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J Biol Chem, Vol. 273, Issue 30, 18705-18708, July 24, 1998
§,
From the The activation of protein kinase B/Akt is thought
to be a critical step in the phosphoinositide 3-kinase pathway that
regulates cell growth and differentiation. Because insulin-like growth
factor 1 stimulates the resumption of meiosis in Xenopus
laevis oocytes via phosphoinositide 3-kinase activation, we
investigated the Akt involvement in this process. Injection of mRNA
coding for a constitutively active Akt in Xenopus oocytes
induced germinal vesicle breakdown (GVBD) to the same extent as
progesterone or insulin treatment. Injection of mRNA coding for the
wild type Akt kinase was less effective in stimulating GVBD, whereas
Akt bearing a lysine mutation in the catalytic domain that abolishes the kinase activity had no effect. A mutant Akt lacking a
membrane-targeting sequence did not induce GVBD, despite high levels of
expression and activity. As previously reported for insulin, induction
of GVBD by Akt was prevented by incubating the oocytes with
cilostamide, an inhibitor specific for the type 3 phosphodiesterase
(PDE3), suggesting that the activity of a PDE is required for Akt
action. That an increase in PDE activity in the oocyte is sufficient to induce meiotic resumption was demonstrated by expression of an active
PDE protein. In addition, the constitutively active Akt caused a 2-fold
increase in the activity of the endogenous PDE. These data demonstrate
that Akt is in the pathway controlling resumption of meiosis in the
Xenopus oocyte and that regulation of the activity of a
PDE3 is a step distal to the kinase activation.
A critical step in growth factor stimulation of the target cell is
the activation of the phosphoinositide 3-kinase
(PI3-K)1 pathway. This
signaling pathway has been implicated in a wide array of cellular
events including mitogenesis, transformation, differentiation, and
regulation of metabolism (1). Recently Akt, also known as protein
kinase B or related to the A and protein kinase C (2-4), was
identified as a kinase distal to PI3-K (1). Three Akt isoforms ( Akt is activated by insulin and IGF-1 (12), and this regulation
mediates the activation of glucose uptake (13), the phosphorylation and
deactivation of GSK3 (14), and activation of p70 S6 kinases (15). Akt
also phosphorylates the proapoptotic protein Bad, promoting its
interaction with 14-3-3 and thereby preventing apoptosis (16).
In somatic cells including adipocytes, insulin regulates the activation
of cGMP-inhibited phosphodiesterase (PDE3) (17). This activation is
mediated by PI3-K because wortmannin, a PI3-K inhibitor, blocks the
insulin-dependent activation of the PDE (18). The PDE3B
isoform expressed in adipocytes is phosphorylated following insulin
stimulation, and the phosphorylation is associated with an increase in
PDE activity (19). This PDE activation and the consequent decrease in
intracellular cAMP are probably responsible for the decrease in the
hormone-sensitive lipase activity (17) and for the antilipolytic
effects of insulin. The properties of the kinase phosphorylating PDE3B
have not been firmly established, although a recent report indicates
that Akt coelutes with a kinase activity that phosphorylates PDE3B in a
cell-free system (20).
In Xenopus laevis oocytes, IGF-1 induces the dissolution of
the nuclear membrane (GVBD) and completion of the first meiotic division (21). Although distinct signaling pathways may be activated, IGF-1, or the physiological stimulus progesterone, activates meiotic resumption by inducing a transient decrease in intraoocyte cAMP levels,
thereby reducing the protein kinase A activity (22, 23). It has been
proposed that activation of a PDE and a decrease in cAMP are crucial
steps for reentry into the cell cycle. The effects of insulin and IGF-1
on meiosis are blocked by cilostamide, a specific inhibitor of the type
3 family of phosphodiesterases (24). Furthermore, insulin treatment or
injection of an activated Ras leads to an increase in the PDE activity
in the Xenopus oocyte (25).
Here we have tested the hypothesis that Akt is involved in the
IGF-1-induced resumption of meiosis by activating a PDE present in the
Xenopus oocyte. Our data demonstrate that Akt is a signal for resumption of meiosis in the Xenopus oocyte and that
activation of a PDE3 is a step distal to the Akt activation.
Construction of mRNA Expression Vectors--
The constructs
encoding the wild type (WT)-Akt In Vitro mRNA Synthesis--
To express Akt, PI3-K, and
PDE4D3 mRNAs, the pSP64(poly(A)) constructs were transcribed using
the SP6 polymerase according to the procedure supplied by the
manufacturer (Message Machine Kit, Promega). The transcribed mRNA
was purified by phenol/chloroform extraction, precipitated at
Injection into Xenopus Oocytes--
Ovary fragments were
surgically removed from pregnant mare serum gonadotropin-primed
X. laevis, and the oocytes were isolated manually. Stage VI
oocytes were selected for all the experiments. Oocyte storage and
experiments were carried out in OR2 solution (82.5 mM NaCl,
2.5 mM KCl, 1.0 mM CaCl2, 1.0 mM MgCl2, 1.0 mM Na2HPO4, 5.0 mM HEPES, pH 7.8).
Oocytes were routinely tested for their insulin responsiveness by
incubation of 10 oocytes in 1 µM insulin in OR2 solution
overnight at room temperature. Oocytes that exhibited an
insulin-stimulated GVBD of 80-100% were used. The mRNA or
H2O (vehicle) was injected using a micromanipulator (Drummond) into defolliculated Xenopus oocytes. Resumption
of meiosis was scored by the appearance of a white spot on the animal pole of the oocyte.
Western Blot Analysis--
Expression of HA-tagged Akt was
analyzed after lysing injected oocytes in 10 µl of lysis buffer (250 mM sucrose, 1 mM KCl, 1 mM MgCl,
0.2 mM phenylmethylsulfonyl fluoride) per oocyte. Oocyte extracts were isolated by centrifugation at 15,000 × g
for 10 min at 4 °C. The lipid supernatant was removed, and the
clarified supernatants were transferred to Eppendorf tubes. Oocyte
extract were analyzed by gel electrophoresis on 8% SDS-polyacrylamide gel electrophoresis (1:30 bis:acrylamide). After transfer,
nitrocellulose membranes were blocked overnight at 4 °C in 5%
bovine serum albumin in Tris-buffered saline with 0.02% Tween 20. Immunostaining to detect Akt-HA expression was performed by incubating
for 1 h with a 1:1000 dilution of an HA.11 polyclonal antibody
(Babco) and subsequently with a 1:5000 dilution of anti-rabbit Ig
conjugated to horseradish peroxidase (Amersham Pharmacia Biotech).
Blots were visualized by the ECL procedure (Amersham Pharmacia
Biotech). Western blot for endogenous ERK2 phosphorylation was
performed as described previously (27).
In Vivo PDE Assay--
PDE activity was measured in
Xenopus oocytes by the method described by Sadler and Maller
(23). In brief, oocytes were first stimulated with insulin or injected
with either PI3-K, K179M-Akt, or myr-Akt mRNA. Three hours later,
they were reinjected with 50 nl of 2 mM
[3H]cAMP to a final concentration of 200 µM
(500-700 cpm/pmol). The reaction products were separated and
quantitated as previously described (23, 28).
Akt Assay--
Oocytes were lysed as described above, and the
expressed Akt was immunoprecipitated and analyzed for activity as
described (12). GSK peptide GRPRTSSFAEG was used as substrate (14). Akt
activity was determined by both densitometry and scintillation counting.
Akt Stimulation of Meiotic Resumption in Xenopus Oocytes--
To
test whether Akt is involved in the resumption of meiosis, an mRNA
encoding a constitutively active Akt (7) was microinjected in
Xenopus oocytes; in this construct, the Akt coding region
with a deletion in the PH domain ( Department of Gynecology and Obstetrics,
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
,
, and 
) with closely related properties have been identified.
These are proteins of approximately 60 kDa containing a pleckstrin
homology (PH) domain (5) and a serine/threonine kinase domain
structurally related to the catalytic domains of protein kinases A and
C. Phosphatidylinositol 3,4-bisphosphate and 3,4,5-trisphosphate, the
products of PI3-K, bind to the PH domain of Akt and serve to anchor the
enzyme to the membrane as well as to induce a conformational change in
the enzyme (6). The subsequent phosphorylation of Akt, at Thr-308 in
the catalytic domain and Ser-473 of the C terminus of Akt, is
crucial for the activation of Akt (7, 8). At least one of the kinases
phosphorylating Akt has been identified as the
3-phosphoinositide-dependent kinase-1 (9-11).
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
, myristoylated (myr)-Akt, K179M-Akt,
and A2myr-Akt have been described (12, 13). These constructs were
transferred from the pECE vector to the XbaI-SalI
sites of the mRNA expression vector pSP64(poly(A)) (Promega,
Madison WI). The pCMV-5 iSH2-p110 (a gift from M. Birnbaum) was
subcloned into pSP64(poly(A)) using XbaI and
BamHI for the p110 subunit and XbaI for the
inter-SH-2 domain (iSH2-I) of p85. The pCMV-5 rat PDE4D3 construct has
been described previously (26). The complete open reading frame of
PDE4D3 was subcloned from pCMV-5 into EcoRI of pBluescript
KSII and then into pSP64(poly(A)) using
AvaI-HindIII. All Akt constructs had the
influenza virus HA epitope tag fused in-frame to the C terminus to
monitor protein expression.
20 °C with 1 volume of isopropyl alcohol, and resuspended in
diethylpyrocarbonate water. The mRNA concentration was measured by
A260, and transcript size was determined by
formaldehyde gel electrophoresis. The mRNA was diluted to 1 mg/ml
in diethylpyrocarbonate water and stored at 70 °C.
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
4-129) is fused to a 14-amino
acid leader containing the myr sequence of src (myr-Akt).
This engineered protein is constitutively active in 3T3L1 cells by
being targeted to the membrane (7). The expression of this
constitutively active Akt caused resumption of meiosis as measured by
GVBD (Fig. 1). The effect of active Akt
expression was comparable with the insulin or progesterone stimulation
(Fig. 1). When the time course of GVBD was monitored after myr-Akt
injection, half-maximal maturation occurred 9.3 ± 1.6 h
after injection (data not shown), whereas progesterone- and
insulin-induced GVBD occurred with half-times of 2.9 ± 0.3 and
4.8 ± 0.7 h, respectively.
View larger version (18K):
[in a new window]
Fig. 1.
Microinjection of mRNA coding for a
constitutively active Akt (myr-Akt) stimulates meiotic resumption in
Xenopus oocytes. Stage VI oocytes were treated with
either 1 µM insulin or 10 µM progesterone
(not injected groups) or injected with 50 nl of H2O or 50 ng (1 mg/ml) of myr-Akt (injected groups). The number in parentheses
denotes the number of oocytes injected for each group. Oocytes were
incubated at 20 °C after treatment, and GVBD was scored after
20 h. Maturation is expressed as the percentage of oocytes
exhibiting GVBD ± S.E. of five independent experiments.
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Activation of a PDE3 Is Distal to Akt in the Maturation Pathway-- Previous work by Sadler (24) had shown that the maturation induced by insulin and IGF-1 is blocked by type 3 PDE inhibitors. To investigate whether a PDE is distal to Akt, different concentrations (0.2, 0.5, and 1.0 mg/ml) of constitutively active Akt (myr-Akt) mRNA were injected into Xenopus oocytes preincubated with inhibitors of PDE3. A 1-h preincubation with 10 µM cilostamide effectively prevented the myr-Akt-induced meiotic resumption (Fig. 3). Under the same conditions, 10 µM cilostamide also blocked insulin-induced maturation (data not shown). Whereas preincubation of oocytes in 10 µM cilostamide reduced the effect of microinjected myr-Akt no inhibition was observed by preincubating oocytes with a type 4 PDE inhibitor, rolipram (data not shown). This suggests that Akt-stimulated resumption of meiosis requires the activity of a PDE3, as does IGF-1-stimulated oocyte maturation (24).
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
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Discussion
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Akt is a serine-threonine kinase in the PI3-K signaling pathway that mediates growth factor regulation of cell differentiation and survival (1). Here we provide evidence for an additional role of this Akt kinase in the control of the cell cycle and meiosis. Akt expression in the Xenopus oocyte causes germinal vesicle breakdown and mitogen-activated protein kinase phosphorylation, hallmarks of the resumption of meiosis. Furthermore, our findings indicate that a PDE3 is distal to Akt activation. This conclusion is supported by both the pharmacological manipulation of the oocyte PDE and by the observation that an increase in PDE activity follows expression of the Akt kinase in oocyte. Finally, the finding that expression of a PDE in the oocyte per se is sufficient to promote a meiotic resumption is consistent with the above conclusions.
Numerous observations have underscored the importance of the lipid PI3-K in insulin and growth factor signaling in somatic cells (1) as well as in Xenopus oocytes. Because insulin and IGF-1 effects on meiosis are inhibited by wortmannin (29), PI3-K has been implicated in oocyte maturation. Injection of a constitutively active PI3-K mRNA causes resumption of meiosis (30), whereas injection of the lipid phosphatase SIP/SHIP blocks the insulin effects (31). Our data extend these observations by identifying downstream steps in the pathway by which IGF-1 induces resumption of meiosis. As for somatic cells where the PI3-K effects are mediated by activation of Akt, we have shown that Akt activates resumption of meiosis in a manner similar to insulin/IGF-1.
Although the myr-Akt efficiently promoted oocyte maturation, a wild type Akt was only partially effective in the oocyte model, consistent with observations in mammalian cells showing that wild type Akt has low basal activity in the absence of insulin or growth factor signals (7, 8, 12, 13). Unlike the myr-Akt, the A2myr-Akt lacking the PH domain and with a mutation in the myristoylation signal was ineffective in inducing resumption of meiosis. This protein was efficiently expressed, and the overall Akt activity recovered in oocyte extracts was comparable with that obtained by expression of myr-Akt. When the activity of A2myr-Akt was corrected for the amount of protein expressed, this mutant kinase had one-fourth the specific activity of the myr-Akt. Because the only difference between A2myr-Akt and myr-Akt is the ability to interact with the lipid bilayer, we can conclude that Akt interaction with lipids is essential for the signaling meiotic resumption.
Our findings that PDE3 inhibitors block the oocyte maturation induced by insulin and Akt, but not progesterone, indicate that a PDE3 is involved in the PI3-K signal transduction pathway activated by IGF-1/insulin. In agreement with this hypothesis, we have shown that insulin, PI3-K, and Akt stimulate the activity of PDE3 endogenous to the oocyte. That Akt activation of PDE3 may be a sufficient signal for meiosis resumption is strongly suggested by the observation that expression of a PDE causes GVBD. Our conclusion is consistent with the finding in adipocytes, where it has been shown that insulin through the PI3-K pathway activates PDE3B (32).
Our data do not exclude the possibility that Akt is at a branch point in the signaling pathway controlling maturation. Upon activation, this kinase may phosphorylate several substrates in addition to a PDE3, and the activation of a PDE3 has only a permissive role on meiotic maturation. According to this model, both the release of a cAMP-dependent blockade and the activation of a distinct signaling cascade may be required for meiotic resumption. For instance, Akt activates the p70 ribosomal S6 kinase, which may be involved in the regulation of mos translation, a crucial step in oocyte meiosis (33).
Regardless of the mechanism of Akt action, our findings indicate a role
for this enzyme in oocyte maturation. Interestingly, the cloning of
3-phosphoinositide-dependent kinase-1, the
kinase-phosphorylating Thr-308 of Akt, has uncovered a structural
and function homology with the Drosophila DSTPK61 kinase.
Although little is known about the function of the PI3-K and Akt
pathway in Drosophila, DSTPK61 kinase may play an important
role in the differentiation of the female and male germ cells (9). Our
data in the Xenopus oocyte are in line with the view that
this pathway is involved in oocyte maturation. The physiological
signals activating the PI3-K and the Akt pathway are unknown at present
but may be a means by which somatic cells control the function of germ
cells. It is worth noting that the PDE3A mRNA (34) and protein are
expressed in mammalian oocytes. Consistent with that shown in
Xenopus, PDE3 inhibitors block the spontaneous resumption of
meiosis in rat and mouse oocytes as well as the maturation induced by
the luteinizing hormone in the intact follicle (34, 35). Recent
in vivo observations have further confirmed the crucial role
of PDE3 for resumption of meiosis in mammals (35). Thus, our data on
the Xenopus oocyte open the possibility that, in the
mammalian follicle, physiological signals promoting resumption of
meiosis regulate the intraoocyte cAMP levels via the PI3-K/Akt
signaling pathway.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jim Ferrell for the polyclonal anti-ERK2 antibody and the helpful discussion; Eric Machleder and Mike Wu for help with microinjections; Morris Birnbaum for iSH2-p110 clone; and Kristina Kovacina and Aimee Kohn for pECE-Akt constructs.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HD20788 and P50 HD31398 Project IV (to M. C.) and DK34976 (to R. A. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by Grant UDL9526 from The Danish Research Academy and Grant 9600503 from The Danish Research Councils.
To whom correspondence should be addressed. Tel.:
650-725-2452; Fax: 650-725-7102; E-mail:
marco.conti{at}forsythe.stanford.edu.
1 The abbreviations used are: PI3-K, phosphatidylinositol 3-kinase; PDE, phosphodiesterase; IGF-1, insulin-like growth factor 1; HA, hemagglutinin; myr, myristoylation; PH, pleckstrin homology; GVBD, germinal vesicle breakdown; WT, wild type.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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