From the University of Manchester, School of Biological Sciences, Manchester, M13 9PT, United Kingdom
Received for publication, December 22, 2000
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ABSTRACT |
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Movement of various cargoes toward microtubule
minus ends is driven by the microtubule motor cytoplasmic dynein (CD).
Many cargoes are motile only during certain cell cycle phases,
suggesting that CD function may be under cell cycle control.
Phosphorylation of the CD light intermediate chain (DLIC) has been
suggested to play a crucial role in modulating CD function during the
Xenopus embryonic cell cycle, where CD-driven organelle
movement is active in interphase but greatly reduced in metaphase. This
down-regulation correlates with hyperphosphorylation of DLIC and
release of CD from the membrane. Here we investigate the role of the
key mitotic kinase, cdc2-cyclinB1, in this process. We show that DLIC
within the native Xenopus CD complex is an excellent
substrate for purified Xenopus cdc2-glutathione
S-transferase (GST) cyclinB1 (cdc2-GSTcyclinB1) kinase.
Mass spectrometry of native DLIC revealed that a conserved cdc2 site
(Ser-197) previously implicated in the metaphase modulation of CD
remains phosphorylated in interphase and so is unlikely to be the key
regulatory site. We also demonstrate that incubating interphase
membranes with cdc2-GSTcyclinB1 kinase results in substantial release
of CD from the membrane. These data suggest that phosphorylation of
DLIC by cdc2 kinase leads directly to the loss of membrane-associated CD and an inhibition of organelle movement.
Cytoplasmic dynein (CD)1
is a multiprotein complex which binds to microtubules and moves along
them using energy from ATP hydrolysis. This movement is directed toward
microtubule minus ends and allows CD to move and/or position other
structures (cargo) within eukaryotic cells (1, 2). CD is implicated in
many important processes within eukaryotic cells during interphase,
including vesicular transport from the endoplasmic reticulum (ER) to
the Golgi apparatus, centripetal movement of lysosomes, nuclear
migration, and maintenance of organelle structure (3). During cell
division, it is likely that organelle movement is inactivated (4),
while CD continues to function in the formation, alignment and
maintenance of mitotic spindles, as well as the movement and
positioning of chromosomes (5, 6).
The CD complex consists of four distinct classes of proteins: heavy,
intermediate, light-intermediate, and light chains. The ~500-kDa
cytoplasmic dynein heavy chain (DHC) is present in the complex as a
dimer and is responsible for microtubule binding, ATP hydrolysis, and
movement along microtubules. Two or three dynein intermediate chains
(DICs) per CD complex bind to a specific region toward the N terminus
of the DHC (7). The DIC also binds to the
p150Glued protein, a subunit of another
multi-protein complex called dynactin (reviewed in Ref. 8), and this
interaction is thought to be required for many if not all of the
cellular functions of CD (9). A variety of dynein light chains have
been identified (10-12) and may have a role in the targeting of
particular CD complexes to distinct cargo (13, 14). Four to five dynein
light-intermediate chains (DLICs) are present per CD complex, binding
to an N-terminal portion of the DHC near to the DIC binding site (15).
DLICs show great diversity owing to the presence of two genes (DLIC1 and DLIC2), multiple spliced variants, and differential phosphorylation (15-21).
Little is known yet about the function of the DLICs. They are
hyperphosphorylated in extracts made from Xenopus eggs
arrested in meiotic metaphase (19). In a well characterized in
vitro motility assay (22), this cell cycle-specific
hyperphosphorylation of the DLIC correlated with a reduction in
CD-driven membrane movement and a reduction in the amount of CD (and
its accessory complex dynactin) on cargo membranes (19). On the basis
of these data it has been postulated that DLICs have the following
regulatory role. Mitotic hyperphosphorylation of the DLICs is proposed
to cause CD to detach from cargo membranes, thus down-regulating membrane movement during mitosis. Dell et al. (23) recently demonstrated that purified cdc2 and cyclinB1 could phosphorylate recombinant chicken DLIC1 in vitro. They identified the
in vitro phosphorylation site, a conserved cdc2-cyclinB1
phosphorylation motif, and showed that mutation of the phosphorylated
serine residue caused the recombinant DLIC1 to become refractory to
phosphorylation in meiotic cytosol (23).
Here we provide further evidence to support the hypothesis that
phosphorylation of DLIC by a mitotically active kinase plays a key role
in metaphase inhibition of membrane movement (19). First, we show that
incubation with purified Xenopus cdc2-GSTcyclinB1 kinase
removes CD from Xenopus interphase membranes in
vitro. Second, we demonstrate for the first time that
Xenopus DLIC within the native CD complex is specifically
phosphorylated by Xenopus cdc2-GSTcyclinB1 kinase. Finally,
we find that this phosphorylation occurs at a site distinct from that
proposed by Dell et al. (23). Our results therefore
implicate alternative conserved phosphorylation sites in the cell-cycle
regulation of CD-driven membrane movement.
Cloning of Xenopus DLIC1 cDNA--
Degenerate primers
DEG2I,
5'-cgggatccga(a/g)aa(a/g)ga(t/c)gc(t/c/a)gtItt(t/c)at(t/c/a)cc-3' and
DEG4REVI and
5'-ggaattcIcc(a/t/g)gg(g/a/t)gaIcc(a/t/g)gt(t/c)tt(t/c)tt(a/g)ct-3' (MWG Biotech Ltd, Milton Keynes, UK) were used to amplify DLIC sequences from a Xenopus oocyte cDNA library (24). The
product was cloned into pBluescriptSK+ (Amersham Pharmacia Biotech;
plasmid pEB3), sequenced (Big-Dye Terminator Cycle Sequencing Ready
Reaction, PE Applied Biosystems, Warrington, UK), and found to encode
part of a DLIC. The pEB3 insert was used as a probe to screen the
Xenopus oocyte cDNA library as per the manufacturer's
instructions (Gene Images, Amersham Pharmacia Biotech). Four
independent clones encoding all or part of the same novel DLIC sequence
were obtained. Clone 70/1 was chosen for subsequent manipulations.
Purification of His6-XDLIC1--
Full-length
N-terminal hexahistidine-tagged Xenopus DLIC1
(His6-XDLIC1) was constructed using primers BgNdLIC,
5'-gaagatctcatatggcatccgggcgtagc-3', and rseq4,
5'-taaccagcttacagaggc-3', and clone 70/1 as a template. The polymerase
chain reaction product was cloned into pQE30 (Qiagen Ltd, Crawley, UK)
to give pEB8. The BamHI/PstI fragment from pEB8 was replaced with the corresponding fragment from clone 70/1 to form
pEB10, and the remaining polymerase chain reaction-generated DNA was
sequenced. His6-XDLIC1 was purified from insoluble
inclusion bodies under denaturing conditions using a His-Trap nickel
column (Amersham Pharmacia Biotech).
Antibody Production and Purification--
The pEB3 insert was
cloned into pGEX-4T-3 (Amersham Pharmacia Biotech) (plasmid pEB5a) to
express an N-terminal GST fusion protein (GST-XDLIC1'). GST-XDLIC1' was
purified as per the manufacturer's instructions (Amersham Pharmacia
Biotech) and used to generate antibodies in sheep (Diagnostics
Scotland, Carluke, UK). Crude serum from all bleeds (but not the
pre-immune serum) recognized Xenopus DLICs. To remove
contaminating anti-GST antibodies, serum was pre-absorbed over a GST
column, then affinity-purified on a GST-XDLIC1' column. Eluted
fractions were stored at 4 °C (with 0.02% sodium azide) and used in
immunoblotting experiments at dilutions of 1/500 to 1/1000. For
experiments where it was critical to remove GST antibodies completely,
an alternative, small scale affinity purification method was used. The
C-terminal 382 amino acids of XDLIC1 were N-terminally
His6-tagged in pQE31 (Qiagen Ltd; pEB7). The fusion protein
(His6-XDLIC') was enriched by isolation of insoluble
inclusion bodies using B-PER (Pierce & Warriner (UK) Ltd., Chester,
UK), subjected to SDS-PAGE, transferred to nitrocellulose membrane
(Protran, Schleicher and Schuell), and visualized using Ponceau S
(0.2% in 3% trichloroacetic acid). The His6-XDLIC' band was excised and used to affinity purify anti-XDLIC antibodies as
described (25). Both types of affinity-purified antibody and the crude
serum were able to immunoprecipitate Xenopus DLICs under
denaturing conditions and the intact CD complex under native conditions.
Sequence Analysis--
Internet sites (NCBI, CNRS, IMGEN,
EXPASY) were used for sequence analysis (26-28).
Xenopus Egg Extracts and Membrane Preparation--
Interphase
and meiotic metaphase II (cytostatic factor-arrested (CSF) extracts)
Xenopus egg extracts were prepared as described (29, 30).
CSF extracts were converted to interphase by incubation with 0.2 mM calcium and 100 µg/ml cycloheximide for 45 min (29, 30). Membranes were floated from 200 µl of interphase extract and
collected in a volume of 50 µl, as described by Lane and Allan (31) except low salt acetate buffer (20 mM potassium
acetate, 3 mM magnesium acetate, 2 mM EGTA, 5 mM HEPES, pH 7.4, with KOH) replaced acetate buffer where
mentioned. For Fig. 2B, membranes were floated from 40 µl
of extract.
Purification of Xenopus Cytoplasmic Dynein and cdc2-GSTcyclinB1
Kinase--
CD was purified from interphase or CSF extracts as
described previously (19). Bacterially expressed GSTcyclinB1 was
purified and used to isolate active cdc2-GSTcyclinB1 kinase from
Xenopus egg extracts as described (32). For membrane
incubation experiments cdc2-GSTcyclinB1 kinase was prepared similarly
except that detergent was omitted from the final two washes and
elution. Histone kinase activity was assayed as described (32).
In Vitro Phosphorylation--
cdc2-GSTcyclinB1 kinase (5 µl; histone kinase activity: 50-100 pmol of phosphate
incorporated into histone per µl of kinase preparation per min) was
incubated with 9 µl of purified CD at room temperature for 20 min in
the presence of 250 µM ATP (containing 5 µCi of
[ Incubation of cdc2-GSTcyclinB1 with Membranes--
Interphase
membranes (~15 µg of total protein) were added to 1 µl of
cdc2-GSTcyclinB1 kinase (activity ~100 pmol/µl/min) in the presence
of 1 mM ATP and 0.5 µM microcystin LR
(Alexis Corp. Ltd, Nottingham, UK) in a total volume of 25 µl of low
salt acetate buffer plus 150 mM sucrose and incubated for
30 min at room temperature. Kinase elution buffer (32) without
detergent was used in place of cdc2-GSTcyclinB1 kinase as a control.
Each incubation was pipetted into 25 µl of ice-cold low salt acetate
buffer containing 150 mM sucrose, 10 µg/ml PIs, and 1 mM dithiothreitol, then layered onto and pelleted through a
150-µl cushion (0.5 M sucrose in low salt acetate buffer
containing 0.5 µM microcystin LR, 10 µg/ml PIs,
and 1 mM dithiothreitol) in a TL-100 benchtop centrifuge (TLS55 rotor, 32,000 rpm, 4 °C, 5 min; Beckman Instruments).
Pelleted membranes were collected by careful removal of the cushion and addition of SDS-PAGE sample buffer, then subjected to SDS-PAGE and
transferred to nitrocellulose. Immunoblotting was carried out with
anti-dynein intermediate chain (monoclonal antibody 1618, Chemicon
International Ltd., Harrow, UK; 1/4,000 dilution) and anti-ribophorin
(CEL5C culture supernatant; a gift from Prof. Birgit Lane, University
of Dundee, Dundee, UK; 1/20) followed by 35S-labeled
anti-mouse IgG secondary antibody (ICN Biomedicals Inc., Basingstoke,
UK), which was diluted 1/250 in Tris-buffered saline (20 mM
Tris-HCl, 150 mM NaCl, pH 7.7) containing 1% TX-100 and 1% skimmed milk powder (Marvel, Premier Brands UK Ltd, Wirral, UK).
Detection was carried out using a BAS2000 imager (Fuji Photo Film Co.,
Tokyo, Japan), then densitometric analysis was carried out (AIDA
200, Version 2.0). Standard curves for DIC and ribophorin antibodies
were obtained using a dilution series of floated interphase Xenopus membranes, which were run beside each experiment.
The relative amount of DIC in each test sample was calculated by
comparison with the DIC standard curve, after which this value was
normalized for the amount of membrane (calculated by comparison with
the ribophorin standard curve).
Analysis of DLIC Phosphorylation by Mass
Spectrometry--
Tryptic digests of recombinant or endogenous DLIC
were analyzed by matrix-assisted laser desorption ionization mass
spectrometry (MALDI-MS) using a model VG TofSpec E mass spectrometer.
Recombinant His6-XDLIC (~15 µg) or purified
Xenopus CD prepared from interphase or CSF extracts were
subjected to SDS-PAGE and silver-stained (33), and the appropriate
bands were excised from the gel. These were digested with trypsin
(Promega Corp.), and the resulting peptides were processed for MALDI-MS
as described (33), except that products of the enzymatic digestion were
passed over Poros resin (Applied Biosystems, Cheshire, UK) before
spotting onto the metal target. To obtain larger amounts of DLIC
peptides for this analysis we also immunoprecipitated CD directly from
crude Xenopus egg extract using anti-XDLIC1 antibody. This
was carried out essentially as described (19) except that
rProteinG-agarose (Zymed Laboratories Inc., San
Francisco, CA) was used to carry the antibody. The peptide profiles of
DLIC immunoprecipitated from extracts were identical to those from
purified CD.
Cloning of Xenopus laevis DLIC1--
Multiple alignments of
published DLIC sequences from rat (17), chicken (16), and human
(GenBankTM accession number AAB88513) were used to design
degenerate primers to the amino acid sequences EKDAVFIP and SKKTGSPG.
These were used to amplify DLIC sequences from a Xenopus
oocyte cDNA library (24). The resulting polymerase chain reaction
product, which showed high similarity to published DLIC sequences, was used to screen the same cDNA library. Four independent clones were
identified, all of which encoded part or all of the same sequence. This
sequence, which was most closely related to DLIC1s from other organisms
(Table I), was called Xenopus
DLIC1 (XDLIC1). The XDLIC1 sequence is presented in Fig.
1 and is available under GenBankTM accession number AF317841.
Multiple Phosphorylation of Xenopus DLIC Proteins and Release from
Membranes--
We have previously observed a single DLIC species by
SDS-PAGE that migrates more slowly by SDS-PAGE in metaphase than
interphase samples (19), probably owing to increased levels of
phosphorylation. Here we have examined the SDS-PAGE mobility pattern of
Xenopus DLICs in more detail (Fig.
2) using modified electrophoresis
conditions that separate multiple DLIC bands and immunoblotting with an
antibody to XDLIC1. Interphase extracts, prepared either by activating a meiotically arrested CSF extract with calcium (Fig. 2A,
aCSF) or by making extracts from interphase eggs (Fig.
2B, left lane, top panel), showed
three DLIC bands from ~63 to 67 kDa, with the middle band being the
major species. This complex pattern is similar to that observed in
species which have two DLIC isoforms, for example bovine (17), chick
(16), and rat (17). This may indicate the presence of two
Xenopus DLIC isoforms even though a Xenopus DLIC2
was not found in our library screen.
CSF extracts (arrested in meiotic metaphase II) showed a major DLIC
band at 70-71 kDa and a minor band at 73 kDa (Fig. 2A, CSF). Interphase Xenopus egg extracts can be
driven into metaphase by the addition of recombinant GSTcyclinB1, which
leads to an activation of cdc2 kinase within 60 min, as monitored by
histone kinase activity (Fig. 2B), and a shift in
molecular weight of GSTcyclinB1 (see faint uppermost band
in Fig. 2B, upper panel, recognized by
residual anti-GST antibodies in the anti-XDLIC1 preparation), probably
owing to its phosphorylation by cdc2 kinase (34). This metaphase state
can be regarded as mitotic, since there is little remaining CSF
activity (a characteristic of meiotic metaphase II extracts). In the
presence of active cdc2-GSTcyclinB1 kinase, the DLIC migrated as a
doublet of ~71 and 68 kDa (Fig. 2B, 60 min,
upper panel), both forms migrating more slowly than the
interphase species (0 min lane). The 68-kDa band was not
observed in CSF extracts (Fig. 2A), perhaps reflecting
incomplete activation of cdc2 after a 60-min incubation with
GSTcyclinB1 (Fig. 2B) or, alternatively, that there are
additional kinases active in CSF extracts such as MAP kinase (35),
which might also phosphorylate DLIC. Since the DLIC bands observed in
both types of metaphase extracts had higher apparent molecular weights
than the interphase bands, all Xenopus DLICs appear to be
phosphorylated in a cell cycle-dependent manner.
Because we observed a 68-kDa DLIC form after incubation of interphase
extracts with GSTcyclinB1 (Fig. 2B) but not in CSF extracts (Fig. 2A), we wanted to determine whether the CD containing
this phospho isoform was released from the membrane. Floated membrane samples were therefore prepared at various times after GSTcyclinB1 addition and analyzed by immunoblotting for DLICs. Thirty minutes after
the addition of GSTcyclinB1, CD remained on the membrane with a
completely interphase-like pattern of phosphorylation (Fig. 2B). By 60 min, histone kinase activity was high, DLIC
migrated as ~71 kDa and 68 kDa bands in the total extract (Fig.
2B, upper panel), and both forms of DLIC had been
lost from the membranes (Fig. 2B, lower panel).
This indicated that release of CD requires cdc2 kinase activity and
rules out a requirement for any other kinases that are active in
meiotic but not in mitotic extracts.
Xenopus cdc2-GSTcyclinB1 Kinase Phosphorylates Xenopus DLICs in the
CD Complex and Removes CD from Membranes in Vitro--
We next tested
specifically whether Xenopus cdc2-GSTcyclinB1 kinase could
phosphorylate Xenopus DLICs while they were part of the
intact CD complex. Active cdc2-GSTcyclinB1 kinase was incubated with interphase CD (both purified from Xenopus egg
extracts) in the presence of
We wondered whether this phosphorylation of DLICs in the CD complex by
cdc2 kinase could be directly responsible for removal of CD from
membranes as extracts enter metaphase. To test this in vitro
we incubated active Xenopus cdc2-GSTcyclinB1 with membranes isolated by flotation from interphase Xenopus egg extracts.
We then pelleted membranes through a sucrose cushion and immunoblotted for the DIC (to show the amount of CD) and ribophorin (an integral membrane protein to control for equal amounts of membrane) using [35S]methionine-labeled secondary antibodies. We
quantitated the blot signal very carefully versus a dilution
series of membrane samples loaded on the same gel to provide a
standard curve for both DIC and ribophorin antibody signals (see
"Experimental Procedures"). Incubation with cdc2-GSTcyclinB1 kinase
caused around half of the CD (51 ± 3.2%, n = 3 ±S.E.) to be displaced from membranes as compared with the buffer only
control (Fig. 3C).
The First of Four Potential cdc2 Phosphorylation Sites Conserved in
DLICs Is Constitutively Phosphorylated in Interphase Xenopus Egg
Extracts--
Dell et al. (23) identify a conserved cdc2
phosphorylation site (site 1; Fig.
4A) as being responsible for
changes in DLIC mobility on SDS-PAGE gels after in vitro
incubation of recombinant chick DLIC1 with cdc2-cyclinB1 (23). The
implication of this finding is that this site is involved in cell cycle
regulation of CD function. However, by comparing the XDLIC1 sequence
with other published DLIC sequences and using consensi described by Pearson and Kemp (36), we have found three additional conserved potential cdc2 phosphorylation sites in DLICs (sites 2-4;
Fig. 4B).
To try to determine whether any of these sites are phosphorylated by
cdc2, we isolated native CD from metaphase and interphase extracts
either by classical microtubule affinity and sucrose-gradient purification or by immunoprecipitation. The DLIC proteins (and bacterially expressed His6-XDLIC1 as a control) were
excised from silver-stained gels and processed for analysis by
MALDI-MS. Bacterially expressed His6-XDLIC1 gave a
consistent pattern of tryptic peptides, most of which were identifiable
in both metaphase and interphase preparations.2 However, we
found that DLICs from both interphase and metaphase CD lacked the
peptide containing site 1 (2124 Da), which was obvious in the control
bacterially expressed sample (Fig. 5).
Instead, both traces showed a peptide 80 Da larger than the expected
size (2208-2211 Da) that was absent from the control, corresponding to
the addition of one phosphate to the site 1 peptide (37). We conclude
that site 1 (Fig. 4A) is phosphorylated in both interphase and metaphase and therefore cannot be responsible for the regulation of
CD function in this system. We therefore propose that cell cycle-dependent regulation of CD-driven membrane movement
is mediated by phosphorylation of DLICs by cdc2 kinase at an
alternative conserved potential phosphorylation site (Fig.
4B). The close proximity of these potential phosphorylation
sites (Fig. 4B) and the lack of convenient protease sites in
this portion of the XDLIC1 meant that we were unable to determine which
of these alternative sites is phosphorylated using MALDI-MS.
Surprisingly little is known about how microtubule-based motors
are regulated. Even in melanophores, where phosphorylation controls the
direction of organelle movement, the molecular details of how this is
achieved are unclear (38, 39). One example where motor phosphorylation
has been directly correlated with altered motor function is the cell
cycle regulation of CD-driven ER movement in Xenopus egg
extracts (19). On conversion from interphase to metaphase, both ER
motility and the amount of membrane-associated CD decreased ~10-fold,
whereas phosphate incorporation into the DLIC increased 12 times (19).
This suggests that DLIC phosphorylation by a cell cycle-regulated
kinase causes CD release from membranes and, hence, an inhibition of
membrane movement. The data obtained in this study support and extend
this model. We have demonstrated that cdc2 kinase not only
phosphorylates DLIC within the native CD complex but also triggers the
release of CD from isolated interphase membranes.
Interestingly, DLIC was the only CD subunit phosphorylated by cdc2
kinase either in vitro (Fig. 3A) or in metaphase
extracts (19) despite the fact that DHC and DIC sequences contain
multiple potential cdc2 kinase phosphorylation sites. We can therefore conclude that there is no phosphate turnover and therefore no 32P incorporation in vitro at any of these sites
in DHC or DIC in metaphase or interphase extracts. Whether these sites
are unused or whether they are stably phosphorylated is not known. The
latter phenomenon has been elegantly demonstrated for DIC, which
becomes hyperphosphorylated upon entry into meiotic metaphase I in
Xenopus oocytes and remains stably phosphorylated through
multiple interphases and metaphases, at least up to stage X embryos
(40). The p150Glued subunit of dynactin
also behaved in this unusual manner (40), and it along with the rest of
the dynactin complex is a poor substrate for cdc2 kinase in
vitro.3 These data raise
interesting questions about potential differential regulation of CD
phosphatases during oogenesis, early embryogenesis, and the somatic
cell cycle.
A key question is which if any of the four obvious potential cdc2
kinase phosphorylation sites in DLIC is used? Dell et al. (23) demonstrate that site 1 in recombinant chick light intermediate chain was efficiently phosphorylated in metaphase Xenopus
egg extracts but not interphase extracts and was also
phosphorylated by purified cdc2 kinase. However, when we used MALDI-MS
to investigate native CD isolated from both interphase and metaphase
extracts, we found that site 1 (Fig. 4) is phosphorylated regardless of cell cycle status (Fig. 5). That this site remains phosphorylated in
interphase, when very little DLIC phosphate turnover takes place (19),
strongly suggests that site 1 is stably phosphorylated in the native
molecule and therefore cannot be responsible for the regulation of CD
binding to membranes. Whether this phosphate turns over during meiotic
metaphase II or whether it becomes stably phosphorylated during meiosis
I, as is the case for DIC (40), remains to be established. In addition,
although site 1 in recombinant protein is used by cdc2 kinase, it is
also a consensus site for MAP kinase (36), and so at present, we can
not conclude which kinase is responsible for its phosphorylation in the
native complex in CSF extracts, which have high MAP kinase activity
(35).
If site 1 can be ruled out, then what about the other three sites? cdc2
kinase sites 2-4 lie in regions that unfortunately have not generated
suitable peptides for MALDI-MS with any of the proteases tested so far,
so we do not yet know which site in DLIC is responsible for the
observed cell cycle regulation. It is interesting to note, however,
that Drosophila has only a single DLIC that appears equally
related to DLIC1s and DLIC2s and that only possesses sites 1 and 3 (Fig. 4), suggesting that site 3 is the most likely candidate. One
method for testing the importance of sites 2-4 is site-directed
mutagenesis, but the difference between our results and those of Dell
et al. (23) raises concerns over that approach. First,
bacterially expressed DLIC may not be in a native conformation, either
because of folding problems or because it is not assembled as part of
the CD complex. Second, our preliminary results suggest that
recombinant DLIC added to extracts does not become incorporated into
existing Xenopus CD molecules.2 Third,
bacterially expressed DLIC will be completely unphosphorylated, which
may leave sites accessible that are normally stably phosphorylated. Fourth, it is well known that some phosphorylation events require other
sites to be phosphorylated first, which could complicate interpretation
of mutagenesis studies. It seems likely then that a combination of
experimental approaches will be needed to investigate fully the
regulation of CD activity by DLIC phosphorylation.
Inhibition of ER movement in metaphase egg extracts correlates with the
loss of CD from the membrane (19). We show here that incubating
interphase egg membranes with Xenopus cdc2-cyclinB1 kinase
leads to the release of about half of the CD, suggesting that cdc2
kinase is directly involved in the cell cycle regulation of ER motility
in Xenopus early embryogenesis. The simplest model is that
DLIC phosphorylation by cdc2 kinase triggers the release of CD,
although it is formally possible that DLIC phosphorylation prevents
re-binding of any CD that detaches from membranes as part of an
equilibrium. How cdc2 activity also leads to the loss of
membrane-associated dynactin (19) is unclear, particularly since the
dynactin complex itself is not a substrate for this kinase. However, we
have not yet tested whether cdc2-dependent phosphorylation
of any membrane components is also important. The fact that treatment
of interphase membranes with cdc2 kinase releases ~50% of CD,
whereas metaphase membranes have lost ~90% of their CD, may simply
suggest that the assay with membranes and kinase alone is not fully
optimized. This is quite likely, since not all of the DLIC underwent
complete molecular weight shifts when purified CD was incubated with
cdc2 kinase (Fig. 3B) compared with when the kinase was
activated by adding GSTcyclinB1 to interphase extracts (Fig.
2B). It is also possible that other cytosolic factors are
needed for full release.
The simple binding or release of CD from membranes is clearly not the
only mechanism for regulating organelle movement however. For instance,
a variety of organelles that are moving using plus end-directed
microtubule motors during interphase also possess CD, presumably
inactive, on their surface (31, 39, 41, 42). Moreover, CD-driven ER
movement in interphase Xenopus egg extracts can be greatly
stimulated without recruiting any extra motors (22).
One interesting question is whether the regulatory mechanism we have
characterized operates during mitosis in somatic cells (rather than
through the embryonic cell cycle, as assessed here and previously), as
there are conflicting reports of whether CD remains associated with its
membranous cargoes (43, 44). Since both DIC and
p150Glued do incorporate phosphate during
somatic mitosis but not during embryonic cycles (40), it seems likely
that the regulatory system is more complex in the former, perhaps
because there are extra functions for dynein in somatic cell division.
It should be stressed that although the membrane movement functions of
CD may be down-regulated in mitosis, it seems certain that there are
other roles for this motor (see Ref. 9 for a review) that have to be
stimulated on entry into mitosis, such as the interaction with
NuMA (45) and those like the interaction between LIC1 and
pericentrin, which may be active at all times (46, 47). To add to this
complexity, there may be distinct CD complexes that contain specific
assortments of subunits, which may be responsible for different CD
functions (44, 47) and which could be differentially regulated. Other microtubule motors, such as Eg5 and MKLP1, may also be regulated by
cdc2 kinase and other cell cycle-regulated kinases (48-51). Understanding how the cell integrates the function of these motors to
generate different cellular processes and architecture throughout the
cell cycle will require knowledge of the regulation of a whole range of
motors, and Xenopus egg extracts offer an excellent model system for such investigations.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP), 10 µg/ml protease inhibitors (PIs: stock
solution 10 mg/ml leupeptin, pepstatin, chymostatin, and aprotinin in
Me2SO) and 1 mM dithiothreitol in a total
volume of 20 µl. Incubations were quenched with 1 ml of
immunoprecipitation buffer (80 mM sodium
-glycerophosphate, 10 mM sodium pyrophosphate, 10 mM Tris/HCl, pH 7.5, 1% Surfact-Amps X-100 (TX100; Pierce
& Warriner (UK) Ltd.), 1 µg/ml PIs), incubated on ice for 10 min,
then mixed with 7 µl of anti-dynein intermediate chain antibody 70.1 (Sigma) pre-bound to 40 µl of rProteinL-agarose (Actigen, Cambridge,
UK). This was rotated at 4 °C for 2 h, then supernatant was
removed, and beads were washed with immunoprecipitation buffer (4 × 500 µl), immunoprecipitation buffer containing 500 mM
NaCl (1 × 500 µl), and 50 mM Tris, pH 7.5, containing PIs and 1 mM dithiothreitol (1 × 500 µl). SDS-PAGE sample buffer was added to the final beads, samples
were subjected to SDS-PAGE, and the dry gel was exposed to x-ray film.
Immunoprecipitation of CD from in vitro phosphorylation
reactions was necessary because GSTcyclinB1 was autophosphorylated by
the cdc2-GSTcyclinB1 kinase and runs close to DLICs on gradient gels.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
XDLIC1 is most closely related to DLIC1 isoforms
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Fig. 1.
Sequence analysis of XDLIC1. cDNA
sequence for XDLIC1. Stop codons (*), XDLIC1 open reading frame
(uppercase), amino acid numbers (left), and
nucleotide numbers (right) are indicated. cdc2 consensus
phosphorylation sites are boxed, and the consensus P-loop
sequence is underlined. The XDLIC' polymerase chain reaction
product sequence runs from nucleotide 1011 to 1440.
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Fig. 2.
Time course of DLIC molecular weight shift
and loss of CD from membranes. A, a meiotic metaphase
extract was incubated for 45 min with buffer (CSF) or with
calcium (aCSF) to trigger entry into interphase, and then
both samples were immunoblotted using affinity-purified antibody to
XDLIC1. Separation of proteins in the 50-80 kDa region was optimized
by running 8% SDS-PAGE gels until proteins under 50 kDa were at the
bottom of the gel. Sizes of protein molecular weight markers are shown
in kDa. B, interphase Xenopus egg extract was
incubated with recombinant GSTcyclinB1. After the indicated times,
total extracts (E) and floated membranes (M) were
analyzed by immunoblot for XDLIC. Molecular mass markers are shown on
the left in kDa. Asterisks mark the position of GSTcyclinB1.
Histone kinase activity (HK) of total extract is indicated
in pmol of phosphate incorporated into histone per µl of extract per
min.
-32P-labeled ATP. Intact CD
complex was immunoprecipitated from this reaction mixture using a
monoclonal antibody to DIC and examined by SDS-PAGE. Fig.
3A shows that a strongly
radioactive protein band was detected at ~71 kDa corresponding to
DLICs that had incorporated labeled phosphate. Despite possessing
potential cdc2kinase phosphorylation sites (see "Discussion"), the
other CD subunits were not substrates for cdc2-GSTcyclinB1 kinase
in vitro. Immunoblotting for DLICs after incubation of CD
with cdc2-GSTcyclinB1 kinase (Fig. 3B) revealed two new
bands at around 71 and 68 kDa, very like the pattern observed 60 min
after GSTcyclinB1 was added to interphase extract (Fig. 2B).
None of the lowest mobility interphase DLIC remained, whereas some DLIC
still co-migrated with the upper interphase DLIC form.
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Fig. 3.
Purified cdc2-GSTcyclinB1 kinase
phosphorylates DLIC and removes CD from membranes in vitro.
A, Xenopus CD and cdc2-GSTcyclinB1 were incubated
together in an in vitro phosphorylation experiment as
described under "Experimental Procedures" (lane 1).
Controls were as follows: lane 2, no antibody for
immunoprecipitation from the reaction mixture; lane 3, no
cdc2-GSTcyclinB1; lane 4, no cdc2-GSTcyclinB1 or antibody;
lane 5, no CD; lane 6, no dynein or antibody. A
5-20% gradient gel was used to ensure resolution of all CD
components, but under these conditions DLIC migrates as one band.
B, a similar in vitro phosphorylation experiment
run on an 8% gel was immunoblotted with anti-XDLIC1 antibody. The
equivalent to lanes 3 ( cdc2) and 1 (+cdc2) in
panel A are shown. Sizes of protein molecular weight markers
are indicated in kDa. C, immunoblotting of membranes
pelleted after incubation with (+) or without (
) cdc2-GSTcyclinB1
kinase was carried out using antibodies to DIC, with ribophorin
(R) as a membrane-loading control. A representative
experiment is shown. The amount of CD on membranes in this experiment
(as a percentage of the buffer only control) is displayed
(CD) and was calculated after normalization to the amount of
membrane present, as described under "Experimental
Procedures."
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Fig. 4.
Conserved potential cdc2 phosphorylation
sites in DLICs. Accession numbers and abbreviations for sequences
are as for Table I except for: DDLIC, Drosophila DLIC
(GenBankTM accession number AAF48011); MDLIC1, mouse DLIC1
(partial translation from GenBankTM accession number
AA146205); and MDLIC2, mouse DLIC2 (partial translation from
GenBankTM accession number A253855). The following
consensus sequences were used to identify potential cdc2
phosphorylation sites: (S/T)PX(K/R); (K/R)(S/T)P; and
(S/T)P(K/R) (36). A, the region corresponding to amino acids
184-210 of XDLIC1 contains site 1, SPQR, which is conserved
in all DLICs (SPVK in Drosophila) (23). B, the
region corresponding to amino acids 379-405 of XDLIC1 contains site 2 SPR in DLIC1s (SPAR in DLIC2s, not present in Drosophila),
site 3 SPR (SPLR in Drosophila) which is conserved in all
DLICs, and site 4 TPxR, which is conserved in all DLIC1s.
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Fig. 5.
Analysis of trypsin-generated fragments of
recombinant and native Xenopus DLICs. Peptide
peaks from the 1900-2500-Da region of a MALDI-MS trace are shown from
recombinant His6-XDLIC1 (A), interphase DLICs
prepared by immunoprecipitation (B), and metaphase DLICs
prepared by microtubule affinity (C). Peaks are labeled with
their molecular mass in Da, and relative intensity of peaks is
displayed at the side. The asterisk marks a peak of around
2113 that was also present in lanes containing antibody
alone.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Dr. David J. Thornton and David Knight for advice on mass spectrometry and Dr. Jon Lane and Pete Brown for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported by Wellcome Trust Grant 048894/Z/96/A, Medical Research Council Co-operative Group Award G9722026, Medical Research Council Senior Fellowship G117/153 (to P. G. W.), and a Senior Fellowship from the Lister Institute of Preventive Medicine (to V. J. A.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF317841.
To whom correspondence should be addressed: University of
Manchester, School of Biological Sciences, 2.205 Stopford Bldg., Oxford
Rd., Manchester, M13 9PT, UK. Tel.: 44 161 275 5646; Fax: 44 161 275 5082; E-mail: viki.allan@man.ac.uk.
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M011628200
2 S. Addinall, S. Doyle, and V. Allan, unpublished results.
3 V. Allan and P. Mayr, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: CD, cytoplasmic dynein; DHC, cytoplasmic dynein heavy chain; DIC, cytoplasmic dynein intermediate chain; DLIC, cytoplasmic dynein light intermediate chain; CSF, cytostatic factor; GST, glutathione S-transferase; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; PI, protease inhibitor; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis.
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