* Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06032; and Institut National de
la Santé et de la Recherche Medicale CJF9207, Faculté de Pharmacie 15, F-34060 Montpellier, France
Although inositol trisphosphate (IP3) functions in releasing Ca2+ in eggs at fertilization, it is not
known how fertilization activates the phospholipase C
that produces IP3. To distinguish between a role for
PLC, which is activated when its two src homology-2 (SH2) domains bind to an activated tyrosine kinase,
and PLC
, which is activated by a G protein, we injected starfish eggs with a PLC
SH2 domain fusion
protein that inhibits activation of PLC
. In these eggs,
Ca2+ release at fertilization was delayed, or with a high
concentration of protein and a low concentration of
sperm, completely inhibited. The PLC
SH2 protein is a
specific inhibitor of PLC
in the egg, since it did not inhibit PLC
activation of Ca2+ release initiated by the
serotonin 2c receptor, or activation of Ca2+ release by
IP3 injection. Furthermore, injection of a PLC
SH2 domain protein mutated at its phosphotyrosine binding
site, or the SH2 domains of another protein (the phosphatase SHP2), did not inhibit Ca2+ release at fertilization. These results indicate that during fertilization of
starfish eggs, activation of phospholipase C
by an SH2
domain-mediated process stimulates the production of IP3 that causes intracellular Ca2+ release.
AT fertilization, the sperm initiates a propagated rise of
Ca2+ in the egg, which is of central importance in
activating the egg to begin development (Jaffe,
1985 IP3 is produced from phosphatidylinositol 4,5-bisphosphate (PIP2), by the action of a phospholipase C (PLC;
Rhee and Choi, 1992 Both PLC Several previous studies have examined whether PLC PLC Recombinant proteins containing the two SH2 domains
of PLC Obtaining Oocytes and Sperm
Starfish (Asterina miniata) were obtained from Marinus, Inc. (Long
Beach, CA). Ovaries and testes were collected through a hole in the dorsal surface of the animal, made with a 3-mm sample corer (Fine Science
Tools, Foster City, CA). Follicle cell-free oocytes were obtained by mincing the ovary in ice-cold, Ca2+-free sea water followed by washing in natural sea water. Oocyte maturation was induced by addition of 1 µM 1-methyladenine (Sigma Chemical Co., St. Louis, MO). Sperm were obtained by
mincing the testis, followed by centrifugation for 1 min at 3,000 g to separate the sperm suspension from other testis tissue. Except as indicated, the
sperm suspension was diluted 1:5,000 before use. All experiments were
performed in natural sea water at 18-20°C.
GST Fusion Proteins and RNA
Plasmid DNAs encoding GST fusion proteins of bovine PLC
GST fusion proteins were produced as described by Gish et al., 1995 DNA for the serotonin 2c receptor, in the vector Bluescript SK Calcium Measurements
Ca2+ measurements were performed using eggs at first meiotic metaphase,
the stage at which fertilization normally occurs. The eggs were injected
with 10 µM Ca-green 10-kD dextran (Molecular Probes, Eugene, OR)
and were held between two coverslips, with ~1 egg diam (~180 µm) between the coverslip edge and the egg surface (Kiehart, 1982 Microinjection
Quantitative microinjection was performed using mercury-filled micropipets (Kiehart, 1982 Delay of Ca2+ Release at Fertilization by
PLC The Ca2+ rise seen in starfish eggs at fertilization consists
of two phases. The initial response is a Ca action potential, resulting from Ca2+ entry through voltage-gated channels
in the plasma membrane (Miyazaki et al., 1975
In eggs injected with a GST fusion protein including the
NH2- and COOH-terminal SH2 domains of PLC Table I.
Effect of PLC
Microinjection of GST alone, at 200 to 1,100 µg/ml, or a
GST fusion protein composed of the tandem NH2- and
COOH-terminal SH2 domains of another protein, the
phosphatase SHP2 (240-1,000 µg/ml), had no effect on the
kinetics or amplitude of Ca2+ release (Fig. 2, A and D; Table I). Fusion proteins composed of the individual NH2- or
COOH-terminal SH2 domains of PLC Injections of the SH2 proteins were performed 28-130 min
before insemination. Within this range, the time of injection
had no effect on the kinetics or amplitude of the fertilization-induced Ca2+ rise. When injections were performed
13-19 min before insemination, PLC At the time of injection of the PLC Imaging of Ca2+ in PLC Imaging of the Ca2+ rise during fertilization of eggs injected with the PLC Table II.
Effect of PLC; Whitaker and Steinhardt, 1985
; Kline, 1988
). In echinoderm as well as vertebrate eggs, the rise in Ca2+ results,
at least in large part, from Ca2+ release from the endoplasmic reticulum in response to a rise in inositol trisphosphate (IP31; Whitaker and Irvine, 1984
; Ciapa and Whitaker, 1986
; Miyazaki et al., 1992
; Mohri et al., 1995
; Jaffe,
1996
). However, it has not been established how the IP3 is
generated at fertilization.
). This family of enzymes includes
,
, and
isoforms. PLC
is activated by G proteins, while
PLC
is activated by tyrosine kinases. The regulation of
PLC
is poorly understood, although the enzymatic activity of all 3 PLC isoforms can be stimulated by an increase
in Ca2+ (Park et al., 1992
; Wahl et al., 1992
; Banno et al.,
1994
). Very likely the generation of IP3 at fertilization results from the activation of one of these isoforms of phospholipase C.
and PLC
pathways are present in eggs. Expression in frog, mammalian, and starfish eggs of exogenous
receptors known to release Ca2+ by a G protein/PLC
pathway, such as serotonin 2c or muscarinic m1 receptors,
allows Ca2+ release in eggs when the corresponding agonists are applied (Kline et al., 1988
; Williams et al., 1992
;
Shilling et al., 1994
). This indicates that functional PLC
and corresponding G proteins are present. Likewise, expression in frog and starfish eggs of exogenous receptors
known to release Ca2+ by a tyrosine kinase/PLC
pathway, such as receptors for EGF or PDGF, allows Ca2+ release in response to these agonists (Shilling et al., 1994
; Yim et al., 1994
). Point-mutated receptors that do not activate PLC
do not cause Ca2+ release (Shilling et al., 1994
;
Yim et al., 1994
). These findings indicate that a functional
PLC
is present. Such experiments have not been carried
out in mammalian eggs, but the presence of PLC
has
been demonstrated by immunoblotting (Dupont et al.,
1996
).
or PLC
pathways cause Ca2+ release at fertilization
(Miyazaki, 1988
; Crossley et al., 1991
; Moore et al., 1994
;
Moore and Kinsey, 1995
; Dupont et al., 1996
). However,
the conclusions from these studies have been uncertain due to questions about the specificity of the pharmacological inhibitors used (see Discussion). To distinguish whether
fertilization involves PLC
- or PLC
-mediated Ca2+ release mechanisms, we injected starfish eggs with a recombinant portion of the PLC
protein that specifically inhibits PLC
but not PLC
activation.
is activated when its two tandem src homology-2
(SH2) domains interact with a specific SH2 binding site on
an activated protein tyrosine kinase, thus bringing PLC
in close contact with the kinase and allowing it to be phosphorylated (Kim et al., 1991
; Rhee and Choi, 1992
). The
kinase can be a transmembrane receptor kinase, such as
the PDGF receptor, or a cytosolic kinase such as Syk (Sillman and Monroe, 1995
). SH2 domain-mediated enzyme activation is widespread and highly specific; for example
the PDGF receptor kinase has at least eight sites that specifically bind particular SH2 domain-containing enzymes
or adaptor proteins (Claesson-Welsh, 1994
). The SH2 domains of each of these proteins are different, and none of
these proteins binds to the same site on the PDGF receptor as PLC
(Claesson-Welsh, 1994
; Gish et al., 1995
; Pawson, 1995
). Similarly for the EGF receptor, the SH2 domains of PLC
show specificity among the several SH2
domain binding sites present on the receptor, with much
higher affinity for one particular site compared to the others (Rotin et al., 1992
).
have been used to disrupt the signalling between
the tyrosine kinase and PLC
in fibroblasts (Roche et al.,
1996
). In an analogous way, we examined the effect of injecting a glutathione-S-transferase (GST) fusion protein
composed of the two SH2 domains of PLC
(NH2- and
COOH-terminal), into eggs of the starfish Asterina miniata. Our findings indicate that the PLC
pathway initiates
Ca2+ release at fertilization in starfish eggs.
Materials and Methods
SH2(N+C),
PLC
SH2(N), and PLC
SH2(C) (Fig. 1) in pGEX2T
6 (Roche et al.,
1996
) were obtained from S. Courtneidge (Sugen, Inc., Redwood City,
CA). DNA for a GST fusion protein of the tandem NH2- and COOH-terminal SH2 domains of the phosphatase SHP2, encoding amino acids 2 to
216 of the murine protein (Feng et al., 1993
; Adachi et al., 1996
), was obtained from T. Pawson (Mt. Sinai Hospital, Toronto, Canada) and was inserted in frame into the BamHI and EcoRI sites of pGEX2T (Pharmacia
Biotech, Piscataway, New Jersey). DNA for SH2(N+C)-wt and SH2
(N+C)-mut GST fusion proteins (Fig. 1) was derived from DNA for wild-type and mutant constructs containing the SH2 and SH3 domains of human PLC
(see Huang et al., 1995
) obtained from P. Huang (Merck Research Laboratories, West Point, PA). To do this, the SH2(N+C) portions
of the SH2(N+C)SH3 constructs were cut out using Bsp 120I, and the
overhangs were filled in with the Klenow fragment of Escherichia coli
DNA polymerase I. The DNA was subsequently digested with BglII, and
the fragments were inserted in frame into the SmaI and BamHI sites of
pGEX2T
6.
Fig. 1.
Bacterial fusion proteins of PLC SH2 domains. The
diagram shows the NH2- and COOH-terminal SH2 domains, the
SH3 domain, and the X and Y catalytic domains of the 1261 amino acids of PLC
, as well as the regions used to make the
GST fusion proteins. Proteins contained the following amino acid
residues as well as GST: (lane 1) SH2(N+C), 547-752; (lane 2)
SH2(N), 547-659; (lane 3) SH2(C), 663-752; (lane 4) GST; (lane
5) SH2(N+C)-mut, 518-771, R586/694K; (lane 6) SH2(N+C)-wt,
518-771. Lanes 1-3 are bovine sequences; lanes 5 and 6 are human sequences. 12% SDS-PAGE gel, 2-3 µg protein per lane,
stained with Coomassie blue.
[View Larger Versions of these Images (21 + 81K GIF file)]
,
purified using glutathione agarose (Sigma Chemical Co.) or glutathione
sepharose (Pharmacia Biotech), dialyzed extensively in PBS (137 mM
NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4), and concentrated to 3-50 mg/ml, using a 10-kD cutoff Ultrafree-4 centrifugal filter
device (Millipore Corp., Bedford, MA). Protein concentrations were determined using a BCA assay (Pierce Chemical Co., Rockford, IL) with
BSA as the standard. The correct folding of the PLC
SH2(N+C) and
SHP2-SH2(N+C) recombinant GST fusion proteins was confirmed by
testing their ability to bind to the activated PDGF receptor (see Roche et
al., 1996
).
, was
obtained from D. Julius (University of California, San Francisco). Synthetic RNA was made as previously described (Shilling et al., 1990
).
). Sperm were
added to the front of the chamber. Ca-green fluorescence was measured
with a photomultiplier (Shilling et al., 1994
) or imaged with a confocal microscope (MRC600; Bio Rad Laboratories, Hercules, CA) with a 20×, 0.5 N.A. neofluar objective (Zeiss, Inc., Thornwood, NY). The video output
from the confocal microscope was stored on an optical memory disk recorder (OMDR; 3038F; Panasonic). Each scan was automatically recorded by using the confocal sync to trigger the OMDR (details available at http://www.uchc.edu/~terasaki/trigger.html). To make the figure, data
from the OMDR was digitized and the images were assembled using NIH
Image (Wayne Rasband, Research Services Branch, National Institutes of
Health, Bethesda, MD) and Photoshop (Adobe Systems, Inc., Mountain
View, CA).
) or micropipets with an oil-filled constriction (Kishimoto, 1986
). These methods allow injection of precisely defined picoliter
volumes. Injected volumes were 1-5% of the egg volume (3,100 pL), except for the PLC
SH2(N+C)-wt and PLC
SH2(N+C)-mut proteins,
which were injected at 5-7% of the egg volume. In general, protein concentrations in the stock solutions were adjusted so that the volume injected was the same for control and test injections in the same series of experiments. Injections of control proteins at these volumes had no
inhibitory effect on Ca2+ release. Protein concentrations given in the text
indicate the final values in the egg cytoplasm.
Results
SH2(N+C)
; Miyazaki
and Hirai, 1979
); this is followed by a much larger Ca2+
rise, resulting from the release of Ca2+ from intracellular
stores in a wave across the egg (Stricker et al., 1994
). The
Ca action potential functions to establish a fast electrical
block to polyspermy (Jaffe, 1976
) and probably occurs simultaneously with sperm-egg fusion (McCulloh and Chambers, 1992
). In photomultiplier records of eggs injected
with Ca-green dextran, the action potential appeared as a
transient deflection preceding the main Ca2+ rise (Fig. 2
A). In confocal microscope images, it appeared as a transient ring of brightness at the egg surface preceding the Ca2+ wave (see Fig. 3). Previous studies in sea urchin eggs
have demonstrated that these Ca2+ signals are due to the
action potential (McDougall et al., 1993
; Shen and Buck,
1993
). In starfish eggs from different animals, the amplitude
and duration of the Ca2+ signal due to the action potential
were variable, with a duration of ~5-10 s (compare Figs. 2
A and 4 A). In the present study, we used the action potential as a time marker, with respect to which we measured
the timing of Ca2+ release from intracellular stores.
Fig. 2.
Delay of Ca2+ release during fertilization of starfish
eggs injected with PLCSH2(N+C) protein. Eggs were co-injected with 10 µM Ca-green dextran; photomultiplier current
indicating Ca-green fluorescence is shown as a function of time.
Each fluorescence trace was normalized to the fluorescence of
the unfertilized egg. Arrows indicate the time of sperm addition
(1:5,000 dilution of the suspension from the testis). (A) An egg
that was injected with 1,100 µg/ml of the GST control protein.
The asterisk indicates the action potential. (B and C) Eggs that
were injected with 105 (B) or 900 (C) µg/ml of the PLC
SH2
(N+C) protein. In C, the lower line shows the continuation of the
same record. (D) An egg that was injected with 1,000 µg/ml of
the SHP2-SH2(N+C) control protein.
[View Larger Version of this Image (9K GIF file)]
Fig. 3.
Ca2+ imaging during fertilization of starfish eggs injected with
PLCSH2(N+C) or GST proteins.
The control egg on the left was injected with 190 µg/ml GST and 10 µM Ca-green dextran. The egg on
the right was injected with 130 µg/ml
PLC
SH2(N+C) and 10 µM Ca-green dextran. Fluorescence intensity
(0-255) is displayed using the 32-color look-up table of NIH Image
(increasing values correspond to purple, blue, green, yellow, orange, red).
Images are shown at 3-s intervals; the
sequence is shown reading down each
column, starting at the upper left.
Sperm (1:5,000 dilution of the suspension from the testis) were added
to the recording chamber 14 s before
the first image. In the second image
in the first column, an action potential occurred in both eggs (increased fluorescence at the egg surface). In
the third image in the first column,
the Ca2+ wave is starting in the control egg. In the first image in the fourth column, a local Ca2+ rise occurs in the PLC
SH2(N+C)-injected
egg but does not propagate across the
egg. In the last image in the fourth
column, another Ca2+ rise occurs in
the PLC
SH2(N+C)-injected egg, and a Ca2+ wave propagates across the egg in the fifth column. The difference in fluorescence intensity between the egg cortex and interior is due at least in part to greater absorbance of light in the center of the egg where the specimen
is thicker; it is also possible that there is a real difference in Ca2+ activity between the cortex and interior. A Quicktime movie sequence from this experiment is available at: http://www.uchc.edu/~terasaki/data/sh2.html. Bar, 200 µm.
[View Larger Version of this Image (125K GIF file)]
Fig. 4.
Inhibition of Ca2+ release during fertilization of starfish
eggs injected with PLCSH2(N+C) protein and exposed to a low
concentration of sperm (1:50,000 dilution of the suspension from
the testis). Photomultiplier current as a function of time. (A) An
egg that was injected with 1,000 µg/ml of the SHP2-SH2(N+C)
control protein. (B) An egg that was injected with 1,000 µg/ml of
the PLC
SH2(N+C) protein. Arrows indicate the time of sperm
addition. The lower lines show the continuation of the same
record. (C and D) Images of eggs injected with 1,000 µg/ml
SHP2-SH2(N+C) and 1,000 µg/ml PLC
-SH2(N+C) and inseminated with a 1:50,000 sperm dilution; images were taken at the
time of first cleavage in uninjected eggs, using the scanning transmission mode of the BioRad confocal microscope. (C) The
SHP2-SH2(N+C)-injected egg showed a Ca-green record like
that in A, elevated a normal fertilization envelope, and cleaved
normally. Photographed at 3.0 h after insemination. (D) The
PLC
SH2(N+C)-injected egg showed a Ca-green record like that in B and did not elevate a fertilization envelope or cleave; however, the presence of two pronuclei in the cytoplasm indicated that it was fertilized. Photographed at 2.6 h after insemination. These pronuclei were similar in appearance to those described by Sluder et al. (1989)
for normal starfish fertilization,
although the time course of their development was not compared
with that in control eggs. Asterisks indicate pronuclei; "oil" indicates the oil drop introduced during microinjection. Bar, 100 µm.
[View Larger Versions of these Images (72 + 10K GIF file)]
(90-220
µg/ml, final intracellular concentration), intracellular Ca2+
release, as detected with photomultiplier records of Ca-green dextran fluorescence, did not begin until an average
of 75 s after the rise of the action potential (Fig. 2 B; Table
I), compared with 6 s in control eggs (Fig. 2 A; Table I).
The magnitude of the delay was concentration dependent;
the lowest concentration at which the delay was significantly
different from the control was ~90 µg/ml (1.8 µM; Table
I). In eggs injected with 90-220 µg/ml PLC
SH2(N+C),
the rise to the peak Ca-green fluorescence often included
several smaller amplitude increases (see Fig. 5 B), and the
peak amplitude was slightly less than in control eggs (Table I). When the concentration of the PLC
SH2(N+C)
protein was increased to 900 to 1,000 µg/ml in the egg cytoplasm, the delay between the action potential and the
initiation of Ca2+ release was increased further, and the
amplitude of the Ca2+ release was also reduced (Fig. 2 C;
Table I). The PLC
SH2(N+C) protein did not change the
Ca2+ level in the unfertilized eggs, the amplitude or duration of the action potential, or the time between sperm addition and the occurrence of the action potential. The cytoplasm of the PLC
SH2(N+C)-injected eggs appeared
normal as observed with transmitted light microscopy.
SH2(N+C) on the Delay between the
Action Potential and the First Detectable Ca2+ Release at
Fertilization
Fig. 5.
Normal Ca2+ release during fertilization of starfish eggs
injected with a point-mutated PLC SH2(N+C) protein. Photomultiplier current as a function of time. Arrows indicate the time
of sperm addition (1:5,000 dilution of the suspension from the
testis). (A) An egg that was injected with 260 µg/ml of the PLC
SH2(N+C)-mut protein. (B) An egg that was injected with 220 µg/ml of the PLC
SH2(N+C)-wt protein.
[View Larger Version of this Image (4K GIF file)]
were also without
effect at the concentrations tested (140-180 µg/ml; Table
I). Although these individual SH2 domains of PLC
can
also bind to kinases, the affinity of the interaction is lower
(Anderson et al., 1990
; Rotin et al., 1992
; Larose et al.,
1995
).
SH2(N+C) delayed
the Ca2+ release, but the delay was smaller (42 ± 9 s for 7 injections of 220 µg/ml versus 100 ± 13 s for 14 injections
of the same amount of protein made >28 min before insemination). The time required to see the full inhibitory
effect may be related to the time for the protein to spread
to the site of sperm interaction at the egg plasma membrane. This time is comparable to the times seen for some
other proteins to spread in the cytoplasm of eggs (Hamaguchi et al., 1985
; Mabuchi et al., 1985
).
SH2(N+C) protein,
oocytes were at either the prophase or first metaphase
stage. When the PLC
SH2(N+C) protein was injected before applying 1-methyladenine to cause the transition
from prophase to first metaphase, there was no effect on
the time of germinal vesicle breakdown, and the delay of
the fertilization-induced Ca2+ release was the same as in
oocytes injected at the metaphase stage.
SH2(N+C)-injected Eggs
SH2(N+C) protein (100-130 µg/ml)
also showed an increased delay between the occurrence of
the action potential, indicated by a Ca2+ rise at the egg
surface, and the initiation of Ca2+ release, seen as a wave
that spread across the egg (Fig. 3; Table II). Multiple local
Ca2+ rises occurred before the initiation of a Ca2+ wave
that crossed the entire egg. In favorable optical sections, the local Ca2+ rises could be seen to occur at sites of sperm
interaction, as indicated by the subsequent appearance of
cytoplasmic protrusions where sperm had entered the egg
("fertilization cones"). Sometimes more than one local Ca2+
rise occurred at the same time, and a wave was initiated
from multiple sites. When the wave was initiated from a single site, such that it was possible to measure the time to
propagate to the opposite pole of the egg, the propagation
time was the same as in control eggs (Fig. 3; Table II).
SH2(N+C) on Kinetics of Ca2+
Release in Eggs Imaged by Confocal Microscopy
Polyspermy in PLCSH2(N+C)-injected Eggs
Eggs injected with 90-220 µg/ml PLCSH2(N+C) underwent normal, but delayed, cortical granule exocytosis, as
indicated by the elevation of a normal fertilization envelope. In a series of experiments in which these eggs were
observed at the time of first cleavage, 24/24 eggs injected
with 100-220 µg/ml PLC
SH2(N+C) were seen to be
polyspermic (first cleavage to multiple cells), while 22/25 control eggs that were injected with GST alone or SHP2-SH2(N+C) (200-1,100 µg/ml) were monospermic (normal
early cleavage). The eggs injected with PLC
SH2(N+C)
presumably became polyspermic because the delay in
Ca2+ release delayed the elevation of the fertilization envelope. Also, although the action potential provides a fast
electrical block to polyspermy, the positive membrane potential that establishes the electrical block is probably not
sustained in the absence of Ca2+ release (Kline et al.,
1986
). Eggs injected with 900-1,000 µg/ml PLC
SH2(N+C)
showed little or no fertilization envelope elevation and
were highly polyspermic; >10 sperm pronuclei were observed in the cytoplasm of these eggs, using transmitted
light microscopy.
Complete Inhibition of Ca2+ Release in
PLCSH2(N+C)-injected Eggs Inseminated with a Low
Concentration of Sperm
The occurrence of polyspermy in the eggs injected with
the PLCSH2(N+C) protein suggested the possibility that
the residual Ca2+ release seen in these eggs might be eliminated if we further reduced the probability of PLC
activation by reducing the number of sperm interacting with
the egg. For the experiments described above, we used a
relatively high concentration of sperm (1:5,000 dilution of
the suspension obtained from the testis). With this sperm
concentration, ~100% of eggs in the recording chambers
were fertilized. When the sperm concentration was reduced 10-fold (1:50,000 dilution), ~80% of the control uninjected eggs in the recording chambers were fertilized. Of
10 eggs injected with 1,000 µg/ml PLC
SH2(N+C) and inseminated with sperm diluted 1:50,000, 9 were fertilized, as
judged by the occurrence of an action potential and the
subsequent observation of at least one sperm pronucleus in the egg cytoplasm (Fig. 4 D). However, during the 30-min recording period, 7/9 of these eggs showed no detectable Ca2+ release after the action potential (Fig. 4 B) and
no fertilization envelope elevation. 2/9 of these eggs
showed an action potential followed by a small and delayed Ca2+ release. In 8/9 control eggs injected with 1,000 µg/ml SHP2-SH2(N+C) and inseminated with a 1:50,000
dilution of sperm, the action potential was followed by
normal Ca2+ release (Fig. 4 A). These results indicate that
under low sperm concentration conditions, inhibition of
PLC
activation can completely inhibit Ca2+ release at
fertilization.
Specificity of the Inhibition of Ca2+ Release by
PLCSH2(N+C)
SH2 domains of various enzymes have specific binding sites
on their target proteins such as the PDGF receptor (Claesson-Welsh, 1994), and therefore inhibition of enzyme activation by isolated SH2 domains should show specificity for
the enzyme from which the domains were derived. However, it was critical to examine whether PLC
SH2(N+C) inhibited other cellular processes besides the activation of PLC
.
As noted above, the PLC
SH2(N+C)-injected eggs appeared normal in all respects except for the release of intracellular Ca2+, and sperm-egg fusion occurred normally
as indicated by the entry of sperm nuclei into the egg cytoplasm. Also as described above, two control proteins, GST
and a GST fusion protein including the SH2 domains of a
different protein (the SHP2 phosphatase), had no inhibitory effect on Ca2+ release at fertilization.
As a general control against the possibility that PLCSH2
(N+C) interfered with some cellular process that was unrelated to SH2-domain signaling, we injected a PLC
SH2
(N+C) fusion protein that had been point mutated at a
particular arginine (Fig. 1) that is conserved in all SH2 domain proteins (the "FLVR" sequence; Koch et al., 1991
).
For several SH2 domain proteins (Mayer et al., 1992
;
Iwashima et al., 1994
), including PLC
(Huang et al., 1995
), substitution of lysine for this arginine eliminates
binding to the target kinase. At the concentrations tested
(180-260 µg/ml), injection of the point-mutated protein
had no effect on the kinetics or amplitude of Ca2+ release
at fertilization (Fig. 5 A; Table I; Fig. 5 B shows the wild-type protein for comparison). Due to precipitation of protein in the concentrator tube when we attempted to prepare a more concentrated protein solution, we were unable
to test the mutant protein at higher concentrations. Nevertheless, our results indicated that the inhibitory effect of
PLC
SH2 was related to its specific SH2 domain properties.
If the inhibitory effect of PLC
SH2 was due to nonspecific
binding to a cytoplasmic protein, this point mutation would
not be expected to alter its biological effect (Itoh et al.,
1996
).
To rule out the possibility that PLCSH2(N+C) directly
interfered with IP3-induced Ca2+ release, we injected IP3
into eggs that had been injected with the PLC
SH2(N+C)
protein. Using an amount of IP3 (1% injection of 1 µM)
that was close to the minimum needed to cause Ca2+ release (Chiba et al., 1990
), we found that 1,000 µg/ml
PLC
SH2(N+C) did not delay or attenuate the response
(Fig. 6, A and B). The time between injection of IP3 and
the initial increase in Ca-green dextran fluorescence was
<2 s for both controls without protein injection (n = 17)
and eggs injected with PLC
SH2(N+C) (n = 11). For
both groups, the peak amplitude of the Ca2+ response was
the same (.94 ± .03, mean ± SEM, peak fluorescence/unstimulated egg fluorescence).
To obtain direct evidence that the PLCSH2(N+C) protein did not interfere with activation of PLC
, a PLC isoform
that lacks SH2 domains, we tested the effect of PLC
SH2
(N+C) on the PLC
pathway stimulated by the serotonin
2c receptor (Julius et al., 1988
; Baxter et al., 1995
). In starfish eggs expressing this exogenously introduced receptor,
application of serotonin causes a Ca2+ rise (Shilling et al.,
1990
, 1994
). When such eggs were injected with PLC
SH2
(N+C) at concentrations that delayed or completely inhibited Ca2+ release at fertilization, the kinetics and amplitude
of the Ca2+ rise in response to serotonin were unaffected
(Fig. 6, C and D; Table III). These control experiments further indicated that the PLC
SH2(N+C) protein is a specific
inhibitor of the activation of PLC
.
Table III.
No Effect of PLC |
Our main finding from these studies is that injection of
starfish eggs with recombinant SH2 domains of PLC can
completely and specifically inhibit Ca2+ release at fertilization. This confirms that IP3-mediated signaling accounts
for the initiation of Ca2+ release at fertilization, and furthermore, indicates that activation of PLC
initiates the
IP3-mediated Ca2+ release. This finding implicates a tyrosine kinase in the upstream signaling pathway at fertilization.
IP3-mediated Signaling Accounts for the Initiation of Ca2+ Release at Fertilization
In hamster eggs, complete inhibition of Ca2+ release at fertilization by an antibody against the IP3 receptor has indicated that the IP3 pathway accounts for the initiation of
Ca2+ release at fertilization (Miyazaki et al., 1992). In frog
eggs, heparin can completely block egg activation at fertilization (Nuccitelli et al., 1993
), supporting the conclusion
that IP3 initiates Ca2+ release at fertilization, although
heparin is not a highly specific inhibitor (Bezprozvanny et
al., 1993
). In sea urchin eggs, initial studies indicated that
the IP3-mediated pathway was only part of the mechanism
by which Ca2+ release is initiated at fertilization (Galione
et al., 1993
; Lee et al., 1993
), but subsequent work has
shown that inhibition of the IP3 receptor by pentosan
polysulfate can completely inhibit Ca2+ release at fertilization (Mohri et al., 1995
). However, like heparin, pentosan
polysulfate is not a specific inhibitor of the IP3 receptor
(Bezprozvanny et al., 1993
). Our results extend this previous work, since the SH2 domains of PLC
, a highly specific inhibitor of IP3 production, can completely inhibit
Ca2+ release at fertilization in starfish eggs.
Activation of PLC Initiates IP3-mediated Ca2+ Release
at Fertilization
Since inhibition of PLC activation by injection of SH2
domains can completely block Ca2+ release at fertilization,
it appears that activation of PLC
initiates the IP3-mediated Ca2+ release. The related question of whether tyrosine kinase activation is necessary for Ca2+ release at
fertilization has been examined in sea urchin eggs, using
pharmacological tyrosine kinase inhibitors. These substances do not inhibit fertilization envelope elevation (Moore
and Kinsey, 1995
), but it is not certain that activation of
PLC
was completely inhibited under the conditions of
these experiments. This study also noted that one of the
inhibitors, genistein, caused polyspermy, suggesting a delay in Ca2+ release. In mammalian eggs, studies with pharmacological inhibitors of protein tyrosine kinases and phospholipase C suggest a role for PLC
in initiating Ca2+ release
at fertilization (Dupont et al., 1996
), but other evidence that GDP-
-S injection completely inhibits Ca2+ release at
fertilization of mammalian eggs indicates that G proteins mediate this signaling (Miyazaki, 1988
; Moore et al., 1994
).
Uncertainty about the specificity of these inhibitory substances complicates the interpretation of these findings
(see also Jaffe, 1990
; Crossley et al., 1991
). Injection of
PLC
SH2(N+C) into mammalian eggs should help to resolve the relative roles of G proteins and tyrosine phosphorylation in initiating Ca2+ release at fertilization in
mammals.
Our findings also indicate that the Ca action potential
preceding the large release of intracellular Ca2+ is not dependent on SH2 domain-mediated activation of PLC.
The opening of the voltage-dependent Ca channels that
produce the action potential presumably results from a
small depolarization of the egg plasma membrane. This
depolarization could be produced by the introduction of
ion channels from the sperm membrane or by opening of
ion channels in the egg membrane (Hagiwara and Jaffe, 1979
; McCulloh and Chambers, 1992
).
Upstream Components in the Signaling Pathway at Fertilization
The involvement of SH2 domains in the activation of
PLC at fertilization indicates that the activator is a tyrosine kinase, either a receptor tyrosine kinase or a cytosolic tyrosine kinase (Rhee and Choi, 1992
). This kinase
could come from either the sperm or the egg; possibilities
include activation of a receptor kinase in the egg membrane by contact with a sperm ligand, activation of a receptor in the egg membrane leading to activation of a cytosolic kinase, introduction of a receptor kinase from the
sperm membrane into the egg membrane as a consequence of sperm-egg fusion, or introduction of a cytosolic
kinase or kinase-activating protein from the sperm into
the egg cytoplasm. At present, there is insufficent information to distinguish between these possibilities. Sperm-egg fusion appears to precede Ca2+ release in both echinoderms (McCulloh and Chambers, 1992
) and mammals
(Lawrence et al., 1997
), although some uncertainty remains (Longo et al., 1994
). However, timing alone does
not identify whether Ca2+ release is initiated by contact of
the sperm with a receptor or by sperm-egg fusion. A hamster sperm-derived protein, oscillin, causes Ca2+ release
when injected into mouse eggs (Parrington et al., 1996
), but there is no evidence how this protein could lead to IP3
production or that the amount of protein in a single sperm
is sufficient to cause Ca2+ release. A completely different
protein derived from mouse sperm, a truncated form of
c-kit, also causes activation when introduced into mouse
eggs (Sette et al., 1997
). This latter finding is particularly relevant to our findings, since c-kit is a receptor tyrosine
kinase. The truncated form of c-kit used in these experiments lacks part of the tyrosine kinase domain, but as discussed by these authors, it might still participate in activating PLC
. In particular, although activation of PLC
by
receptor tyrosine kinases is known to be caused by phosphorylation of PLC
, there is also evidence that a noncatalytic interaction with a receptor tyrosine kinase can activate PLC
in vitro (Hernandez-Sotomayor and Carpenter, 1993
). Whether a protein that activates eggs upon injection can be purified from echinoderm sperm is unknown,
although activation of sea urchin and nemertean eggs by
injection of sperm extracts has been reported (Dale et al.,
1985
; Stricker, 1997
).
Tyrosine kinase activity in sea urchin eggs increases before Ca2+ release (Ciapa and Epel, 1991; Abassi and Foltz,
1994
), and one of the proteins that is phosphorylated has a
molecular mass of ~138 kD, consistent with PLC
, although its identity is unknown (Ciapa and Epel, 1991
).
Several cytoplasmic protein tyrosine kinases that could
possibly activate PLC
have been found in sea urchin eggs
(Moore and Kinsey, 1994
; Kinsey, 1995
, 1996
), but so far none of these has been found to show increased kinase activity before Ca2+ release. It may be possible to use the
GST fusion proteins of the SH2 domains of PLC
as a
means to look for such a fertilization-activated kinase in
either sperm or egg extracts (Gish et al., 1995
; Sillman and
Monroe, 1995
), and thus to search for upstream components in the signaling pathway at fertilization.
Received for publication 13 May 1997 and in revised form 7 July 1997.
Address all correspondence to David J. Carroll or Laurinda A. Jaffe, Department of Physiology, University of Connecticut Health Center, Farmington, CT 06032. Tel.: (860) 679-2661. Fax: (860) 679-1661. E-mail: ljaffe{at}neuron.uchc.eduWe thank S. Courtneidge, S. Rayter, P. Huang, L. Davis, T. Pawson, and D. Julius for providing DNA, and R. Kado and D. Serwanski for building equipment. We are also happy to acknowledge useful discussions with W. Kinsey, L. Runft, and F. Shilling, and to thank K. Foltz for advice about making GST fusion proteins and for insightful comments on the manuscript.
This work was supported by grants from the National Institutes of Health and the National Science Foundation to L.A. Jaffe, a grant from the Patrick and Catherine Weldon Donaghue Medical Research Foundation to M. Terasaki, and a National Research Service Award from the National Institutes of Health to L.M. Mehlmann.
GST, glutathione-S-transferase; IP3, inositol trisphosphate; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate.
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