From the
Bacterially expressed, dual specificity phosphatase VHR protein
induced germinal vesicle breakdown (GVBD) when microinjected into
Xenopus oocytes, albeit with slower kinetics than that
observed in progesterone- or insulin-induced maturation. A mutant VHR
protein missing an essential cysteine residue for its in vitro phosphatase activity completely lacked activity in injected
oocytes. VHR injection done in conjunction with progesterone or insulin
treatment resulted in highly synergized GVBD responses showing much
faster kinetics than that produced by VHR or either hormone alone. The
delayed kinetics of VHR-induced GVBD and the synergistic responses
obtained in the presence of hormones suggested that this protein may be
promoting G
Various experimental observations
are consistent with such a role for the injected VHR in oocytes: 1) as
opposed to hormone-treated oocytes, histone H1 kinase activation is not
preceded by MAPK activation in the process of GVBD in VHR-injected
oocytes; 2) incubation of purified VHR with highly concentrated
cell-free extracts of untreated oocytes resulted in activation of
histone H1 kinase activity in the lysates; 3) coinjection of VHR with
activated Ras proteins resulted in synergized responses, faster than
those produced by either protein alone; 4) coinjection of VHR with the
purified amino-terminal SH2 domain of the p85 subunit of
phosphatidylinositol 3-kinase (which blocks insulin-induced GVBD) does
not affect VHR-induced maturation.
The biological actions of VHR in
oocytes clearly distinguish it from other dual specificity
phosphatases, which have shown inhibitory effects when tested in
oocytes. We speculate that VHR may represent a dual specificity
phosphatase responsible for activation of cdk-cyclin complex(es) at a
still undetermined stage of the cell cycle.
The process of signal transduction from surface tyrosine kinase
receptors to the nucleus is mainly regulated by mechanisms of
reversible protein phosphorylation and dephosphorylation. Increasing
attention has been focused recently on the family of protein tyrosine
phosphatases involved in signaling pathways and regulation of cell
proliferation
(1, 2) . Dual specificity phosphatases
constitute a novel subfamily of protein tyrosine phosphatases
characterized by their ability to dephosphorylate not only Tyr but also
Ser/Thr residues in substrates in vitro. The first described
member in this subfamily, cdc25, is known to activate the
p34cdc2-cyclin kinase complex that regulates entry into the M phase,
through dephosphorylation of specific Tyr and Thr residues
(3) .
A number of other dual specificity phosphatases have been recently
described (4-10). While some of these have been shown to
dephosphorylate MAP
VHR is a dual specificity phosphatase
recently isolated in our laboratory by means of expression
cloning
(14) . Searches of available data bases have failed to
identify significant similarity between VHR and other protein tyrosine
phosphatases, except for a motif, HCXXXXGR, which seems to
constitute the catalytic core common to all protein tyrosine
phosphatases
(9, 14, 15) . In fact, cdc25 is more
similar in sequence to VH1 and VHR than to the other
phosphatases
(16) . Also, in contrast to most other members of
the dual specificity protein tyrosine phosphatase subfamily, VHR
exhibits broad substrate specificity in vitro(14) . No
physiological role is yet known for this newly identified protein
tyrosine phosphatase.
Xenopus oocytes are arrested at the
G
Xenopus oocytes possess at least two independent pathways of MPF
activation through phosphorylation. In one, progesterone leads to
decreased adenylate cyclase activity with a resulting drop in overall
cAMP levels and protein kinase A-dependent phosphorylations. In the
other, insulin or insulin-like growth factor-1 triggers a cascade of
phosphorylations initiated by tyrosine phosphorylation of their
receptors
(17, 18) . Ras and other oncogenic proteins
also have been shown to induce meiotic maturation when microinjected
into Xenopus oocytes
(21, 23, 24, 25, 26, 27, 28) .
Several lines of evidence indicate that Ras proteins are essential
components in insulin-induced
maturation
(29, 30, 31) . The universality of MPF
and the possibility of activating it by microinjection of various
oncogene products into Xenopus oocytes makes this a very
useful experimental model for the analysis of the role of various
cellular gene products in progression through the eukaryotic cell
cycle. In the present study we utilized the Xenopus oocyte
system in order to investigate possible in vivo function(s) of
VHR.
For
induction of meiotic maturation, groups of 10-30 oocytes were
incubated in ND-96 without KCl in the presence of progesterone (15
µM; Sigma) or insulin (7.5 µM; Sigma) or were
microinjected into the cytoplasm with 30-60 nl of normal or
mutant VHR (1 mg/ml, in 20 mM Tris-HCl, pH 7.5). Controls were
microinjected with buffer. Meiotic maturation was assayed by scoring
the disappearance of the nucleus (GVBD) in oocytes fixed with 10%
trichloroacetic acid. In most cases, the absence of the nucleus
correlated with the appearance of a white spot in the animal pole.
Concentrated, cell-free lysates
from untreated oocytes for in vitro assays were prepared
essentially as previously described (34), with some minor
modifications. Stage VI oocytes were washed 10 times in cytostatic
factor extraction buffer (CSF-XB, 100 mM KCl, 0.1 mM
CaCl
Protein kinase activation in highly concentrated cell-free extracts
incubated in the presence of exogenous VHR was assayed basically as
follows. 6 µl of purified VHR protein (1 mg/ml) were incubated with
22 µl of extract prepared as described above and and 1 µl of
ATP-regenerating buffer (20 µM creatine phosphate, 2
mM ATP, 5 µg/ml creatine phosphokinase; Sigma). After
mixing on ice, extract samples were incubated at 21 °C, and equal
aliquots were removed at different times and diluted with an equal
volume of extraction buffer buffer B (EB; 80 mM
Histone H1 kinase and MBP kinase assays were
performed following described procedures
(36) after diluting the
samples 60 or 100 times, respectively, in buffer B. MAP kinase
activation was also determined by assays of mobility shift in Western
immunoblots (10% SDS-PAGE gels) done using anti-rat MAP kinase R2
antibodies (Upstate Biotechnology, Inc. catalog no. 06-182).
In order to
determine if VHR could interact with components of any of these
pathways, we microinjected VHR in oocytes that were also treated with
either insulin or progesterone. As shown in Fig. 1B,
microinjected VHR produced a marked acceleration in the rate of GVBD
induced by either progesterone or insulin at a variety of
concentrations of either hormone. The synergistic effect of VHR was
most obvious when suboptimal concentrations of either hormone were used
(Fig. 1B presents results obtained using 1/10 and 1/100
of the optimal concentrations for insulin and progesterone,
respectively). The synergism between hormone-induced maturation and VHR
is in contrast to the behavior shown by several other phosphatases,
which block hormone-induced GVBD and/or kinase
activation
(6, 37) . The GVBD-inducing activity of VHR
and its cooperation with both progesterone and insulin are consistent
with a model whereby VHR might act at a late biochemical step, common
to both progesterone- and insulin-initiated pathways of GVBD.
We sought to analyze
activation of early and late acting kinases during the process of GVBD
induced by microinjected VHR. Fig. 2presents results obtained in
experiments in which the kinetics of GVBD (Panel A), the
kinetics of activation of the MAP kinase (Panels D and
E) and the p34cdc2 kinase (assayed as histone H1 kinase
activity, Panels B and C) were analyzed in parallel.
The process of VHR-induced GVBD was also accompanied by the
significant activation of MAP kinase activity that could be detected
either by direct kinase assays of oocyte lysates
(Fig. 2D) or by mobility-shift assays using specific
antibodies against the 42-kDa MAP kinase molecule
(Fig. 2E). In contrast to hormone-treated- or
Ras-injected oocytes, in which the peak of MAP kinase activation
clearly precedes histone H1 kinase activation
(21) , the peak of
MAP kinase activation in VHR-injected oocytes (Fig. 2C)
occurred several hours after maximal histone H1 kinase activity was
already achieved, coinciding, in fact, with a minimum in the histone H1
kinase activity (compare Fig. 2, B and C).
Under experimental
conditions similar to those used with Ras and Mos (11, 22, 38, 40), we
observed that addition of purified VHR to highly concentrated oocyte
lysates resulted in the rapid in vitro activation of the
soluble cytosolic kinase activities (Fig. 3). In particular,
histone H1 kinase showed significant activation very shortly after the
start of incubation in the presence of VHR, reaching a maximum at
6-9 h (Fig. 3, A and B). The pattern of
activation of MAP kinase activity paralleled the activation of histone
H1 activity, with a maximum at 6-9 h, although it did not decline
to base-line values afterward (Fig. 3C). Since it has
been shown that MAPK can be a substrate for
MPF
(41, 42, 43) , we interpret these results as
an indication that the MAP kinase activation observed may be due to the
histone H1 kinase activity.
We tested those predictions by coinjecting VHR
with activated Ras (Lys-12) protein
(21) (Fig. 4A), which induces GVBD by acting in
the insulin pathway, upstream of MPF
activation
(29, 30, 31) . We also coinjected VHR
with a peptide corresponding to the amino-terminal SH2 domain of the
p85 subunit of phosphatidylinositol 3-kinase (Fig. 4B),
which blocks insulin-induced GVBD
(44) by acting at a step near
the activated receptor tyrosine kinase and upstream of Ras activation
by insulin. Fig. 4A shows that coinjection of activated
Ras (Lys-12) and VHR proteins resulted in a significant acceleration of
the onset of GVBD, as compared to injection with either VHR or Ras
(Lys-12) alone. In contrast, the amino-terminal SH2 p85 peptide
completely blocked insulin-induced GVBD (Fig. 4B)
without affecting progesterone-induced GVBD (not shown). Of note, the
kinetics of GVBD observed in oocytes coinjected with both VHR and the
p85 SH2 peptide were no different from that of oocytes injected with
VHR alone (Fig. 4B), indicating that the peptide could
not inhibit maturation induced by VHR. All of our findings support a
model (Fig. 5) whereby microinjected VHR weakly mimics the
physiological phosphatase activity of cdc25, activating the MPF kinase
complex and triggering the process of meiotic maturation in Xenopus oocytes.
In Xenopus oocytes, factors that control cell
proliferation and/or regulate the cell cycle (including hormones,
growth factors and their receptors, and oncogenes) can be readily
analyzed functionally. Oocytes offer models for adenylate cyclase and
protein kinase A-dependent (progesterone-induced GVBD) and receptor
tyrosine kinase-dependent (insulin or insulin-like growth
factor-1-induced GVBD) signaling pathways leading to meiotic maturation
(17-20).
The distinct cascades of phosphorylation initiated by
either progesterone (protein kinase A-dependent) or insulin receptor
tyrosine kinase-dependent) eventually converge in the activation of MPF
kinase activity, the universal regulator of M phase transition in
eukaryotic cells
(19, 20) (Fig. 5). Because the
receptor tyrosine kinase-dependent and protein kinase A-dependent
signaling pathways have been at least partially characterized,
injection of suspected intermediates in some of these pathways may help
pinpoint their sites of action in those signaling pathways (see
Fig. 5
).
Our results showed that the purified VHR dual
specificity phosphatase protein induced GVBD in the microinjected
oocytes. These results were not expected in view of recent reports
showing that other tyrosine phosphatases or dual specificity
phosphatases block the process of meiotic maturation or of activation
of kinase cascades in Xenopus oocytes
(6, 37) .
GVBD induction by VHR was slow compared to maturation in the presence
of physiological concentrations of progesterone or pharmacological
doses of insulin. In addition, its ability to induce GVBD was dependent
on the presence of large amounts of VHR, with no observable effects
when less than 30 ng were injected. Nevertheless, the VHR effects were
reproducible and specific since a phosphatase defective VHR mutant did
not produce the effect. The slow kinetics and low rate of GVBD, as well
as the need for high amounts of exogenous VHR, suggest that VHR-induced
GVBD is mediated via its potential functional similarity with cdc25,
the dual specificity phosphatase that physiologically promotes
transition from G
The GVBD-inducing
activity of VHR was also unexpected in view of the very high tyrosine
phosphatase activity shown by this protein in vitro on a
variety of substrates including, for example, the insulin
receptor
(14) . It is clear that after microinjection of VHR into
the cytoplasm of oocytes, multiple potential targets for
dephosphorylation exist between the point of hormone contact at the
surface and the activation of MPF. In fact, dephosphorylation of one or
more of such targets must account for the inhibitory effects on GVBD
exhibited by other phosphatases such as 1B or
CL100
(6, 37) . For example, microinjected phosphatase 1B
(37) has been reported to dephosphorylate at least the insulin
receptor and possibly other targets, while CL100
(6) dephoshorylated and inactivated MAP kinase, which is
absolutely required for insulin induced GVBD
(22) .
The fact
that VHR did not block hormone-induced GVBD but actually potentiated it
and that the pattern of overall phosphorylation was similar in injected
and uninjected oocytes (not shown) indicate that the injected VHR is
not indiscriminately exerting its phosphatase activity. These findings
suggest that after injection, VHR is directed to a compartment where
substrates whose dephosphorylation results in inhibition of GVBD are
not accessible. The nucleus is one such candidate compartment, a
possibility that can be tested when specific antibodies become
available.
All of our findings are compatible with a model (see
Fig. 5
) in which the injected VHR protein, through a low degree
of functional similarity to cdc25 phosphatase, activates the cdc2
complex of MPF, by faintly mimicking the effect of the physiological
activator, cdc25. The slow kinetics and low rate of the GVBD induced by
VHR would be compatible with such a hypothesis. The observation that
VHR consistently activates the histone H1 kinase in vitro in
oocyte extracts further supports this hypothesis. It is of note that
exogenous Ras and Mos can only consistently activate MAP kinase, but
not MPF kinase, in similar in vitro experiments with oocyte
extracts
(11, 38, 40) .
Even if VHR were
distinct from cdc25 and most likely not normally involved in the
process of G
/M transition by weakly mimicking the action of
cdc25, the dual specificity phosphatase that physiologically activates
the maturation promotion factor.
(
)
kinase in vivo or
in vitro(10, 11, 12) , others appear to
form complexes with cdk molecules involved in regulating the cell cycle
(reviewed in Ref. 13).
/M transition of the first meiotic prophase. Progesterone
or insulin induces these cells to enter metaphase and undergo meiotic
maturation involving germinal vesicle breakdown (GVBD), in a process
that involves significant changes in cellular
phosphorylation
(17, 18) . Most of the phosphorylations
associated with GVBD are triggered by activation of maturation
promoting factor (MPF), a universal regulator of M phase transition in
eukaryotic cells
(19, 20) . MPF is is a complex composed
of p34cdc2 (a serine-threonine protein kinase) and B
cyclin
(18, 19, 20) whose kinase activity is
up-regulated by cdc25, a dual specificity phosphatase
(3) .
Recent evidence indicates also that MAP kinase kinase and MAP kinase
are essential elements in the cascade of phosphorylations culminating
in MPF activation and oocyte maturation (21, 22).
Oocyte Preparation and Microinjection
Adult
female Xenopus laevis were obtained from Nasco (Fort Atkinson,
WI) and stimulated to ovulate by microinjecting 50 units of pregnant
mare serum gonadotropin (Calbiochem) 3 days before oocyte extraction.
Ovarian fragments were surgically removed from frogs anesthetized by
hypothermia. Fully grown stage VI oocytes were manually dissected into
ND-96 medium (5 mM Hepes, 96 mM NaCl, 1 mM
MgCl, 2 mM KCl, 1.8 mM CaCl
, pH 7.8,
and 10 µg/ml each of penicillin and streptomycin sulfate). The
oocytes were allowed to recover overnight in the same buffer before
further treatment and were always maintained at 20 °C.
VHR Protein Expression and Purification
Plasmid
pGEX-VHR, a pGEX-KT-derived expression vector
(32) , was used to
express wild-type and mutant VHR as a fusion protein with glutathione
S-transferase (GST). Expression and purification of GST-VHR
were essentially as previously described
(14, 33) .
GST-VHR and GST-VHR (C124S) were >98% pure as determined by
Coomassie Blue staining of SDS-polyacrylamide gels. VHR proteins
obtained by thrombin cleavage of purified GST fusion protein as
described elsewhere
(14) were then dialyzed extensively against
20 mM Tris-HCl, pH 7.5, and stored at high protein
concentration at -80 °C. No loss of enzymatic activity
occurred during long term storage under those conditions. For
microinjection, the proteins were used at a concentration of 1 mg/ml in
all assays.
Preparation of Oocyte Lysates
Lysates of
VHR-microinjected oocytes were prepared as previously
described
(21) . Oocytes were homogenized in ice-cold buffer (20
µl/oocyte) containing 20 mM Hepes, pH 7.4, 10 mM
EGTA, 15 mM MgCl, 1 mM dithiothreitol,
100 mM
-glycerophosphate, 2 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10
µg/ml aprotinin and leupeptin.
, 2 mM MgCl
, 10 mM
Hepes-potassium, pH 7.7, 50 mM sucrose, 5 mM EGTA, pH
7.7) and then twice in the same buffer containing protease inhibitors
(leupeptin, pepstatin, and chymostatin, 10 µg/ml each). The oocytes
were then transferred to SW50.1Ti ultracentrifuge tubes (Beckman)
containing CSF-XB buffer plus protease inhibitors and 100 µg/ml
cytochalasin B and spun at 1000 rpm at room temperature for 60 s and
then 30 s at 2000 rpm. After removing all CSF-XB, a crushing spin was
performed at 30,000 rpm for 20 min at 4 °C. The cytoplasmic layer
was then collected by using a needle and syringe via side puncture.
Protease inhibitors (leupeptin, pepstatin, and chymostatin, 10
µg/ml each), 100 µg/ml cytochalasin B, 0.05 volume of
CSF-energy mix (150 mM creatine phosphate, 20 mM ATP,
2 mM EGTA, pH 7.7, 20 mM MgCl
) and 0.2
M sucrose were then added to this cytoplasmic fraction. The
final protein concentration of these extracts was approximately 80
mg/ml.
Protein Kinase Assays
For measurement of
GVBD-associated kinases in VHR-microinjected oocytes, histone H1 kinase
was assayed essentially as described elsewhere
(21) . 5 µl of
clarified extracts were incubated in a final volume of 15 µl with 3
µg of exogenous histone (type III-S calf thymus; Sigma), 100
µM cold ATP, and 4 µCi of
[-
P]ATP (3000 Ci/mmol, Amersham Corp.) for
15 min at room temperature. Phosphorylated histone H1 was visualized by
autoradiography after SDS-PAGE on 10-20% gels, and radioactivity
in the histone H1 bands was quantitated on the whole gels with a
PhosphorImager (Molecular Dynamics). Myelin basic protein (MBP) kinase
assay was performed as described elsewhere
(35) , but using 1
mg/ml of MBP peptide substrate (UBI) in the presence of protein kinase
A inhibitor peptide (7 µM; Life Technologies, Inc.). 5
µl of oocyte extracts were incubated with 35 µl of reaction
buffer at 30 °C for 10 min and then spotted on P81 filters
(Whatman), washed, and counted using Biofluor as scintillation liquid.
-glycerophosphate, 20 mM EGTA, 15 mM MgCl
in 10 mM Hepes-potassium, pH 7.7) before performing
kinase assays.
Microinjected VHR Protein Induces Slow Meiotic
Maturation of Xenopus Oocytes
A purified, bacterially expressed
20-kDa polypeptide encoded by the VHR gene
(14) was
injected into full-grown X. laevis oocytes, and its biological
effects were evaluated. The injected protein was highly stable and
detectable in the cytosol of the injected oocytes using specific
antibodies, even after 30 h of microinjection (not shown).
Interestingly, we observed that injection of VHR resulted in GVBD after
rather prolonged incubation times (Fig. 1A). Typically,
the start of GVBD induced by VHR was significantly delayed (by about 10
h) as compared to progesterone or insulin. In addition, VHR-induced
GVBD was variable in intensity, occasionally reaching 100% maturation,
but most often remaining in the range 60-70%
(Fig. 1A). The induction of GVBD by microinjected VHR
was reproducible and dependent on its enzymatic activity since
injection of buffer or an inactive mutant (Cys
Ser) that lacks phosphatase activity (14) failed to produce maturation
under similar experimental conditions (Fig. 1A).
Figure 1:
Effect of purified VHR phosphatase on
Xenopus oocyte maturation. A, induction of GVBD in
Xenopus oocytes by microinjected VHR. Time course of GVBD in
oocytes microinjected with purified, wild-type, and mutant (C124S) VHR
protein, or treated with concentrations of insulin or progesterone
producing fast, optimal induction of meiotic maturation. Symbols:
, 50 ng purified wild-type VHR protein;
, 50 ng of
mutant C124S VHR-purified protein;
, 7.5 µM insulin;
, 20 µM progesterone. Data represented in this panel
are the average of 10 separate experiments where the standard
deviations was always lower than 20% of values plotted for each time
point. B, synergism between injected VHR and hormones insulin
or progesterone in the induction of Xenopus oocyte maturation.
Time course of GVBD in oocytes treated with low concentrations of
insulin or progesterone producing suboptimal (delayed) induction of
meiotic maturation, alone or in the presence of microinjected, purified
VHR protein. Symbols:
, oocytes treated with 0.75 µM
insulin alone;
, oocytes treated with 0.2 µM of
progesterone alone;
, oocytes microinjected with 50 ng of VHR
and incubated in the presence of 0.75 µM of insulin;
, oocytes microinjected with 50 ng VHR and incubated in the
presence of 0.2 µM progesterone. Results presented in this
panel correspond to a representative experiment, and similar
synergistic results were obtained in three additional, separate
experiments.
The
process of GVBD induced by VHR was also highly dependent on the
concentration of VHR present in the injected oocytes. No GVBD was
observed when less than 30 ng of purified VHR were injected (not
shown).
Synergism between Hormones and VHR in Induction of GVBD
in X. laevis Oocytes
Xenopus oocytes possess at least
two distinct pathways that eventually converge in activation of the MPF
kinase complex and subsequent GVBD. In one, progesterone down-regulates
the oocyte adenylate cyclase activity and subsequent protein kinase
A-dependent phosphorylations. In the other, insulin or insulin-like
growth factor-1 triggers cascades of phosphorylation initiated by
tyrosine phosphorylation of the specific
receptor
(17, 18) . Several lines of evidence indicate
the participation of Ras in insulin-induced
maturation
(29, 30, 31) .
In Vivo Activation of Intracellular Kinases in
VHR-microinjected Oocytes
It has been shown previously that
cascades of phosphorylation involving activation of MAP kinase and the
cdc2 kinase component of MPF are essential for the process of GVBD
induced by insulin or progesterone
(21, 22, 38) .
We showed that microinjected Ras proteins can activate these kinases,
even in the absence of protein synthesis, in a process in which the
peak of activation of MAP kinase precedes the peak of activation of the
p34cdc2 kinase component of MPF (19, 20), as measured as histone H1
kinase activity
(21) . In fact, purified Ras proteins have been
shown to activate MAP kinase in vitro when added to oocyte
cell-free extracts
(19, 38) .
Figure 2:
Kinetics of in vivo activation of
histone H1 and MAP kinase in oocytes microinjected with VHR.
Representative experiment determining the kinetics of GVBD (Panel
A) and parallel activation of cytoplasmic kinases (histone H1
kinase, Panels B and C; MBP kinase, Panels D and E) in VHR-injected oocytes. Similar results were
obtained in two more separate experiments. A, time course of
GVBD in oocytes microinjected with 50 ng of VHR. B, histone H1
kinase assay in cytosolic extracts of oocytes microinjected with VHR
(15 oocytes used per each time point). Kinase activity is shown in
arbitrary units, normalized to the activity (PhosphorImager signal)
detected in control untreated oocytes at time 0. The maximal H1 kinase
peak is coincident with the beginning of oocyte maturation (see
Panel A). For comparison purposes, control oocytes had 4314
PhosphorImager (Molecular Dynamics) units, with a blank of 186
PhosphorImager units for the same sample. C, autoradiogram of
the same H1 kinase assays analyzed in Panel B, performed on a
4-20% PAGE-SDS minigel. Mobility position of histone H1 protein
band is indicated by the arrow. D, MBP kinase in 5 µl of
clarified extract (15 oocytes used per each time point), performed as
described under ``Experimental Procedures.'' Results
presented as -fold increase, normalizing all values to radioactivity
incorporated in sample of control, untreated oocytes at time 0. Blank
values substracted from all samples were obtained by performing the
kinase assays in the absence of the exogenously added substrate. For
comparison purposes, control untreated oocytes gave 5154 cpm, with a
blank of 3528 cpm for the same sample. E, Western blot
analysis with anti-MAP-2 kinase antibodies of the same cytosolic
extracts of analyzed in Panel D.
As described for hormone-treated
oocytes
(19, 20, 21) , we observed that the rapid
increase in H1 kinase activity coincided with the onset of oocyte
maturation triggered by microinjected VHR (compare Fig. 2,
A, with B and C). Also as described
previously for hormone-matured oocytes
(39) , H1 kinase activity
of oocytes induced to mature by VHR peaked at GVBD and then fell to a
minimum before gradually rising again, suggesting that the VHR-injected
oocytes underwent a complete meiotic maturation process
(39) .
In Vitro Activation of Kinases by VHR Protein Exogenously
Added to Cell-free Oocyte Lysates
The linkage of the
GVBD-inducing ability of VHR to its phosphatase activity (Fig. 1)
and the unusual kinetics of kinase activation accompanying this process
(Fig. 2) suggested that VHR might be acting directly at the level
of activation of MPF, mimicking in a sense the action of cdc25, another
dual specificity phosphatase known to be the physiological activator of
the p34cdc2 kinase activity in the MPF complex during this
G/M transition
(3, 19, 20) . In order
to test this hypothesis, we to sought determine whether purified VHR
could activate MPF kinase in vitro, when added to highly
concentrated cell-free oocyte extracts.
Figure 3:
Assays of in vitro histone H1 and
MAP kinase activities in oocyte cytosolic extracts incubated in the
presence of VHR. Kinase assay activity of highly concentrated oocyte
cytosolic extracts after incubation with VHR phosphatase at room
temperature. Equal aliquots were removed at the indicated times and
assayed for the kinases as described under ``Experimental
Procedures.'' A, autoradiogram of histone H1 kinase
activity analyzed using SDS-PAGE as described under ``Experimental
Procedures.'' B, profile of histone H1 kinase activity
detected using a PhosphorImager on the gel shown in Panel A.
Kinase activity is shown in arbitrary units (as -fold increase),
normalized to the activity (PhosphorImager signal) detected in control,
untreated extracts at time 0. For comparison purposes, control extracts
at time 0 of treatment yielded 12,108 PhosphorImager units, with a
blank of 73 PhosphorImager units. C, MBP kinase activity of
samples removed at the indicated times and assayed as described under
``Experimental Procedures.'' Control untreated extract: 7880
cpm, with a blank of 2834 cpm for the same
sample.
Effect of GVBD Inducers and Blockers on Meiotic
Maturation Produced by Microinjected VHR Protein
The above
results implied a slow, direct activation of the MPF kinase by VHR,
acting at a late biochemical step of the GVBD process, common to both
progesterone- and insulin-initiated pathways. If that were the case, it
would be expected that GVBD-promoting agents acting upstream of the
point of convergence of progesterone and insulin pathways should also
synergize with VHR-induced GVBD. However, GVBD-blocking agents acting
upstream of such a convergence point would not be expected to inhibit
VHR-induced GVBD.
Figure 4:
Effects of inducers and blockers of GVBD
on meiotic maturation promoted by VHR. A, effect of
transforming Ras (Lys-12). Representative experiment showing time
course of GVBD in oocytes microinjected with: , 50 ng of
purified VHR;
, 10 ng of transforming Ras (Lys-12) protein;
[
, 50 ng of VHR and 10 ng of transforming Ras. Similar
results were obtained in two separate experiments. B, effect
of the amino-terminal SH2 domain of the p85 subunit of of
phosphatidylinositol 3-kinase (N-SH2 p85). Kinetics of GVBD in oocytes
treated as follows:
, incubated with 7.5 µM insulin
alone;
, microinjected with 30 ng of N-SH2-p85 of
phosphatidylinositol 3-kinase and incubated in the presence of 7.5
µM insulin;
, microinjected with 50 ng of VHR;
, coinjected with both 50 ng of VHR and 30 ng of N-SH2-p85. Data
presented for each time point correspond to the average of four
separate experiments where the S.D. was always lower than 15% of values
plotted here.
Figure 5:
Tentative model for VHR action in
Xenopus oocytes. GVBD in Xenopus oocytes can be
driven through separate cascades of phosphorylation initiated by either
progesterone (protein kinase A-dependent) or insulin (receptor tyrosine
kinase-dependent) that eventually converge in activation of MPF kinase,
the universal regulator of M phase transition in eukaryotic cells (19,
20). Activation of the MAP kinase kinase/MAP kinase cascade is a
requirement for MPF activation in vivo (21, 22, 38), although
MAP kinase can also be a substrate for retrophosphorylation by
activated MPF (41-43). Ras proteins are essential components in
insulin-induced maturation (29-31) and can also induce activation
of MAP kinase, but not MPF, in oocyte lysates in vitro (38).
Microinjected Ras proteins, even in the absence of protein synthesis,
lead to GVBD through a process where MAP kinase activation precedes MPF
kinase activation (21). A model explaining the results observed here
postulates that VHR acts by mimicking the function of cdc25, the
physiological phosphatase activator of MPF (3). This model predicts
that agents acting upstream of cdc25 in the pathway of GVBD induction
will synergize with microinjected VHR if they exert a positive
regulatory effect (progesterone, insulin, microinjected Ras) but will
not inhibit the VHR effect if they have a negative regulatory role
(N-SH2-PI3K). Another prediction is that the microinjected VHR must
localize to a compartment where its phosphatase activity exerts a
positive regulatory role instead of the inhibitory effect that would be
expected if its high phosphatase activity was exerted randomly
throughout the oocyte.
to M phase in Xenopus oocytes
(19, 20) and can also induce quick GVBD
when injected into the oocytes
(45) .
/M transition in oocytes, this mammalian dual
specificity phosphatase may be a physiological component of the
machinery involved in modulation of the kinase activity of cdk-cyclin
complexes involved in different steps of the cell cycle. The existence
of multiple forms of these complexes acting at different steps of the
cell cycle has been recently
reported
(13, 46, 47) . For example, a dual
specificity phosphatase (KAP) with significant sequence similarity to
VHR has been recently shown to form complexes with cdk in mammalian
cells
(9) . Characterization of the effects of VHR exogenously
expressed in transfected mammalian cells may help in determining
whether such a hypothesis is correct.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.