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Involvement of phospholipase Cgamma1 in mouse egg activation induced by a truncated form of the C-kit tyrosine kinase present in spermatozoa.

Sette C, Bevilacqua A, Geremia R, Rossi P - J. Cell Biol. (1998)

Bottom Line: A GST fusion protein containing the SH3 domain of PLCgamma1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective.A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit-induced egg activation, showing that the effect of the SH3 domain of PLCgamma1 is specific.These data indicate that tr-kit activates PLCgamma1, and that the SH3 domain of PLCgamma1 is essential for tr-kit-induced egg activation.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Sanitá Pubblica e Biologia Cellulare, Sezione di Anatomia, Universitá di Roma Tor Vergata, Rome, Italy.

ABSTRACT
Microinjection of a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa (tr-kit) activates mouse eggs parthenogenetically, and tr-kit- induced egg activation is inhibited by preincubation with an inhibitor of phospholipase C (PLC) (Sette, C., A. Bevilacqua, A. Bianchini, F. Mangia, R. Geremia, and P. Rossi. 1997. Development [Camb.]. 124:2267-2274). Co-injection of glutathione-S-transferase (GST) fusion proteins containing the src-homology (SH) domains of the gamma1 isoform of PLC (PLCgamma1) competitively inhibits tr-kit- induced egg activation. A GST fusion protein containing the SH3 domain of PLCgamma1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective. A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit-induced egg activation, showing that the effect of the SH3 domain of PLCgamma1 is specific. Tr-kit-induced egg activation is also suppressed by co-injection of antibodies raised against the PLCgamma1 SH domains, but not against the PLCgamma1 COOH-terminal region. In transfected COS cells, coexpression of PLCgamma1 and tr-kit increases diacylglycerol and inositol phosphate production, and the phosphotyrosine content of PLCgamma1 with respect to cells expressing PLCgamma1 alone. These data indicate that tr-kit activates PLCgamma1, and that the SH3 domain of PLCgamma1 is essential for tr-kit-induced egg activation.

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The SH3 domain, and not the SH2 domains, of PLCγ1 inhibits tr-kit–induced cortical  granules exocytosis and pronuclear formation in  mouse eggs. Eggs were co-injected with cell extracts (200 μg/ml) containing recombinant tr-kit  and 500 μg/ml (final concentration in the egg:  ∼10 μg/ml) of either GST-PLCγ1-SH3 (A) or  GST-PLCγ1-SH2SH2 (B) and fixed 4 h after injection. Tr-kit–induced cortical granule reaction  was inhibited by co-injection of GST-PLCγ1-SH3, but not by GST-PLCγ1-SH2SH2, with a  similar rate as for pronuclear formation (see Table I) in three separate experiments. Double  staining of chromatin (Hoechst dye) and cortical  granules (TRITC-labeled lectin) was performed  as described under Materials and Methods. Bar,  30 μm.
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Figure 4: The SH3 domain, and not the SH2 domains, of PLCγ1 inhibits tr-kit–induced cortical granules exocytosis and pronuclear formation in mouse eggs. Eggs were co-injected with cell extracts (200 μg/ml) containing recombinant tr-kit and 500 μg/ml (final concentration in the egg: ∼10 μg/ml) of either GST-PLCγ1-SH3 (A) or GST-PLCγ1-SH2SH2 (B) and fixed 4 h after injection. Tr-kit–induced cortical granule reaction was inhibited by co-injection of GST-PLCγ1-SH3, but not by GST-PLCγ1-SH2SH2, with a similar rate as for pronuclear formation (see Table I) in three separate experiments. Double staining of chromatin (Hoechst dye) and cortical granules (TRITC-labeled lectin) was performed as described under Materials and Methods. Bar, 30 μm.

Mentions: To further investigate which SH domain of PLCγ1 is involved in tr-kit–induced egg activation, we co-injected tr-kit with GST fusion proteins containing either the tandem SH2 domains (GST-PLCγ1-SH2SH2) or the SH3 domain (GST-PLCγ1-SH3) of the protein (Fig. 3). Co-injection of GST-PLCγ1-SH2SH2 only slightly reduced tr-kit–induced egg activation (53% versus 70%; Table I). However, co- injection of GST-PLCγ1-SH3 inhibited egg activation as efficiently as the entire SH2-SH2-SH3 region, reducing the activation rate to 14% (Table I). Since we have previously reported that tr-kit–induced egg activation is also associated with early events of egg activation, such as the Ca2+-dependent cortical granules (CGs) exocytosis (Sette et al., 1997), the effect of the GST-PLCγ1 SH2 and SH3 fusion proteins on CGs release was investigated. Co-injection of GST-PLCγ1-SH3 inhibited both cortical granule release (Fig. 4 A) and polar body extrusion (not shown) with a rate similar to that observed for pronuclear formation (Fig. 4 A; see Table I for rate of inhibition) while the GST-PLCγ1-SH2SH2 protein was much less effective (Fig. 4 B; see Table I for rate of inhibition). Co-injection of 10-fold diluted GST-PLCγ1-SH3, at a final concentration in the egg of ∼1 μg/ml, resulted in an almost equally efficient inhibition of tr-kit– induced pronuclear formation (Table I). To test for the specificity of the SH3 domain of PLCγ1, we co-injected tr-kit together with a GST fusion protein containing the SH3 domain of the adaptor protein Grb2. Although the SH3 domains of Grb2 and PLCγ1 can bind to common targets, such as dynamin (Gout et al., 1993), they have been reported to direct the corresponding GST fusion proteins to different cell compartments when microinjected in NIH3T3 fibroblasts (Bar-Sagi et al., 1993), implying that the Grb2 and PLCγ1 SH3 domains can also recognize different targets. As shown in Table I, co-injection of GST-Grb2-SH3 was not able to affect tr-kit–induced egg activation. These data indicate that competition for targets of the SH3 domain specific to PLCγ1 impairs tr-kit–induced egg activation.


Involvement of phospholipase Cgamma1 in mouse egg activation induced by a truncated form of the C-kit tyrosine kinase present in spermatozoa.

Sette C, Bevilacqua A, Geremia R, Rossi P - J. Cell Biol. (1998)

The SH3 domain, and not the SH2 domains, of PLCγ1 inhibits tr-kit–induced cortical  granules exocytosis and pronuclear formation in  mouse eggs. Eggs were co-injected with cell extracts (200 μg/ml) containing recombinant tr-kit  and 500 μg/ml (final concentration in the egg:  ∼10 μg/ml) of either GST-PLCγ1-SH3 (A) or  GST-PLCγ1-SH2SH2 (B) and fixed 4 h after injection. Tr-kit–induced cortical granule reaction  was inhibited by co-injection of GST-PLCγ1-SH3, but not by GST-PLCγ1-SH2SH2, with a  similar rate as for pronuclear formation (see Table I) in three separate experiments. Double  staining of chromatin (Hoechst dye) and cortical  granules (TRITC-labeled lectin) was performed  as described under Materials and Methods. Bar,  30 μm.
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Figure 4: The SH3 domain, and not the SH2 domains, of PLCγ1 inhibits tr-kit–induced cortical granules exocytosis and pronuclear formation in mouse eggs. Eggs were co-injected with cell extracts (200 μg/ml) containing recombinant tr-kit and 500 μg/ml (final concentration in the egg: ∼10 μg/ml) of either GST-PLCγ1-SH3 (A) or GST-PLCγ1-SH2SH2 (B) and fixed 4 h after injection. Tr-kit–induced cortical granule reaction was inhibited by co-injection of GST-PLCγ1-SH3, but not by GST-PLCγ1-SH2SH2, with a similar rate as for pronuclear formation (see Table I) in three separate experiments. Double staining of chromatin (Hoechst dye) and cortical granules (TRITC-labeled lectin) was performed as described under Materials and Methods. Bar, 30 μm.
Mentions: To further investigate which SH domain of PLCγ1 is involved in tr-kit–induced egg activation, we co-injected tr-kit with GST fusion proteins containing either the tandem SH2 domains (GST-PLCγ1-SH2SH2) or the SH3 domain (GST-PLCγ1-SH3) of the protein (Fig. 3). Co-injection of GST-PLCγ1-SH2SH2 only slightly reduced tr-kit–induced egg activation (53% versus 70%; Table I). However, co- injection of GST-PLCγ1-SH3 inhibited egg activation as efficiently as the entire SH2-SH2-SH3 region, reducing the activation rate to 14% (Table I). Since we have previously reported that tr-kit–induced egg activation is also associated with early events of egg activation, such as the Ca2+-dependent cortical granules (CGs) exocytosis (Sette et al., 1997), the effect of the GST-PLCγ1 SH2 and SH3 fusion proteins on CGs release was investigated. Co-injection of GST-PLCγ1-SH3 inhibited both cortical granule release (Fig. 4 A) and polar body extrusion (not shown) with a rate similar to that observed for pronuclear formation (Fig. 4 A; see Table I for rate of inhibition) while the GST-PLCγ1-SH2SH2 protein was much less effective (Fig. 4 B; see Table I for rate of inhibition). Co-injection of 10-fold diluted GST-PLCγ1-SH3, at a final concentration in the egg of ∼1 μg/ml, resulted in an almost equally efficient inhibition of tr-kit– induced pronuclear formation (Table I). To test for the specificity of the SH3 domain of PLCγ1, we co-injected tr-kit together with a GST fusion protein containing the SH3 domain of the adaptor protein Grb2. Although the SH3 domains of Grb2 and PLCγ1 can bind to common targets, such as dynamin (Gout et al., 1993), they have been reported to direct the corresponding GST fusion proteins to different cell compartments when microinjected in NIH3T3 fibroblasts (Bar-Sagi et al., 1993), implying that the Grb2 and PLCγ1 SH3 domains can also recognize different targets. As shown in Table I, co-injection of GST-Grb2-SH3 was not able to affect tr-kit–induced egg activation. These data indicate that competition for targets of the SH3 domain specific to PLCγ1 impairs tr-kit–induced egg activation.

Bottom Line: A GST fusion protein containing the SH3 domain of PLCgamma1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective.A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit-induced egg activation, showing that the effect of the SH3 domain of PLCgamma1 is specific.These data indicate that tr-kit activates PLCgamma1, and that the SH3 domain of PLCgamma1 is essential for tr-kit-induced egg activation.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Sanitá Pubblica e Biologia Cellulare, Sezione di Anatomia, Universitá di Roma Tor Vergata, Rome, Italy.

ABSTRACT
Microinjection of a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa (tr-kit) activates mouse eggs parthenogenetically, and tr-kit- induced egg activation is inhibited by preincubation with an inhibitor of phospholipase C (PLC) (Sette, C., A. Bevilacqua, A. Bianchini, F. Mangia, R. Geremia, and P. Rossi. 1997. Development [Camb.]. 124:2267-2274). Co-injection of glutathione-S-transferase (GST) fusion proteins containing the src-homology (SH) domains of the gamma1 isoform of PLC (PLCgamma1) competitively inhibits tr-kit- induced egg activation. A GST fusion protein containing the SH3 domain of PLCgamma1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective. A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit-induced egg activation, showing that the effect of the SH3 domain of PLCgamma1 is specific. Tr-kit-induced egg activation is also suppressed by co-injection of antibodies raised against the PLCgamma1 SH domains, but not against the PLCgamma1 COOH-terminal region. In transfected COS cells, coexpression of PLCgamma1 and tr-kit increases diacylglycerol and inositol phosphate production, and the phosphotyrosine content of PLCgamma1 with respect to cells expressing PLCgamma1 alone. These data indicate that tr-kit activates PLCgamma1, and that the SH3 domain of PLCgamma1 is essential for tr-kit-induced egg activation.

Show MeSH