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INTRODUCTION |
Fully grown (stage VI) Xenopus oocytes are arrested in
a G2-like state. Exposure to progesterone releases oocytes
from this arrest and causes meiotic maturation. The maturing oocyte
undergoes germinal vesicle breakdown
(GVBD),1 forms a meiotic
spindle, segregates its homologous chromosomes, completes the first
meiotic division, enters meiosis 2, and then arrests in metaphase of
meiosis 2.
Oocyte maturation depends upon the activation of Cdc2-cyclin B
complexes (1), just as entry into mitotic M phase does (reviewed in
Refs. 2-4). In the Xenopus oocyte, Cdc2 activation is
tightly linked to activation of p42 mitogen-activated protein kinase
(MAPK) and its upstream activators Mek-1 (a MAPK kinase) and Mos (a
MAPK kinase kinase) (5-7). Interfering with the activation of Mos (8),
Mek-1 (9), or p42 MAPK (10) inhibits progesterone-induced Cdc2
activation, and overexpression of Mos (11, 12) or introduction of
active forms of Mek-1 (13) or p42 MAPK (14) can cause activation in the
absence of progesterone. p42 MAPK is activated in an all-or-none fashion (15), ensuring that the oocyte's decision to carry out maturation is decisive and irrevocable.
Relatively little is known about how the activation of MAPK and Cdc2
brings about the dramatic cell biological changes of maturation. Some
of the relevant substrates of these kinases are undoubtedly involved in
entry into mitosis as well; others must be specific for meiosis or
maturation. Expression cloning studies have identified a number of
proteins that become phosphorylated at the onset of mitosis (16-18),
and work is under way to determine whether and how their mitotic
phosphorylation affects their function.
Here we have approached the identification of meiotic phosphoproteins
by antiphosphotyrosine immunoblotting, the strategy that implicated p42
MAPK in Xenopus oocyte maturation (19-22). We looked for
immunoreactive bands that increased in intensity during oocyte
maturation (as does p42 MAPK), decreased in intensity (as does Cdc2),
or changed in their electrophoretic mobility.
This paper focuses on the first of these proteins, p83, a band that
shifts up in its apparent molecular weight during oocyte maturation but
does not change substantially in intensity. Through purification and
peptide sequencing, we have identified p83 as a component of the
minus-end-directed microtubule motor dynein, the cytoplasmic dynein
intermediate chain (dynein IC). We found that dynein IC comigrates with
p83 and undergoes mobility shifts that exactly parallel those of p83;
that the mobility shift of dynein IC is due to phosphorylation; and
that dynein IC cross-reacts with various phosphotyrosine antisera but
is phosphorylated mainly at serine in vivo. Dynein IC was
found to undergo hyperphosphorylation just prior to germinal vesicle
breakdown and to remain hyperphosphorylated throughout maturation
and early embryogenesis.
In addition, we examined the phosphorylation of the
p150Glued subunit of dynactin, because of the physical and
functional association between dynein and dynactin (23-28). We found
that p150Glued also becomes hyperphosphorylated during
oocyte maturation and, like dynein IC, remains in a shifted form during
early embryogenesis. Finally, we found that both dynein IC and
p150Glued exhibit mobility shifts in nocodazole-treated
XTC-2 cells and HeLa cells. These findings demonstrate that multiple
components of the dynein/dynactin system undergo coordinated
phosphorylation changes at the G2/M transitions of both
meiosis and mitosis.
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EXPERIMENTAL PROCEDURES |
Isolation of Xenopus Oocytes, Eggs, and
Embryos--
Xenopus ovarian tissue was obtained surgically
and defolliculated with 2 mg/ml collagenase as described (29). Stage VI
oocytes were sorted manually and incubated at 16 °C for at least
8 h. Eggs were obtained by dorsal lymph sac injection of female
frogs with human chorionic gonadotropin. Eggs were fertilized and
dejellied as described (30).
Oocyte Maturation--
Immature stage VI oocytes were treated
with progesterone (5 µg/ml) in modified Barth's saline containing
Ca2+ and bovine serum albumin at room temperature for
various lengths of time to induce maturation. GVBD was inferred from
the appearance of a distinct white dot at the oocyte's animal pole.
Groups of oocytes were quick-frozen on dry ice.
Preparation of Oocyte, Egg, and Embryo Lysates--
Ten oocytes,
eggs, or embryos were added to 100 µl of ice-cold extract buffer (250 mM sucrose, 100 mM NaCl, 2.5 mM
MgCl2, 20 mM HEPES, pH 7.2) containing 0.5 mM Na3VO4 and protease inhibitors (10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml chymostatin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride)
and lysed by pipetting through a 200-µl pipette tip. Lysates were centrifuged for 5 min in a microcentrifuge with a right angle rotor.
Samples of clarified cytoplasm were removed from the overlying lipid
and underlying yolk protein. Cytoplasm was frozen on dry ice and stored
at
80 °C.
Preparation of Concentrated Oocyte and Egg
Extracts--
Concentrated cell-free Xenopus oocyte
extracts were prepared essentially as described previously (29).
Cycling egg extracts were prepared as described (32).
Protein Purification--
G2 phase oocyte lysates
containing approximately 600 mg of protein were subjected to a 20-35%
ammonium sulfate cut. The precipitate was resuspended in 60 ml of
buffer A (50 mM MES, pH 6.5, 20 mM NaCl, 0.1 mM EGTA, 1 mM dithiothreitol, and 10%
glycerol) and centrifuged at 10,000 rpm for 10 min at 4 °C to remove
insoluble proteins. The supernatant was loaded on an S-Sepharose Fast
Flow column (Amersham Pharmacia Biotech) equilibrated with buffer A. The column was washed extensively with buffer A, and proteins were
eluted with buffer B (50 mM MES, pH 6.5, 1 M
NaCl, 0.1 mM EGTA, 1 mM dithiothreitol, and
10% glycerol). The eluent was diluted with a 10× volume of buffer C
(20 mM Tris, pH 7.6, 1 mM dithiothreitol, and
10% glycerol) and then spun at 10,000 rpm for 15 min at 4 °C. The
supernatant was filtered through a 0.22-µm filter. The filtrate was
applied to a 5-ml FPLC HiTrap Blue column (Amersham Pharmacia Biotech)
equilibrated with buffer D (buffer C plus 10 mM NaCl).
Protein was eluted with a 35-ml linear gradient from buffer C to buffer
E (buffer C plus 1.5 M NaCl and 2% ethylene glycol).
Fractions were collected and assayed for p83 by antiphosphotyrosine immunoblotting. The 83-kDa doublet bands were excised from the SDS-PAGE
and sent to the W. M. Keck Foundation Biotechnology Resource Laboratory (Yale University) for sequencing.
32P Labeling in Vivo--
For in vivo
32P labeling, groups of 50 oocytes were placed in a
12-well plate containing 2 mCi of [32P]orthophosphate/ml
in modified Barth's saline containing Ca2+ and bovine
serum albumin as described above and incubated with or without
progesterone (5 µg/ml). Once the progesterone-treated oocytes reached
GVBD, both groups of oocytes were transferred to nonradioactive
modified Barth's saline solution, washed four times, and frozen on dry
ice. Lysates were prepared as described above.
Immunoblotting--
Samples were separated on 7.5 or 10%
low-bis polyacrylamide SDS gels (acrylamide:bisacrylamide ratio 100:1)
and transferred to Immobilon P (Millipore Corp.) blotting membranes.
Proteins were detected with polyclonal antiphosphotyrosine antiserum
(21), dynein IC monoclonal antibody (clone 70.1; Sigma), or
p150Glued monoclonal antibody (Transduction Laboratories),
followed by 125I-protein A for phosphotyrosine detection or
alkaline phosphatase-conjugated secondary antibodies (Sigma) for dynein
IC and p150Glued detection.
Immunoprecipitation--
Oocyte lysates were diluted with 1 volume of IP buffer (25 mM Tris, pH 8.0, 10 mM
MgCl2, 15 mM EGTA, 0.1% Triton X-100, 0.5 mM NaF, 0.5 mM Na3VO4,
60 mM 2-glycerolphosphate, and 0.1% bovine serum albumin).
The solution was incubated with dynein IC antibody 70.1 or
p150Glued antiserum at 4 °C for 2-4 h. Anti-mouse IgM
agarose (for dynein IC) (Sigma) or anti-mouse IgG agarose (for
p150Glued) (Sigma) was added and incubated for another
2 h at 4 °C. Immune complexes were washed four times with
immunoprecipitation buffer lacking NaF, Na3VO4,
and 2-glycerolphosphate. The resulting pellets were resuspended either
in SDS sample buffer for SDS-PAGE analysis or
-phosphatase buffer
(50 mM Tris, pH 7.5, 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij 35, and 2 mM
MnCl2).
-Phosphatase Treatment--
Dynein IC or
p150Glued immunoprecipitates were incubated with 400 units
of
-phosphatase (New England BioLabs) in
-phosphatase buffer at
30 °C for 30 min. This was followed by adding an additional 400 units of
-phosphatase for a further 60 min at 30 °C.
Phosphoamino Acid Analysis--
32P-Labeled lysates
were subjected to immunoprecipitation with dynein or
p150Glued antibodies followed by SDS-PAGE and transfer to
Immobilon P membranes as described above. Dynein IC and
p150Glued bands were excised and subjected to partial acid
hydrolysis (33). The hydrolysates were mixed with phosphoamino acid
standards and subjected to two-dimensional (pH 1.9 followed by pH 3.5)
electrophoresis on thin layer cellulose plates (34).
Cell Culture and Synchronization--
XTC-2 cells were grown in
70% L-15 medium supplemented with 10% fetal calf serum. HeLa cells
were grown in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum. Cells were arrested in interphase (S phase) by
double thymidine block. Cells were treated with 2 mM
thymidine for 24 h, grown in regular medium for 12 h, and
then grown again for 24 h in medium containing 2 mM
thymidine. Cells were arrested in M phase by release from double thymidine block followed by treatment with nocodazole (100 ng/ml) for
12-16 h. Mitotic cells were recovered by gentle shake off. Mitotic
index was determined by staining an aliquot of the cells with Hoechst
33342 dye followed by epifluorescence microscopy. Samples for
immunoblotting were prepared by suspending washed pelleted cells in
phosphate-buffered saline and lysing with SDS sample buffer.
Recombinant Proteins and Other Chemicals--
Bacterially
expressed wild-type malE-Mos protein was purified as described (12).
Okadaic acid was purchased from Life Technologies, Inc.
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RESULTS |
Hyperphosphorylation of p83 during Oocyte Maturation--
We used
antiphosphotyrosine immunoblotting to search for proteins that undergo
phosphorylation changes during Xenopus oocyte maturation,
with the aim of identifying new regulators or effectors of meiosis. In
agreement with previous reports (19, 21, 22), the most prominent
antiphosphotyrosine-reactive bands were a ~42-kDa band corresponding
to p42 MAP kinase, which appeared just prior to GVBD, and a ~33-kDa
band corresponding to Cdc2, which disappeared just prior to GVBD (Fig.
1A). In addition, we
identified five other bands recognized by at least one
antiphosphotyrosine antiserum that changed in intensity or mobility
during maturation, with apparent molecular masses of 83, 95, 100, 116, and 140 kDa (Fig. 1A and data not shown). Early attempts to
identify these proteins by testing plausible candidates proved to be
unsuccessful. We therefore purified each of the proteins from
Xenopus oocytes or eggs, using antiphosphotyrosine
immunoblotting as an assay.

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Fig. 1.
Tyrosine phosphorylation of p83.
A, antiphosphotyrosine immunoblot of G2 and M
phase oocyte lysates. Stage VI oocytes were treated with progesterone
to induce maturation. G2 and M phase oocyte lysates were
subjected to SDS-PAGE, transferred to a blotting membrane, and probed
with antiphosphotyrosine antiserum. Migration of molecular weight
standards is indicated on the left. B,
antiphosphotyrosine immunoblot of Xenopus extracts treated
with okadaic acid or Mos. Cell-free G2 phase oocyte
extracts were treated with okadaic acid (5 µM) or Mos (40 nM). At the indicated times (top), samples were
removed and analyzed by antiphosphotyrosine immunoblotting.
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Here we shall focus on p83, which migrated as a doublet in
G2 phase with the upper band becoming more prominent during
maturation (Fig. 1A). Both p83 bands were recognized by
several antiphosphotyrosine antisera (PY20, 4G10, and the polyclonal
serum used in the blots shown herein), and their recognition was
blocked by phenylphosphate (40 mM) or phosphotyrosine (1 mM) but not by phosphothreonine (1 mM) or
phosphoserine (1 mM) (data not shown).
Both p83 bands were detected in G2 phase Xenopus
extracts and shifted to the upper band in extracts treated with okadaic
acid, a phosphatase inhibitor, and Mos, an activator of the MAP kinase cascade (Fig. 1B). p83 also shifted in response to added
active Cdc2-cyclin B (Fig. 4 and data not shown).
Purification of p83 and Identification as Dynein Intermediate
Chain--
Initial attempts to immunopurify p83 with
antiphosphotyrosine antibodies were unsuccessful; we therefore used
classical protein purification techniques. G2 phase oocyte
lysates were subjected to ammonium sulfate precipitation (taking a
20-35% cut) followed by S-Sepharose cation exchange chromatography
and HiTrap Blue dye affinity chromatography. The HiTrap Blue column
profile is shown in Fig. 2. p83 eluted as
a doublet in fractions 31-35 as judged by antiphosphotyrosine
immunoblotting (Fig. 2C). Corresponding bands were seen by
Coomassie staining (Fig. 2B).

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Fig. 2.
HiTrap Blue column fractionation of p83 from
G2 phase Xenopus oocyte lysates.
G2 phase lysates were fractionated by ammonium sulfate
precipitation and S-Sepharose cation exchange chromatography as
described under "Experimental Procedures." Fractions containing p83
were diluted, applied to a HiTrap Blue column, and eluted with a linear
salt gradient. Fractions were collected and analyzed by SDS-PAGE
(B) and immunoblotting with antiphosphotyrosine antiserum
(C). A, elution profile from the HiTrap Blue
column. Fractions 31-35 contained p83. Migration of an 85-kDa
molecular mass standard is indicated on the left.
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To address whether the two p83 bands were derived from the same
protein, both bands were cut out from SDS-polyacrylamide gels and
subjected to tryptic digestion and reversed phase microbore HPLC
analysis. The patterns of tryptic peptides from the two bands were
nearly identical (Fig. 3A).
All of the major peaks were present in both samples, indicating that
these two proteins were probably closely related. To further test this
conclusion, four peptide peaks from each of the samples were subjected
to matrix-assisted laser desorption ionization mass spectrometry
(MALDI-MS). The masses of the upper and lower band peptides were very
similar (Fig. 3B), again supporting the idea that the upper
and lower p83 bands were derived from a single protein. These findings
also suggested that both of the two p83 bands were essentially pure (or, possibly, that both bands were contaminated by the same non-p83 protein).

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Fig. 3.
Tryptic analysis of the upper and lower p83
bands. Purified p83 was eluted from an SDS-polyacrylamide gel,
digested with trypsin, and subjected to microbore reversed phase HPLC.
A, elution profiles of tryptic peptides from the upper and
lower p83 bands. B, MALDI-MS analysis of peaks 1-4 from
A. The mass/charge ratio (M/Z) of each peak is shown. Peak 4 contained two peptides. C, sequence alignments. The protein
sequence of tryptic peptide 5 from the p83 lower band (A)
and a similar tryptic peptide from rat cytosolic dynein intermediate
chain are shown.
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The MALDI-MS analysis was used to identify peptide peaks that would be
suitable for gas phase microsequencing. One such peak (peak 5, from the
p83 lower band) was sequenced and found to comprise VTQVDFAPR. This
peptide was similar (8 of 9 identities) to a predicted tryptic peptide
from rat cytoplasmic dynein intermediate chain (dynein IC) (Fig.
3C). The predicted molecular mass of rat dynein IC is 74 kDa
(31), close to the apparent molecular weight of Xenopus p83.
We therefore hypothesized that p83 was Xenopus dynein IC.
To test this hypothesis, we made use of a monoclonal dynein IC antibody
that cross-reacts with the Xenopus protein. G2
phase oocyte extracts were obtained and treated with buffer, Mos,
okadaic acid, or Cdc2-cyclin B. Aliquots of the extracts were subjected to immunoblotting with the dynein IC antibody 70.1. Dynein IC was found
to migrate as a closely spaced 83-kDa doublet in G2 phase
extracts (Fig. 4A), and dynein
IC shifted to a single upper band in response to Mos, okadaic acid, or
Cdc2-cyclin B (Fig. 4A). Thus, dynein IC (Fig.
4A) and p83 (Fig. 1B) migrate similarly and
respond similarly to various stimuli. Immunoprecipitated dynein IC was
recognized on immunoblots by antiphosphotyrosine antibodies (Fig.
4B). Taken together, the common properties of the two
proteins identify p83 as dynein IC.

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Fig. 4.
Effects of Mos, Cdc2-cyclin B, and okadaic
acid on dynein intermediate chain phosphorylation. Cell-free
Xenopus extracts were treated with extract buffer, Mos (40 nM), okadaic acid (5 µM), or Cdc2-cyclin B
(50 units/µl). Samples were taken at various times and analyzed by
immunoblotting with dynein antibody 70.1 (A) or by
immunoprecipitation with dynein antibody 70.1 followed by
immunoblotting with antiphosphotyrosine antiserum (B).
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The Dynein IC Upper Band Is Hyperphosphorylated--
Next, we
addressed whether the upper dynein IC band represented a
hyperphosphorylated form of the lower band. Dynein IC was immunoprecipitated from G2 and M phase lysates.
Immunoprecipitates were then treated with extract buffer,
-phosphatase (a dual specificity phosphatase), or
-phosphatase
plus vanadate (an inhibitor of
-phosphatase), and the products were
subjected to electrophoresis and dynein IC immunoblotting.
G2 phase dynein IC migrated as a doublet (Fig.
5A, lane
1), and the upper band was eliminated by treatment with
-phosphatase (lane 2) but not by treatment
with
-phosphatase plus vanadate (lane 3).
Dynein IC isolated from M phase lysates migrated as a single upper band
(lane 4) and was shifted to a lower band by
treatment with
-phosphatase (lane 5) but not
by treatment with
-phosphatase plus vanadate (lane 6). These findings establish the dynein IC upper band to be
a hyperphosphorylated form of the lower band.

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Fig. 5.
The phosphorylation state of the upper and
lower dynein intermediate chain bands. A, effect of
phosphatase treatment on dynein IC electrophoretic mobility.
G2 or M phase lysates were immunoprecipitated by using
dynein IC antibody 70.1. The immunoprecipitates were treated with
extract buffer (lanes 1 and 4),
-phosphatase (lanes 2 and 5), or
-phosphatase plus Na3VO4 (lanes
3 and 6). Samples were then analyzed by the
SDS-PAGE and immunoblotting with dynein antibody 70.1. B,
phosphoamino acid analysis of in vivo labeled dynein
intermediate chain. Stage VI oocytes, treated with or without
progesterone, were metabolically labeled in vivo with
[32P]orthophosphate. Labeled oocytes were lysed and
immunoprecipitated with dynein antibody 70.1. Two-dimensional
phosphoamino acid analyses of 32P-dynein intermediate chain
immunoprecipitates from G2 (lower and
upper bands) and M phases are shown. The
positions of nonradioactive phosphoamino acid standards are shown on
the right. S, phosphoserine; T,
phosphothreonine; Y, phosphotyrosine. C, lack of
effect of phosphatase treatment on dynein IC antiphosphotyrosine
immunoreactivity. Dynein IC was immunoprecipitated from M phase lysates
and treated with -phosphatase with or without
Na3VO4.
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Phosphoamino Acid Analysis of in Vivo Labeled Dynein IC--
The
fact that both the upper and lower bands of dynein IC were recognized
by antiphosphotyrosine antibodies (Fig. 1) suggested that both forms
were phosphorylated at tyrosine. However, previous phosphoamino acid
analysis of axonemal dynein reportedly yielded only phosphoserine (35).
Consequently, we examined further whether dynein IC is phosphorylated
on tyrosine.
Dynein IC was labeled with [32P]orthophosphate in
vivo, subjected to immunoprecipitation, and transferred to a
blotting membrane. Radiolabel was found to be incorporated into
G2 phase dynein IC (both bands) and M phase dynein IC, and
the overall level of labeling of G2 and M phase dynein IC
was similar (data not shown). The dynein IC bands were excised from the
blot and subjected to partial acid hydrolysis and thin layer
electrophoresis. The two G2 phase dynein IC bands and the M
phase dynein IC band all yielded primarily phosphoserine (Fig.
5B), consistent with a previous report (35), and contrary to
the antiphosphotyrosine immunoblotting results (Figs. 1, 2, and 4), no
phosphotyrosine was detected. This finding suggests that the
recognition of dynein IC by antiphosphotyrosine antibodies might have
been spurious. We also immunoprecipitated dynein IC from M phase
oocytes and treated the immunoprecipitates with
-phosphatase. The
-phosphatase caused dynein IC to shift completely to the lower band
without decreasing its antiphosphotyrosine immunoreactivity (Fig.
5C). The simplest interpretation of these data is that the
antiphosphotyrosine antibodies recognize dynein IC in a phosphorylation
state-independent fashion.
Dynein IC Phosphorylation during Maturation, Mitosis in Extracts,
and Embryogenesis--
To determine whether dynein IC
hyperphosphorylation was specific to meiosis or occurred in other M
phases, we examined in detail the timing of dynein IC
hyperphosphorylation during maturation, mitosis in cycling extracts,
and the mitotic cycles of fertilized eggs and early embryos. Dynein IC
shifted to its hyperphosphorylated form just prior to GVBD (Fig.
6A), at about the time when
Cdc2 and p42 MAPK become activated (data not shown). Dynein IC remained hyperphosphorylated throughout the rest of meiosis 1 and into meiosis
2. There was no detectable decrease in dynein IC phosphorylation during
the period before meiosis 2 when Cdc2 activity drops (Fig. 6A and data not shown; see also Ref. (62).

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Fig. 6.
Dynein intermediate chain phosphorylation
during Xenopus oocyte maturation, in cycling egg
extracts, and during early embryogenesis. A, dynein IC
phosphorylation during progesterone-induced oocyte maturation. Stage VI
oocytes were treated with progesterone for the times indicated. Cell
cycle progression was monitored by the appearance of a white dot and by
Cdc2-cyclin B activity (not shown). Dynein IC was detected with
antibody 70.1. B, dynein IC phosphorylation in cycling egg
extracts. Extracts from electrically activated eggs were warmed to room
temperature to initiate cycling, and samples were taken at various
times. Demembranated sperm chromatin (~500 nuclei/µl) was added to
the cycling extract to allow monitoring of cell cycle progression.
C, dynein phosphorylation in fertilized Xenopus
eggs. Eggs were fertilized and dejellied, and samples were taken at
various times after fertilization. Unfertilized eggs (t = 0), and stage X embryos are also shown. Cleavages occurred at 90, 125, and 155 min.
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Next, we assessed dynein IC phosphorylation in cycling egg extracts,
with cell cycle progression assessed by observation of nuclei formed
from added sperm chromatin. As shown in Fig. 6B, the
extracts were initially in interphase and then entered mitosis at about
60 min and exited mitosis at about 80 min. During all of these cell
cycle phases, dynein IC remained in its hyperphosphorylated form (Fig.
6B).
Finally, we examined dynein IC phosphorylation during the completion of
meiosis 2 and the first three mitotic cycles of fertilized eggs. Once
again, dynein IC remained constitutively in its hyperphosphorylated form (Fig. 6C). Dynein IC was also found to be
hyperphosphorylated in stage X (gastrula) embryos. Taken together,
these results show that dynein IC goes from partially
hyperphosphorylated to fully hyperphosphorylated at the
G2/meiosis 1 transition during oocyte maturation, and then
remains constitutively hyperphosphorylated throughout the remainder of
meiosis, mitosis, and early embryogenesis.
p150Glued Undergoes Cell Cycle-dependent
Phosphorylation Changes--
Dynein IC physically interacts with the
p150Glued component of dynactin complex (27, 28). Since
dynein IC underwent cell cycle-dependent hyperphosphorylation during oocyte maturation, we set out to determine whether p150Glued did as well. As shown in Fig.
8A, p150Glued progressively shifted from a lower
apparent molecular weight band to a higher one during oocyte
maturation. The mobility shift was due to hyperphosphorylation, since
-phosphatase treatment caused M phase p150Glued to shift
from the higher band to the lower one (Fig.
7A). In vivo
32P labeling and phosphoamino acid analysis of
p150Glued showed that G2 phase
p150Glued was essentially nonphosphorylated, and M phase
p150Glued was phosphorylated predominantly at serine (Fig.
7B). Like dynein IC, p150Glued
hyperphosphorylation persisted throughout meiosis and early
embryogenesis (Fig. 8, A,
B, and D), and p150Glued became
hyperphosphorylated in Mos-, okadaic acid-, and Cdc2-cyclin B-treated
G2 phase oocyte extracts (although less rapidly and less
completely than did dynein IC; Fig. 8C versus
Fig. 4A). Okadaic acid also caused p150Glued to
shift to a third more highly shifted band that we have not detected
during the normal cell cycles of oocytes and eggs (Fig. 8C).

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Fig. 7.
Phosphorylation of p150Glued in
G2 and M phase oocytes. A, effect of
phosphatase treatment on p150Glued electrophoretic
mobility. G2 or M phase lysates were immunoprecipitated
with p150Glued antiserum. The immunoprecipitates were
treated with extract buffer (lanes 1 and
4), -phosphatase (lanes 2 and
5), or -phosphatase plus Na3VO4
(lanes 3 and 6). Samples were then
analyzed by the SDS-PAGE followed by immunoblotting with
p150Glued antiserum. B, phosphoamino acid
analysis of in vivo labeled p150Glued. Stage VI
oocytes, treated with or without progesterone, were metabolically
labeled in vivo with [32P]orthophosphate.
Labeled oocytes were lysed and immunoprecipitated with
p150Glued antiserum. Two-dimensional phosphoamino acid
analyses of 32P-dynein intermediate chain
immunoprecipitates from G2 and M phases are shown. The
positions of nonradioactive phosphoamino acid standards are shown on
the right. S, phosphoserine; T,
phosphothreonine; Y, phosphotyrosine.
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Fig. 8.
p150Glued phosphorylation during
Xenopus oocyte maturation, in cycling egg extracts, in
G2 phase oocyte extracts, and during early
embryogenesis. A, p150Glued phosphorylation
during progesterone-induced oocyte maturation. Stage VI oocytes were
treated with progesterone for the times indicated. Lysates were
subjected to SDS-PAGE and immunoblotting with p150Glued
antiserum. The percentage of oocytes with a white dot is shown
below the blot. B,
p150Glued phosphorylation in cycling egg extracts. Extracts
from electrically activated eggs were warmed to room temperature to
initiate cycling, and samples were taken at various times.
Demembranated sperm chromatin (~500 nuclei/µl) was added to the
cycling extract to allow monitoring of cell cycle progression.
G2 phase and M phase lysates are included for comparison.
C, effects of Mos, Cdc2-cyclin B, and okadaic acid on
p150Glued phosphorylation. Cell-free Xenopus
extracts were treated with extract buffer, Mos (40 nM),
okadaic acid (5 µM), or Cdc2-cyclin B (50 units/µl).
Samples were taken at the indicated times and subjected to
immunoblotting with p150Glued antiserum. D,
p150Glued phosphorylation in fertilized Xenopus
eggs. Eggs were fertilized and dejellied, and samples were taken at
various times after fertilization. Unfertilized eggs (t = 0) and stage X embryos are also shown. Cleavages occurred at 90, 120, and 140 min.
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Mitotic Phosphorylation of Dynein IC and p150Glued in
XTC-2 Cells and HeLa Cells--
Finally, we assessed whether dynein IC
and p150Glued were hypophosphorylated in interphase (as was
the case for oocytes) or hyperphosphorylated in interphase (as was the
case for early embryos) in two somatic cell lines, Xenopus
tadpole XTC-2 cells and human HeLa cells, and whether there was any
change in their phosphorylation when the cells entered mitosis.
Interphase cells were obtained after double thymidine block; mitotic
cells were obtained by releasing the double-thymidine blocked cells,
arresting them in M phase by nocodazole treatment, and harvesting the
mitotic cells by gentle shake off. This treatment yielded approximately
80% cells in mitosis as judged by chromosome staining.
In XTC-2 cells, dynein IC migrated as three bands in interphase (Fig.
9). In mitosis, the bottom band
disappeared, and the proportion of the dynein IC in the highest band
increased (Fig. 9). In HeLa cells, a single interphase band was
detected, and most of the dynein IC shifted to a higher band in M
phase. Thus dynein IC undergoes hyperphosphorylation both in meiosis in
oocytes and in mitosis in these two cell lines. Similar results were
found when unsynchronized XTC-2 cells were compared with
nocodazole-treated cells (data not shown).

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Fig. 9.
p150Glued and dynein intermediate
chain phosphorylation in S phase and M phase XTC-2 cells and HeLa
cells. S phase cells were obtained by double thymidine block. M
phase cells were obtained by releasing the S phase cells from the
double thymidine block in the presence of nocodazole (100 ng/ml). Cell
lysates were blotted with p150Glued (top) and
dynein IC (bottom) antibodies.
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p150Glued migrated as two bands in interphase XTC-2 cells,
with most of the p150Glued found in the lower band (Fig.
9). Most of the p150Glued shifted to the higher band in
mitosis (Fig. 9). Similar results were found with HeLa cells (Fig. 9).
Likewise, similar results were found when unsynchronized XTC-2 cells
were compared with nocodazole-treated cells (data not shown). Thus,
both p150Glued and dynein IC become hyperphosphorylated
during mitosis in XTC-2 and HeLa cells.
 |
DISCUSSION |
Initially, we identified p83 as a band on an antiphosphotyrosine
immunoblot that shifts from a lower to a higher apparent molecular mass
during oocyte maturation (Fig. 1). We purified both the upper and lower
p83 bands (Fig. 2) and demonstrated by peptide sequencing and mass
spectroscopy that they most likely represent the intermediate chain of
cytoplasmic dynein (Fig. 3). In support of this identification, we have
shown that dynein IC runs as two bands that co-migrate with the two p83
bands (Fig. 4 and data not shown) and that immunoprecipitated dynein IC
is recognized by antiphosphotyrosine antibodies on immunoblots (Fig. 4). Moreover, stimuli that cause p83 to shift to the upper band also
cause dynein IC to shift to the upper band (Fig. 4). We conclude that
p83 is dynein IC.
Both the upper and lower dynein IC bands are phosphorylated in
vivo (Fig. 5B). The upper band is converted to the
lower band by
-phosphatase treatment (Fig. 5A),
indicating that it represents a hyperphosphorylated form. Both bands
are phosphorylated predominantly at serine; no phosphotyrosine is
detected by in vivo labeling and phosphoamino acid analysis
(Fig. 5B). Moreover, the antiphosphotyrosine reactivity of
dynein IC is not diminished by
-phosphatase treatment (Fig.
5C). Thus, although the antiphosphotyrosine reactivity of p83/dynein IC is what originally allowed us to identify and purify the
protein, we believe that dynein IC is not phosphorylated at tyrosine.
However, it is the serine phosphorylation of dynein IC, not the
(apparent) tyrosine phosphorylation, that changes at the
G2/M transition, so it is the serine phosphorylation that is of particular interest. Thus, we examined under what circumstances the serine hyperphosphorylation occurs.
Dynein IC becomes hyperphosphorylated during oocyte maturation at about
the time of germinal vesicle breakdown and remains hyperphosphorylated
throughout maturation and early embryogenesis (Fig. 6). Dynein IC
undergoes a shift from hypo- to hyperphosphorylated forms in
nocodazole-treated M phase XTC cells and HeLa cells (Fig. 9). Thus,
dynein IC represents a novel M phase phosphoprotein that remains
constitutively in its M phase form during the rapid cell cycles of
early embryogenesis.
This is an unusual phosphorylation pattern; most M phase
phosphoproteins and M phase kinases cycle in their
phosphorylation/activity during early embryogenesis. However, one of
the kinases responsible for the phosphorylation of cyclin B2 (a kinase
of uncertain identity that phosphorylates N-terminal site(s) different
from serine 90) is activated during oocyte maturation and remains
active throughout early embryogenesis (36). It will be of interest to
determine whether this cyclin B2 kinase is also responsible for dynein
IC phosphorylation.
Because dynein IC physically and functionally interacts with the
p150Glued component of dynactin, we examined
p150Glued phosphorylation as well. We found that
p150Glued, like dynein IC, becomes phosphorylated during
oocyte maturation and remains hyperphosphorylated throughout maturation
and early embryogenesis (Fig. 8). It also shifts to a
hyperphosphorylated form in M phase in XTC cells and is less subtle in
this shift than is dynein IC (Fig. 9). Thus, p150Glued is
another novel M phase phosphoprotein and undergoes phosphorylation changes similar to those of its binding partner dynein IC.
Phosphorylation of Dynein IC and p150Glued in Other
Systems--
Pfister and co-workers have previously demonstrated that
dynein IC is a phosphoprotein, and that different cellular pools of
dynein exhibit differences in the relative phosphorylation of various
dynein subunits (35, 37, 38). p150Glued has also been
previously shown to be a phosphoprotein, and its phosphorylation has
been shown to be affected by activators of protein kinases A and C
(39). The present work extends these observations by showing that
dynein IC and p150Glued undergo marked changes in their
phosphorylation during meiotic and mitotic M phases. This suggests the
hypothesis that dynein IC and p150Glued phosphorylation
contribute to the dramatic changes in microtubule function that occur
at the G2/M transition. The demonstration that dynein and
p150Glued phosphorylation can be manipulated in
Xenopus oocyte extracts (Figs. 1, 4, 8) establishes a
powerful system for future work aimed at testing this hypothesis.
Mitotic Phosphorylation of Other Dynein Components--
Niclas and
co-workers (40) previously examined dynein phosphorylation in cycling
Xenopus egg extracts. They found a ~12-fold increase in
the phosphorylation of dynein light intermediate chain during M phase.
Thus, there are two different temporal patterns for the M phase
phosphorylation of dynein and dynactin: that shown by dynein IC and
p150Glued, with the proteins attaining their M phase form
during oocyte maturation and remaining locked in that state throughout
early embryogenesis and in cycling extracts, and that shown by dynein light intermediate change, with the protein cycling between M phase and
interphase forms in cycling extracts.
M Phase Functions of Cytoplasmic Dynein and
Dynactin--
Cytoplasmic dynein is a multisubunit complex consisting
of heavy chains, intermediate chains, light intermediate chains, and light chains (23, 41-43). Dynein can transport cargo along
microtubules toward the minus-end. Dynein IC can directly interact with
the p150Glued component of dynactin, which connects dynein
to organelles and other structures that are to be transported.
Dynein and dynactin have been implicated in a number of the dramatic
changes in cellular organization that occur at the G2/M transition. During mitosis, cytoplasmic dynein is associated with kinetochores, spindle, and centrosomes in mammalian cells in culture (44, 45), placing it in an appropriate location for control of
chromosomes or the spindle. Dynactin localization appears to be very
similar to that of dynein (46, 47).
Moreover, genetic studies and immunoneutralization studies have
functionally implicated dynein and dynactin in spindle assembly and
positioning. Budding yeasts with mutations in dynein show defects in
spindle orientation (48, 49). Immunodepletion or microinjection of
dynein antibodies interferes with spindle pole formation and
positioning (50-52), as does immunodepletion with antibodies to the
dynein-associated protein NuMA (53). Overexpression of p50 dynamitin, a
subunit of dynactin, results in distortion of the spindle and
dissociation of the dynactin complex from kinetochores (54).
Finally, biochemical studies have shown that phosphorylation has the
potential of regulating kinetochore associated plus- and
minus-end-directed microtubule motors (55) and that phosphorylation has
important effects on a variety of dynein- and kinesin-associated spindle components (56-60). It will clearly be of interest to
determine whether and how the M phase phosphorylation of dynein IC and
p150Glued described here contributes to these events. In
addition, minus-end-directed organelle transport decreases during M
phase in cycling extracts (61), apparently as a result of a
phosphorylation-associated decrease in the binding of dynein to its
membranous cargo (40). It is plausible that M phase phosphorylation of
dynein IC and p150Glued might be involved in shutting
down interphase processes that depend upon dynein and dynactin.