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Src kinase induces calcium release in Xenopus egg extracts via PLCγ and IP3-dependent mechanism
Research
Cell Calcium (2002) 32(1), 11–20 0143-4160/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0143-4160(02)00078-7, available online at http://www.idealibrary.com on
Src kinase induces calcium release in Xenopus egg extracts via PLC␥ and IP3-dependent mechanism A. A. Tokmakov, 1 K. -I. Sato, 2,3 T. Iwasaki, 2 Y. Fukami 3 1
Genomic Sciences Center, RIKEN Yokohama Institute, Yokohama, Japan Research Center for Environmental Genomics, Kobe University, Kobe, Japan 3 Department of Biology, Faculty of Science and Graduate School of Science and Technology, Kobe University, Kobe, Japan 2
Summary Mobilization of intracellular calcium is an indispensable step of fertilization-induced egg activation. Recently, this process has been shown to require the sequential activation of Src family tyrosine kinases, phospholipase C␥ (PLC␥), and inositol-1,4,5-trisphosphate (IP3 )-dependent receptor of endoplasmic reticulum. In the present study, we made an attempt to recapitulate the early events of egg activation by stimulating Src kinase activity in the cell-free extracts of Xenopus eggs. We found that enhanced Src kinase activity can initiate calcium response of low magnitude in cytostatic factor (CSF)-arrested mitotic extracts without releasing them into interphase. The addition of catalytically active recombinant Src kinase, as well as the activation of endogenous Xenopus Src family kinase by hydrogen peroxide (H2 O2 ), increased total tyrosine phosphorylation, tyrosine phosphorylation of PLC␥, and IP3 production in the extracts. The treatment with the Src family kinase-specific inhibitor, PP1, or PLC inhibitor, U73122, or IP3 receptor antagonist, heparin, prevented calcium release in the extracts. We conclude, therefore, that possible mechanism of Src/H2 O2 action in the extracts might involve tyrosine phosphorylation and activation of PLC␥, accompanied by the increase in IP3 content and subsequent calcium release from IP3 -regulated calcium stores. These results also suggest that monitoring calcium signals induced in the Xenopus egg extracts by various components of signaling pathways may provide a particularly useful approach to investigating their role in the signal transduction. © 2002 Elsevier Science Ltd. All rights reserved. INTRODUCTION The mobilization of intracellular calcium that originates from the sperm entry point and spreads throughout the entire egg cytoplasm is an early universal event of fertilization-induced egg activation [1–3]. The basal level of free calcium in the nonstimulated eggs and extracts falls in the range of 200–500 nM [4–6]. Fertilization or the injection of inositol-1,4,5-trisphosphate (IP3 ) into eggs, as well as the addition of IP3 to the egg extracts, sharply increases calcium concentration to micromolar range [7–10], and releases eggs and extracts from cytostatic factor (CSF)-mediated metaphase arrest into interphase. Received 6 December 2001 Revised 21 February 2002 Accepted 17 April 2002 Correspondence to: Alexander A. Tokmakov, PhD, Genomic Sciences Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan. Tel.: +81-45-503-9204; fax: +81-45-503-9201; e-mail: [email protected]
Although the initial triggering signal of fertilization inside the egg is not known, stimulation of tyrosine phosphorylation, and specifically, activation of Src family kinases, seems to be one of the earliest events of egg activation that plays a crucial role in mediating the calcium transient. Fertilization stimulates tyrosine phosphorylation of numerous egg proteins suggesting that multiple tyrosine kinases might be activated upon fertilization. The overexpression or artificial stimulation of tyrosine kinase activity can bring about egg activation. Thus, PDGF has been demonstrated to activate starfish eggs overexpressing a chimera receptor comprising extracellular portion of PDGF receptor and intracellular portion of FGF receptor [11]. Also, the ectopic expression of EGF receptor in Xenopus oocytes makes possible their parthenogenetic activation by EGF [12]. The treatment of sea urchin eggs with aminoguanide stimulates tyrosine phosphorylation and induces calcium release, that can be blocked by the tyrosine kinase inhibitors [13]. 11
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Activation of Src family kinases can be detected within 1 min following fertilization and specific inhibitor of Src family kinases, PP1, can block the fertilization-induced egg activation [14–16]. The importance of Src activation for the initiation of intracellular signaling has also been confirmed in the experiments with the Src-mediated parthenogenetic egg activation. The injection of catalytically active c-Src kinase into starfish eggs is able to induce calcium release [17]. On the other hand, recombinant Src SH2 domain which blocks the function of Src family kinases by inhibiting their interactions with other proteins has been demonstrated to suppress the fertilization-induced calcium release [16,18]. Most recently, H2 O2 has been shown to induce Src family tyrosine kinase-dependent activation of Xenopus egg [19]. It was accompanied by the enhanced production of IP3 and sustained calcium release, thus mimicking the early events of fertilization-induced egg activation.
Recently, we described periodic oscillations of free calcium in the cell cycle of Xenopus egg cycling extracts and identified the source of calcium in the extracts as a particulate fraction containing egg intracellular IP3 -regulated calcium stores [10]. In the present study, we used cell-free extracts prepared from metaphase-arrested Xenopus eggs to investigate the mechanism of calcium release in the extracts. These extracts retain high activity of CSF and can be released into interphase upon the addition of exogenous calcium [20]. Here, we show that calcium signal can be initiated in the extracts upon the activation of Src family kinases. Calcium response in the extracts was prevented by the treatment with the Src family kinase-specific inhibitor PP1, or PLC inhibitor U73122, or IP3 receptor antagonist heparin. The addition of catalytically active recombinant Src kinase, as well as the activation of endogenous Xenopus Src family kinase by H2 O2 , increased total tyrosine phosphorylation, tyrosine phosphorylation
Fig. 1 Effect of Src kinase on tyrosine phosphorylation and IP3 production in the Xenopus egg CSF-arrested extracts. Time course of total tyrosine phosphorylation in the extracts after the addition of active Src kinase and its quantification are presented in panels (A) and (B), respectively. Time course of tyrosine phosphorylation of PLC␥ is presented in panels (C) and (D). IP3 level in the extracts (E) was determined in duplicates using radiolabeled IP3 assay kit after sample extraction with TCA and diethyl ether. Data points in panels (B) and (D) represent the mean ± S.D. of four experiments. Cell Calcium (2002) 32(1), 11–20
© 2002 Elsevier Science Ltd. All rights reserved.
Src kinase-induced calcium release
of phospholipase C␥ (PLC␥), and IP3 production in the extracts, thus, reconstituting the early events of fertilization-induced egg activation. However, the magnitude of this calcium signal was not sufficient to overcome CSF-mediated metaphase arrest.
MATERIALS AND METHODS Egg extract preparation CSF-arrested extracts of Xenopus eggs were prepared from the unfertilized eggs in the presence of EGTA, essentially as described earlier [20]. Frogs were primed with pregnant mare serum, gonadotropin (50 U per animal; Biogenesis) before inducing ovulation by the injection of human chorionic gonadotropin (500 U per animal; Teikokuzoki, Japan). Eggs were squeezed from the frogs, dejellied with 2% cysteine (Sigma), and washed extensively with the extract buffer, containing 100 mM KCl, 0.1 mM CaCl2 , 1 mM MgCl2 , 5 mM EGTA, 50 mM sucrose, and 10 mM potassium HEPES (pH 7.7). Then, the eggs were trans-
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ferred to the centrifuge tubes containing extract buffer plus 100 g/ml cytochalasin B (Sigma) and 10 g/ml each of leupeptin, pepstatin, and chymostatin. Tubes were centrifuged for 30 s at 1000 rpm, then for 30 s at 1500 rpm at 4 ◦ C. All buffer was removed from the top of the tubes and eggs were crushed by the centrifugation for 15 min at 12,000 rpm. The cytoplasmic layer was collected and subjected to the second clarifying centrifugation under the same conditions. Cytochalasin B, protease inhibitors, and energy mix (1/20 volume of 150 mM creatine phosphate, 20 mM ATP, and 20 mM MgCl2 ) were added to the extracts, that were kept on ice until use. All experiments with the extracts were performed at 23 ◦ C within 3 h after preparation. Measurement of calcium For the calcium measurements, ratiometric calcium indicator, Fura-2 (Molecular Probes), was added to the Xenopus egg extracts at a final concentration of 2 M. The basal level of fluorescent signal was monitored over several
Fig. 2 Src-induced calcium response in Xenopus egg extracts and its inhibition by PP1, U73122, and heparin. The experiment was repeated six times using different extracts with the consistent detection of calcium release. The results of typical experiment are shown. (A) Calcium response in the CSF-arrested extracts after the addition of indicated compounds (0.2 U/l Src, 10 M PP1, 10 M U73122, and 1 mg/ml heparin) was monitored continuously in the presence of 2 M Fura-2 by ratio-imaging microscopy using CCD imaging ARGUS/HISCA system from Hamamtsu Photonics (Japan). The morphology of demembranated sperm nuclei incubated for 40 min in CSF-arrested extracts after the addition of active (0.2 U/l), or inactive Src kinase (control), or calcium (0.5 mM) is shown in panel (B). © 2002 Elsevier Science Ltd. All rights reserved.
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minutes, then the effectors were administered. The final concentration of recombinant Src kinase (p60c-src ; Upstate Biotechnology) employed to induce calcium response in the extracts was 0.2 U/l and the final concentration of H2 O2 (Santoku Chemical Industries, Japan) was 10 mM. As a control (Fig. 2), the enzyme, thermoinactivated at 95 ◦ C for 3 min, was used. Ratio-imaging fluorescent microscopy was performed as described earlier [10]. Protein kinase assays Total tyrosine kinase activity of the H2 O2 -treated extracts (Fig. 3C and D) was measured with poly (Glu, Tyr) (4:1; Sigma), as a protein substrate. The samples of extracts
were fivefold diluted with a kinase dilution buffer (80 mM -glycerophosphate (pH 7.5), 20 mM EGTA, 15 mM MgCl2 , 1 mM DTT, 0.1 mM NaF, 1 mM Na3 VO4 , 0.2 mM APMSF, 10 g/ml leupeptin, and 10 g/ml aprotonin). The activity of immunoprecipitated Xenopus Src family kinase (Fig. 3E and F) was estimated with cdc2 peptide, that was synthesized and purified as described previously [21]. Prior to the kinase assay, immunoprecipitated samples (5 l of beads) were washed with the kinase dilution buffer. The reaction mixture of protein kinase assay (20 l) contained 50 mM Tris–HCl (pH 7.5), 5 mM MgCl2 , 1 mM dithiothreitol, 0.5 mg/ml poly (Glu, Tyr) or 1 mg/ml cdc2 peptide, 2 M [␥-32 P]ATP (1 Ci), and 5 l of diluted or immunoprecipitated extracts. Samples were incubated for 10 min
Fig. 3 Effect of H2 O2 on tyrosine phosphorylation in Xenopus egg CSF-arrested extracts. Time course of tyrosine phosphorylation in the extracts after the addition of 10 mM H2 O2 was analyzed by western blotting with anti-phosphotyrosine antibody (A and B), in a protein kinase assay with poly (Glu, Tyr) as a substrate (C and D), and in a protein kinase assay with cdc2 peptide in the extracts immunoprecipitated with anti-Src family kinase antibody (E and F). The final concentration of a Src family kinase-specific inhibitor, PP1, and its inactive analog, PP3, in the kinase assay was 10 M. Data points in panels (B) and (F) represent the mean ± S.D. of four experiments. Cell Calcium (2002) 32(1), 11–20
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Src kinase-induced calcium release
at 30 ◦ C, then the reaction was terminated by the addition of concentrated SDS–PAGE sample buffer [22]. After electrophoresis, the radioactive bands of phosphorylated products were visualized and quantified by BAS2000 image analyzer (FUJI Film). Immunoprecipitation Twenty-microliter aliquots of the extracts were 10-fold diluted with the homogenization buffer (20 mM Tris–HCl (pH 7.5), 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM -mercaptoethanol, 1 mM sodium vanadate, 10 g/ml leupeptin, and 20 M APMSF), then sonicated for 2 min on ice with a TOMY UD-201 ultrasonic disrupter (Tomy Seiko, Tokyo). Samples were centrifuged for 10 min at 15,000 rpm, then supernatants were incubated for 2 h at 4 ◦ C with 10 g of anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology) or with 5 g of anti-Xenopus Src family (anti-Xyk) antibody, raised against a synthetic peptide corresponding to the residues 410–428 of chicken c-Src, as described earlier [14]. To collect immune complexes, protein A-Sepharose was added to the samples at a final concentration of 10% for 1 h. Nonspecifically bound proteins were washed with the buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS. The samples were treated with SDS–PAGE sample buffer and subjected to the immunoblotting analysis described as follows. Immunoblotting Aliquots of the extracts were 10-fold diluted with the homogenization buffer, sonicated as described earlier under “Immunoprecipitation” section, and mixed with a concentrated SDS–PAGE sample buffer. Proteins were separated by the electrophoresis on 10% polyacrylamide gels and transferred to PVDF membranes using a semi-dry blotting device (Bio-Rad). Membranes were blocked with T–TBS buffer (20 mM Tris–HCl (pH, 7.5), 150 mM NaCl, and 0.05% Tween 20) containing 3 mg/ml bovine serum albumin and incubated for 2 h with either 100-fold diluted anti-phosphotyrosine mouse monoclonal antibody PY99 (Santa Cruz) or with 200-fold diluted mixed mouse monoclonal anti-PLC␥-1 antibody (Upstate Biotechnology). After washing, the membranes were treated with anti-mouse IgG rabbit polyclonal antibody at a 500-fold dilution, then with a 1000-fold diluted alkaline phosphatase-conjugated goat polyclonal antibody against rabbit IgG. Membranes were thoroughly washed with T–TBS buffer and incubated in the developing buffer (100 mM Tris–HCl (pH 9.5), 5 mM MgCl2 , 100 mM NaCl, 50 g/ml 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt, and 150 g/ml nitro blue tetrazolium) to vizualize the immune complexes. © 2002 Elsevier Science Ltd. All rights reserved.
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Extraction and assay of IP3 Twenty-microliter aliquots of the extracts were treated on ice with 15% trichloracetic acid in a final volume of 100 l, then centrifuged for 20 min at 7000 × g, 4 ◦ C to remove the precipitated material. The resulting supernatants were extracted four times with 1 ml of water-saturated diethyl ether to remove traces of trichloracetic acid. After neutralization with 1 M NaHCO3 , the content of IP3 in the samples was measured with the use of commercially available D-myo-IP3 [3s H] assay system (TRK 1000, Amersham Pharmacia) according to the manufacturer’s protocol. Other methods Detection of nuclear morphology of demembranated sperm nuclei was carried out as described earlier [10]. Protein content in the samples was determined spectrophotometrically using a protein assay kit (Bio-Rad).
RESULTS Effect of Src kinase on tyrosine phosphorylation and IP3 production in Xenopus egg CSF-arrested extracts The addition of Src kinase to Xenopus egg CSF-arrested extracts, increased the total content of tyrosine-phosphorylated proteins, as revealed by blotting with phosphotyrosine-specific antibody (Fig. 1A and B). This increase could be detected already after 1-min treatment and reached the level of 150% by 10 min (the estimation does not include the prominent band of autophosphorylated Src detected at around 60 kDa). We found that the tyrosine phosphorylation of PLC␥ was almost twofold increased in the extracts treated with Src kinase, whereas the amount of PLC␥ remained unchanged (Fig. 1C and D). No increase in the tyrosine phosphorylation of PI3-kinase was detected (data not shown), and the phosphorylation state of MAPK was not affected by the addition of Src kinase (Fig. 1A). Considering that tyrosine phosphorylation of PLC␥ is known to activate the enzyme, next we investigated the IP3 production in the extracts treated with Src kinase and found that the content of IP3 was significantly elevated in these extracts (Fig. 1E). Thus, these results suggested that IP3 -mediated calcium release from IP3 -regulated calcium stores might be initiated under these conditions. Src kinase-induced calcium release in the extracts Calcium content in the extracts was monitored with the use of ratiometric calcium indicator Fura-2. The addition of catalytically active Src kinase to the CSF-arrested extracts of Xenopus eggs resulted in a noticeable increase of the ratio signal, which started to elevate in about 1 min after the beginning of treatment (Fig. 2A). The magnitude of Cell Calcium (2002) 32(1), 11–20
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ratio signal change comprised 0.01 ratio unit. This value corresponds to about 50 nM increase in the concentration of free calcium in the extract, as estimated with the calibration curve obtained by the addition of the known concentrations of calcium to the extracts, as described earlier [10]. Remarkably, the addition of thermoinactivated Src kinase failed to initiate calcium signaling. Also, the treatment with the specific inhibitor of Src activity, PP1, prevented calcium response, indicating that catalytic activity of Src is essential for its calcium-releasing effect. Src-induced calcium signal in the extracts was also prevented by the treatment with the inhibitor of PLC activity, U73122, or with IP3 receptor antagonist, heparin, (Fig. 2A), suggesting that PLC␥-mediated production of IP3 should occur upstream of calcium release. Calcium signal of fertilization is known to promote the transition of CSF-arrested eggs from metaphase to interphase. Also CSF-arrested egg extracts can be released into interphase by the addition of exogenous calcium [20]. Nevertheless, Src kinase-treated extracts remained in metaphase even after 40-min incubation, although the exogenous calcium induced their transition to the interphase state, as judged by the nuclear morphology of demembranated sperm nuclei (Fig. 2B). Effect of H2 O2 on tyrosine phosphorylation and IP3 production in Xenopus egg CSF-arrested extracts Recently, the treatment with H2 O2 has been shown to stimulate tyrosine phosphorylation and upregulate Src
family kinase of Xenopus egg leading to the calcium release and egg activation [19]. Therefore, to confirm the calcium-releasing effect of Src kinase, next we employed H2 O2 to alternatively activate the endogenous Xenopus Src family kinase in the extracts. First, we confirmed that H2 O2 could stimulate tyrosine phosphorylation in the CSF-arrested Xenopus egg extracts. The treatment with H2 O2 increased the total content of tyrosine-phosphorylated proteins in the extracts, as revealed by blotting with phosphotyrosine-specific antibody (Fig. 3A and B). The enhancement of tyrosine phosphorylation in the treated extracts was also detected in the protein kinase assay with poly (Glu, Tyr), a nonspecific substrate of tyrosine kinases (Fig. 3C and D). The kinase assay in the presence of specific inhibitor of Src family kinases, PP2, revealed that the increase of the specific Src family kinase activity in the extracts also takes place (Fig. 3C and D). Similar results were obtained in the kinase assay with cdc2 peptide, a specific substrate of Src family kinases, in the extracts immunoprecipitated with anti-Src family kinase-specific antibody (Fig. 3E and F). It should be noted, however, that 20-min long incubation with peroxide led to the decrease of tyrosine-phosphorylating activity of the extracts even below the level of untreated control. Furthermore, we examined whether H2 O2 can stimulate the tyrosine phosphorylation of PLC␥ and IP3 production in the extracts. We detected almost twofold increase in the level of PLC␥ phosphorylation after 5-min treatment with peroxide (Fig. 4A and B). Correspondingly,
Fig. 4 Effect of H2 O2 on tyrosine phosphorylation of PLC␥ and IP3 production in the Xenopus egg CSF-arrested extracts. Time course of tyrosine phosphorylation of PLC␥ in the extracts after the addition of 10 mM H2 O2 and its quantification are presented in panels (A) and (B). IP3 level in the extracts (C) was determined in duplicates using radiolabeled IP3 assay kit after sample extraction with TCA and diethyl ether. Data points in panel (B) represent the mean ± S.D. of four experiments. Cell Calcium (2002) 32(1), 11–20
© 2002 Elsevier Science Ltd. All rights reserved.
Src kinase-induced calcium release
Fig. 5 H2 O2 -induced calcium response in the Xenopus egg extracts and its inhibition by PP1, U73122, and heparin. The data are presented as described in Fig. 2. Final concentration of H2 O2 in the extracts was 10 mM. Representative of three experiments with different extracts is shown.
the content of IP3 was also elevated in the treated extracts (Fig. 4C). H2 O2 -induced calcium release in the extracts We found that H2 O2 could initiate calcium signal of approximately the same magnitude as that registered upon the addition of recombinant Src to the Xenopus egg CSF-arrested extracts (Fig. 5A). Similarly, H2 O2 -induced calcium release in the extracts was prevented by the addition of the specific inhibitor of Src activity, PP1, indicating that the activation of Src family kinase is a prerequisite event upstream of calcium release. Also calcium release could be abolished in the presence of the PLC inhibitor, U73122, or IP3 receptor antagonist, heparin (Fig. 5A). Like Src kinase-treated extracts, the extracts loaded with H2 O2 remained in the metaphase after 40-min incubation, although the exogenous calcium induced their transition to the interphase state (Fig. 5B).
DISCUSSION Xenopus oocytes and eggs represent an established model system for studying calcium signaling. Recently, © 2002 Elsevier Science Ltd. All rights reserved.
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fertilization-induced calcium release in the eggs has been shown to require the sequential activation of Src family tyrosine kinases, PLC␥, and IP3 -dependent receptor of endoplasmic reticulum [15,16,18,23,24]. Thus, in the present study, we tried to reconstitute the signaling events leading to the calcium release by stimulating Src kinase activity in the egg extracts. We found that both catalytically active recombinant Src kinase and H2 O2 increased total tyrosine phosphorylation, tyrosine phosphorylation of PLC␥, and IP3 production resulting in calcium release in the extracts. However, the magnitude of this response (50 nM) was rather low when compared to the magnitude of the calcium signal triggered in eggs by fertilization. Evidently, the signal induced in the extracts by the stimulation of Src kinase activity was not strong enough to overcome CSF arrest in the extracts that remained in metaphase after the release of calcium (Figs. 2 and 5). CSF extracts prepared by the described conventional method should contain the contaminant EGTA, which might buffer the level of the endogenous calcium and affect the magnitude of calcium release in the extracts. The inclusion of EGTA in the extraction buffer is crucial for obtaining CSF-arrested extracts that maintain metaphase arrest [20]. Considering the procedure of the extract preparation, the level of residual EGTA in the extracts should fall in the micromolar range. However, the earlier reports indicate that the addition of calcium chelators, such as EGTA and BAPTA, even at millimolar concentrations did not dramatically reduce the level of free calcium in Xenopus egg extracts [5,6]. We also found that EGTA can effectively deplete calcium in the extracts only when added at millimolar but not at micromolar or submillimolar concentrations (Fig. 6A). Moreover, the residual EGTA in the extracts cannot effectively suppress transient changes in calcium concentration; calcium release of micromolar magnitude can be registered in the extracts treated with 10 M IP3 , as reported earlier [10]. We conclude, therefore, that the presence of residual EGTA cannot account for the low magnitude of calcium release in the extracts. The main reason for the insufficient calcium release seems to be insufficient stimulation of IP3 production in the extracts. We found that in the control nonstimulated extracts of CSF-arrested eggs, the level of IP3 was 35 ± 12 nM (n = 6). Although the treatment with Src kinase or H2 O2 increased IP3 content in the extracts more than four times, it might not be enough to promote a full-scale calcium release. Indeed, when IP3 was added to the extracts at the concentration below 200 nM, only a limited calcium response could be registered (Fig. 6B). The sigmoid curve is indicative of a cooperativity in the response supporting the suggestion that multiple molecules of IP3 should bind to the IP3 receptor before its activation [25]. Earlier, the similar positively cooperative response (Hill coefficient = 3) with rather little calcium release Cell Calcium (2002) 32(1), 11–20
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Fig. 6 Concentration-dependent effects of EGTA and IP3 on the calcium level and calcium response in the Xenopus egg extracts. (A) The extract was prepared in the absence of EGTA in the extraction buffer. The level of free calcium was monitored simultaneously at two different fields of the extract after the addition of EGTA at various concentrations (1–10 mM). (B) The extent of calcium response (mean ± S.D. of four measurements) is expressed as the integrated area of the peakwise ratio signal induced by the addition of IP3 (100–800 nM).
below 200 nM IP3 was observed in the intracellular calcium stores of hepatocytes [26]. The IP3 receptor of Xenopus oocytes is a large tetrameric complex almost identical to the type I receptor of mammalian cells. The measured dissociation constant for IP3 receptor of Xenopus oocytes falls below 100 nM [27], suggesting that the physiological concentration of IP3 lies in the 10-nM order. Indeed, the physiological concentration of IP3 in the resting Xenopus oocytes was reported to be 40 nM, and almost 50-fold increase to the level of 1.8 M could be registered upon oocyte activation with lysophosphatidic acid [28]. On the other hand, only fivefold increase in the intracellular IP3 content from 53 to 261 fmol per cell was reported upon Xenopus egg fertilization [29]. Still, considering the ordered cellular compartmentalization of the living egg, it is possible that the local concentration of IP3 in the vicinity of IP3 receptor might considerably surpass its average intracellular level. In the extracts which lack ordered compartmentalization, much higher distributed concentration of IP3 might be necessary to release calcium from IP3 -regulated calcium stores. Another possibility is that in the intact egg fertilization might stimulate the alternative signaling pathways that cooperate to the calcium release and egg activation. One probable candidate for this is G-protein-mediated signaling. The ectopic expression of G-protein-associated receptors makes possible egg activation with the corresponding hormone [30]. Also, the activation of G-proteins with the nonhydrolisable analog of GTP, GTP␥S, results in parthenogenic activation of eggs [31–33], whereas blockCell Calcium (2002) 32(1), 11–20
ing G-proteins with GDPS inhibits fertilization-induced calcium release [34]. Although these facts strongly suggest the involvement of G-proteins in the fertilization signaling, the direct measurements of G-protein activation upon fertilization have not been performed yet. The exposure of cells to H2 O2 has been reported to increase the activity of Src family kinases [19,35,36]. The treatment of T cells with H2 O2 increased the catalytic activity of Lck kinase and induced tyrosine phosphorylation of the enzyme at the autophosphorylation site. Using mutated enzyme it has been demonstrated that tyrosine in the activation loop is required for the peroxide-induced activation of Lck [35]. In the cells, it is not clear, however, whether this activation is direct or not. As H2 O2 is shown to inhibit tyrosine phosphatases by the inactivation of their catalytic center [37], the possibility exists that the inhibition of phosphatase activity might account for the detected increase of phosphotyrosine. Recently, sulfhydryl group modification-mediated mechanism of Src activation by nitric oxide has been proposed, which suggests that intramolecular S–S bond formation destabilizes Src structure for autophosphorylation-dependent regulation [38]. The similar mechanism might also be realized upon Src activation by H2 O2 , however, more studies are necessary to clarify this point. In conclusion, our data suggest that Xenopus egg extracts may be a very useful assay system for investigating the role of various components of signaling pathways in the signal transduction based on the detection of calcium signals induced in the extracts by these compounds. © 2002 Elsevier Science Ltd. All rights reserved.
Src kinase-induced calcium release
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kinase-dependent activation of Xenopus eggs. Dev Growth Differ 2001; 43: 55–72. Murray AW. Cell cycle extracts. Methods Cell Biol 1991; 36: 581–605. Fukami Y, Sato K-I, Ikeda K, Kamisango K, Koizumi K, Matsuno T. Evidence for autoinhibitory regulation of the c-src gene product: a possible interaction between the Src homology 2 domain and autophosphorylation site. J Biol Chem 1993; 268: 1132–1140. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680–685. Dupont G, Mc Guinness OM, Johnson MH, Berrige MJ, Borgese F. Phospholipase C in mouse oocytes: characterization of beta and gamma isoforms and their possible involvement in sperm-induced Ca2+ -spiking. Biochem J 1996; 316: 583–591. Carroll DJ, Ramarao CS, Mehlmann LM, Roche S, Terasaki M, Jaffe LA. Calcium release at fertilization in starfish eggs is mediated by phospholipase C␥. J Cell Biol 1997; 138: 1303–1311. Kaftan EF, Ehrlich BE, Watras J. Inositol 1,4,5-trisphosphate (InsP3 ) and calcium interact to increase the dynamic range of InsP3 receptor-dependent calcium signaling. J Gen Physiol 1997; 110: 529–538. Marchant JS, Chang Y-T, Chung S-K, Irvine RF, Taylor CW. Rapid kinetic measurements of 45 Ca2+ mobilization reveal that Ins(2,4,5)P3 is a partial agonist at hepatic InsP3 receptors. Biochem J 1997; 321: 573–576. Parys JB, Sernett SW, DeLisle S, Snyder PM, Welsh MJ, Campbell KP. Isolation, characterization, and localization of the inositol 1,4,5-trisphosphate receptor protein in Xenopus laevis oocytes. J Biol Chem 1992; 267: 18776–18782. Luzzi V, Sims CE, Soughayer JS, Allbritton NL. The physiologic concentrations of inositol 1,4,5-trisphosphate in the oocytes of Xenopus laevis. J Biol Chem 1998; 273: 28657–28662. Stith BJ, Goalstone M, Silva S, Jaynes C. Inositol 1,4,5-trisphosphate mass changes from fertilization through first cleavage in Xenopus laevis. Mol Biol Cell 1993; 4: 435–443. Moore GD, Kopf GS, Schultz RM. Complete mouse egg activation in the absence of sperm by stimulation of an exogenous G protein-coupled receptor. Dev Biol 1993; 159: 669–678. Turner PR, Jaffe LA, Primakoff P. A cholera toxin-sensitive G-protein stimulates exocytosis in sea urchin eggs. Dev Biol 1987; 120: 577–583. Kline D, Simoncini L, Mandel G, Maue RA, Kado RT, Jaffe LA. Fertilization events induced by neurotransmitters after injection of mRNA in Xenopus egg. Science 1988; 241: 464–467. Miyazaki S. Inositol 1,4,5-trisphosphate-induced calcium release and guanine nucleotide binding protein-mediated periodic calcium rises in golden hamster eggs. J Cell Biol 1988; 106: 345–353. Moore GD, Ayabe T, Visconti PE, Schultz RM, Kopf GS. Roles of heterotrimeric and monomeric G proteins in sperm-induced activation of mouse eggs. Development 1994; 120: 3313–3323. Hardwick JS, Sefton BM. Activation of the Lck tyrosine protein-kinase by hydrogen peroxide requires the phosphorylation of Tyr-394. Proc Natl Acad Sci USA 1995; 92: 4527–4531. Cell Calcium (2002) 32(1), 11–20
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36. Yan SR, Berton G. Regulation of Src family tyrosine kinase activities in adherent human neutrophils. Evidence that reactive oxygen intermediates produced by adherent neutrophils increase the activity of the p58c-fgr and p53/56lyn tyrosine kinases. J Biol Chem 1996; 271: 23464–23471. 37. Denu JM, Tanner KM. Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide:
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evidence for a sulfenic acid intermediate and implications for redox regulation. Biochemistry 1998; 37: 5633– 5642. 38. Akhand AK, Pu MY, Senga T et al.. Nitric oxide controls Src activity through a sulfhydryl group modification-mediated Tyr-527-independent and Tyr-416-linked mechanism. J Biol Chem 1999; 274: 25821–25826.
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Cell Calcium (2002) 32(1), 11–20 0143-4160/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0143-4160(02)00078-7, available online at http://www.idealibrary.com on
Src kinase induces calcium release in Xenopus egg extracts via PLC␥ and IP3-dependent mechanism A. A. Tokmakov, 1 K. -I. Sato, 2,3 T. Iwasaki, 2 Y. Fukami 3 1
Genomic Sciences Center, RIKEN Yokohama Institute, Yokohama, Japan Research Center for Environmental Genomics, Kobe University, Kobe, Japan 3 Department of Biology, Faculty of Science and Graduate School of Science and Technology, Kobe University, Kobe, Japan 2
Summary Mobilization of intracellular calcium is an indispensable step of fertilization-induced egg activation. Recently, this process has been shown to require the sequential activation of Src family tyrosine kinases, phospholipase C␥ (PLC␥), and inositol-1,4,5-trisphosphate (IP3 )-dependent receptor of endoplasmic reticulum. In the present study, we made an attempt to recapitulate the early events of egg activation by stimulating Src kinase activity in the cell-free extracts of Xenopus eggs. We found that enhanced Src kinase activity can initiate calcium response of low magnitude in cytostatic factor (CSF)-arrested mitotic extracts without releasing them into interphase. The addition of catalytically active recombinant Src kinase, as well as the activation of endogenous Xenopus Src family kinase by hydrogen peroxide (H2 O2 ), increased total tyrosine phosphorylation, tyrosine phosphorylation of PLC␥, and IP3 production in the extracts. The treatment with the Src family kinase-specific inhibitor, PP1, or PLC inhibitor, U73122, or IP3 receptor antagonist, heparin, prevented calcium release in the extracts. We conclude, therefore, that possible mechanism of Src/H2 O2 action in the extracts might involve tyrosine phosphorylation and activation of PLC␥, accompanied by the increase in IP3 content and subsequent calcium release from IP3 -regulated calcium stores. These results also suggest that monitoring calcium signals induced in the Xenopus egg extracts by various components of signaling pathways may provide a particularly useful approach to investigating their role in the signal transduction. © 2002 Elsevier Science Ltd. All rights reserved. INTRODUCTION The mobilization of intracellular calcium that originates from the sperm entry point and spreads throughout the entire egg cytoplasm is an early universal event of fertilization-induced egg activation [1–3]. The basal level of free calcium in the nonstimulated eggs and extracts falls in the range of 200–500 nM [4–6]. Fertilization or the injection of inositol-1,4,5-trisphosphate (IP3 ) into eggs, as well as the addition of IP3 to the egg extracts, sharply increases calcium concentration to micromolar range [7–10], and releases eggs and extracts from cytostatic factor (CSF)-mediated metaphase arrest into interphase. Received 6 December 2001 Revised 21 February 2002 Accepted 17 April 2002 Correspondence to: Alexander A. Tokmakov, PhD, Genomic Sciences Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan. Tel.: +81-45-503-9204; fax: +81-45-503-9201; e-mail: [email protected]
Although the initial triggering signal of fertilization inside the egg is not known, stimulation of tyrosine phosphorylation, and specifically, activation of Src family kinases, seems to be one of the earliest events of egg activation that plays a crucial role in mediating the calcium transient. Fertilization stimulates tyrosine phosphorylation of numerous egg proteins suggesting that multiple tyrosine kinases might be activated upon fertilization. The overexpression or artificial stimulation of tyrosine kinase activity can bring about egg activation. Thus, PDGF has been demonstrated to activate starfish eggs overexpressing a chimera receptor comprising extracellular portion of PDGF receptor and intracellular portion of FGF receptor [11]. Also, the ectopic expression of EGF receptor in Xenopus oocytes makes possible their parthenogenetic activation by EGF [12]. The treatment of sea urchin eggs with aminoguanide stimulates tyrosine phosphorylation and induces calcium release, that can be blocked by the tyrosine kinase inhibitors [13]. 11
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Activation of Src family kinases can be detected within 1 min following fertilization and specific inhibitor of Src family kinases, PP1, can block the fertilization-induced egg activation [14–16]. The importance of Src activation for the initiation of intracellular signaling has also been confirmed in the experiments with the Src-mediated parthenogenetic egg activation. The injection of catalytically active c-Src kinase into starfish eggs is able to induce calcium release [17]. On the other hand, recombinant Src SH2 domain which blocks the function of Src family kinases by inhibiting their interactions with other proteins has been demonstrated to suppress the fertilization-induced calcium release [16,18]. Most recently, H2 O2 has been shown to induce Src family tyrosine kinase-dependent activation of Xenopus egg [19]. It was accompanied by the enhanced production of IP3 and sustained calcium release, thus mimicking the early events of fertilization-induced egg activation.
Recently, we described periodic oscillations of free calcium in the cell cycle of Xenopus egg cycling extracts and identified the source of calcium in the extracts as a particulate fraction containing egg intracellular IP3 -regulated calcium stores [10]. In the present study, we used cell-free extracts prepared from metaphase-arrested Xenopus eggs to investigate the mechanism of calcium release in the extracts. These extracts retain high activity of CSF and can be released into interphase upon the addition of exogenous calcium [20]. Here, we show that calcium signal can be initiated in the extracts upon the activation of Src family kinases. Calcium response in the extracts was prevented by the treatment with the Src family kinase-specific inhibitor PP1, or PLC inhibitor U73122, or IP3 receptor antagonist heparin. The addition of catalytically active recombinant Src kinase, as well as the activation of endogenous Xenopus Src family kinase by H2 O2 , increased total tyrosine phosphorylation, tyrosine phosphorylation
Fig. 1 Effect of Src kinase on tyrosine phosphorylation and IP3 production in the Xenopus egg CSF-arrested extracts. Time course of total tyrosine phosphorylation in the extracts after the addition of active Src kinase and its quantification are presented in panels (A) and (B), respectively. Time course of tyrosine phosphorylation of PLC␥ is presented in panels (C) and (D). IP3 level in the extracts (E) was determined in duplicates using radiolabeled IP3 assay kit after sample extraction with TCA and diethyl ether. Data points in panels (B) and (D) represent the mean ± S.D. of four experiments. Cell Calcium (2002) 32(1), 11–20
© 2002 Elsevier Science Ltd. All rights reserved.
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of phospholipase C␥ (PLC␥), and IP3 production in the extracts, thus, reconstituting the early events of fertilization-induced egg activation. However, the magnitude of this calcium signal was not sufficient to overcome CSF-mediated metaphase arrest.
MATERIALS AND METHODS Egg extract preparation CSF-arrested extracts of Xenopus eggs were prepared from the unfertilized eggs in the presence of EGTA, essentially as described earlier [20]. Frogs were primed with pregnant mare serum, gonadotropin (50 U per animal; Biogenesis) before inducing ovulation by the injection of human chorionic gonadotropin (500 U per animal; Teikokuzoki, Japan). Eggs were squeezed from the frogs, dejellied with 2% cysteine (Sigma), and washed extensively with the extract buffer, containing 100 mM KCl, 0.1 mM CaCl2 , 1 mM MgCl2 , 5 mM EGTA, 50 mM sucrose, and 10 mM potassium HEPES (pH 7.7). Then, the eggs were trans-
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ferred to the centrifuge tubes containing extract buffer plus 100 g/ml cytochalasin B (Sigma) and 10 g/ml each of leupeptin, pepstatin, and chymostatin. Tubes were centrifuged for 30 s at 1000 rpm, then for 30 s at 1500 rpm at 4 ◦ C. All buffer was removed from the top of the tubes and eggs were crushed by the centrifugation for 15 min at 12,000 rpm. The cytoplasmic layer was collected and subjected to the second clarifying centrifugation under the same conditions. Cytochalasin B, protease inhibitors, and energy mix (1/20 volume of 150 mM creatine phosphate, 20 mM ATP, and 20 mM MgCl2 ) were added to the extracts, that were kept on ice until use. All experiments with the extracts were performed at 23 ◦ C within 3 h after preparation. Measurement of calcium For the calcium measurements, ratiometric calcium indicator, Fura-2 (Molecular Probes), was added to the Xenopus egg extracts at a final concentration of 2 M. The basal level of fluorescent signal was monitored over several
Fig. 2 Src-induced calcium response in Xenopus egg extracts and its inhibition by PP1, U73122, and heparin. The experiment was repeated six times using different extracts with the consistent detection of calcium release. The results of typical experiment are shown. (A) Calcium response in the CSF-arrested extracts after the addition of indicated compounds (0.2 U/l Src, 10 M PP1, 10 M U73122, and 1 mg/ml heparin) was monitored continuously in the presence of 2 M Fura-2 by ratio-imaging microscopy using CCD imaging ARGUS/HISCA system from Hamamtsu Photonics (Japan). The morphology of demembranated sperm nuclei incubated for 40 min in CSF-arrested extracts after the addition of active (0.2 U/l), or inactive Src kinase (control), or calcium (0.5 mM) is shown in panel (B). © 2002 Elsevier Science Ltd. All rights reserved.
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minutes, then the effectors were administered. The final concentration of recombinant Src kinase (p60c-src ; Upstate Biotechnology) employed to induce calcium response in the extracts was 0.2 U/l and the final concentration of H2 O2 (Santoku Chemical Industries, Japan) was 10 mM. As a control (Fig. 2), the enzyme, thermoinactivated at 95 ◦ C for 3 min, was used. Ratio-imaging fluorescent microscopy was performed as described earlier [10]. Protein kinase assays Total tyrosine kinase activity of the H2 O2 -treated extracts (Fig. 3C and D) was measured with poly (Glu, Tyr) (4:1; Sigma), as a protein substrate. The samples of extracts
were fivefold diluted with a kinase dilution buffer (80 mM -glycerophosphate (pH 7.5), 20 mM EGTA, 15 mM MgCl2 , 1 mM DTT, 0.1 mM NaF, 1 mM Na3 VO4 , 0.2 mM APMSF, 10 g/ml leupeptin, and 10 g/ml aprotonin). The activity of immunoprecipitated Xenopus Src family kinase (Fig. 3E and F) was estimated with cdc2 peptide, that was synthesized and purified as described previously [21]. Prior to the kinase assay, immunoprecipitated samples (5 l of beads) were washed with the kinase dilution buffer. The reaction mixture of protein kinase assay (20 l) contained 50 mM Tris–HCl (pH 7.5), 5 mM MgCl2 , 1 mM dithiothreitol, 0.5 mg/ml poly (Glu, Tyr) or 1 mg/ml cdc2 peptide, 2 M [␥-32 P]ATP (1 Ci), and 5 l of diluted or immunoprecipitated extracts. Samples were incubated for 10 min
Fig. 3 Effect of H2 O2 on tyrosine phosphorylation in Xenopus egg CSF-arrested extracts. Time course of tyrosine phosphorylation in the extracts after the addition of 10 mM H2 O2 was analyzed by western blotting with anti-phosphotyrosine antibody (A and B), in a protein kinase assay with poly (Glu, Tyr) as a substrate (C and D), and in a protein kinase assay with cdc2 peptide in the extracts immunoprecipitated with anti-Src family kinase antibody (E and F). The final concentration of a Src family kinase-specific inhibitor, PP1, and its inactive analog, PP3, in the kinase assay was 10 M. Data points in panels (B) and (F) represent the mean ± S.D. of four experiments. Cell Calcium (2002) 32(1), 11–20
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at 30 ◦ C, then the reaction was terminated by the addition of concentrated SDS–PAGE sample buffer [22]. After electrophoresis, the radioactive bands of phosphorylated products were visualized and quantified by BAS2000 image analyzer (FUJI Film). Immunoprecipitation Twenty-microliter aliquots of the extracts were 10-fold diluted with the homogenization buffer (20 mM Tris–HCl (pH 7.5), 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM -mercaptoethanol, 1 mM sodium vanadate, 10 g/ml leupeptin, and 20 M APMSF), then sonicated for 2 min on ice with a TOMY UD-201 ultrasonic disrupter (Tomy Seiko, Tokyo). Samples were centrifuged for 10 min at 15,000 rpm, then supernatants were incubated for 2 h at 4 ◦ C with 10 g of anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology) or with 5 g of anti-Xenopus Src family (anti-Xyk) antibody, raised against a synthetic peptide corresponding to the residues 410–428 of chicken c-Src, as described earlier [14]. To collect immune complexes, protein A-Sepharose was added to the samples at a final concentration of 10% for 1 h. Nonspecifically bound proteins were washed with the buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS. The samples were treated with SDS–PAGE sample buffer and subjected to the immunoblotting analysis described as follows. Immunoblotting Aliquots of the extracts were 10-fold diluted with the homogenization buffer, sonicated as described earlier under “Immunoprecipitation” section, and mixed with a concentrated SDS–PAGE sample buffer. Proteins were separated by the electrophoresis on 10% polyacrylamide gels and transferred to PVDF membranes using a semi-dry blotting device (Bio-Rad). Membranes were blocked with T–TBS buffer (20 mM Tris–HCl (pH, 7.5), 150 mM NaCl, and 0.05% Tween 20) containing 3 mg/ml bovine serum albumin and incubated for 2 h with either 100-fold diluted anti-phosphotyrosine mouse monoclonal antibody PY99 (Santa Cruz) or with 200-fold diluted mixed mouse monoclonal anti-PLC␥-1 antibody (Upstate Biotechnology). After washing, the membranes were treated with anti-mouse IgG rabbit polyclonal antibody at a 500-fold dilution, then with a 1000-fold diluted alkaline phosphatase-conjugated goat polyclonal antibody against rabbit IgG. Membranes were thoroughly washed with T–TBS buffer and incubated in the developing buffer (100 mM Tris–HCl (pH 9.5), 5 mM MgCl2 , 100 mM NaCl, 50 g/ml 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt, and 150 g/ml nitro blue tetrazolium) to vizualize the immune complexes. © 2002 Elsevier Science Ltd. All rights reserved.
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Extraction and assay of IP3 Twenty-microliter aliquots of the extracts were treated on ice with 15% trichloracetic acid in a final volume of 100 l, then centrifuged for 20 min at 7000 × g, 4 ◦ C to remove the precipitated material. The resulting supernatants were extracted four times with 1 ml of water-saturated diethyl ether to remove traces of trichloracetic acid. After neutralization with 1 M NaHCO3 , the content of IP3 in the samples was measured with the use of commercially available D-myo-IP3 [3s H] assay system (TRK 1000, Amersham Pharmacia) according to the manufacturer’s protocol. Other methods Detection of nuclear morphology of demembranated sperm nuclei was carried out as described earlier [10]. Protein content in the samples was determined spectrophotometrically using a protein assay kit (Bio-Rad).
RESULTS Effect of Src kinase on tyrosine phosphorylation and IP3 production in Xenopus egg CSF-arrested extracts The addition of Src kinase to Xenopus egg CSF-arrested extracts, increased the total content of tyrosine-phosphorylated proteins, as revealed by blotting with phosphotyrosine-specific antibody (Fig. 1A and B). This increase could be detected already after 1-min treatment and reached the level of 150% by 10 min (the estimation does not include the prominent band of autophosphorylated Src detected at around 60 kDa). We found that the tyrosine phosphorylation of PLC␥ was almost twofold increased in the extracts treated with Src kinase, whereas the amount of PLC␥ remained unchanged (Fig. 1C and D). No increase in the tyrosine phosphorylation of PI3-kinase was detected (data not shown), and the phosphorylation state of MAPK was not affected by the addition of Src kinase (Fig. 1A). Considering that tyrosine phosphorylation of PLC␥ is known to activate the enzyme, next we investigated the IP3 production in the extracts treated with Src kinase and found that the content of IP3 was significantly elevated in these extracts (Fig. 1E). Thus, these results suggested that IP3 -mediated calcium release from IP3 -regulated calcium stores might be initiated under these conditions. Src kinase-induced calcium release in the extracts Calcium content in the extracts was monitored with the use of ratiometric calcium indicator Fura-2. The addition of catalytically active Src kinase to the CSF-arrested extracts of Xenopus eggs resulted in a noticeable increase of the ratio signal, which started to elevate in about 1 min after the beginning of treatment (Fig. 2A). The magnitude of Cell Calcium (2002) 32(1), 11–20
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ratio signal change comprised 0.01 ratio unit. This value corresponds to about 50 nM increase in the concentration of free calcium in the extract, as estimated with the calibration curve obtained by the addition of the known concentrations of calcium to the extracts, as described earlier [10]. Remarkably, the addition of thermoinactivated Src kinase failed to initiate calcium signaling. Also, the treatment with the specific inhibitor of Src activity, PP1, prevented calcium response, indicating that catalytic activity of Src is essential for its calcium-releasing effect. Src-induced calcium signal in the extracts was also prevented by the treatment with the inhibitor of PLC activity, U73122, or with IP3 receptor antagonist, heparin, (Fig. 2A), suggesting that PLC␥-mediated production of IP3 should occur upstream of calcium release. Calcium signal of fertilization is known to promote the transition of CSF-arrested eggs from metaphase to interphase. Also CSF-arrested egg extracts can be released into interphase by the addition of exogenous calcium [20]. Nevertheless, Src kinase-treated extracts remained in metaphase even after 40-min incubation, although the exogenous calcium induced their transition to the interphase state, as judged by the nuclear morphology of demembranated sperm nuclei (Fig. 2B). Effect of H2 O2 on tyrosine phosphorylation and IP3 production in Xenopus egg CSF-arrested extracts Recently, the treatment with H2 O2 has been shown to stimulate tyrosine phosphorylation and upregulate Src
family kinase of Xenopus egg leading to the calcium release and egg activation [19]. Therefore, to confirm the calcium-releasing effect of Src kinase, next we employed H2 O2 to alternatively activate the endogenous Xenopus Src family kinase in the extracts. First, we confirmed that H2 O2 could stimulate tyrosine phosphorylation in the CSF-arrested Xenopus egg extracts. The treatment with H2 O2 increased the total content of tyrosine-phosphorylated proteins in the extracts, as revealed by blotting with phosphotyrosine-specific antibody (Fig. 3A and B). The enhancement of tyrosine phosphorylation in the treated extracts was also detected in the protein kinase assay with poly (Glu, Tyr), a nonspecific substrate of tyrosine kinases (Fig. 3C and D). The kinase assay in the presence of specific inhibitor of Src family kinases, PP2, revealed that the increase of the specific Src family kinase activity in the extracts also takes place (Fig. 3C and D). Similar results were obtained in the kinase assay with cdc2 peptide, a specific substrate of Src family kinases, in the extracts immunoprecipitated with anti-Src family kinase-specific antibody (Fig. 3E and F). It should be noted, however, that 20-min long incubation with peroxide led to the decrease of tyrosine-phosphorylating activity of the extracts even below the level of untreated control. Furthermore, we examined whether H2 O2 can stimulate the tyrosine phosphorylation of PLC␥ and IP3 production in the extracts. We detected almost twofold increase in the level of PLC␥ phosphorylation after 5-min treatment with peroxide (Fig. 4A and B). Correspondingly,
Fig. 4 Effect of H2 O2 on tyrosine phosphorylation of PLC␥ and IP3 production in the Xenopus egg CSF-arrested extracts. Time course of tyrosine phosphorylation of PLC␥ in the extracts after the addition of 10 mM H2 O2 and its quantification are presented in panels (A) and (B). IP3 level in the extracts (C) was determined in duplicates using radiolabeled IP3 assay kit after sample extraction with TCA and diethyl ether. Data points in panel (B) represent the mean ± S.D. of four experiments. Cell Calcium (2002) 32(1), 11–20
© 2002 Elsevier Science Ltd. All rights reserved.
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Fig. 5 H2 O2 -induced calcium response in the Xenopus egg extracts and its inhibition by PP1, U73122, and heparin. The data are presented as described in Fig. 2. Final concentration of H2 O2 in the extracts was 10 mM. Representative of three experiments with different extracts is shown.
the content of IP3 was also elevated in the treated extracts (Fig. 4C). H2 O2 -induced calcium release in the extracts We found that H2 O2 could initiate calcium signal of approximately the same magnitude as that registered upon the addition of recombinant Src to the Xenopus egg CSF-arrested extracts (Fig. 5A). Similarly, H2 O2 -induced calcium release in the extracts was prevented by the addition of the specific inhibitor of Src activity, PP1, indicating that the activation of Src family kinase is a prerequisite event upstream of calcium release. Also calcium release could be abolished in the presence of the PLC inhibitor, U73122, or IP3 receptor antagonist, heparin (Fig. 5A). Like Src kinase-treated extracts, the extracts loaded with H2 O2 remained in the metaphase after 40-min incubation, although the exogenous calcium induced their transition to the interphase state (Fig. 5B).
DISCUSSION Xenopus oocytes and eggs represent an established model system for studying calcium signaling. Recently, © 2002 Elsevier Science Ltd. All rights reserved.
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fertilization-induced calcium release in the eggs has been shown to require the sequential activation of Src family tyrosine kinases, PLC␥, and IP3 -dependent receptor of endoplasmic reticulum [15,16,18,23,24]. Thus, in the present study, we tried to reconstitute the signaling events leading to the calcium release by stimulating Src kinase activity in the egg extracts. We found that both catalytically active recombinant Src kinase and H2 O2 increased total tyrosine phosphorylation, tyrosine phosphorylation of PLC␥, and IP3 production resulting in calcium release in the extracts. However, the magnitude of this response (50 nM) was rather low when compared to the magnitude of the calcium signal triggered in eggs by fertilization. Evidently, the signal induced in the extracts by the stimulation of Src kinase activity was not strong enough to overcome CSF arrest in the extracts that remained in metaphase after the release of calcium (Figs. 2 and 5). CSF extracts prepared by the described conventional method should contain the contaminant EGTA, which might buffer the level of the endogenous calcium and affect the magnitude of calcium release in the extracts. The inclusion of EGTA in the extraction buffer is crucial for obtaining CSF-arrested extracts that maintain metaphase arrest [20]. Considering the procedure of the extract preparation, the level of residual EGTA in the extracts should fall in the micromolar range. However, the earlier reports indicate that the addition of calcium chelators, such as EGTA and BAPTA, even at millimolar concentrations did not dramatically reduce the level of free calcium in Xenopus egg extracts [5,6]. We also found that EGTA can effectively deplete calcium in the extracts only when added at millimolar but not at micromolar or submillimolar concentrations (Fig. 6A). Moreover, the residual EGTA in the extracts cannot effectively suppress transient changes in calcium concentration; calcium release of micromolar magnitude can be registered in the extracts treated with 10 M IP3 , as reported earlier [10]. We conclude, therefore, that the presence of residual EGTA cannot account for the low magnitude of calcium release in the extracts. The main reason for the insufficient calcium release seems to be insufficient stimulation of IP3 production in the extracts. We found that in the control nonstimulated extracts of CSF-arrested eggs, the level of IP3 was 35 ± 12 nM (n = 6). Although the treatment with Src kinase or H2 O2 increased IP3 content in the extracts more than four times, it might not be enough to promote a full-scale calcium release. Indeed, when IP3 was added to the extracts at the concentration below 200 nM, only a limited calcium response could be registered (Fig. 6B). The sigmoid curve is indicative of a cooperativity in the response supporting the suggestion that multiple molecules of IP3 should bind to the IP3 receptor before its activation [25]. Earlier, the similar positively cooperative response (Hill coefficient = 3) with rather little calcium release Cell Calcium (2002) 32(1), 11–20
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Fig. 6 Concentration-dependent effects of EGTA and IP3 on the calcium level and calcium response in the Xenopus egg extracts. (A) The extract was prepared in the absence of EGTA in the extraction buffer. The level of free calcium was monitored simultaneously at two different fields of the extract after the addition of EGTA at various concentrations (1–10 mM). (B) The extent of calcium response (mean ± S.D. of four measurements) is expressed as the integrated area of the peakwise ratio signal induced by the addition of IP3 (100–800 nM).
below 200 nM IP3 was observed in the intracellular calcium stores of hepatocytes [26]. The IP3 receptor of Xenopus oocytes is a large tetrameric complex almost identical to the type I receptor of mammalian cells. The measured dissociation constant for IP3 receptor of Xenopus oocytes falls below 100 nM [27], suggesting that the physiological concentration of IP3 lies in the 10-nM order. Indeed, the physiological concentration of IP3 in the resting Xenopus oocytes was reported to be 40 nM, and almost 50-fold increase to the level of 1.8 M could be registered upon oocyte activation with lysophosphatidic acid [28]. On the other hand, only fivefold increase in the intracellular IP3 content from 53 to 261 fmol per cell was reported upon Xenopus egg fertilization [29]. Still, considering the ordered cellular compartmentalization of the living egg, it is possible that the local concentration of IP3 in the vicinity of IP3 receptor might considerably surpass its average intracellular level. In the extracts which lack ordered compartmentalization, much higher distributed concentration of IP3 might be necessary to release calcium from IP3 -regulated calcium stores. Another possibility is that in the intact egg fertilization might stimulate the alternative signaling pathways that cooperate to the calcium release and egg activation. One probable candidate for this is G-protein-mediated signaling. The ectopic expression of G-protein-associated receptors makes possible egg activation with the corresponding hormone [30]. Also, the activation of G-proteins with the nonhydrolisable analog of GTP, GTP␥S, results in parthenogenic activation of eggs [31–33], whereas blockCell Calcium (2002) 32(1), 11–20
ing G-proteins with GDPS inhibits fertilization-induced calcium release [34]. Although these facts strongly suggest the involvement of G-proteins in the fertilization signaling, the direct measurements of G-protein activation upon fertilization have not been performed yet. The exposure of cells to H2 O2 has been reported to increase the activity of Src family kinases [19,35,36]. The treatment of T cells with H2 O2 increased the catalytic activity of Lck kinase and induced tyrosine phosphorylation of the enzyme at the autophosphorylation site. Using mutated enzyme it has been demonstrated that tyrosine in the activation loop is required for the peroxide-induced activation of Lck [35]. In the cells, it is not clear, however, whether this activation is direct or not. As H2 O2 is shown to inhibit tyrosine phosphatases by the inactivation of their catalytic center [37], the possibility exists that the inhibition of phosphatase activity might account for the detected increase of phosphotyrosine. Recently, sulfhydryl group modification-mediated mechanism of Src activation by nitric oxide has been proposed, which suggests that intramolecular S–S bond formation destabilizes Src structure for autophosphorylation-dependent regulation [38]. The similar mechanism might also be realized upon Src activation by H2 O2 , however, more studies are necessary to clarify this point. In conclusion, our data suggest that Xenopus egg extracts may be a very useful assay system for investigating the role of various components of signaling pathways in the signal transduction based on the detection of calcium signals induced in the extracts by these compounds. © 2002 Elsevier Science Ltd. All rights reserved.
Src kinase-induced calcium release
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