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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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Tr-kit stimulates tyrosine phosphorylation of PLCγ1 in  transfected COS cells. Cells were transfected with a PLCγ1 expression vector, either alone or together with the tr-kit expression vector. (A) Cell extracts were immunoprecipitated using a  mixture of anti-PLCγ1 antibodies (see Materials and Methods).  Immunoprecipitated proteins were analyzed in Western blot using either the anti-PLCγ1bd antibody (αPLCγ1, left panel) or an  anti-phosphotyrosine antibody (αPY, right panel). These images  are representative of six separate experiments, which gave similar  results. (B) Western blot analysis with anti-PLCγ1 antibody  (αPLCγ1) and anti-phosphotyrosine antibody (αPY) of total cell  extracts (50 μg in each lane) from PLCγ1- and PLCγ1/tr-kit– transfected COS cells.
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Figure 6: Tr-kit stimulates tyrosine phosphorylation of PLCγ1 in transfected COS cells. Cells were transfected with a PLCγ1 expression vector, either alone or together with the tr-kit expression vector. (A) Cell extracts were immunoprecipitated using a mixture of anti-PLCγ1 antibodies (see Materials and Methods). Immunoprecipitated proteins were analyzed in Western blot using either the anti-PLCγ1bd antibody (αPLCγ1, left panel) or an anti-phosphotyrosine antibody (αPY, right panel). These images are representative of six separate experiments, which gave similar results. (B) Western blot analysis with anti-PLCγ1 antibody (αPLCγ1) and anti-phosphotyrosine antibody (αPY) of total cell extracts (50 μg in each lane) from PLCγ1- and PLCγ1/tr-kit– transfected COS cells.

Mentions: Since tyrosine phosphorylation is often associated with activation of PLCγ1 (Rhee and Bae, 1997), we tested whether an increase in phosphotyrosine content of PLCγ1 is detectable in tr-kit–expressing cells. In cells transfected with a PLCγ1 expression vector alone, PLCγ1 was found to be already tyrosine-phosphorylated, but a significant increase in its phosphotyrosine content was observed in tr-kit/PLCγ1 cotransfected cells (Fig. 6 A, right panel). We routinely observed an increase in immunoprecipitated PLCγ1 from PLCγ1/tr-kit cotransfected cells (Fig. 6 A, left panel), but this does not reflect higher PLCγ1 expression in these cells as shown by the Western-blot analysis of total cell extracts (Fig. 6 B, left panel; see also Fig. 5 C). Densitometric analysis indicated that the tyrosine phosphorylation of PLCγ1 (normalized for PLCγ1 content of the immunoprecipitates) was approximately threefold higher in tr-kit cotransfected cells, with respect to cells transfected with PLCγ1 alone (not shown). This effect was selective since tr-kit expression did not induce a general increase in the tyrosine phosphorylation pattern of total cell extracts (Fig. 6 B, right panel).


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)

Tr-kit stimulates tyrosine phosphorylation of PLCγ1 in  transfected COS cells. Cells were transfected with a PLCγ1 expression vector, either alone or together with the tr-kit expression vector. (A) Cell extracts were immunoprecipitated using a  mixture of anti-PLCγ1 antibodies (see Materials and Methods).  Immunoprecipitated proteins were analyzed in Western blot using either the anti-PLCγ1bd antibody (αPLCγ1, left panel) or an  anti-phosphotyrosine antibody (αPY, right panel). These images  are representative of six separate experiments, which gave similar  results. (B) Western blot analysis with anti-PLCγ1 antibody  (αPLCγ1) and anti-phosphotyrosine antibody (αPY) of total cell  extracts (50 μg in each lane) from PLCγ1- and PLCγ1/tr-kit– transfected COS cells.
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Figure 6: Tr-kit stimulates tyrosine phosphorylation of PLCγ1 in transfected COS cells. Cells were transfected with a PLCγ1 expression vector, either alone or together with the tr-kit expression vector. (A) Cell extracts were immunoprecipitated using a mixture of anti-PLCγ1 antibodies (see Materials and Methods). Immunoprecipitated proteins were analyzed in Western blot using either the anti-PLCγ1bd antibody (αPLCγ1, left panel) or an anti-phosphotyrosine antibody (αPY, right panel). These images are representative of six separate experiments, which gave similar results. (B) Western blot analysis with anti-PLCγ1 antibody (αPLCγ1) and anti-phosphotyrosine antibody (αPY) of total cell extracts (50 μg in each lane) from PLCγ1- and PLCγ1/tr-kit– transfected COS cells.
Mentions: Since tyrosine phosphorylation is often associated with activation of PLCγ1 (Rhee and Bae, 1997), we tested whether an increase in phosphotyrosine content of PLCγ1 is detectable in tr-kit–expressing cells. In cells transfected with a PLCγ1 expression vector alone, PLCγ1 was found to be already tyrosine-phosphorylated, but a significant increase in its phosphotyrosine content was observed in tr-kit/PLCγ1 cotransfected cells (Fig. 6 A, right panel). We routinely observed an increase in immunoprecipitated PLCγ1 from PLCγ1/tr-kit cotransfected cells (Fig. 6 A, left panel), but this does not reflect higher PLCγ1 expression in these cells as shown by the Western-blot analysis of total cell extracts (Fig. 6 B, left panel; see also Fig. 5 C). Densitometric analysis indicated that the tyrosine phosphorylation of PLCγ1 (normalized for PLCγ1 content of the immunoprecipitates) was approximately threefold higher in tr-kit cotransfected cells, with respect to cells transfected with PLCγ1 alone (not shown). This effect was selective since tr-kit expression did not induce a general increase in the tyrosine phosphorylation pattern of total cell extracts (Fig. 6 B, right panel).

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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