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Mol Biol Cell
1994 Aug 01;58:921-32. doi: 10.1091/mbc.5.8.921.
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Expression and activity of p40MO15, the catalytic subunit of cdk-activating kinase, during Xenopus oogenesis and embryogenesis.
Brown AJ
,
Jones T
,
Shuttleworth J
.
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Threonine 161 phosphorylation of p34cdc2 and its equivalent threonine 160 in p33cdk2 by cdk-activating kinase (CAK) is essential for the activation of these cyclin-dependent kinases. We have studied the expression and associated kinase activity of p40MO15, the catalytic subunit of CAK, during Xenopus oogenesis, meiotic maturation, and early development to understand in more detail how cdk kinases are regulated during these events. We find that p40MO15 is a stable protein with a half-life > 16 h that is accumulated during oogenesis. p40MO15 protein and its associated CAK activity are localized predominantly to the germinal vesicle; however, a small but significant proportion is found in the cytoplasm. The amount of p40MO15 detected in stage VI oocytes remains unchanged through meiotic maturation, fertilization, and early embryogenesis. Significantly, p40MO15 was found to be constitutively active during oogenesis, meiotic maturation, and the rapid mitotic cycles of early development. This suggests that regulation of p34cdc2 and p33cdk2 activity during cell cycle progression does not involve changes in the level or activity of p40MO15/CAK.
Figure 1. The synthesis and stability of p40M"l" during oogenesis
and meiosis. (A) Proteins from oocytes labeled for 4 h using 35Smethionine
were immunoprecipitated with MO15-specific immune
serum and analyzed by SDS-PAGE. The relative amounts of labeled
p40Mol5 in 30 stage II (lane 1), 20 stage IV (lane 2), five stage VI (lane
3), five dissected stage VI oocyte GVs (lane 4), and five dissected stage
VI oocyte cytoplasms (lane 5) are shown. Also shown is protein immunoprecipitated
from five labeled stage VI oocytes using preimmune
serum (lane 6). (B) Oocytes were labeled for 4 h with 35S-methionine
then incubated for 16 h in excess cold methionine. In addition, a
number of 35S-methionine labeled stage VI oocytes were incubated
for 16 h in excess cold methionine in the presence of progesterone.
Some of these progesterone-matured stage VI oocytes were then prickactivated
and incubated for a further 90 min. Proteins were then
immunoprecipitated and analyzed as described in A. The relative
amounts of labeled p40MOl5 in 30 stage II (lane 1), 20 stage IV (lane
2), five stage VI (lane 3), five progesterone-matured stage VI oocytes
(lane 4), five prick-activated, mature stage VI oocyte GVs (lane 6),
and five dissected stage VI oocyte cytoplasms (lane 7) are shown. The
positions of molecular weight markers (in kDa) are indicated.
Figure 2. Steady-state levels of p40MOlS in oocytes and embryos.
(A) Proteins from 25 stage II oocytes (lane 1), 12.5 stage IV oocytes
(lane 2), and 2.5 stage VI oocytes were separated by SDS-PAGE and
analyzed by Western blotting using M015-specific immune serum
and ECL detection (bottom). The relative amounts of p40MOI5 present
in each sample were quantitated by densitometry and plotted using
an arbitrary scale of 1-100. (B) Proteins from five progesterone-matured
stage VI oocytes (lane 1), and five stage 2, 6, 8, 10, 12, 16, 22,
and 30 embryos (lanes 2-9) were separated by SDS-PAGE and analyzed
by Western blotting using M015-specific immune serum and
ECL detection as described in A. The relative amounts of p40MOl5
present in each sample were quantitated by densitometry and plotted
using an arbitrary scale of 1-100.
Figure 3. The relative levels of M015 mRNA in oocytes and embryos.
(A) M015 cRNA (lane 1) and 10 ug of total RNA extracted
from stage VI oocytes (lane 2), unfertilized eggs (lane 3), and stage 8,
10, 12, 16, and 30 embryos (lanes 4-8) were annealed to a 689-nt
32P-labeled antisense probe from the 5'-end of M015. After RNase
digestion the products were analyzed on a 4% acrylamide/urea denaturing
gel. Protected fragments of the predicted size of 579 nt and
shorter (504 nt and 488 nt) are indicated. (B) Two micrograms of the
total RNA preparations used in A from stage VI oocytes (lane 1),
unfertilized eggs (lane 2), and stage 8, 10, 12, and 16 embryos (lanes
3-7) were annealed to a 454-nt 2P-labeled antisense ornithine decarboxylase
RNA probe. After RNase digestion the products were
analyzed on a 4% acrylamide/urea denaturing gel. The protected
fragment of the predicted size of 369 nt is indicated.
Figure 4. Biochemical localization of
p40MOlS in stage VI oocytes. Proteins from five dissected stage VI oocyte GVs
(lane 1), five dissected stage VI oocyte
cytoplasms (lane 2), and five total stage
VI oocytes (lane 3) were separated by
SDS-PAGE and analyzed by Western
blotting using M015-specific immune
serum and ECL detection (bottom). The relative amounts of p40Mol` present
were quantitated by densitometry and plotted using an arbitrary scale OF 1-100
Figure 5. Indirect immunofluorescent localization of p4O-MO15 in stage VI oocytes. Acetone-fixed cryosections of stage VI oocytes were incubated with either M015-specific rabbit polyclonal antibody, together with a mouse monoclonal antibody to nucleoplasmin ( A and B) preimmune rabbit serum together with a mouse monoclonal antibody to nucleoplasmin (C and D). Rabbit antibodies were detected using alkaline phosphatase conjugated goat. antirabbit IgG, followed by incubation with Fast Red substrate and fluorescence viewed using a Rhodamine filter (A and C). Mouse antibodies were detected using FITC-conjugated goat antimouse IgG and fluorescence viewed using a fluorescein filter (B and D).
Figure 6. The activity of
p4OMOI5 during oogenesis and
resumption of meiosis. (A)
p40Mo°5 immunoprecipitates
prepared from 25 stage II oocytes (lane 1), 12.5 stage IV oocytes (lane 2), and 2.5 stage VI
oocytes (lanes 3 and 4) were incubated with 1 jg of GSTcdk2
(lanes 1-3) or GSTcdk2T16OA
(lane 4) and [y_-32P]ATP for 45
min at 23°C. Supematants were
recovered, and phosphorylated
GSTcdk2 protein was analyzed
by SDS-PAGE and autoradiography (bottom). The relative
amounts of 32P associated with
GSTcdk2 in each reaction were
subsequently quantitated by
phosphorimage analysis and
were plotted on an arbitrary
scale of 1-100. (B) Immuno-
precipitates were prepared from
five dissected stage VI oocyte
GVs (lane 1), five dissected
stage VI cytoplasms (lane 2),
and five total stage VI oocytes
(lanes 3-6) using MO15-specific
immune serum (lanes 1-4) or
preimmune serum (lane 5). The
relative amounts of CAK in
each sample were assayed as
described in A using 1 ,Ag of GSTcdk2 (lanes 1-3 and 5) or GSTcdk2/
T160A (lane 4). (C) p40MO15 immunoprecipitates were prepared from
stage VI oocytes before (lane 1) and 0 (lane 2), 2 (lane 3), and 17 h
(lane 4) after the addition of progesterone. The relative amounts of
CAK in each sample were assayed as described in A using 1 ,ug of
GSTcdk2.
Figure 7. The activity of p4OMOI5 during early embryogenesis. (A)
p40M"lS immunoprecipitates were prepared from five unfertilized eggs
(lane 1) and five cleaving embryos at 205 (lane 2), 312 (lane 3), 322
(lane 4), 333 (lane 5), 343 (lane 6), and 359 min (lane 7) after fertilization
and were incubated with 1 ,g of GSTcdk2 and [_-32P]ATP for
45 min at 23°C. Supematants were recovered, and phosphorylated
GSTcdk2 protein was analyzed by SDS-PAGE and autoradiography
(bottom). The relative amounts of 32P associated with GSTcdk2 in
each reaction were subsequently quantitated by phosphorimage analysis
and plotted on an arbitrary scale of 1-100. Mitosis 5 (M5) occurred
after 312 min, and mitosis 6 (M6) occurred after 343 min. (B) p40M"OI
immunoprecipitates were prepared from five mature stage VI oocytes
(lane 1) and five stage 2, 6, 8, 10, 12, 16, and 30 embryos (lanes 2-
8), then assayed for CAK activity as described in A.
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