Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305, USA
* Author for correspondence (e-mail: marco.conti{at}stanford.edu)
Accepted 22 July 2004
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SUMMARY |
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Key words: Xenopus oocyte, Cyclic AMP, Meiotic resumption, Tyrosine phosphatase
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Introduction |
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The signaling network controlling meiotic resumption includes early
components proximal to plasma membrane receptors and distal components, which
are centered around the regulation of the Cdc2/cyclinB complex (maturation
promoting factor, MPF) (Ferrell,
1999a; Ferrell,
1999b
). The distal components are well characterized
(Karaiskou et al., 2001
;
Nebreda and Ferby, 2000
;
Schmitt and Nebreda, 2002b
).
Extensive data are available on the control of MPF activity and the most
immediate partners, including Cdc25
(Gautier et al., 1991
;
Kumagai and Dunphy, 1991
;
Rime et al., 1994
) and Myt1
(Palmer et al., 1998
).
Activation of MAP kinase cascade (Gotoh et
al., 1995
; Nebreda and Hunt,
1993
) and de novo synthesis of Ringo/Speedy
(Ferby et al., 1999
;
Lenormand et al., 1999
) are
additional components involved in the activation of MPF and resumption of
meiosis.
Whereas the early events causing resumption of meiosis in mammalian oocytes
are still a matter of debate, at least two receptor-activated pathways have
been shown to cause meiotic resumption in Xenopus oocytes. One
pathway involves progesterone binding and activation of a putative membrane
steroid receptor that is coupled to a decrease in adenylyl cyclase (AC)
activity and cAMP (Masui,
1967; Schuetz,
1967a
; Schuetz,
1967b
; Zhu et al.,
2003a
; Zhu et al.,
2003b
). In a distinct pathway of less clear physiological
significance, insulin-like growth factor 1 (IGF1) binds and activates a
tyrosine kinase receptor that most likely signals through activation of a
phosphatidylinositol 3 kinase (PI3K) pathway
(Liu et al., 1995
;
Sadler and Maller, 1987
;
Sadler and Maller, 1989
). In
this pathway, PKB/AKT is activated and a phosphodiesterase (PDE) is one of the
downstream targets of the kinase producing a decrease in cyclic AMP (cAMP)
(Andersen et al., 1998
;
Andersen et al., 2003
). It is
then possible that both the progesterone and the IGF1 pathways converge on
cAMP regulation.
Convincing evidence is available that cAMP plays an important role in
maintaining meiotic arrest in both mammals and amphibians
(Conti et al., 2002;
Maller et al., 1979
).
Inhibition of the activity of Gs in mice and frogs induces oocyte maturation
(Gallo et al., 1995
;
Mehlmann et al., 2002
),
suggesting that G
s or Gß
maintain the oocyte AC in an
active state leading to a constitutive production of cAMP. In agreement with
this view, it has been reported recently that AC3 and AC9 are expressed in
mouse oocytes and that inactivation of the Adcy3 gene causes a leaky
meiotic arrest and possibly infertility
(Horner et al., 2003
).
Downstream from cAMP, protein kinase A (PKA) is probably involved in the
maintenance of meiotic arrest. Early studies have demonstrated that injection
of a PKA catalytic subunit completely blocks hormonal induction of oocyte
maturation (Maller and Krebs,
1977). Conversely, injection of the regulatory subunit of PKA or
the heat stable inhibitor of PKA (PKI) causes meiotic resumption in
Xenopus and mouse oocytes
(Bornslaeger et al., 1986
;
Huchon et al., 1981
;
Maller and Krebs, 1977
). More
recent data by Schmitt et al. indicate that an inactive PKA catalytic subunit
also blocks progesterone-induced Xenopus oocyte maturation
(Schmitt and Nebreda, 2002a
),
suggesting that PKA may prevent oocyte maturation by PKA catalytic-dependent
and -independent mechanisms.
In contrast to the above findings, little is known about the mechanisms by
which an active PKA maintains meiotic arrest. It has been hypothesized that
PKA phosphorylates and maintains key components of the meiotic machinery in an
inactive state, but evidence for direct substrates of PKA is scant. Recently,
it has been proposed that Xenopus Cdc25 phosphorylation fulfills
these criteria (Duckworth et al.,
2002). PKA phosphorylates Cdc25 in a cell-free system in S287,
which is thought to be an inhibitory site also involved in 14-3-3 interaction.
However, other PKA substrates are probably present in the cascade controlling
meiotic arrest. For example, it is unclear how the translation machinery is
maintained in an inactive state during meiotic arrest. Here, we have used a
small-pool strategy to identify PKA substrates that function in the signaling
pathways involved in meiotic control.
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Materials and methods |
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Plasmids and mutagenesis
Isolated mouse C-terminal PTPN13 (amino acid 1917-2460) was subcloned into
a pCMV-HA vector for tagging the influenza virus HA epitope on the N terminus.
HA-tagged C-PTPN13 was then inserted into the pSP64-poly (A) vector (Promega)
for mRNA transcription. Mutagenesis of mouse C terminus PTPN13 (Cys2374Ser)
was performed by using a Quick change site-directed mutagenesis kit
(Stratagene, La Jolla, CA) according to the protocol provided by the
manufacturer. The mutation was checked by sequence analysis. The pSP64-poly
(A)-PDE3A construct was prepared as described previously
(Andersen et al., 2003).
Full-length mouse Cdc25B cDNA was amplified from mouse lung total RNA by
RT-PCR using forward, 5'-GCCGGATCCGCCACGATGGAGGT-3' and reverse,
5'-TGGCAGCAGGCTCATCATCACT-3' primers. Amplified PCR products were
then inserted into pcDNA3.1/V5-His-TOPO TA cloning vector (Invitrogen), and
the insert excised by BamHI and subcloned into pSP64-poly (A) vector
(Promega Corp.). The insert was checked by sequence analysis.
Reverse transcription and polymerase chain reaction
For semiquantitative measurements of gene expression, total RNA was
extracted from Xenopus oocytes using TRIzol solution (Invitrogen).
Total RNA was then treated with DNase I (Invitrogen) to remove genomic DNA.
For reverse transcription, first-strand cDNA was synthesized from 2 µg of
total RNA with random hexanucleotides as primer (SuperScript First-Strand
Synthesis System for RT-PCR, Invitrogen). PCR incubation mixtures contained 50
mM KCl, 20 mM Tris/HCl (pH 8.4), 1.5 mM MgCl2, 200 µM of
deoxynucleoside triphosphate, 0.5 µM of each primer and 2.5 units of
Taq DNA Polymerase (Invitrogen). The PCR conditions were as follows:
denaturation at 94°C for 2 minutes; followed by 27 cycles of denaturation
at 94°C for 1 minute, annealing at 55°C for 1 minute, and extension at
72°C for 1 minute, and final extension at 72°C for 10 minutes. The
numbers of cycles were chosen by the pilot experiments that showed the
reactions were in the exponential phase. Specific primers were used to amplify
cDNAs: xPTPN13 forward, 5'-AAGTCAGGACCCATCATTACA-3'; xPTPN13
reverse, 5'-CTTGATAGCAGAAAATATATT-3'; xGAPDH forward,
5'-CTCCTCTCGCAAAGGTCATC-3'; xGAPDH reverse,
5'-GGAAAGCCATTCCGGTTATT-3' (Stanford PAN Facility, Stanford, CA).
PCR products were analyzed by 1% agarose gel electrophoresis. RNA without RT
did not yield any amplicons indicating there is no contamination of genomic
DNA.
For sequence analysis of the PTPN13 catalytic subunit, primers were designed from EST clones (GenBank accession number BJ063819 and BJ070269). Specific primers were used to amplify cDNA coding tyrosine phosphatase domain of PTPN13: (forward primers) xPTPN13F1, 5'-GTCATCCAATCTCTACTAG-3'; xPTPN13F2, 5'-GAGGACACAGACTGTGATG-3'; (reverse primers) xPTPN13R1, 5'-TGCCTCTGAAGTCGCATTG-3'; xPTPN13R2, 5'-GAGTATAACTTGATAGCAG-3'. Four different combinations of the primers (F1 and R1, F1 and R2, F2 and R1, F2 and R2) were used for RT-PCR and amplified PCR products were subcloned into pcDNA3.1/V5-His-TOPO TA cloning vector (Invitrogen) followed by sequence analysis.
Immunohistochemistry of mouse ovaries
PTPN13 protein expression was visualized by immunohistochemical detection
with the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA).
Ovaries were fixed in Bouin's solution, embedded in paraffin and then cut into
5 µm sections. Deparaffinized sections were rehydrated and rinsed in PBS,
and endogenous peroxidases were blocked by incubation in hydrogen peroxide
followed by incubation in normal horse serum. The sections were then incubated
overnight in an anti-mouse PTPN13 antibody (1:80; Santa Cruz Biotechnology) in
a humidified chamber at 4°C. The distribution of the primary antibody was
revealed with a biotinylated horse anti-goat secondary antibody, and the
avidin-biotin-peroxidase complex was visualized with DAB. Sections were rinsed
in PBS between each step. The specificity of the PTPN13 staining was checked
by replacing the antibody with nonimmune IgG (Santa Cruz Biotechnology).
In vitro mRNA synthesis
For in vitro mRNA synthesis, the pSPORT-P or pSP64-poly (A) constructs were
transcribed using the T7 or SP6 polymerases according to the procedure
supplied by the manufacturer (mMessage mMachine Kit, Ambion, Austin, TX). The
transcribed mRNA was purified by phenolchloroform extraction, precipitated at
20°C with one volume isopropanol and subsequently resuspended in
DEPC water. The mRNA concentration was measured by OD260 and
agarose gel electrophoresis. The mRNA was diluted to a concentration of 1
mg/ml in DEPC water and stored at 80°C.
Injection into Xenopus oocytes
Ovary fragments were surgically removed from PMSG-primed Xenopus
laevis and defolliculated oocytes were isolated after treatment with
collagenase (2.5 mg/ml) in MBS buffer [10 mM HEPES (pH 7.4), 88.0 mM NaCl, 1
mM KCl, 0.82 mM MgSO4, 2.4 mM Na2 HCO3] for 1-1.5 hours.
Dumont Stage VI oocytes were selected for all experiments. Oocyte storage and
experiments were carried out in OR2 solution [5 mM HEPES (pH 7.8), 82.5 mM
NaCl, 2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM
Na2HPO4] supplemented with 0.1% BSA. Oocytes were
routinely tested for their progesterone responsiveness by incubation of 10
oocytes in 500 nM of progesterone in OR2 solution overnight at 16°C.
Oocytes that exhibited a progesterone-stimulated GVBD of 80-100% were used.
The mRNAs, siRNAs or H2O (vehicle) were injected using a
micromanipulator (Narishige USA, Long Island, NY) into defolliculated
Xenopus oocytes. The oocyte maturation was induced by stimulating
0-500 nM of progesterone, or injecting Cdc25B mRNA (1 ng/oocyte) or PDE3A mRNA
(20 ng/oocyte). 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 clones and intracellular signaling molecules was
analyzed after lysing injected oocytes in 10 µl lysis buffer (250 mM
sucrose, 1 mM KCl, 1 mM MgCl2, 0.2 mM PMSF) per oocyte. The oocyte
extract was isolated by centrifugation in a Beckman model B centrifuge for 4
minutes at 4°C. The lipid supernatant was removed and the clarified
supernatants were transferred to new microtubes. Approximately 25 µl of
oocyte extract was analyzed by gel electrophoresis on 8% SDS-PAGE (1:30 bis:
acrylamide). After transfer, PVDF membranes (Immobilon, Millipore, Bedford,
MA) were blocked overnight at 4°C in 0.2% blocking grades BSA (BioRad
Laboratories, Hercules, CA) in Tris buffered saline with 0.1% Tween 20.
Immunostaining to detect HA-clone expression was performed by incubating 1
hour with a 1:1000 dilution of an anti-mouse PTPN13 antibody (Santa Cruz
Biotechnology, Santa Cruz, CA), anti-human PTPN13 antibody (ScienceReagent, El
Cajon, CA), HA.11 monoclonal antibody (COVANCE, Princeton, NJ),
anti-Xenopus Mos antibody (Santa Cruz Biotechnology),
antiphospho-p42/p44 antibody (Cell Signaling, Beverly, MA), DC3 MAP kinase
monoclonal antibody (gift from Dr James J. Ferrell, Jr), antiphospho-Cdc2
antibody (Cell Signaling) or anti-Cdc2 antibody (Oncogene Research Products,
San Diego, CA). Specific proteins were visualized after subsequent 1:5000
dilution of anti-mouse, -rabbit, or -goat IgG conjugated to horseradish
peroxidase (Amersham-Pharmacia Biotech, Santa Cruz Biotechnology) and ECL
procedure (Amersham Pharmacia Biotech).
In vitro phosphorylation of PTPN13
HEK293 cells were cultured until 50% confluency in 6 ml of DMEM
(Invitrogen) + 10% FBS media using 60 mm plates. After 6 hours of serum
starvation of the cells using serum-free DMEM, cells were harvested by using
50 mM Tris (pH 7.6), 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 4 µg/ml aprotinin,
0.7 µg/ml pepstatin, 0.2 mM PMSF, 0.5 µg/ml leupeptin, 1 µM
microcystin-LR), immunoprecipitated with 2 µg of anti-human PTPN13
antibodies (ScienceReagent) or 2 µg of nonimmune IgG (Santa Cruz
Biotechnology) and protein G-sepharose (Amersham Pharmacia Biotech).
Immunoprecipitated PTPN13 was duplicated then half were incubated with a
kinase assayed solution (50 mM Tris, pH 7.6, 5 mM MgCl2, 1 mM ATP,
4 µg/ml aprotinin, 0.7 µg/ml pepstatin, 0.2 mM PMSF, 0.5 µg/ml
leupeptin and 500 µCi/ml [-32P]ATP (3000 Ci/mmol,
Amersham Pharmacia Biotech)) with or without 0.1 unit of PKA catalytic subunit
at 30°C for 0, 5, 15 or 30 minutes. The reactions were terminated by
adding 4xLaemmeli's sample buffered solution and boiling for 5 minutes.
The samples then were subjected to 6% SDS-PAGE, followed by autoradiography
for 12 hours. The remaining half was used for western blot analysis using
anti-human PTPN13 antibodies (ScienceReagent).
Metabolic labeling of HEK293 cells
HEK293 cells were cultured until 50% confluency in 6 ml of DMEM
(Invitrogen) + 10% FBS media using 60 mm plates (Corning, Corning, NY). For
the metabolic labeling, the cell medium was exchanged to 3 ml of
phosphate-free DMEM for 1 hour, then 500 µCi of 32P
orthophosphate (Phosphorus 32P, Amersham Pharmacia Biotech) was
added in each plate for 6 hours. Metabolically labeled cells were stimulated
with 100 µM forskolin, harvested by using 1 ml of Tris-NP40 lysis buffered
solution [50 mM Tris (pH 7.6), 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 4 µg/ml
aprotinin, 0.7 µg/ml pepstatin, 0.2 mM PMSF, 0.5 µg/ml leupeptin, 1
µM microcystin-LR), and immunoprecipitated with 2 µg of anti-human
PTPN13 antibody (ScienceReagent) and protein G-sepharose (Amersham Pharmacia
Biotech). The immunoprecipitated PTPN13 was duplicated and half were subjected
to 6% SDS-PAGE, followed by autoradiography for 12 hours. The remaining half
was used for western blot analysis by anti-human PTPN13 antibodies.
Measurement of protein tyrosine phosphatase activity
Myelin basic protein (MBP; Sigma) and constitutively active EGF receptor
(Calbiochem) were incubated at 30°C for 6 hours in 40 mM imidazole-HCl (pH
7.2) containing 50 mM NaCl, 15 mM Mg(CH3COOH)2, 100 mM
MgCl2, 100 µM Na3VO4, 200 µM EDTA,
0.05% (vol/vol) Triton-X 100, 3% glycerol, and 40 µM
[-32P]ATP. At the end of the incubation, the sample was
subjected to gel filtration on a Sephadex G-15 fine column (20 ml column
volume; Amersham Pharmacia Biotech). Fractions containing the
32P-labeled MBP were pooled and stored at 4°C before use in
protein-tyrosine phosphatase (PTPase) assay. The PTPN13 immunoprecipitates
from HEK293 cell extracts were prepared as described above. Immunoprecipitates
were then subjected to in vitro phosphorylation with 1 unit of PKA catalytic
subunit (PKA; Promega Corp.). The following controls were used: PKA + protein
kinase A inhibitor peptide (PKI; Sigma), heat inactivated PKA or 1 µM
sodium orthovanadate for 30 minutes, followed by washing three times with
Tris-NP40 lysis buffered solution. The washed immunoprecipitates were
incubated with 32P-labeled MBP in 25 mM imidazole (pH 7.4),
containing 1 mg/ml BSA and 0.1%(vol/vol) 2-mercaptoethanol at 30°C for 15
minutes. The reaction was terminated by applying the aliquots to P81 cellulose
filter paper, followed by washing once at 4°C in 10% trichloroacetic acid
(TCA) and then three times in 5% TCA. The paper filters were dried with
methanol, and counted using a liquid scintillation counter. Tyrosine
phosphatase activity was calculated by subtracting the counts from each sample
from the total count (without immunoprecipitates). The experiments were
repeated at least three times and representative data are reported.
siRNA preparation
Four sets of primers from EST Xenopus PTPN13 sequences (GenBank
Accession Number BJ063819 and BJ070269) corresponding to the mouse PTPN13
sequence (2020-2446) were used for generating small interference RNA (siRNA)
targeting Xenopus PTPN13. Twenty-one nucleotide DNA oligos for target
sequences, with an additional T7 promoter binding site, were chemically
synthesized (Stanford PAN Facility). The siRNA sequences targeting PTPN13 mRNA
are shown in Table 1. SiRNAs
were made according to the procedure supplied by the manufacturer (Silencer
siRNA construction kit, Ambion). Concentration of synthesized siRNAs was
determined by measuring the OD260. Aliquoted siRNA (1 mg/ml) was
kept at 80°C until use.
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Results |
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Discussion |
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Protein tyrosine phosphatase nonreceptor type 13 (PTPN13; also known as
FAP1, PTPL1, PTP1E, PTP-BL or PTP-BAS) is a 250 kDa protein tyrosine
phosphatase expressed in most cells
(Hendriks et al., 1995). The
protein is composed of a C-terminal catalytic domain and a large N terminus.
The N terminus contains one FERM and five PDZ domains that are most probably
involved in protein/protein interaction. This protein was identified by yeast
two-hybrid screening as a molecule that interacts with the intracellular
domain of Fas (APO1/CD95) (Sato et al.,
1995
), and several reports have explored the anti- or proapoptotic
role of this protein. Tumor cell lines expressing a high level of PTPN13
protein are resistant to Fas-mediated apoptosis
(Meinhold-Heerlein et al.,
2001
). Likewise, Jurkat cells transfected with PTPN13 cDNA become
resistant to Fas-mediated apoptosis, and downregulation of PTPN13 correlates
with sensitization to Fas-mediated apoptosis in interleukin 2 (IL2)-activated
T cells and myelodysplastic transferred cells (MDS)
(Mundle et al., 1999
).
However, Bompard et al. showed the expression of PTPN13 has a negative effect
on the insulin-like growth factor 1 (IGF1)-induced antiapoptotic effect
(Bompard et al., 2002
).
Therefore, further studies are required to assess the role of PTPN13 in oocyte
apoptosis.
Constitutive activation of PTPN13 modulates Xenopus oocyte maturation
Recently, it has been shown that PTPN13 is present in the centrosomal area
during interphase/metaphase and moves to the spindle mid-zone in anaphase,
underscoring the possibility that PTPN13 functions during the cell cycle
(Herrmann et al., 2003).
In the functional studies we report here, we found that progesterone-induced oocyte maturation is significantly accelerated by the overexpression of C-terminal constitutively active PTPN13 (C-PTPN13). Because a construct containing only the catalytic domain was effective, we conclude that neither the PDZ domains nor the FERM domain is necessary for the meiotic promoting effects. Nevertheless, it is unclear at present whether full-length PTPN13 would affect progesterone-induced maturation because very little accumulation of PTPN13 protein was obtained even after injecting large amounts of full-length PTPN13 mRNA. Moreover, injection of the phosphatase-dead mutant indicated phosphatase activity is required for accelerating oocyte maturation induced by progesterone. These findings suggest that dephosphorylation of an unknown PTPN13 substrate transduce signal(s) is important for meiotic resumption.
It is well established that the dual-specific phosphatase Cdc25 is involved
in oocyte maturation by controlling the phosphorylation state of one of the
components of the maturation promoting factor (MPF), Cdc2
(Gautier et al., 1991;
Kumagai and Dunphy, 1991
;
Lincoln et al., 2002
;
Rime et al., 1994
). Therefore,
one possibility is that PTPN13 substitutes for Cdc25 by dephosphorylating
Tyr15 on Cdc2. However, in the absence of progesterone, little
dephosphorylation of Cdc2 was observed after overexpression of C-PTPN13 (data
not shown). In addition, preliminary experiments incubating immunoprecipitated
PTPN13 with phosphorylated Cdc2 did not show appreciable dephosphorylation
(data not shown). These findings suggest that C-PTPN13 does not directly
dephosphorylate Cdc2 and that other intermediates are dephosphorylated on
tyrosine residues. Identification of these physiological substrates of PTPN13
should clarify the role of this phosphatase in meiotic control. At least two
candidate substrates for PTPN13, ephrin B and I
B, already have been
identified (Nakai et al.,
2000
; Palmer et al.,
2002
). It is well established that I
B is involved in
NF
B signaling, but the significance of the phosphorylation of I
B
in NF
B regulation is unclear, nor is information available on the role
of NF
B in meiosis. Ephrin B was identified as a membrane-bound ligand
for the Eph receptor, and not only the Eph receptor but also the ephrin B can
transduce intracellular signals. Cowan and Henkemeyer reported that several
cytoskeletal regulators were recruited by the tyrosine phosphorylation of
ephrin B (Cowan and Henkemeyer,
2002
); however, the expression and function of ephrin B in oocytes
is unknown.
PTPN13 is required for progesterone-induced Xenopus oocyte meiotic maturation
RNA interference (RNAi) demonstrated that downregulation of the PTPN13 mRNA
produces a meiotic blockade. Our efforts to generate antibodies specific for
the Xenopus PTPN13 have been unsuccessful and the available
antibodies against the human or mouse PTPN13 do not cross-react with the
Xenopus protein. Therefore, we have been unable to verify that the
PTPN13 protein is also downregulated by siRNA treatment. However, several
findings suggest that the siRNA treatment is indeed specific. The ability to
produce meiotic blockades varied among the different siRNA produced and there
was a proportionality between decreased mRNA and inhibition of maturation.
Furthermore, all scrambled siRNAs were ineffective in inducing maturation.
That the effect of the siRNA is selective is confirmed by the observation that
expression of several proteins involved in meiotic control, including Cdc2 and
Erk, is not affected by the siRNA. Finally, the siRNA blockade could be
overcome by the expression of Cdc25, indicating that the arrest is not due to
nonspecific toxic effects and that direct activation MPF causes resumption of
meiosis. The whole of these data suggest that downregulation of PTPN13
interferes with the signaling pathway activated by progesterone and therefore
blocks meiotic resumption.
Based on the above findings, we propose that PTPN13 plays a role in the
control of the meiotic cell cycle. The observation that the mammalian PTPN13
localizes in the centrosomal area or on the spindle of dividing HeLa cells is
consistent with this hypothesis (Herrmann
et al., 2003). In the report involving PTPN13 in mitosis, it was
proposed that PTPN13 also may function during anaphase or more likely during
cytokinesis. An important difference between our data and the previous report
is that in our case the phosphatase activity is necessary for the effect on
meiosis, whereas the phosphatase inactive mutant was still effective in
disrupting cytokinesis in Hela cells. Without excluding a possible effect
later in the cell cycle, our data suggest that PTPN13 may have an additional
function earlier during the prophase/metaphase or G2/M transition. The
experiment manipulating cAMP levels by PDE overexpression and Cdc25 rescue
suggest that PTPN13 acts at a step distal to cAMP and PKA, but upstream of
Cdc25. Taken together with our finding that PTPN13 is inactivated by PKA
phosphorylation in vitro, we propose that the inactivation of PKA that follows
the progesterone-induced decrease in cAMP causes PTPN13 activation; this in
turn causes dephosphorylation of substrates critical for MPF activation. This
may not be the primary event, however, because PTPN13 does not function by
itself.
Recently, it has been proposed that microtubules play a role in nuclear
envelope breakdown (Aitchison and Rout,
2002) and that the FERM domain in PTPN13 has been shown to be
necessary for microtubule interaction
(Herrmann et al.,
2003
). Thus, it is possible that PTPN13 localizes to the
microtubules and controls the phosphorylation of a microtubule regulatory
protein required for nuclear envelope breakdown. We cannot exclude, however,
the possibility that the downregulation of PTPN13 by siRNA also disrupts
macromolecular complexes crucial for the progression of the cell cycle.
In conclusion, our findings strongly suggest that PTPN13 is a phosphatase that functions downstream of cAMP signaling and plays an essential role in controlling meiotic resumption in Xenopus oocytes. Identification of the tyrosine substrate dephosphorylated by PTPN13 will help to uncover novel regulatory circuits involved in meiotic, and perhaps mitotic, control.
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ACKNOWLEDGMENTS |
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