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© The Rockefeller University Press,
0021-9525/1997//1303 $5.00
The Journal of Cell Biology, Volume 138, Number 6,
, 1997 1303-1311
Article |
Calcium Release at Fertilization in Starfish Eggs Is Mediated by Phospholipase C
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 PLCβ, 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 PLCSH2 protein is a specific inhibitor of PLC in the egg, since it did not inhibit PLCβ 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.
Abbreviations used in this paper: GST, glutathione-S-transferase; IP3, inositol trisphosphate; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate.
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; 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.
IP3 is produced from phosphatidylinositol 4,5-bisphosphate (PIP2), by the action of a phospholipase C (PLC; Rhee and Choi, 1992). This family of enzymes includes β, 
, and isoforms. PLCβ 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.
Both PLCβ 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/PLCβ 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 PLCβ 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).
Several previous studies have examined whether PLCβ 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 PLCβ-mediated Ca2+ release mechanisms, we injected starfish eggs with a recombinant portion of the PLC protein that specifically inhibits PLC but not PLCβ activation.
PLC 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).
Recombinant proteins containing the two SH2 domains of PLC 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.
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GST Fusion Proteins and RNA
Plasmid DNAs encoding GST fusion proteins of bovine PLCSH2(N+C), PLCSH2(N), and PLCSH2(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.
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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). DNA for the serotonin 2c receptor, in the vector Bluescript SK–, was obtained from D. Julius (University of California, San Francisco). Synthetic RNA was made as previously described (Shilling et al., 1990).
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). 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 20x, 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).
Microinjection
Quantitative microinjection was performed using mercury-filled micropipets (Kiehart, 1982) 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 PLCSH2(N+C)-wt and PLCSH2(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.
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SH2(N+C)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.
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(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 PLCSH2(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 PLCSH2(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 PLCSH2(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 PLCSH2(N+C)-injected eggs appeared normal as observed with transmitted light microscopy.
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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).
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, PLCSH2(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).
At the time of injection of the PLCSH2(N+C) protein, oocytes were at either the prophase or first metaphase stage. When the PLCSH2(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.
Imaging of Ca2+ in PLCSH2(N+C)-injected Eggs
Imaging of the Ca2+ rise during fertilization of eggs injected with the PLCSH2(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).
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SH2(N+C)-injected EggsSH2(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 PLCSH2(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 PLCSH2(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 PLCSH2(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 PLCSH2(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 PLCSH2(N+C) inhibited other cellular processes besides the activation of PLC. As noted above, the PLCSH2(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 PLCSH2 (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 PLCSH2 was related to its specific SH2 domain properties. If the inhibitory effect of PLCSH2 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 PLCSH2(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 PLCSH2(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 PLCSH2(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).
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SH2(N+C) protein did not interfere with activation of PLCβ, a PLC isoform that lacks SH2 domains, we tested the effect of PLCSH2 (N+C) on the PLCβ 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 PLCSH2 (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 PLCSH2(N+C) protein is a specific inhibitor of the activation of PLC.
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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-β-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 PLCSH2(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.
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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.
Submitted: 13 May 1997
Revised: 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.edu
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