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J. Biol. Chem., Vol. 276, Issue 37, 34753-34758, September 14, 2001
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,From the Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington 98195
Received for publication, July 16, 2001, and in revised form, July 18, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Mitotic centromere-associated kinesin (MCAK) is a
microtubule depolymerizer that is consistent with its role in promoting chromosome segregation during mitosis. Here we show that the conserved motor domain of MCAK is necessary but not sufficient for microtubule depolymerization in cells or in vitro. The addition of only
30 amino acids N-terminal to the motor restores depolymerization activity. Furthermore, dimerization studies revealed that the smallest
functional MCAK deletion constructs are monomers. These results define
a highly conserved domain within MCAK and related (KIN I) kinesins that
is critical for depolymerization activity and show that this
depolymerization is not dependent on MCAK dimerization.
Kinesins are molecules that convert chemical energy into physical
work to perform tasks such as vesicle transport, chromosome segregation, and organization of the mitotic spindle. All
kinesin-related motors share a conserved motor domain that contains a
nucleotide and a microtubule-binding site (1). The position of the
motor domain within the primary sequence of the protein predicts the direction that the kinesin travels along microtubules (2, 3). However,
directionality is conferred by conformational interactions between the
motor and other domains within the protein that impose a directional
bias in the choice of the next tubulin-binding site (2-5). Structural
variations such as these permit the conserved motor domain of kinesin
to perform diverse functions in the cell.
Mitotic centromere-associated kinesin
(MCAK)1 (6) belongs to a
third subfamily of kinesin motors, the Kin I subfamily (I for
internal). This family of homodimeric kinesins has the conserved motor
domain in the interior of the protein rather than at either end. Unlike
most kinesins, which walk along the surface of microtubules, the Kin I
kinesins depolymerize them (7, 8). Purified XKCM1 (the
Xenopus homologue of MCAK) depolymerizes stabilized
microtubules in the presence of ATP (7) and promotes microtubule
depolymerization in Xenopus egg extracts (8, 9). In
vivo, overexpression of MCAK protein in CHO cells results in a
loss of mitotic spindle microtubules during mitosis, and the
replacement of functional MCAK at the centromere with a motorless
version delays the migration of chromosomes to the spindle poles at
anaphase (10). This e+ is consistent with the in vitro
results reported above because chromosomes are normally attached to
depolymerizing microtubules during anaphase (11). Together, these
results firmly establish MCAK and its homologues as microtubule
depolymerizers whose activity is relevant to cellular functions.
The structural differences between kinesins that allow one to travel
along a microtubule surface and another to induce its depolymerization
are not understood. One possibility is that some feature of the protein
other than the motor domain is responsible for inducing
depolymerization. Another possibility is that a small change within the
conserved motor itself produces the transformation to depolymerization.
Finally, microtubule depolymerization may be dependent on the
quaternary structure of MCAK. This report addresses these issues by
describing the minimal amount of the MCAK structure necessary to induce
microtubule depolymerization. Here we show that the conserved
kinesin-like motor domain of MCAK is required but is insufficient to
cause depolymerization. An additional 30 amino acids N-terminal to the
motor region are required to restore this activity. In addition, it is
demonstrated via three independent methods that the smallest functional
MCAK constructs exist as monomers. This indicates that the microtubule
depolymerization activity of MCAK is not dependent on its quaternary
structure. These results improve our understanding of what makes a
kinesin motor behave as a microtubule depolymerizer by elucidating the structural requirements responsible for depolymerization.
Cell Transfection and Immunofluorescence--
Cell culture was
performed using CHO cells as described in Wordeman and Mitchison (6).
The cells were transfected for 4 h using LipofectAMINE (Life
Technologies, Inc.) and fixed at either 18 or 42 h
post-transfection depending on the experiment. For immunoprecipitations, the cells were transfected on 100-mm plates and
lysed at 42 h post-transfection. Where paclitaxel was used, it was
added at a concentration of 15 µM 2 h into the
transfection until fixation. No GFP-expressing cells were ever observed
at the 2-h time point using visual fluorescent inspection. The cells were fixed in Immunoprecipitation--
Two plates of CHO cells were
transfected with either GFP-MCAK and Myc-MCAK or GFP-A182 and Myc-A182.
The cells were lysed for 30 min on ice with 1 ml of chilled lysis
buffer (150 mM NaCl, 100 mM KCl, 50 mM Tris-HCl, pH 8.0, 1% Triton X-100) and protease inhibitors (pepstatin, leupeptin, aprotinin, antipain,
phenylmethylsulfonyl fluoride). The cells were scraped, Dounce
homogenized, and then spun to remove debris from the lysate.
Myc-antibody-agarose beads (CLONTECH) were added to
the lysate and incubated for 3 h at 4 °C. The beads were
collected and washed three times with lysis buffer. The protein was
eluted in 100 mM glycine, pH 2.5, at room temperature for
1 h and neutralized by the addition of MCAK DNA Constructs--
All GFP constructs were created using
the TOPO/TA GFP cloning kit (Invitrogen). When expressed, these
constructs contain the MCAK sequence listed in the name of the
construct, linker residues from the TOPO vector, and the GFP protein at
the N or C terminus, depending on the TOPO vector used. All of the
pictures in the paper are from constructs using an N-terminal GFP with
the exception of Glu201-Ser583 which
has a C-terminal GFP. Myc-tagged constructs were made using the
CLONTECH c-Myc eukaryotic expression vector and
polymerase chain reaction-generated MCAK fragments. Bacterial
expression was achieved by cloning MCAK fragments into the pTrcHis TOPO
vector (Invitrogen). Two-hybrid constructs were made using the
CLONTECH pGBKT7 and pADT7 vectors and polymerase
chain reaction-generated MCAK fragments. Motorless MCAK was made as
described in Maney et al. (10) and inserted into the
two-hybrid vectors listed above. Polymerase chain reaction was
performed using Pfu Turbo polymerase (Stratagene) and
oligonucleotides from Genset (La Jolla, CA). Construct sequences were
verified by DNA sequencing. The specific amino acids in each construct
are listed in the text. Amino acid numbers correspond to the MCAK
peptide and not MCAK fusion peptides.
The Yeast Two-hybrid Assay--
The yeast two-hybrid assay was
performed by cloning MCAK deletion constructs in the pGBKT7 vector
(DNA-binding domain fusion) and cloning motorless MCAK into the pADT7
vector (activation domain fusion). The constructs were then transformed
into yeast strain AH109 (purchased from CLONTECH)
pretransformed with the motorless-activation domain fusion construct.
The transformed cells were plated on plates lacking leucine and
tryptophan to select for presence of both plasmids. After 3 days of
growth at 30 °C, the colonies were streaked onto plates lacking
adenine, histidine, leucine, and tryptophan to select for protein
interactions. Plates selecting for interaction were allowed to grow for
15 days. The positive results reported in the paper were seen no later
than 5 days following plating on the restrictive media. All media were
prepared using the Yeast Protocol Handbook from
CLONTECH (PT3024-1). False positive tests were
conducted with the MCAK motorless construct in each vector. Motorless
MCAK was incapable of inducing growth when transfected alone, in
conjunction with an empty complementing vector or with a complementing
vector fused to a random protein (SV40 large T-antigen in pADT7 or
lamin C in pGBKT7).
Hydrodynamic Analysis--
Full-length MCAK was expressed and
purified as described by Maney et al. (10). Sucrose density
sedimentation and size exclusion chromatography of Ala182
and Asp246 were performed as described by Maney et
al. (10). Molecular weights were calculated from the apparent
Stoke's radius (rS) and sedimentation
co-efficient (S20,W) using the following
relationship: Mr = S20,WNa6 Microtubule Depolymerization Assays--
The depolymerization of
paclitaxel-stabilized microtubules was performed essentially as
described in Desai et al. (7) except that 70 mM
KCl was omitted in some assays. Motor concentration was determined from
the proportion of active ATP-binding sites in the preparation (12).
Equivalent amounts of either baculovirus expressed full-length MCAK
(10) or bacterially expressed Ala182 or Asp246
was added to paclitaxel-stabilized microtubules. The reactions were
incubated in the presence or absence of 1.5 mM ATP for 10 min at room temperature and then centrifuged in the top speed of an
airfuge for 10 min. Supernatants and pellets were assayed for the
presence of tubulin on Coomassie-stained SDS-polyacrylamide gels
(Novex) and quantified using NIH Image.
In Vivo Depolymerization Activity of MCAK and MCAK Deletion
Constructs--
CHO cells were transfected with GFP-MCAK and a series
of GFP-MCAK deletion constructs to determine what domains of MCAK were important for in vivo microtubule depolymerization.
Transfection of full-length GFP-MCAK results in significant microtubule
loss after 18 h (Fig. 1,
A and B). Deletion of the entire portion of MCAK
C-terminal to the motor domain does not negatively affect this activity
(not shown). Deleting amino acids 1-181 (construct Ala182-Ser583 hereafter called "A182")
results in similar microtubule polymer loss at the same time points as
full-length MCAK (Fig. 1, C and D). Deleting the
N terminus to Glu201 (construct
Glu201-Ser583 hereafter called "E201")
also produces a construct capable of decreasing polymer levels in cells
(Fig. 2), although this construct is not
as effective as A182.
Deletion of the entire N terminus abolishes the microtubule
depolymerizing ability of MCAK (Fig. 2). The construct
Glu232-Ser583 (hereafter called "E232"),
which represents only the conserved MCAK motor domain, was not able to
depolymerize microtubules in cells even though it can still bind them
as judged by a microtubule binding/extraction assay (data not shown).
Overexpression of GFP alone did not produce any visible microtubule
depolymerization (Fig. 1, E and F).
To control for possible effects of GFP on the quaternary structure of
the expressed protein, these experiments were repeated with Myc-tagged
constructs of MCAK, A182 and E201, with identical results. The A182,
E201, and E232 constructs were also tested with GFP fused to either end
with no change in results (data not shown).
One concern with expressing GFP-MCAK constructs in living cells is that
the observed microtubule loss may not be a result of direct
MCAK-dependent depolymerization. The reduction in polymer may simply be a result of free tubulin sequestration by an association with the overexpressed MCAK. To address this issue, transfected CHO
cells were incubated with 15 µM paclitaxel to stabilize
microtubules. This concentration of paclitaxel renders the microtubules
less sensitive to depolymerization triggered by a decrease in the
concentration of free tubulin dimers (13). The cells were transfected
with GFP-MCAK, GFP-MCAK deletion constructs, and the microtubule
depolymerizing protein stathmin and expressed for 2 days in the
presence of paclitaxel. GFP-MCAK is capable of depolymerizing
paclitaxel stabilized microtubules (Fig. 1, G-L). The
amount of depolymerization is not as significant as without paclitaxel
but is still dramatic compared with the microtubule density of
untransfected cells (see untransfected cells in Fig. 1, J
and L, arrows). In addition to depolymerizing microtubules, the GFP-MCAK construct became associated with the microtubules in the cells treated with paclitaxel (Fig. 1,
G-J, insets). It is interesting
that lattice binding of MCAK is promoted in the presence of paclitaxel
(see "Discussion"). All of the deletion constructs that could
depolymerize microtubules in the absence of paclitaxel could also do so
in the presence of paclitaxel, but to varying degrees.
The A182 and the E201 mutants were both functional, but it is clear
that some depolymerization activity is lost between them (Fig. 2).
GFP-stathmin depolymerizes microtubules in the assay without paclitaxel
(data not shown) but is unable to depolymerize microtubules stabilized
with 15 µM paclitaxel when transfected at similar levels
as the GFP-MCAK constructs (Fig. 1, K and L). This is consistent with previous reports indicating that stathmin cannot depolymerize taxol-stabilized microtubules. GFP alone had no
effect on paclitaxel-stabilized microtubules (data not shown). Myc-tagged versions of these proteins were also able to depolymerize microtubules (data not shown).
The results of these in vivo depolymerization experiments
are summarized in Fig. 2. From these experiments, it is clear that the
conserved kinesin-related motor domain of MCAK is not sufficient for
microtubule depolymerization in cells. An additional 31 amino acids are
necessary to achieve significant depolymerization using this visual
assay. An additional 19 more are necessary to reach levels equal to or
better than full-length MCAK. These results also demonstrate that the
N- and C-terminal regions of MCAK important for centromere binding are
unnecessary for its depolymerizing activity.
Analysis of MCAK Deletion Construct Dimerization--
Native MCAK
appears to be a homodimer (10). Therefore, it is essential to test
whether the depolymerization activity exhibited by some of the
constructs is dependent on the quaternary structure of the native
expressed protein. One possible explanation for the return of
depolymerization activity in the A182 and E201 mutants is that
multimerization is a prerequisite for depolymerization and these 30-50
amino acids added to the N terminus of the MCAK motor domain promote
dimer formation. To determine whether functional motor constructs are
multimers, the yeast two-hybrid assay, immunoprecipitation, and
hydrodynamic assays were performed.
The constructs used in the yeast two-hybrid assay consisted of the same
amino acids used in the depolymerization assay with the exception of
the motor domain (Fig. 3A).
The motor domain was left off of these constructs because the motor is
toxic to the yeast cells. All of the N-terminal MCAK fragments to be
tested were cloned into the "bait" vector and transformed into
yeast containing motorless MCAK cloned into the "prey" vector.
Motorless MCAK was also cloned into the "bait" vector as a positive
control. Interaction between an MCAK N-terminal fragment and motorless MCAK should result in colony growth. As shown in Fig. 3, the yeast cells expressing motorless MCAK in each vector exhibited a large amount
of growth, indicating strong dimerization between these constructs.
This demonstrates that the motor domain is not important for multimer
formation. The fragment containing the complete N terminus of MCAK
(M1-K231) also promoted growth, although an extra 2 days of incubation
was required. Deleting a significant portion of the N terminus (the
S150 construct) severely reduced growth to where only a few colonies
were present after 5 days of incubation. This amount of growth is
possibly spurious but may represent a weak interaction. The next
constructs tested (Ala182-Lys231 and
Glu201-Lys231), both of which exhibit
microtubule depolymerization activity when fused to the MCAK motor, did
not exhibit any growth even after 15 days of incubation. These results
suggest that the smallest functional deletion constructs used in the
depolymerization experiments are not dimers. Finally, the C terminus of
the motorless MCAK construct was tested. This construct produced a very
low, but consistent, amount of growth after 5 days incubation,
suggesting a role for the C terminus in MCAK dimerization in addition
to the N terminus.
The multimerization question was further analyzed in vivo by
performing co-immunoprecipitation assays with differentially tagged
MCAK constructs. Because the focus of this paper is whether or not the
minimal functional MCAK fragments can operate as monomers, we chose to
analyze only the A182 mutant rather than perform
co-immunoprecipitations with each construct. The A182 construct was
chosen over the E201 construct because its paclitaxel-stabilized
microtubule depolymerization activity rivaled that of wild-type MCAK.
CHO cells were transfected with Myc and GFP-tagged versions of MCAK or
Myc and GFP-tagged versions of the A182 deletion mutant. After
expressing these constructs, the cells were lysed, and the Myc-tagged
proteins were immunoprecipitated (Fig. 3B, lanes
1-4). These pellets were then tested to see whether they
contained any GFP-tagged MCAK, indicating dimerization (Fig. 3B, lanes 5-8). Full-length GFP-tagged MCAK was
pulled down with the Myc-tagged MCAK, indicating dimerization between
these proteins (Fig. 3B, lane 7). The GFP-A182
construct, however, did not co-immunoprecipitate with Myc-tagged A182,
suggesting that these proteins are not dimerizing (Fig. 3B,
lane 8). This result is consistent with the two-hybrid data
described above.
Size exclusion chromatography and sucrose gradient centrifugation was
performed on bacterially expressed A182, which exhibits strong
microtubule depolymerization activity in vivo. The same analysis was performed on Asp246-Glu581
(hereafter called "D246") as a nondepolymerizing control. This control construct consists of the minimal catalytic core motor domain
for kinesin-related as defined by the crystal structure (14). The
results of the size exclusion chromatography are shown in Fig.
4. The measured Stoke's radius and
sedimentation co-efficient for both A182 and D246 are consistent with
both of these proteins existing as monomers in solution (Table
I). The molecular weight for A182 can be
calculated from the apparent Stoke's radius (3.1 nm) and the
sedimentation co-efficient (S20,w = 3.6) as
43,800 (see "Experimental Procedures"). Interestingly, both
the Stoke's radius and the sedimentation co-efficient for D246 were
slightly larger than those measured for A182. This suggests that D246
may have a slightly different shape or hydration state than A182. D246
is known from previous studies on similar sized constructs of kinesin
heavy chain to be a monomer in solution (15). Our hydrodynamic
measurements for D246 are also consistent with a monomeric quaternary
structure. The predicted molecular weights for A182 and D246 including
the His tag and leader sequences are 49,500 and 41,800, respectively
(Table I).
In Vitro Depolymerization Activity of MCAK and MCAK Deletion
Constructs--
The bacterially expressed proteins used for these
hydrodynamic analyses were tested in vitro for
depolymerizing activity. Purified motor was added to
paclitaxel-stabilized microtubules in the presence or absence of
ATP (Fig. 5A). The ratio of
motor heads to tubulin dimers was at least 1:16. This would be expected to more than saturate microtubule ends (average microtubule length equal to 8 microns) if the ends were preferentially bound by motor (7).
The reaction progressed at room temperature for 10 min. After 10 min
the microtubules were centrifuged, and the pellets and supernatants
were assayed for the presence of tubulin. The reactions were not
allowed to go to completion (complete depolymerization of all
microtubule polymer), but they were capable of complete depolymerization if the reaction progressed for 30 min at room temperature (data not shown). It can be seen that tubulin was liberated
into the supernatant in the presence of MCAK plus ATP and in the
presence of the monomeric A182 construct plus ATP (Fig. 5A,
seventh lane). However, little or no tubulin was liberated into the supernatant by D246 in the presence of ATP (Fig.
5A, eleventh lane). These results demonstrate
that a monomeric form of MCAK motor is capable of depolymerizing
microtubules both in vitro and in vivo.
To directly compare the depolymerization efficiency of A182 to
full-length MCAK it was necessary to measure the proportion of motor
heads that can bind ATP (active sites) in our preparations and then
compare the number of active motor heads required to release an
equivalent concentration of tubulin dimer. The results of this
experiment are shown in Fig. 5B. In BRB80, a standard buffer
for assaying kinesin motility, MCAK and A182 show equivalent ATP-dependent microtubule depolymerizing activity over the
course of 10 min at room temperature. In BRB80 plus an additional 70 mM KCl (7), the activities of both motors suffer, but A182 appears more sensitive to added salt than MCAK.
We have prepared four DNA constructs consisting of the "neck"
region of MCAK fused to the motor domain of KIF5B and successively increasing portions of the MCAK motor domain. Each of these constructs displayed no microtubule depolymerizing activity when expressed in
cultured cells (data not shown). This suggests that regions within the
core MCAK motor domain are required for full depolymerizing activity in
addition to the neck region of MCAK.
In this report, we have shown that the conserved catalytic motor
domain of MCAK is necessary but not sufficient for microtubule depolymerization in cells and in vitro. Depolymerization
activity can be partially restored with the addition of 30 amino acids of the N-terminal neck domain and fully restored with the addition of
20 more. These results define a minimal domain required for microtubule
depolymerization and dissociate centromere localization and
depolymerization activities of MCAK. We have constructed four MCAK-KIF5B chimeric motors that do not depolymerize microtubules in
cells, suggesting that properties intrinsic to the MCAK motor must also
be important for depolymerization. We also demonstrate, using three
different procedures, that these functional deletion constructs are
monomers and that the monomer can be as effective at depolymerizing
microtubules as the dimer.
One obvious question posed by these results is the nature of the domain
that restores depolymerization activity to the motor. This segment can
be divided into two regions: the Gly201-Lys231
region (segment II) that restores partial activity and the
Ala182-Glu200 region (segment I) that restores
full activity when added with segment II (Fig.
6). This area of MCAK has been called the
neck region of the protein as it connects the motor to the rest of the
molecule (1). Both of these regions are predicted to be helical in
nature but are not predicted to be coiled-coils, either alone or in
series (16). These predictions are in line with our current data
because no dimerization is seen with these domains. Both segments are
highly conserved between other Kin I kinesins but not as conserved with
the dimerization-conferring coiled-coil neck of the minus
end-directed kinesin NCD (Ref. 17 and Fig. 6). If these segments
are not involved in dimerization, then how do they participate in motor
activity? One possibility is that the neck interacts with microtubules.
The positively charged neck region may interact with negatively charged
microtubule polymer to either tether MCAK to the microtubule or wedge
apart protofilaments or promote tubulin dimer curling. Another
possibility is that the neck directly interacts with the motor domain
and affects its conformation. Examples of neck/motor interactions exist
for both conventional kinesin and NCD and were shown to be responsible for motor directionality and speed of translocation along the microtubule (3, 5, 18, 19). Regions of the NCD neck that interact with
the motor map spatially to the area of MCAK represented by segment II
(5). Interestingly, few of the NCD amino acid pairs responsible for the
neck/motor interaction are conserved in MCAK. Perhaps these differences
in NCD and MCAK neck/motor interactions assist in the transformation of
MCAK to a depolymerizer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C methanol with 1% paraformaldehyde for 1 min. To view microtubules, the coverslips were labeled with the rat YL
anti-tubulin antibody. Myc-containing cells were labeled with an
anti-Myc antibody from CLONTECH. Ten fields of
~20-100 transfected cells were scored by eye for assembled
microtubules per construct. The transfection efficiencies and GFP
expression levels were consistent from coverslip to coverslip, allowing
rapid assignment of MT polymer loss based on the fluorescence level of
assembled microtubules for hundreds of cells. In non-paclitaxel-treated
cells, the extent of polymer loss corresponded to the following formula
(one focal plane): ++++, no MTs to less than 10/cell; +++, 10-30
MTs/cell; ++, 30-70 MTs/cell; +, greater than 70 MTs but fewer than
control cells; , MT polymer level equivalent to control cells. In
paclitaxel-treated cells individual MTs were not discernible because of
bundling, but fluorescence levels of assembled MTs were normalized
relative to control cells as compared with untreated transfectants. The samples were observed using a Nikon FX-A photomicroscope and
photographed using either a Sensys Digital Camera or Kodak Technical
Pan film. Images were processed using Adobe Photoshop 5.5.
rS/(1-
)
where = solvent viscosity, = calculated protein
density from the amino acid composition, and = solvent density. Gels were scanned on a UMAX scanner and quantified using NIH Image.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Depolymerization of microtubules by MCAK and
MCAK fragments in vivo. A,
C, E, G, I, and
K show GFP expression, and B, D,
F, H, J, and L show tubulin
staining in the same cells. A-F, CHO cells transfected with
GFP-MCAK and GFP-MCAK deletion constructs. Overexpression of
full-length GFP-MCAK (A and B) results in a
decrease of microtubule polymer. Overexpression of deletion constructs
containing the entire MCAK motor domain in addition to 65 amino acids
of the N terminus also produce loss of polymer (C and
D). Transfection GFP alone (E and F)
has no visible effect on microtubules. G-L,
paclitaxel-treated CHO cells transfected with GFP-MCAK and various
GFP-MCAK deletion constructs. Paclitaxel-stabilized microtubules are
partially depolymerized by GFP-MCAK (G and H) or
A182-S583 (I and J). These constructs bind
microtubules in paclitaxel-treated cells (G-J,
insets). GFP-stathmin is unable to depolymerize paclitaxel
microtubules (K and L). The arrows
(I and L) show untransfected cells with large
amounts of tubulin polymer. Bars, 10 microns.
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Fig. 2.
Microtubule depolymerization by GFP-MCAK
deletion constructs in the presence and absence of paclitaxel.
Fields of transfected cells were scored immunofluorescently for MT
polymer loss (see "Experimental Procedures"). Complete microtubule
loss was scored as ++++, and MT polymer levels indistinguishable from
control cells were scored as . DP, depolymerizing
activity; DP+T, depolymerizing activity in
paclitaxel-treated cells.
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Fig. 3.
Dimerization and analysis of MCAK
deletions. A, yeast two-hybrid analysis of MCAK
fragments. The Ala182-Lys231 and
Glu201-Lys231 MCAK fragments produced no
growth of transformed yeast, suggesting that there is no interaction
between these protein fragments and motorless MCAK. B,
co-immunoprecipitation analysis of MCAK and A182-S583. CHO cell lysates
containing GFP-MCAK and Myc-MCAK (MCAK columns) or
GFP-Ala182-Ser583 and
Myc-Ala182-Ser583 (A182 columns)
were immunoprecipitated with anti-Myc antibody-couple agarose beads.
Post-immune lysate (L) and eluate from the pelleted beads
(P) are shown probed with anti-Myc (Myc) or
Anti-GFP (GFP) antibodies. All four proteins were
successfully expressed (lanes 1, 2, 5,
and 6) and Myc-MCAK and Myc-A182 were present in large
amounts in the bead pellets (lanes 3 and 4).
GFP-MCAK was also present in the Myc antibody-agarose bead pellet
(lane 7), indicating an interaction between Myc-MCAK and
GFP-MCAK. In contrast, GFP-Ala182-Ser583 was
not seen in the pellet with Myc-Ala182-Ser583
(lane 8), suggesting that these proteins do not interact.
The bands in all four lanes of the Myc panel
represent proteins in the lysate that cross-react with the Myc antibody
that were then concentrated in the pellet fraction.
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Fig. 4.
Hydrodynamic analysis of MCAK motor fragments
in vitro. The upper two graphs show
the size exclusion chromatography for A182 and D246. Stoke's radii
(angstroms) are plotted relative to the elution volume
(Ve/Vo). The lower
two graphs show the sucrose gradient sedimentation profile of A182
and D246. Known sedimentation co-efficients (S) for the
marker proteins are plotted relative to the distance migrated in the
centrifuge tube. Control marker proteins are 
-amylase, bovine serum
albumin (BSA), and carbonic anhydrase (CA).
Hydrodynamic measurements for MCAK and MCAK truncation proteins
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Fig. 5.
Depolymerizing activity of MCAK motor
fragments in vitro. A, A182 can
depolymerize microtubules in vitro.
Paclitaxel-stabilized microtubules were incubated with,
respectively, no motor, full-length MCAK, A182, and D246. MCAK and A182
were incubated in the presence and the absence of ATP, whereas D246 was
incubated in the presence of ATP. MCAK and A182 exhibit
ATP-dependent microtubule depolymerizing activity in
vitro. B, comparison of MCAK and A182 activity in the
presence and the absence of additional KCl. A182 and MCAK exhibit
comparable activity on a mol/mol basis, but A182 is more sensitive to
salt.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Sequences involved in conferring
microtubule-depolymerizing activity to MCAK. A,
alignment of the helical necks of Kin I kinesins and NCD. These
alignments are of the helical neck regions N-terminal to the motor
domain of each motor. Of the Kin I motors, XKIF2 and XKCM1 are from
Xenopus, and MCAK is from hamster. NCD is from
Drosophila melanogaster. Boxed amino acids
represent identities shared between all three Kin I motors. NCD
identities to these amino acids are also boxed. Shaded
boxes represent amino acids with similar positive or negative
charges or functional groups. Segment II constitutes the domain needed
to restore partial depolymerization activity to the MCAK motor domain.
Segment I, together with segment II, restored complete activity as
judged by our visual assay. The LLRC segment below NCD is a portion of
NCD removed during alignment. The asterisks below the NCD
sequence label amino acids important for the neck interaction with the
NCD motor domain.
Our data demonstrate that MCAK in monomer form can depolymerize microtubules. Under standard motility assay conditions the monomeric from of MCAK is as effective as the dimeric form. This indicates that depolymerization does not occur via coupled coordination between the heads of a dimeric motor. Kin I kinesins accumulate at both ends of microtubules under certain conditions (7). In theory there are 13 potential MCAK-binding sites at each end of the microtubule. It is possible that independent heads may cooperate at the end of a microtubule to depolymerize a paclitaxel-stabilized microtubule. We hypothesize that if this is the case, it would occur not necessarily though a physical coupling between the heads that introduces strain in the protofilament structure but through the combined action of several heads, each contributing a subcritical perturbation at the microtubule end.
The monomeric minimal depolymerizing motor (A182) is as effective or even more effective at depolymerizing microtubules in standard motility buffer and also in cells (Fig. 2, DP+T). However, the addition of 70 mM KCl to the depolymerization assay causes the monomer to lose depolymerizing activity faster than the dimer. There are two possible explanations for this observation. Either the second head may bind an adjacent tubulin dimer, thus increasing the overall affinity of the MCAK molecule for the microtubule in higher salt, or alternatively regions within the N- or C-terminal tail of MCAK are useful in promoting depolymerization or increasing microtubule affinity under these conditions irrespective of the quaternary structure, perhaps by adding more microtubule-binding sites. Our KIF5B-MCAK chimera studies suggest that, in addition to the neck, differences between the kinesin and MCAK motor domains may contribute to the different functions of the proteins. Interestingly, recent studies have revealed microtubule-depolymerizing activity in the HIV-1 REV protein. A search for regions of homology with other microtubule depolymerizers revealed significant homology with the microtubule-binding domain at the C terminus of the motor domain of XKCM1 (20). We are devising further experiments to test these possibilities.
Elegant studies by Desai et al. (7) have shown that
depolymerization by the Kin I family of kinesins induces
protofilament curling similar to that seen during microtubule
depolymerization but without the coincident hydrolysis of GTP. They
also demonstrated that Kin I kinesins will bind specifically to
microtubule ends and trigger initial protofilament peeling in the
presence of AMP-PNP. These data suggest that a high affinity binding
step occurs when the motor is in the ATP-bound state and that this
binding site can be accessed at either microtubule end but not in the
lattice. Because the Kin I kinesins can depolymerize microtubules from both ends (7), this suggests that the high affinity binding site may be
between protofilaments rather than at the end of the microtubule. Such
a site would tend to be exposed preferentially at the ends where the
lateral interactions between the protofilaments are easiest to break.
We have produced an MCAK mutant that cannot hydrolyze ATP and binds
uniformly along the lattice of the microtubules (21). This mutant has
no effect on microtubules in cells. The MCAK motor domain may cycle
between high affinity binding, which breaks the lateral bonds between
protofilaments at the end of the microtubule, and benign association
with the lattice depending on the ATP hydrolysis state of the motor
head. Specifically, our data demonstrate that microtubule
depolymerizing activity coupled to ATP hydrolysis is intrinsic to a
monomeric motor domain.
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ACKNOWLEDGEMENTS |
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We thank Marla Feinstein, Andy Hunter, Ayana Moore, Yulia Ovechkina, Steve Carlson, Henry Hess, and Joe Howard for many helpful discussions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM53654A (to L. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Public Health Service National Research Service Award
T326M07270. Present address: Dept. of Cell and Molecular Pharmacology
and Experimental Therapeutics, Medical University of South Carolina,
Charleston, SC 29425.
§ To whom correspondence should be addressed: Box 357290, Dept. of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA 98195. E-mail: worde@u.washington.edu.
Published, JBC Papers in Press, July 20, 2001, DOI 10.1074/jbc.M106626200
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ABBREVIATIONS |
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The abbreviations used are: MCAK, mitotic centromere-associated kinesin; CHO, Chinese hamster ovary; GFP, green fluorescent protein; MT, microtubule; HIV-1, human immunodeficiency virus, type 1.
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REFERENCES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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