* Dipartimento di Sanitá Pubblica e Biologia Cellulare, Sezione di Anatomia, Universitá di Roma Tor Vergata, Rome, Italy; and Dipartimento di Istologia ed Embriologia Medica and Dipartimento di Psicologia, Universitá di Roma La Sapienza, Rome, Italy
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Abstract |
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Microinjection of a truncated form of the c-kit
tyrosine kinase present in mouse spermatozoa (tr-kit)
activates mouse eggs parthenogenetically, and tr-kit-
induced egg activation is inhibited by preincubation with
an inhibitor of phospholipase C (PLC) (Sette, C., A. Bevilacqua, A. Bianchini, F. Mangia, R. Geremia, and P. Rossi. 1997. Development [Camb.]. 124:2267-2274). Co-injection of glutathione-S-transferase (GST) fusion proteins containing the src-homology (SH) domains of the
1 isoform of PLC (PLC
1) competitively inhibits tr-kit-
induced egg activation. A GST fusion protein containing
the SH3 domain of PLC
1 inhibits egg activation as
efficiently as the whole SH region, while a GST fusion
protein containing the two SH2 domains is much less effective. A GST fusion protein containing the SH3
domain of the Grb2 adaptor protein does not inhibit
tr-kit-induced egg activation, showing that the effect of
the SH3 domain of PLC
1 is specific. Tr-kit-induced egg
activation is also suppressed by co-injection of antibodies raised against the PLC
1 SH domains, but not against
the PLC
1 COOH-terminal region. In transfected COS
cells, coexpression of PLC
1 and tr-kit increases diacylglycerol and inositol phosphate production, and the
phosphotyrosine content of PLC
1 with respect to cells
expressing PLC
1 alone. These data indicate that tr-kit activates PLC
1, and that the SH3 domain of PLC
1 is
essential for tr-kit-induced egg activation.
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Introduction |
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AFTER sperm-egg fusion, sperm cytosolic factors are
released into the egg cytoplasm, and recent evidence obtained in a number of animal systems
suggests that such a factor may trigger the series of events
culminating in cell cycle resumption and the first mitotic
division of the zygote (Stice and Robl, 1990; Swann, 1990
;
Homa and Swann, 1994
; Dozortsev et al., 1995
; Wu et al., 1997
; Stricker, 1997
). In many species, a series of Ca2+
transients is the early event triggered by the sperm at fertilization (Whitaker and Swann, 1993
), and the increase in
intracellular Ca2+ is required for several of the events that
accompany egg activation (Kline and Kline, 1992
). In the
mouse, it has been shown that sperm-egg fusion precedes
the onset of these Ca2+ oscillations (Lawrence et al., 1997
),
suggesting that a factor released by the sperm is responsible for the fertilization-associated Ca2+ mobilization. However, the nature of such factor in mammals is still uncertain.
A possible candidate is oscillin, a glucosamine 6-phosphate deaminase that has been localized in the equatorial segment of the hamster sperm head (Parrington et al.,
1996
). However, whereas the protein fraction containing
oscillin induces Ca2+ transients when microinjected into
mouse eggs (Parrington et al., 1996
), neither recombinant
nor highly purified oscillin has oscillogenic activity, even
though they maintain glucosamine 6-phosphate deaminase
activity (Wolosker et al., 1998
). Thus, it is possible that either oscillin requires additional factors to elicit egg activation or a different protein of the sperm is responsible for
such function.
An additional candidate for a soluble sperm factor inducing the early events of fertilization is tr-kit, an alternative product of the c-kit gene (Sette et al., 1997). Tr-kit is
encoded by an mRNA specifically expressed in the haploid phase of mouse spermatogenesis (Sorrentino et al.,
1991
; Rossi et al., 1992
). Tr-kit mRNA is transcribed in
late spermiogenesis under the control of an intronic promoter, as demonstrated by the tr-kit promoter driven expression of a reporter gene in transgenic mice (Albanesi
et al., 1996
). The open reading frame (ORF)1 of tr-kit encodes a 23-kD protein that contains only part of the cytoplasmic portion of the c-kit receptor tyrosine kinase (Rossi
et al., 1992
). This region corresponds to the c-kit phosphotransferase catalytic domain, but lacks the inter-kinase region, the ATP-binding site, the transmembrane and the
extracellular domains. The tr-kit protein has an apparent
molecular size of 24-28 kD, is expressed in elongating
spermatids (Albanesi et al., 1996
), and immunofluorescence experiments indicate that it is localized in the residual cytoplasm of mouse epididymal spermatozoa (Sette et al.,
1997
). We have previously reported that microinjection of
either lysates from cells expressing a recombinant tr-kit
protein or synthetic tr-kit RNA into metaphase II (MII)-
arrested mouse oocytes triggers the set of events associated with egg activation, from cortical granule exocytosis
to pronuclear formation and progression through cleavage stages (Sette et al., 1997
).
Tr-kit action is blocked by chelation of egg intracellular
Ca2+ or by preincubation of eggs with an inhibitor of phospholipase C (PLC) (Sette et al., 1997), suggesting that tr-kit mediates Ca2+ mobilization through activation of a
PLC isoform(s). PLCs are a family of enzymes that catalyze hydrolysis of phosphatidylinositol 4,5 bisphosphate
(PIP2), with production of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3) (Berridge, 1993
). DAG is a
powerful stimulator of various protein kinase C (PKC)
isoforms, and it has been suggested that PKC activity is required for sperm-induced egg activation (Colonna et al.,
1997
; Gallicano et al., 1997a,b). On the other hand, InsP3
binds to receptors coupled to channels responsible for the
release of Ca2+ from intracellular stores (Berridge, 1993
).
An increase in InsP3 production is required for the Ca2+
wave at fertilization in Xenopus oocytes (Nuccitelli et al., 1993
) and InsP3 receptors have been found to play an essential role in mammalian egg activation at fertilization
(Miyazaki et al., 1992
, 1993
; Xu et al., 1994
; Berridge,
1996
). Furthermore, the involvement of InsP3 produced by
PLC in mammalian fertilization is also supported by the
observation that a PLC inhibitor can block the sperm-
induced Ca2+ spiking at fertilization in mouse eggs (Dupont et al., 1996
).
PLC1 may represent the most likely PLC isoform involved in tr-kit action inside the egg for the following reasons: (a) PLC
1 has been shown by immunoblotting of
ovulated mouse oocytes (Dupont et al., 1996
); (b) PLC
1
is activated after physical interaction with tyrosine kinases
(Lee and Rhee, 1995
; Kamat and Carpenter, 1997
; Rhee
and Bae, 1997
), and it has been found to interact with the
activated c-kit receptor (Herbst et al., 1991
; Lev et al., 1991
; Rottapel et al., 1991
); (c) mutation of a tyrosine residue (Y936) of the COOH-terminal portion of the human
c-kit receptor impairs association with PLC
1 (Herbst et al.,
1995
), and the homologous residue is also present in the
mouse c-kit receptor (Y934) and in tr-kit (Y161); and (d) it
has been recently reported that PLC
is essential for the
sperm-induced Ca2+ mobilization at fertilization in starfish
eggs (Carroll et al., 1997
).
Both physical interaction with tyrosine kinases and tyrosine phosphorylation of PLC1 correlate with PLC
1
activation and subsequent stimulation of PIP2 hydrolysis
(Lee and Rhee, 1995
; Kamat and Carpenter, 1997
; Rhee
and Bae, 1997
). In addition to catalytic domains, PLC
1
contains several regulatory regions, and in particular src-homology 2 (SH2) and SH3 domains, which mediate its interaction with upstream and downstream effectors (Cohen
et al., 1995
; Pawson, 1995
). The SH2 domains of the protein directly bind specific phosphotyrosine residues present in
cytoplasmic domains of receptor tyrosine kinases (RTKs)
(Mohammadi et al., 1991
), whereas the targets of the SH3
domain are proline-rich sequences present in proteins
such as the microtubule-associated GTPase dynamin
(Gout et al., 1993
).
In the present study, we demonstrate that PLC1 is actually involved in tr-kit-induced parthenogenetic egg activation and that the SH3 domain of PLC
1 is essential for
this process. Using biochemical approaches in transfected
COS cells, we also show that coexpression of PLC
1 and
tr-kit stimulates an increase in tyrosine phosphorylation of
PLC
1, together with production of DAG and inositol
phosphates (InsPs). These data strongly suggest that the
mechanism of mouse egg activation triggered by tr-kit microinjection involves PLC
1-mediated hydrolysis of PIP2.
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Materials and Methods |
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Expression of Recombinant Tr-kit Protein
Subconfluent COS cell monolayers were cultured in 90-mm dishes (Corning Glass Works, Corning, NY) and processed for CaPO4 transfection
with either 20 µg of the pCMV5 eukaryotic expression vector containing
the tr-kit cDNA (pCMV5-tr-kit) or no DNA (mock) as previously described (Albanesi et al., 1996). 48 h after transfection, mock- and tr-kit-
transfected COS cells were harvested in microinjection buffer (20 mM
Hepes, pH 7.5, 120 mM KCl, 0.1 mM EGTA, 10 mM
-glycerophosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin), homogenized, and then centrifuged for 10 min at 14,000 g at 4°C. Aliquots of supernatant fractions were
immediately frozen at
80°C. Tr-kit expression was monitored by Western blot analysis before microinjection experiments.
Quantification of Tr-kit in Mouse Spermatozoa and in COS Cell Extracts
Spermatozoa from the cauda of the epididymis of 12- to 15-wk-old CD1 mice were collected in MEM (GIBCO BRL, Gaithersburg, MD) supplemented with 30 mg/ml BSA (Sigma Chemical Co., St. Louis, MO). After a 2-h incubation at 37°C, spermatozoa were collected by centrifugation at 3,000 g at 4°C, washed twice with PBS, and then lysed in SDS-PAGE sample buffer. Lysates were sonicated, for three cycles of 20 s at 4°C, boiled for 5 min, and then centrifuged for 10 min at 10,000 g at 4°C. Soluble material was analyzed by Western blot.
Cell lysate from 3 × 106 spermatozoa and 50 µg of proteins from mock- and tr-kit-transfected COS cell extracts were separated on a 10% SDS-PAGE gel under denaturing conditions, blotted onto a nitrocellulose membrane, and then analyzed by Western blot using an anti-c-kit antibody as described below. Intensity of the bands corresponding to tr-kit were quantified by optical densitometry using the Molecular Analyst program and a GS-700 Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA). 50 µg of tr-kit-transfected COS cell extracts contained an amount of tr-kit threefold higher than that present in 3 × 106 spermatozoa (see Fig. 1). In microinjection experiments we injected 5 pl of a 0.2-0.4 µg/µl solution of tr-kit cell extracts (1-2 pg of proteins), an amount corresponding to 0.2-0.4 sperm equivalents of tr-kit.
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Oocyte Collection, Microinjection and In Vitro Culture
MII-arrested oocytes were collected from hormonally primed (Hogan et al.,
1994) 4- to 6-wk-old CD1 female mice (Charles River, Calco, Italia) 15 h
after hCG (Sigma Chemical Co.) injection. Ovulated oocytes were freed
from cumulus cells by a brief incubation in 0.5 mg/ml hyaluronidase
(Sigma Chemical Co.) in M2 medium (Hogan et al., 1994
), washed with
M2 medium, and then immediately processed for microinjection as described (Sette et al., 1997
). Groups of 20 MII oocytes were transferred to
50-µl drops of M2 under mineral oil (Sigma Chemical Co.) and subjected
to intracytoplasmic injections using a Nikon invertoscope (Nikon Corp.,
Tokyo, Japan) equipped with Hoffman modulation contrast optics (Modulation Optics, Inc., Greenvale, NY) and two Leitz mechanical micromanipulators (Leica AG, Heerbrugg, Switzerland). A quantification of the
approximate volume of solution microinjected into a single oocyte, was
performed in repeated experiments as follows: a known amount of injection solution (usually 100 pl) was drawn in the injection pipette and used
completely for a series of microinjections under the same routinary conditions. The average number of oocytes microinjected with 100 pl of solution was 17. Considering the loss of small amounts of solution between injections, the injected volume per oocyte was ~5 pl. After injection,
surviving oocytes were cultured at 37°C in M16 medium (Hogan et al.,
1994
) under a humidified atmosphere of 5% CO2 in air for
7 h, and then
scored for pronuclei formation by phase-contrast microscopy. To confirm
the score, in most experiments eggs were fixed in 4% PFA in PBS 7 h after the injection, and stained with 10 µg/ml Hoechst 33342 (Sigma Chemical
Co.) for 5 min. After five washes in M2, eggs were mounted in 30% glycerol in PBS on glass slides with coverslip compression, sealed, and then
analyzed by fluorescence microscopy.
For cortical granule staining, microinjected eggs were fixed after 1-4 h, and processed as described below.
Cortical Granule and Chromosome Staining
1-4 h after microinjection, cultured oocytes were freed from the zona pellucida by acidic tyrode solution (Hogan et al., 1994) and fixed in 4% PFA
in PBS for 30 min at room temperature. After three washes in M2 (blocking solution), oocytes were incubated with 0.1% Triton X-100 in the same
medium for 5 min and transferred to blocking solution for 60 min at room
temperature. Oocytes were then treated for 60 min at room temperature
with 0.1 mg/ml TRITC-labeled lectin from Lens Culinaris (Sigma Chemical Co.) in blocking solution (Ducibella et al., 1988
), washed four times for
5 min in blocking solution, incubated for 5 min with 10 µg/ml Hoechst
33342 dye in blocking solution, and washed again. Oocytes were then
mounted in 30% glycerol in PBS as described above and analyzed by fluorescence microscopy.
Glutathione-S-Transferase-PLC1 Fusion Proteins and
Antibodies Used in Microinjection Experiments
The glutathione-S-transferase (GST)-encoding plasmid pGEX3X was obtained from Pharmacia Biotech, Inc. (Piscataway, NJ). Plasmid DNAs encoding for GST fusion proteins of bovine PLC1 SH2-SH2 and human
PLC
1 SH3 (see Fig. 3) in pGEX2T'6 were a kind gift from S. Courtneidge (Sugen, Inc., Redwood City, CA).
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Affinity-purified GST-PLC1 SH region fusion protein (GST-PLC
1-SH2SH2SH3) (No. sc4019), and GST-Grb2-SH3 (residues 156-199; No.
sc4036) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Control GST protein, GST-PLC
1 SH2-SH2, and GST-PLC
1 SH3 fusion proteins were produced as described by Gish et al. (1995)
, affinity purified on glutathione Sepharose, extensively dialyzed against PBS, and
concentrated to 10 mg/ml using a 10-kD cutoff Centricon (Millipore
Corp., Bedford, MA). Protein concentration was determined according to
Bradford (1976)
using BSA as a standard.
The anti-PLC1bd antibody (No. 426; Santa Cruz Biotechnology) consists
of affinity-purified rabbit polyclonal IgGs directed against the SH region of
rat PLC
1 ("binding domain"; amino acids 530-850). The anti-PLC
1ct antibody (No. 81; Santa Cruz Biotechnology) consists of affinity-purified rabbit
polyclonal IgGs directed against a peptide in the COOH terminus of bovine
PLC
1 (amino acids 1,249-1,262). Both antibodies recognize mouse PLC
1
in Western blot and immunoprecipitation and do not cross-react with other PLC isoforms. As a control, affinity-purified normal rabbit IgGs were used.
For tr-kit co-injection experiments in mouse eggs, GST fusion proteins were diluted in the injected solution to 500 µg/ml, whereas affinity-purified rabbit polyclonal IgGs were diluted to 10 µg/ml. Since we injected 5 pl of protein solution in the oocytes, considering the average volume of mouse eggs equal to 270 pl, the final concentration inside the injected eggs of all microinjected proteins was ~50-fold lower (10 µg/ml for the GST fusion proteins and 0.2 µg/ml for the antibodies), unless otherwise specified in the Results section. Control experiments using the Santa Cruz glycerol buffer (vehicle of GST fusion proteins) in addition to tr-kit did not interfere with egg activation (Sette, C., and A. Bevilacqua, unpublished observations).
Measurement of DAG and InsP Production in COS Cells
For measurement of DAG production, subconfluent COS cell monolayers
in 90-mm dishes were processed for CaPO4 transfection with either 20 µg
pRK-PLC1 (expression vector for PLC
1, a generous gift from Dr. A. Ullrich, Max-Planck Institut, Martinsried, Germany) alone, or with 20 µg
pRK-PLC
1 and 20 µg pCMV5-tr-kit (see Albanesi et al., 1996
). 18 h after transfection, cells were washed with PBS and cultured for additional
2 h in DME containing 10% FCS (GIBCO-BRL) and 0.5 mCi/ml
[3H]arachidonic acid. At the end of the incubation, cells were washed twice with cold PBS and harvested in 0.5 ml PBS/dish. The pH of the cell
suspensions was lowered to 2-3 by addition of HCl (30 mM final concentration). Lipids were extracted by addition of 4 vol of chloroform/methanol (1:2) in glass tubes according to the method of Boukhchache and
Lagarde (1982)
. Neutral lipids were separated by thin layer chromatography on silicagel plates (Merck, Darmstadt, Germany) using a solution of
hexane/diethyl ether/acetic acid (50:50:1) for the migration. Plates were
stained with 0.3 mg/ml Coomassie brilliant blue R250 (Bio-Rad Laboratories) in 0.15 M NaCl containing 20% methanol. The DAG bands were
identified on the plates based on the migration of known lipid standards (Sigma Chemical Co.), scraped off, mixed with Picofluor (Packard), and
their radioactivity was determined by liquid scintillation counting. DAG-associated radioactivity was expressed as cpm incorporated in DAG
versus 103 CPM incorporated in total lipids (neutral lipids bands plus
phospholipids at the origin of the chromatogram). DAG-associated radioactivity ranged between 1,200 and 3,000 cpm; the average amount of CPM in total lipids was ~200,000.
For measurement of InsPs production, subconfluent COS cell monolayers in 35-mm dishes were transfected with either 4 µg pRK-PLC1 alone, or 4 µg pRK-PLC
1 and 4 µg pCMV5-tr-kit. Immediately after transfection cells were transferred to DME containing 10% FCS and 5 µCi/ml D-myo-[3H]inositol, and cultured for additional 12-24 h. We selected two time points after transfection (12 and 24 h) to investigate
whether cells had reached steady state of phosphoinositides labeling and InsPs accumulation. The tr-kit-induced InsPs accumulation measured at 24 h is only slightly higher than that observed at 12 h, suggesting that
cells had reached steady-state levels. During the final 60 min of incubation, 10 mM LiCl was added to the medium. Incubation was stopped by
washing three times with PBS and adding ice-cold 10% TCA to the cells.
[3H]Inositol-labeled InsPs were extracted, separated by ion exchange chromatography on Dowex 1×8-200 and counted as described by Adamo et al. (1985)
. InsPs were expressed as cpm incorporated in InsPs fractions
per mg of total protein resuspended after TCA precipitation.
Immunoprecipitation and GST-PLC Coprecipitation Experiments
Subconfluent COS cell monolayers in 90-mm dishes were processed for CaPO4 transfection with the appropriate plasmids as described above. Cells transfected with pCMV5-c-kit (obtained by subcloning c-kit cDNA from pCDM8-c-kit [a generous gift from Dr. P. Besmer, Sloan Kettering Cancer Center, New York, NY] into pCMV5) were treated for 10 min with or without 100 ng/ml stem cell factor (SCF) in the presence of 250 µM sodium orthovanadate in complete medium before harvesting. 24 h after transfection, cells were rinsed with PBS, harvested in lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 15 mM MgCl2, 15 mM EGTA, 250 µM sodium orthovanadate, 10% glycerol, 1% Triton X-100, 0.1% SDS, 10 µg/ ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin), and incubated on ice for 5 min. Detergent-soluble extracts were separated by 10-min centrifugation at 15,000 g at 4°C.
For immunoprecipitation experiments, either a rabbit anti-kit antiserum raised against the 13 COOH-terminal amino acids common to the
mouse c-kit and tr-kit proteins (1:100 dilution; Albanesi et al., 1996) or a
mixture of 1 µg anti-PLC
1bd and 1 µg anti-PLC
1ct IgGs were preincubated for 60 min with protein A-Sepharose beads (Sigma Chemical Co.).
At the end of the incubation, the beads were washed once with 20 mM
Tris-HCl, pH 7.8, containing 0.5 M NaCl, twice with 20 mM Tris-HCl, pH
7.8, and then incubated for 90 min at 4°C with the detergent-soluble extracts under constant shaking. Protein A-Sepharose-bound immunocomplexes were rinsed three times with PBS containing 0.05% BSA, twice
with PBS, and finally eluted in SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% (wt/vol) SDS, 0.7 M 2-mercaptoethanol,
and 0.0025% (wt/vol) bromophenol blue). For GST-PLC coprecipitation experiments, 10 µg of affinity-purified GST-PLC (SH2-SH2-SH3) protein
were added to detergent-soluble extracts. After 30 min, samples were incubated with glutathione-agarose beads for additional 90 min at 4°C under
constant shaking. At the end of the incubation, glutathione-agarose-bound
protein complexes were rinsed three times with PBS before elution in 50 mM Tris-HCl, pH 8.0, containing 5 mM reduced glutathione. Eluted proteins were diluted in SDS-PAGE sample buffer for Western-blot analysis.
Western Blot Analysis
For detection of recombinant proteins, samples from transfected cells,
from immunoprecipitation, and from coprecipitation with GST-PLC protein, were separated on 10% SDS-PAGE, transferred onto nitrocellulose
membrane (Amersham) and subjected to Western blot analysis with different antibodies as previously described (Albanesi et al., 1996). In brief,
first antibody incubation (90 minutes at room temperature) was carried
out with 1:1,000 dilution of a polyclonal anti-kit antiserum (Albanesi et al.,
1996
), or affinity-purified polyclonal anti-PLC
1bd IgGs described above, or affinity-purified mouse anti-phosphotyrosine mAb (No. 508, Santa
Cruz Biotechnology). Second antibody incubation was carried out with
1:10,000 dilution of either anti-rabbit or anti-mouse IgGs antibody conjugated to horseradish peroxidase (Amersham Corp., Arlington Heights,
IL). Immunostained bands were detected by the enhanced chemiluminescence method (Amersham Corp.). Tyrosine phosphorylation of immunoprecipitated PLC
1 was quantified as the ratio of the optical density detected by the anti-phosphotyrosine antibody (
PY) versus that detected by
the anti-PLC
1 antibody (
PLC
1) (mean ± SD of six separate experiments). Optical Densitometry was performed using the Molecular Analyst
program and a GS-700 Imaging Densitometer (Bio-Rad Laboratories).
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Results |
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Kinetics of Tr-kit-induced Parthenogenetic Activation of Mouse Eggs
We have previously shown that microinjection of either
cell extracts expressing recombinant tr-kit or synthetic tr-kit mRNA is able to induce complete parthenogenetic activation of mouse eggs and cleavage to the two-cell stage
(Sette et al., 1997). To compare the amount of recombinant
tr-kit capable of activating mouse eggs with the amount
carried by a single mouse sperm, we performed Western
blot analysis (Fig. 1). Densitometric analysis of immunoreactive bands indicated that the amount of tr-kit protein obtained from extracts of 3 × 106 sperm was nearly threefold smaller than that from 50 µg of extracts of tr-kit-
expressing COS cells (see also Materials and Methods). As
shown in Fig. 2 and Table I, injection of ~0.2-0.4 sperm
equivalents of recombinant tr-kit is sufficient to exert activation of 60-70% mouse eggs. Injection of ~0.1 sperm
equivalents of tr-kit reduced the activation rate to 40-50%,
and injection of ~0.01 sperm equivalents did not result in
significant activation of mouse eggs above the background
reported in Table I (not shown). Although the timing of egg
activation triggered by microinjection of recombinant tr-kit is not completely synchronous, the typical time course and
pattern of the cell cycle events (Fig. 2) closely resemble
those observed at fertilization of the mouse eggs (Mori et
al., 1988
). 1 h after injection, 60-70% of the eggs underwent
metaphase-anaphase transition and initiated polar body extrusion (Fig. 2, A and B). At 4 h, the polar body was extruded in all activated eggs, chromosome decondensation
had begun and an incipient pronucleus was evident (Fig. 2
C). The size of the pronucleus progressively increased from
the time of appearance to reach its full size after 6-7 h from
the injection in all the activated eggs (Fig. 2 D).
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A GST Fusion Protein Containing the
PLC1-SH3 Domain Inhibits Tr-kit-induced
Parthenogenetic Activation of Mouse Eggs
To test the hypothesis that PLC1 is involved in tr-kit-
induced parthenogenetic activation of mouse eggs, we subjected MII-arrested oocytes, which express PLC
1 (Fig. 3
A; Dupont et al., 1996
), to co-injection of extracts from
COS cells expressing a recombinant tr-kit protein, and a
purified GST fusion protein containing the entire SH2-SH2-SH3 region of PLC
1 (GST-PLC
1-SH2SH2SH3)
(Fig. 3 B). The SH region mediates interaction of PLC
1
with proteins involved in enzyme regulation (Lee and
Rhee, 1995
; Kamat and Carpenter, 1997
; Rhee and Bae,
1997
), and competition to this region of PLC
1 has been
shown to prevent enzyme activation (Chen et al., 1994
). Injection of recombinant tr-kit alone, or co-injection of tr-kit together with a control GST protein resulted in activation of 63-70% of the eggs, as monitored by formation of a
pronucleus 4-7 h after injection (Table I). Co-injection of
tr-kit and GST-PLC
1-SH2SH2SH3 significantly reduced
the activation rate to 15%, suggesting a role of PLC
1 in
tr-kit action in the egg cytoplasm. Only 5-8% of spontaneous activation was obtained in either non-injected eggs
(Table I) or in eggs injected with extract from mock-transfected cells (data not shown; Sette et al., 1997
).
To further investigate which SH domain of PLC1 is involved in tr-kit-induced egg activation, we co-injected tr-kit with GST fusion proteins containing either the tandem
SH2 domains (GST-PLC
1-SH2SH2) or the SH3 domain
(GST-PLC
1-SH3) of the protein (Fig. 3). Co-injection of
GST-PLC
1-SH2SH2 only slightly reduced tr-kit-induced
egg activation (53% versus 70%; Table I). However, co-
injection of GST-PLC
1-SH3 inhibited egg activation as
efficiently as the entire SH2-SH2-SH3 region, reducing the
activation rate to 14% (Table I). Since we have previously
reported that tr-kit-induced egg activation is also associated with early events of egg activation, such as the Ca2+-dependent cortical granules (CGs) exocytosis (Sette et al.,
1997
), the effect of the GST-PLC
1 SH2 and SH3 fusion
proteins on CGs release was investigated. Co-injection of
GST-PLC
1-SH3 inhibited both cortical granule release
(Fig. 4 A) and polar body extrusion (not shown) with a rate
similar to that observed for pronuclear formation (Fig. 4 A;
see Table I for rate of inhibition) while the GST-PLC
1-SH2SH2 protein was much less effective (Fig. 4 B; see Table
I for rate of inhibition). Co-injection of 10-fold diluted GST-PLC
1-SH3, at a final concentration in the egg of ~1 µg/ml,
resulted in an almost equally efficient inhibition of tr-kit-
induced pronuclear formation (Table I). To test for the specificity of the SH3 domain of PLC
1, we co-injected tr-kit together with a GST fusion protein containing the SH3
domain of the adaptor protein Grb2. Although the SH3 domains of Grb2 and PLC
1 can bind to common targets, such
as dynamin (Gout et al., 1993
), they have been reported to
direct the corresponding GST fusion proteins to different
cell compartments when microinjected in NIH3T3 fibroblasts (Bar-Sagi et al., 1993
), implying that the Grb2 and
PLC
1 SH3 domains can also recognize different targets. As
shown in Table I, co-injection of GST-Grb2-SH3 was not
able to affect tr-kit-induced egg activation. These data indicate that competition for targets of the SH3 domain specific
to PLC
1 impairs tr-kit-induced egg activation.
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An Antibody Directed Against the SH2-SH2-SH3
Region of PLC1 Blocks Tr-kit-induced Activation of
Mouse Eggs
The role of the SH region of PLC1 in tr-kit-mediated egg
activation was also investigated by microinjection experiments using antibodies raised against different regions of
PLC
1. The anti-PLC
1bd antibody is directed against the
SH region of PLC
1 and we hypothesized that its binding
would prevent PLC
1 interaction with effector proteins.
The anti-PLC
1ct antibody is directed against the COOH terminus of PLC
1, a region of the enzyme that is not
known to be involved in catalytic activity and/or interaction of PLC
1 with other proteins (Lee and Rhee, 1995
).
These antibodies are specific for PLC
1, and they do not
cross-react with other PLC isoenzymes. The anti-PLC
1bd
(Fig. 3 A) and the anti-PLC
1ct antibodies (not shown)
recognize PLC
1 in Western blots from extracts of both
PLC
1-overexpressing COS cells and of MII-arrested
mouse oocytes. Microinjection experiments showed that
the anti-PLC
1bd antibody almost completely suppresses
tr-kit-induced mouse egg activation, resembling the effect
obtained by co-injection of tr-kit and either the GST-PLC
1-SH2SH2SH3 or the GST-PLC
1-SH3 fusion proteins,
whereas nonimmune antibodies at the same concentration are ineffective (Table II). On the other hand, co-injection of recombinant tr-kit and the anti-PLC
1ct antibody did not
significantly inhibit egg activation, showing that binding of
antibodies to the COOH terminus of PLC
1 does not impair
interaction with factors required for egg activation (Table II).
The inhibition obtained with the anti-PLC
1bd antibody
confirms that PLC
1 is involved in parthenogenetic egg activation triggered by tr-kit and that an essential role in such
pathway is played by the SH region of PLC
1.
Tr-kit Stimulates PIP2 Hydrolysis in Transfected COS Cells
The ability of tr-kit to stimulate the catalytic activity of
PLC1 was investigated by cotransfection experiments in
COS cells. Cells were transfected with a tr-kit expression
vector, or with a PLC
1 expression vector, or cotransfected with both plasmids, labeled with either [3H]arachidonic acid or [3H]inositol, and processed for assays of diacylglycerol production or InsP production, respectively.
As shown in Fig. 5 A, when cells were transfected with
tr-kit alone, a slight increase in DAG production was observed, likely indicating activation of endogenous PLCs.
As expected, an increase in DAG production was also observed in cells transfected with PLC1 versus mock-transfected cells. Coexpression of tr-kit and PLC
1 was reproducibly accompanied by a much higher activation of DAG
production, suggesting that, in cotransfection experiments, tr-kit is able to stimulate DAG production by activating
PLC
1. Cotransfection of PLC
1 with the full-length c-kit
receptor did not induce any increase in DAG production
with respect to cells transfected with PLC
1 alone. Moreover, stimulation of c-kit-transfected cells with the c-kit
ligand (SCF) did not induce any increase in DAG production with respect to both unstimulated cells and cells transfected with PLC
1 alone (Fig. 5 A), even though PLC
1 associates with c-kit after SCF stimulation (see below, Fig.
7 A). The inability of autophosphorylated c-kit to activate
PLC
1 is in agreement with our previous observation that
SCF treatment fails to activate MII-arrested oocytes
(Sette et al., 1997
), which express the c-kit receptor
(Manova et al., 1990
; Horie et al., 1991
; Yoshinaga et al.,
1991
). As shown for the closely related PDGF
receptor
(Valius et al., 1995
), the simultaneous binding to the c-kit
receptor to a particular blend of other signal transduction molecules might interfere with PLC
1 activation.
|
|
Since DAG can be produced by other metabolic routes,
such as phosphatidylcholine hydrolysis by phospholipase
D and conversion of the resulting phosphatidic acid into
DAG by a specific phosphatidate phosphohydrolase (Exton, 1997), we set out to measure the activity of PLC
1 by
assaying InsPs production, a more specific marker of PIP2
hydrolysis. In agreement with the increase in DAG production, we found that coexpression of tr-kit and PLC
1 in
COS cells induced a 2.5-fold increase in InsPs production
compared with cells transfected with PLC
1 alone (Fig. 5
B). Similar to DAG production, transfection of tr-kit
alone induced only a slight increase in InsPs production versus mock-transfected cells (Fig. 5 B), however, cotransfection with PLC
1 amplifies tr-kit-induced InsPs production. Western blot analysis of extracts from these cells indicated that similar amounts of PLC
1 were expressed in
the PLC
1 transfected cells (Fig. 5 C). Therefore, the concomitant stimulation of DAG and InsPs production in
COS cells coexpressing tr-kit and PLC
1 is likely due to
posttranslational activation, rather than increased expression, of PLC
1. These results indicate that in COS cells tr-kit is able to induce activation of PLC
1.
Tr-kit Stimulates Tyrosine Phosphorylation of PLC1
in Transfected COS Cells
Since tyrosine phosphorylation is often associated with activation of PLC1 (Rhee and Bae, 1997
), we tested whether
an increase in phosphotyrosine content of PLC
1 is detectable in tr-kit-expressing cells. In cells transfected with a
PLC
1 expression vector alone, PLC
1 was found to be
already tyrosine-phosphorylated, but a significant increase
in its phosphotyrosine content was observed in tr-kit/PLC
1 cotransfected cells (Fig. 6 A, right panel). We routinely observed an increase in immunoprecipitated PLC
1 from
PLC
1/tr-kit cotransfected cells (Fig. 6 A, left panel), but
this does not reflect higher PLC
1 expression in these cells
as shown by the Western-blot analysis of total cell extracts
(Fig. 6 B, left panel; see also Fig. 5 C). Densitometric analysis indicated that the tyrosine phosphorylation of PLC
1
(normalized for PLC
1 content of the immunoprecipitates) was approximately threefold higher in tr-kit cotransfected
cells, with respect to cells transfected with PLC
1 alone
(not shown). This effect was selective since tr-kit expression
did not induce a general increase in the tyrosine phosphorylation pattern of total cell extracts (Fig. 6 B, right panel).
|
PLC1 Activation Does Not Require a Stable
Association with Tr-kit
Tr-kit shares with c-kit the 190 COOH-terminal residues
(Rossi et al., 1992), a region thought to mediate the interaction of activated c-kit with PLC
1. Indeed, mutation of
tyrosine 936 to phenylalanine in the human c-kit receptor
impairs docking of PLC
1 (Herbst et al., 1995
), and this
residue is conserved in mouse tr-kit (tyr161). SCF-induced
autophosphorylation of the c-kit receptor creates docking
sites for several signaling proteins (Herbst et al., 1991
; Lev
et al., 1991
; Rottapel et al., 1991
; Koike et al., 1993
; Blume-Jensen et al., 1994
; Herbst et al., 1995
), and presumably,
phosphorylation of tyrosine 936 creates a binding site for
the SH2 domains of PLC
1 or of intercalated adaptor proteins. It is therefore conceivable that also tr-kit, if phosphorylated on tyr161, can bind to PLC
1.
To test this hypothesis we expressed tr-kit in COS cells
and purified the cell extracts on a GST-PLC1-SH2SH2SH3
fusion protein bound to glutathione-agarose beads. We
could not detect any binding of tr-kit to the GST-PLC
1
(Fig. 7 A), indicating that tr-kit does not interact directly,
or at least does not stably associate, with PLC
1. Under
these conditions, although tr-kit was efficiently precipitated by the anti-c-kit antibody (not shown), no tyrosine phosphorylation of tr-kit was detected (Fig. 7, B and C),
even though we cannot rule out the possibility that this
was due to the sensitivity limits of the assay conditions. As
a control for the coprecipitation experiment shown in Fig.
7 A, we used cell extracts from c-kit-expressing COS cells
that had been previously incubated for 10 min with or
without SCF to induce autophosphorylation of the receptor. As expected, SCF induced tyrosine phosphorylation of the c-kit receptor (Fig. 7, B and C), and c-kit was copurified with the GST-PLC
1 fusion protein only after SCF
treatment, while almost no receptor was bound in its resting state (Fig. 7 A) indicating that tyrosine phosphorylated
c-kit binds to PLC
1. Similar results were obtained by immunoprecipitation of cell extracts with anti-kit or anti-PLC
1 antibodies followed by Western blot analysis using anti-PLC
1 or anti-kit antibodies, respectively (not shown).
Thus, cotransfection experiments in COS cells indicate
that tr-kit stimulates tyrosine phosphorylation and activation of PLC
1 in the absence of a direct association with it.
![]() |
Discussion |
---|
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---|
Entrance of a sperm factor(s) into the egg cytoplasm after
sperm-egg fusion is thought to trigger a series of events
starting with Ca2+ release from intracellular stores and
culminating in completion of the meiotic cell cycle and the
onset of embryonic development (Whitaker and Swann,
1993). Recently, it has been reported that activation of
PLC
is required for the sperm-induced Ca2+ rise observed in starfish eggs at fertilization, and that the two
SH2 domains of PLC
and their ability to bind phosphotyrosines are important for PLC
activation and the onset of
Ca2+ rise (Carroll et al., 1997
). Here we report that PLC
1
mediates the parthenogenetic activation of mouse eggs induced by microinjection of recombinant tr-kit, a protein
present in the residual cytoplasm of mouse spermatozoa.
The SH3 domain of PLC
1 plays a fundamental role in tr-kit-induced egg activation, being required for both cortical granule exocytosis and cell cycle resumption. This role is specific since the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit action inside the egg. We also
show that tr-kit is able to activate recombinant PLC
1
when coexpressed in a heterologous system.
Activation of PLC results in hydrolysis of PIP2 with production of DAG and InsP3 (Berridge, 1993), and both
these second messengers are likely to play a major role in
mammalian egg activation at fertilization. DAG and/or
synthetic PKC activators are able to trigger resumption of
cell cycle in MII-arrested oocytes (Colonna et al., 1997
;
Gallicano et al., 1997a,b). Microinjection of InsP3 into
mammalian eggs is able to trigger Ca2+ transients, cortical
granule exocytosis, and pronuclear formation, and microinjection of antibodies directed against the InsP3 receptor blocks sperm-induced egg activation (Miyazaki et al.,
1992
, 1993
; Xu et al., 1994
; Berridge, 1996
). Furthermore,
inhibition of PIP2 hydrolysis with the specific PLC inhibitor U73122 blocks the sperm-induced Ca2+ spiking at fertilization in mouse eggs (Dupont et al., 1996
). The present
data, showing that tr-kit acts through activation of PLC
1,
are in agreement with the previous observation that U73122 blocks parthenogenetic egg activation triggered
by tr-kit (Sette et al., 1997
). The whole of these data indicates that tr-sit is a sperm factor that might play a physiological role in triggering early mouse embryonic development. Further support to this hypothesis is the observation
that activation of mouse eggs is elicited by microinjection
of an amount of recombinant tr-kit comparable to that
carried by a single mouse sperm.
PLC1 is activated by tyrosine kinase-dependent pathways (Lee and Rhee, 1995
; Kamat and Carpenter, 1997
;
Rhee and Bae, 1997
) and data suggest that tyrosine kinase
activity is involved in egg activation in different species.
Stimulation of artificially expressed RTKs can initiate egg
activation in Xenopus (Yim et al., 1994
) and in starfish
eggs (Shilling et al., 1994
). Moreover, endogenous soluble
src-related tyrosine kinases are activated shortly after fertilization both in Xenopus and in sea urchin (Sato et al.,
1996
; Kinsey, 1996
). In addition, a membrane-associated c-abl-related tyrosine kinase is also activated at fertilization in sea urchin eggs (Moore and Kinsey, 1994
). Although
the activation of these tyrosine kinases does not always
precede the Ca2+ rise, these data indicate that tyrosine
phosphorylation is involved in the early events of fertilization. Furthermore, the involvement of PLC
in Ca2+ rise
at fertilization in starfish eggs (Carroll et al., 1997
) suggests that one or more tyrosine kinases play a role in the upstream signaling pathway at fertilization in several species. Indeed, experiments with specific inhibitors have
shown that tyrosine kinase activity is important for both
block of polyspermy and late events of starfish egg activation (Moore and Kinsey, 1995
). In the mouse, it has been
shown that inhibitors of both tyrosine kinases and PLC
can impair very early events, such as sperm-induced Ca2+
spiking, associated with egg activation at fertilization (Dupont et al., 1996
).
Although tr-kit lacks an ATP-binding site, and thus it
should not present intrinsic tyrosine kinase activity (Rossi
et al., 1992), the possibility exists that tr-kit interacts with
either RTKs or non-receptor tyrosine kinases (NRTKs)
present in the egg, which in turn phosphorylate tr-kit itself,
or other proteins mediating PLC
1 activation. The full-length c-kit RTK is present in ovulated mouse oocytes
(Manova et al., 1990
; Horie et al., 1991
; Yoshinaga et al.,
1991
); however, we have previously shown that SCF fails to induce cortical granule exocytosis, meiosis resumption
and pronuclear formation in MII-arrested oocytes (Sette
et al., 1997
). In agreement with those observations, we
show here that the SCF-stimulated c-kit receptor binds
PLC
1 but does not stimulate its enzymatic activity in
transfected COS cells, as previously reported in other cellular systems (Lev et al., 1991
; Koike et al., 1993
; Blume-Jensen et al., 1994
, Kozawa et al., 1997
). The results herein
presented also indicate that tr-kit is able to stimulate both
DAG and InsPs production when coexpressed with PLC
1
in COS cells. Since activation of PIP2 hydrolysis does not
seem to require a stable physical interaction between tr-kit
and PLC
1, intercalated proteins may mediate the activation of PLC
1.
The SH region of PLC1 plays an essential role in tr-kit-
mediated activation of mouse eggs, as shown by direct competition experiments with either a GST-PLC
1-SH2SH2SH3
fusion protein or an antibody specifically directed against
this region of the enzyme. Since a GST-PLC
1-SH2SH2
fusion protein inhibits sperm-induced activation of starfish
eggs (Carroll et al., 1997
), our results suggest that SH-mediated activation of PLC
1 is an evolutionary conserved
mechanism of egg activation. On the other hand, a GST-PLC
1-SH3 fusion protein is much more effective than a
GST-PLC
1-SH2SH2 fusion protein in inhibiting tr-kit action in mouse eggs. These results are somehow surprising,
since the interaction of the SH2 domains of PLC
1 with
phosphotyrosine residues present in activated RTKs or
NRTKs is thought to be an essential step for tyrosine phosphorylation, translocation, and activation of PLC
1
(Lee and Rhee, 1995
; Kamat and Carpenter, 1997
; Rhee
and Bae, 1997
).
Tyrosine phosphorylation of PLC1 has been shown to
correlate with activation of the enzyme (Kim et al., 1991
).
We found that, although PLC
1 is already tyrosine-phosphorylated when overexpressed in COS cells, coexpression of tr-kit induces an increase in PLC
1 phosphotyrosine content together with activation of PIP2 hydrolysis. Since we have observed that the SH3 domain instead of
the SH2 domains competes for PLC
1 activation in mouse
eggs, it is possible that additional mechanisms, beside tyrosine phosphorylation of PLC
1, are involved in the regulation of the activity of the enzyme by tr-kit. Indeed, alternative routes of PLC
1 activation have been described
(Rhee and Bae, 1997
). For instance, activation of tyrosine
phosphorylated PLC
1 by PDGF in a fibroblast cell line
requires interaction of the pleckstrin homology (PH) domain of the enzyme with phosphatidylinositol 3,4,5-trisphosphate (PIP3) (Falasca et al., 1998
). Moreover, activation of the T cell receptor causes phosphorylation of
PLC
1, but enzyme activation also requires tyrosine phosphorylation of Grb2-associated proteins (Motto et al.,
1996
). Ultimately, cytosolic PLC
1 has to reach the particulate compartments of the cell to exert its enzymatic function. Translocation of PLC
1 to the membrane and/or the
cytoskeleton might bring the enzyme in close proximity to
other agents, such as phosphatidic acid (Jones and Carpenter, 1993
), arachidonic acid in concert with microtubule-associated tau proteins (Hwang et al., 1996
), and PIP3
(Bae et al., 1998
; Falasca et al., 1998
), which have been reported to stimulate its hydrolytic activity also independently from tyrosine phosphorylation. The SH3 domain of
PLC
1 has been shown to direct the enzyme to the cytoskeleton in proximity of the plasma membrane (Bar-Sagi et
al., 1993
), whereas the PH domain is required for the stable interaction of PLC
1 with membrane lipids (Falasca et
al., 1998
). According to this model, our data suggest that
tr-kit triggers activation of PLC
1 by allowing its interaction with effector proteins in the particulate compartment of the egg via the SH3 domain.
The SH3 domain might also be directly involved in the
modulation of PLC1 enzymatic activity. Indeed, microinjection of a catalytically inactive PLC
1 into quiescent
NIH3T3 fibroblasts induces a mitogenic response, and the
SH3 domain of the protein is required for this effect
(Huang et al., 1995
), suggesting that the SH3 domain of
PLC
1 is the target of inhibitory proteins. Titration of
these proteins with exogenous PLC
1-SH3 domains might allow activation of endogenous PLC
1, leading to the mitogenic response. Deletion experiments suggest that the
SH region of PLC
1 exerts an inhibitory role on the enzyme, probably impairing the correct folding of the two X
and Y catalytic domains (Horstman et al., 1996
). Presumably, tyrosine phosphorylation of the enzyme produces a
conformational modification and relieves this negative influence (Kamat and Carpenter, 1997
). However, it is possible that other interactions within the SH region, such as
binding of proteins to the SH3 domain, are able to induce
similar modifications and derepress PLC
1 enzyme activity. Intercalated proteins might mediate the interaction between tr-kit and PLC
1 causing the consequent activation
of the enzyme. Indeed, it is known that SH2-containing, tyrosine-phosphorylated, adaptor proteins, such as the Syp
tyrosine phosphatase, can indirectly couple other signaling
proteins to tyrosine-phosphorylated RTKs (Li et al.,
1994
). Tyrosine phosphorylation induced by tr-kit interaction with a kinase present in the egg cytoplasm might create docking sites for intercalated adaptor proteins, which
in turn may activate PLC
1 by association with its SH3 domain.
Recent findings highlight the importance of SH3 domains in cell signaling. In Xenopus oocytes, the ras-GAP
pathway is involved in germinal vesicle breakdown, and it
has been shown that both an antibody directed against the
SH3 domain of GAP, or peptides encompassing this region of the enzyme, are able to block germinal vesicle
breakdown induced by oncogenic ras (Duchesne et al.,
1993). The role of SH3 domains in regulating enzyme activity has been demonstrated in the case of some NRTKs.
Interaction of proline-rich targets with the SH3 domain of
src-related kinases results in enzyme activation, as demonstrated for Nef-mediated activation of Hck (Moarefi et al.,
1997
). Furthermore, the SH3 domain of Itk (a Tec-related
kinase) interacts with a proline-rich region of the enzyme
resulting in intramolecular inhibition, suggesting that
binding of other proline-rich proteins to this SH3 domain might result in Itk activation (Andreotti et al., 1997
).
Experiments are underway to identify proteins possibly
interacting with tr-kit and PLC1 inside the egg cytoplasm
and to investigate the physiological role played by tr-kit at
fertilization. Mutagenesis experiments will clarify whether
the phosphotransferase domain, or discrete tyrosine residues, or other structural elements present in tr-kit are involved in PLC
1 stimulation and consequent egg activation.
![]() |
Footnotes |
---|
Received for publication 28 January 1998 and in revised form 13 July 1998.
Address all correspondence to P. Rossi, Dipartimento di Sanitá Pubblica e Biologia Cellulare, Sezione di Anatomia, Universitá di Roma Tor Vergata, via O. Raimondo 8, 00173, Rome, Italy. Tel.: 39-6-72596272. Fax: 39-6-72596268. E-mail: pellegrino.rossi{at}med.uniroma2.itWe thank Drs. S. Courtneidge (Sugen, Inc., Redwood City, CA), P. Besmer (Sloan Kettering Cancer Center, New York), and A. Ullrich (Max-Planck Institut, Martinsried, Germany) for generous gift of plasmids; Dr. L.A. Jaffe (University of Connecticut, Farmington, CT) for reagents and for useful discussion; Drs. F. Naro (University "La Sapienza," Rome, Italy) and G. Nemoz (INSERM, Lyon, France) for their help and technical advice with the PLC activity experiments; Prof. F. Mangia (University "La Sapienza") for his advice and support in this study.
This work was supported by World Health Organization special project for Research Development and Research Training in Human Reproduction, by Consiglio Nazionale Delle Ricerche strategic projects Cell Cycle and Apoptosis and Oxidative and Cellular Stress, by Ministero Per l'Universitá e la Ricerca Scientifica e Tecnologica, by Agenzia Spaziale Italiana, and by Fondazione Istituto Pasteur-Cenci Bolognetti.
![]() |
Abbreviations used in this paper |
---|
DAG, diacylglycerol; GST, glutathione-S-transferase; InsP, inositol phosphate; InsP3, inositol 1,4,5-trisphosphate; MII, metaphase II; NRTK, non-receptor tyrosine kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PKC, protein kinase C; PLC, phospholipase C; RTK, receptor tyrosine kinase; SCF, stem cell factor; SH, src-homology; SH2, src-homology 2; SH3, src-homology 3; tr-kit, truncated c-kit.
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