemes was
approximately 0.3 µm.
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Results |
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To determine if the first 57 residues of Op18 are sufficient for functional
interaction with tubulin, modulation of tubulin GTP hydrolysis was compared
with that of wild-type Op18 (Op18-wt) and the C-terminally truncated
Op18(1-99) derivative (Fig.
1B). The result reveals the expected threefold stimulation of
tubulin GTPase hydrolysis in the presence of Op18-wt. This stimulation has
previously been shown to be independent from the N-terminal 46 residues of
Op18 (Larsson et al., 1999),
and the mechanism may involve GTPase-productive interactions between the E
site of ß-tubulin and the catalytic loop of
-tubulin within the
tandem tubulin dimer complex (Steinmetz et
al., 2000
). Moreover, we also show that the Op18(1-99) derivative,
which lacks the second helical repeat, has the opposite activity, namely
inhibition of the basal GTP hydrolysis of soluble tubulin. Inhibition of basal
tubulin GTP hydrolysis by C-terminally truncated Op18 has previously been
shown to be a consequence of loss of cooperative binding of the two tubulin
heterodimers. Thus, the truncated Op18 derivative is inefficient in forming
tandem tubulin dimer complexes and binds preferentially to single tubulin
heterodimers, which in turn inhibit the basal GTP hydrolysis of tubulin in
solution (Segerman et al.,
2000
). Given that the isolated first helical repeat by itself is
inactive [see Op18(46-99), Fig.
1B], it appears that the N-terminus is essential for the observed
inhibition by Op18(1-99).
Taken together, the data in Fig. 1 indicate that the N-terminus of Op18 retains functional interactions with tubulin. The low affinity of the Op18(1-57)-NR-helix, as indicated by the dose response, suggests that the first helical repeat of Op18 serves to increase the affinity of the isolated N-terminus towards tubulin heterodimers. It follows that the Op18(1-99) derivative can be functionally dissected into an N-terminus that blocks basal GTP hydrolysis and the first helical repeat that increases the otherwise very low binding affinity of the N-terminus to single tubulin heterodimers. The presence of both the first and second helical repeat, as in Op18-wt, further increases the tubulin-binding affinity by two-site positive cooperative binding along two tubulin heterodimers, which in turn result in a GTPase-productive complex.
Catastrophe-promoting properties of Op18(1-57)-NR-helix during MT
assembly in vitro
Op18 acts as a specific catastrophe factor during MT assembly in vitro at
pH 7.5, a property that requires an intact N-terminus
(Howell et al., 1999a). To
evaluate whether the N-terminus alone is sufficient for catastrophe promotion,
individual MTs were analyzed in the presence or absence of Op18-NR-helix
derivatives. Low concentrations of these derivatives (2 and 10 µM) were
without significant activity (data not shown), which was not unexpected given
that the N-terminal fragment appeared to have very low tubulin-binding
affinity as evaluated by modulation of tubulin GTP hydrolysis (see
Fig. 1B). However, at 30 and 60
µM, the Op18(1-56)-NR-helix caused a twofold and sixfold increase in the
rate of catastrophe (Fig. 2,
lower panel). At these concentrations Op18(1-57)-NR-helix also caused a slight
but significant reduction in growth rate
(Fig. 2, upper panel). This
effect on growth rates may represent some steric interference with tubulin
polymerization at the high concentrations used, which would be expected from
low-affinity inhibition of basal GTP hydrolysis of soluble tubulin shown in
Fig. 1B (similar levels of
inhibition were obtained at pH 6.8 and pH 7.5, data not shown). Importantly,
doubling the concentration of Op18(1-57)-NR-helix did not further decrease the
growth rate but did increased the catastrophe rate almost threefold. This
suggests that a major part of the observed catastrophe promotion at the
highest concentration is specific and not due to growth rate inhibition via a
simple sequestering-type mechanism. As a control for specificity, a derivative
lacking the first 24 residues of the N-terminus (Op18(25-57)-NR-helix) was
analyzed at 60 µM. This derivative appeared to be essentially inactive
since it has no effect on the growth rate and only a minor effect on the
catastrophe rate. Hence, the data suggest that high concentrations of the
N-terminus alone are sufficient for catastrophe promotion in vitro.
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The N-terminus of Op18 is sufficient for phosphorylation-regulated
MT-destabilizing activity in intact cells
It is shown in Fig. 3A that
all the Op18-NR-helix derivatives tested can be rapidly induced from the
hMTIIa promotor in transfected KA8 cells to produce 35- to 50-fold higher
expression levels than endogenous Op18. This is substantially higher than the
level of native Op18 expressed from the same expression system, which
indicates the high stability of the Op18-NR-helix protein
(Fig. 3A). Native PAGE of Op18
can be used to determine the stoichiometry of Op18 phosphorylation owing to
the contribution of two negative charges from each phosphate group, which has
a strong impact on the migration of a small protein. It is shown in
Fig. 3B that the majority of
Op18(1-57)-NR-helix is phosphorylated on up to three distinct sites in cells
blocked in mitosis by the MT-destabilizing agent nocodazole. As expected, the
phosphorylation-site-deficient Op18-triA(1-57)-NR-helix derivative migrates at
the position of the non-phosphorylated Op18(1-57)-NR-helix. Hence, we conclude
that replacement of the first and second helical repeats of Op18 with the
NR-helix allows high level expression and specific phosphorylation of all
three physiologically relevant sites at the N-terminus of Op18.
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Inducible and robust overexpression of Op18-NR-helix derivatives in a human
leukemia cell line allows analysis of a phenotype on the level of the MT
system. As shown in Fig. 4,
shortly after induced expression Op18-wt causes essentially complete
destabilization of MTs, whereas Op18(1-99) is somewhat less efficient. It
should be noted that native Op18 and Op18 with the second helical repeat
removed, that is Op18(1-99), are expressed at similar levels
(Larsson et al., 1999), which
are twofold to threefold lower than the high expression levels of
NR-helix-fused derivatives (Fig.
3A). It is evident from Fig.
4 that expression of either Op18(1-57)-NR-helix or
non-phosphorylatable Op18-triA(1-57)-NR-helix at these high levels causes a
less dramatic but still significant destabilization of MTs. The destabilizing
activity of Op18(1-57)-NR-helix is sensitive to a 24-residue deletion from the
N-terminal end as evidenced by the Op18(25-57)-NR-helix derivative, which
appears as inactive as the NR-helix alone. The observation that
Op18(1-57)-NR-helix is less active than Op18(1-99) in intact cells is in line
with in vitro data on inhibition of basal GTP hydrolysis of soluble tubulin
(Fig. 1B).
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It is evident from the flow cytometric analysis shown in Fig. 4B that, although the Op18(1-57)-NR-helix derivative is much less efficient than wild-type Op18, 6 hours of expression of either derivative causes a homogeneous decrease in MT content in the cell population. A 6 hour period is too short a time to detect a mitotic block caused by interference with mitotic spindle MTs, and it follows that the results at this early time point reflect the MT content in interphase cells.
Earlier studies have shown that Op18 is phosphorylated at multiple site
with high stoichiometry at mitosis, which results in its inactivation
(Larsson et al., 1997;
Larsson et al., 1995
). Hence,
owing to mitotic phosphorylation, ectopic expression of wild-type Op18
destabilizes the interphase array of MTs without interfering with spindle
formation during mitosis. However, mutants with the N-terminal Ser
phosphorylation sites substituted with Ala are non-phosphorylatable and
consequently constitutively active, and such mutants block formation of the
mitotic spindle (Marklund et al.,
1996
). The data in Fig.
4 shows that the overexpressed N-terminus of Op18 is sufficient to
significantly destabilize the interphase array of MTs. To interpret these data
it was important to know if the N-terminus of Op18, which has been taken out
of its normal context of the first and second helical repeat, is (i) still
regulated by phosphorylation and (ii) is sufficient to block formation of the
mitotic spindle. For this purpose the cell cycle profiles of transfected cells
were evaluated after 24 hours of induced expression
(Fig. 5). As expected from the
low degree of leakage from the hMTIIa promotor, no significant alteration of
the DNA profile is observed prior to induced expression. However, after 24
hours of induced expression, it is evident that the
phosphorylation-site-deficient Op18-triA(1-57)-NR-helix derivative causes a
major accumulation of cells in the G2/M phase, whereas the Op18(1-57)-NR-helix
causes only a minor shift in the DNA profile. Since cells overexpressing
Op18(1-57)-NR-helix readily accumulate in mitosis in the presence of the
MT-directed drug nocodazole (see insert on
Fig. 5), it is clear that the
data are not biased by a potential general cell cycle block or interference
with a metaphase checkpoint by this derivative. Finally, as expected from the
inability of the Op18(25-57)-NR-helix and NR-helix to interfere with
interphase MT, these two derivatives did not cause any detectable alteration
of the DNA profile.
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To evaluate how the non-phosphorylatable Op18-triA(1-57)-NR-helix
derivative causes accumulation of cells with G2/M content of DNA, mitotic
cells and mitotic spindle morphologies were manually inspected and graded into
three classes (normal and abnormal type I and II) described and depicted in
Fig. 6. The percentage of
normal versus abnormal spindles was quantified and normalized to the frequency
of mitotic cells (Table 1). The
data show that only the Op18-triA(1-57)-NR-helix causes a significant increase
in the frequency of mitotic cells. It should be noted that K562 cells have
poor spindle assembly checkpoint control, so that interference with the
mitotic spindle only results in a transient block in metaphase, which is
followed by entry into a tetraploid pseudo-G1 state with G2/M content of DNA
(Marklund et al., 1996). In
the present study we have employed the chicken integrin
8 subunit
expressing KA8 subclone of K562 (Muller et
al., 1995
), since these cells spread on plastic coated with a
integrin-ß1-activating ligand and thereby facilitate morphological
examination of mitotic cells. However, owing to the very transient metaphase
checkpoint of the KA8 subclone, only about 10% of all cells are blocked at
metaphase after 24 hours of induced expression
(Table 1). Nevertheless, it is
still clear that overexpression of Op18-triA(1-57)-NR-helix interferes
dramatically with spindle assembly since the majority of metaphase cells (96%)
show severe spindle abnormalities. Taken together, the results in
Fig. 6 and
Table 1 show that
non-phosphorylatable Op18-triA(1-57)-NR-helix causes accumulation of cells
with G2/M content of DNA owing to potent interference with the mitotic
spindle. As expected by mitosis-specific phosphorylation and inactivation of
Op18-wt (Larsson et al.,
1997
), the Op18(1-57)-NR-helix derivative is extensively
phosphorylated in M-blocked cells (Fig.
3B) and shows minimal interference with the mitotic spindle
(Table 1). Since
destabilization of interphase MTs is not dependent on mutated N-terminal
phosphorylation sites, it follows that the N-terminus of Op18 can be regulated
by mitotic phosphorylation independently from the major tubulin-binding
regions represented by the first and second helical repeats. From this finding
it can be deduced that Op18 phosphorylation can regulate Op18 activity by a
mechanism that is independent from formation of the tandem tubulin dimer
complex. Finally, the morphological appearance of the majority of spindles in
Op18-triA(1-57)-NR-helix-expressing cells (i.e. type II represented by asters
containing dense short MTs) is compatible with autonomous high-level
catastrophe-promoting activity by the overexpressed non-phosphorylatable
Op18-N-terminus.
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Discussion |
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A fragment containing only the first 57 residues of Op18 has only low
binding activity towards tubulin, as evidenced by the high concentrations
required for inhibition of basal tubulin GTPase activity
(Fig. 1B). The low
tubulin-binding affinity of the N-terminal portion precluded direct analysis
of binding; however, derivatives containing both the N-terminus and the entire
first helical repeat [i.e., Op18(1-99)] bind tubulin with an appreciable
affinity (Larsson et al.,
1999). Given the 10- to 20-fold difference in dose response
between Op18(1-57) and Op18(1-99), and the fact that the first helical repeat
in isolation is devoid of activity (see inhibition of basal GTP hydrolysis,
Fig. 1B), it seems likely that
the first helical repeat simply stabilizes binding of the N-terminus.
The present study demonstrates that the N-terminal region of Op18 expresses
autonomous microtubule-destabilizing activity in intact cells. It has been
shown that Op18 is inactivated by phosphorylation at four Ser residues during
mitosis, which allows formation of the mitotic spindle in Op18-overexpressing
cells (Larsson et al., 1997).
The present study shows that the N-terminus expressed in the absence of tandem
helical repeats can also be phosphorylation-inactivated during mitosis. Thus,
expression of the Op18-triA(1-57)-NR-helix resulted in a mitotic block,
whereas the corresponding phosphorylatable wild-type sequence allowed
productive spindle formation during mitosis. Although overexpression of the
N-terminus alone is sufficient for destabilization of interphase MTs and the
mitotic spindle, the first helical repeat increases the potency of the effect
of the N-terminus in intact cells (see Fig.
4). Thus, it seems likely that the first helical repeat stabilizes
the interaction between the N-terminus and
-tubulin in intact cells in
an analogous manner to that discussed above for the in vitro situation.
Previous work has shown that Op18 derivatives truncated at either the
N-terminus or the C-terminus exhibit distinct defects in intact cells. Both
types of truncated Op18 proteins destabilize interphase MTs to a similar
extent but differ completely in their action during mitosis
(Holmfeldt et al., 2001). For
example, whereas non-phosphorylatable (i.e. Ser to Ala substitutions)
Op18-tetra-A(1-99) blocked formation of functional mitotic spindles as
expected, overexpression of a non-phosphorylatable Op18-triA(25-149)
derivative allowed formation of normal spindles and subsequent cell division.
The N-terminal truncated Op18-triA(25-149) derivative, containing the complete
helical region of Op18, is more efficient in destabilizing interphase MTs than
the Op18-triA(1-57)-NR-helix derivative used here. Since the latter
derivative, but not the tandem tubulin dimer complex forming the
Op18-triA(25-149) derivative, blocks cells in mitosis
(Table 1), this result
emphasizes the importance of the N-terminus for Op18-mediated destabilization
of the mitotic spindle (Holmfeldt et al.,
2001
).
If the two mechanisms by which Op18 regulates the MT system operate
independently from each other, it follows that they may be independently
regulated by phosphorylation. We have recently analyzed the phenotype of a
`pseudo-phosphorylation' derivative of Op18 with four Ser to Glu substitutions
at phosphorylation sites (denoted Op18-tetraE)
(Holmfeldt et al., 2001). It
is noteworthy that three of these substitutions are located within the
unstructured N-terminus. Consistent with an altered function of the
N-terminus, it was found that Op18-tetraE does not promote catastrophes in
vitro, and detailed analysis of tubulin-directed activities indicated the Glu
substitutions at phosphorylation sites have a similar functional outcome to
N-terminal truncations. Accordingly, Op18-tetraE efficiently destabilizes the
interphase array of MTs but, as predicted by its lack of catastrophe activity,
does not interfere with formation of the mitotic spindle. Hence, it seems
likely that phosphorylation of N-terminal sites primarily results in
attenuation of N-terminal-mediated functional activities, such as catastrophe
promotion.
As outlined above, both studies in vitro and in intact cells have indicated
a functional dichotomy of Op18. Examples include (i) sequestering versus
catastrophe-promoting activity during MT assembly in vitro
(Howell et al., 1999a), (ii)
various types of modulation of tubulin GTP turnover in vitro
(Larsson et al., 1999
), (iii)
differential MT-directed activities during interphase and mitosis
(Holmfeldt et al., 2001
) and
(iv) efficient suppression by Op18 derivatives with intact tubulin
sequestering activity but not by MT-destabilizing catastrophe-proficient Op18
derivatives that are deficient in tubulin sequestering activity of microtubule
stabilization by the MAP4 protein
(Holmfeldt et al., 2002
).
Functional dichotomy of Op18 was originally suggested by analysis of
MT-directed activities of truncated Op18 derivatives, which demonstrated that
the N-terminus is required for catastrophe promotion but not
tubulin-sequestering activity, which only requires the first and second
helical repeats of Op18 (Howell et al.,
1999b
). This report was subsequently extended by studies on
modulation of tubulin GTP hydrolysis by truncated Op18 derivatives, which
demonstrated that the first and second helical repeats are both sufficient and
necessary for efficient formation of a ternary tubulin complex and to
stimulate a low-rate GTP hydrolysis within this complex
(Larsson et al., 1999
). Since
the C-terminally truncated Op18(1-99) derivative promotes catastrophes, but
not formation of GTPase-productive tandem tubulin dimer complexes, GTPase
stimulation within the ternary tubulin complex is clearly not involved in
catastrophe promotion.
The present data together with recent structural and functional information
suggest a simple model of multiple functional Op18-tubulin interactions, which
all contribute to the final tubulin-binding affinity of native Op18. This
model seems relevant for the understanding of the functional dichotomy of Op18
since it implies two distinct types of Op18 interactions with tubulin
heterodimers, both of which involve multiple stabilizing contact points. One
type of interaction can be described as cooperative binding along the
longitudinally arranged tandem tubulin dimers via the first and second helical
repeats of Op18 (Gigant et al.,
2000), which is essential for sequestering of tubulin in
GTPase-productive tandem tubulin dimers
(Larsson et al., 1999
). A
second type can be described as an independent interaction between the
N-terminus of Op18 and most probably a longitudinal surface on the
-tubulin. This can be viewed as a variant of the `capping model', which
was proposed on the basis of the digital image analysis of electron
micrographs of Op18-tubulin complexes
(Steinmetz et al., 2000
) and
subsequent cross-linking experiments
(Muller et al., 2001
). With
respect to binding affinity, these two types of interaction are synergistic,
with the N-terminus of Op18 mediating very low-affinity binding in the absence
of the first helical repeat. If the most C-terminal helical repeat of Op18 is
removed, this results in a switch from two-site positive cooperative binding
of two heterodimers to non-cooperative binding of a single heterodimer
(Larsson et al., 1999
;
Segerman et al., 2000
).
Truncation of either the N- or C-terminus results in loss of contact points
and therefore a decrease in tubulin affinity, as suggested by both binding
analysis (Larsson et al.,
1999
) and structural studies
(Gigant et al., 2000
;
Muller et al., 2001
). However,
the functional outcome of the interaction(s) with the N-terminus of Op18 is
clearly very different from the functional consequences of formation of a
tandem tubulin dimer complex by the first and second helical repeat. Thus, as
outlined above, interaction of the Op18 N-terminus with a longitudinal surface
on the
-tubulin end of the ternary complex appears essential for
catastrophe promotion but not tubulin-sequestering activity. Moreover, it
appears that this interaction with
-tubulin is also manifested by
inhibition of the basal GTP hydrolysis of soluble tubulin. Since the first and
second helical repeats allow two-site positive cooperativity in binding, and
thereby facilitate interactions between the Op18 N-terminus and
-tubulin, it probably allows Op18 N-terminal-dependent catastrophe
promotions at lower Op18 concentrations. Such synergistic interplay between
the N-terminus and the first and second helical repeats of Op18 for
catastrophe promotion is supported by the present evidence for specific
catastrophe promotion by high concentrations of the Op18(1-57)-NR-helix
(Fig. 2).
Since it has been suggested that the N-terminus of Op18 interacts with the
-tubulin end of the
/ß heterodimer
(Muller et al., 2001
), it
seems unlikely that this region of Op18 promotes plus-end-specific
catastrophes by direct interaction with the ß-subunits exposed at the MT
plus-end tip. It follows that the mechanism for MT destabilization by the
N-terminus of Op18 may involve interactions with soluble
/ß
heterodimers or possibly even MT polymers. Since catastrophe promotion can
clearly be dissociated from tubulin sequestering by distinct activities of
truncated Op18 derivatives (Holmfeldt et
al., 2002
; Holmfeldt et al.,
2001
; Howell et al.,
1999b
), it seems to exclude the possibility that the
catastrophe-promoting activity can be explained by tubulin sequestering. Thus,
the mechanism behind Op18-mediated catastrophe promotion remains elusive.
However, the present model of how distinct types of tubulin interaction
contribute to the functional dichotomy of Op18 should provide a framework for
future functional studies.
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Acknowledgments |
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References |
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