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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 does not stably associate with  PLCγ1. (A) COS cells were transfected with no  DNA (mock), or 20 μg/dish pCMV5-c-kit (c-kit),  or 20 μg/dish pCMV5-tr-kit (tr-kit). C-kit–transfected cells were incubated for the final 10 min  with or without 100 ng/ml SCF. Cell extracts were  either analyzed immediately in Western blot (50  μg in each lane) with an anti-kit antibody (right  side of the panel), or incubated for 2 h with a  GST-PLCγ1-SH2SH2SH3 fusion protein linked  to glutathione–agarose beads. Proteins bound to  the beads were eluted as described under Materials and Methods and analyzed in Western blot using an anti-kit antibody (left side of the panel). (B)  Cells were transfected as described in A with tr-kit or c-kit expression vectors. Cell extracts were  immunoprecipitated using an anti-kit antibody  preadsorbed to protein A–Sepharose beads. Immunoprecipitated proteins were analyzed in  Western blot using an anti-phosphotyrosine antibody. The band recognized by the anti-phosphotyrosine antibody with a molecular size similar to  the one expected for tr-kit is present in all the  samples, regardless of tr-kit presence, indicating  that this band is due to a different tyrosine-phosphorylated protein present in the anti-kit immunoprecipitates from COS cells. (C) Cell extracts  (50 μg) from the same samples shown in B were  analyzed in Western blot using an anti-kit antibody. All panels are representative of at least  three separate experiments.
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Figure 7: Tr-kit does not stably associate with PLCγ1. (A) COS cells were transfected with no DNA (mock), or 20 μg/dish pCMV5-c-kit (c-kit), or 20 μg/dish pCMV5-tr-kit (tr-kit). C-kit–transfected cells were incubated for the final 10 min with or without 100 ng/ml SCF. Cell extracts were either analyzed immediately in Western blot (50 μg in each lane) with an anti-kit antibody (right side of the panel), or incubated for 2 h with a GST-PLCγ1-SH2SH2SH3 fusion protein linked to glutathione–agarose beads. Proteins bound to the beads were eluted as described under Materials and Methods and analyzed in Western blot using an anti-kit antibody (left side of the panel). (B) Cells were transfected as described in A with tr-kit or c-kit expression vectors. Cell extracts were immunoprecipitated using an anti-kit antibody preadsorbed to protein A–Sepharose beads. Immunoprecipitated proteins were analyzed in Western blot using an anti-phosphotyrosine antibody. The band recognized by the anti-phosphotyrosine antibody with a molecular size similar to the one expected for tr-kit is present in all the samples, regardless of tr-kit presence, indicating that this band is due to a different tyrosine-phosphorylated protein present in the anti-kit immunoprecipitates from COS cells. (C) Cell extracts (50 μg) from the same samples shown in B were analyzed in Western blot using an anti-kit antibody. All panels are representative of at least three separate experiments.

Mentions: As shown in Fig. 5 A, when cells were transfected with tr-kit alone, a slight increase in DAG production was observed, likely indicating activation of endogenous PLCs. As expected, an increase in DAG production was also observed in cells transfected with PLCγ1 versus mock-transfected cells. Coexpression of tr-kit and PLCγ1 was reproducibly accompanied by a much higher activation of DAG production, suggesting that, in cotransfection experiments, tr-kit is able to stimulate DAG production by activating PLCγ1. Cotransfection of PLCγ1 with the full-length c-kit receptor did not induce any increase in DAG production with respect to cells transfected with PLCγ1 alone. Moreover, stimulation of c-kit–transfected cells with the c-kit ligand (SCF) did not induce any increase in DAG production with respect to both unstimulated cells and cells transfected with PLCγ1 alone (Fig. 5 A), even though PLCγ1 associates with c-kit after SCF stimulation (see below, Fig. 7 A). The inability of autophosphorylated c-kit to activate PLCγ1 is in agreement with our previous observation that SCF treatment fails to activate MII-arrested oocytes (Sette et al., 1997), which express the c-kit receptor (Manova et al., 1990; Horie et al., 1991; Yoshinaga et al., 1991). As shown for the closely related PDGFβ receptor (Valius et al., 1995), the simultaneous binding to the c-kit receptor to a particular blend of other signal transduction molecules might interfere with PLCγ1 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)

Tr-kit does not stably associate with  PLCγ1. (A) COS cells were transfected with no  DNA (mock), or 20 μg/dish pCMV5-c-kit (c-kit),  or 20 μg/dish pCMV5-tr-kit (tr-kit). C-kit–transfected cells were incubated for the final 10 min  with or without 100 ng/ml SCF. Cell extracts were  either analyzed immediately in Western blot (50  μg in each lane) with an anti-kit antibody (right  side of the panel), or incubated for 2 h with a  GST-PLCγ1-SH2SH2SH3 fusion protein linked  to glutathione–agarose beads. Proteins bound to  the beads were eluted as described under Materials and Methods and analyzed in Western blot using an anti-kit antibody (left side of the panel). (B)  Cells were transfected as described in A with tr-kit or c-kit expression vectors. Cell extracts were  immunoprecipitated using an anti-kit antibody  preadsorbed to protein A–Sepharose beads. Immunoprecipitated proteins were analyzed in  Western blot using an anti-phosphotyrosine antibody. The band recognized by the anti-phosphotyrosine antibody with a molecular size similar to  the one expected for tr-kit is present in all the  samples, regardless of tr-kit presence, indicating  that this band is due to a different tyrosine-phosphorylated protein present in the anti-kit immunoprecipitates from COS cells. (C) Cell extracts  (50 μg) from the same samples shown in B were  analyzed in Western blot using an anti-kit antibody. All panels are representative of at least  three separate experiments.
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Figure 7: Tr-kit does not stably associate with PLCγ1. (A) COS cells were transfected with no DNA (mock), or 20 μg/dish pCMV5-c-kit (c-kit), or 20 μg/dish pCMV5-tr-kit (tr-kit). C-kit–transfected cells were incubated for the final 10 min with or without 100 ng/ml SCF. Cell extracts were either analyzed immediately in Western blot (50 μg in each lane) with an anti-kit antibody (right side of the panel), or incubated for 2 h with a GST-PLCγ1-SH2SH2SH3 fusion protein linked to glutathione–agarose beads. Proteins bound to the beads were eluted as described under Materials and Methods and analyzed in Western blot using an anti-kit antibody (left side of the panel). (B) Cells were transfected as described in A with tr-kit or c-kit expression vectors. Cell extracts were immunoprecipitated using an anti-kit antibody preadsorbed to protein A–Sepharose beads. Immunoprecipitated proteins were analyzed in Western blot using an anti-phosphotyrosine antibody. The band recognized by the anti-phosphotyrosine antibody with a molecular size similar to the one expected for tr-kit is present in all the samples, regardless of tr-kit presence, indicating that this band is due to a different tyrosine-phosphorylated protein present in the anti-kit immunoprecipitates from COS cells. (C) Cell extracts (50 μg) from the same samples shown in B were analyzed in Western blot using an anti-kit antibody. All panels are representative of at least three separate experiments.
Mentions: As shown in Fig. 5 A, when cells were transfected with tr-kit alone, a slight increase in DAG production was observed, likely indicating activation of endogenous PLCs. As expected, an increase in DAG production was also observed in cells transfected with PLCγ1 versus mock-transfected cells. Coexpression of tr-kit and PLCγ1 was reproducibly accompanied by a much higher activation of DAG production, suggesting that, in cotransfection experiments, tr-kit is able to stimulate DAG production by activating PLCγ1. Cotransfection of PLCγ1 with the full-length c-kit receptor did not induce any increase in DAG production with respect to cells transfected with PLCγ1 alone. Moreover, stimulation of c-kit–transfected cells with the c-kit ligand (SCF) did not induce any increase in DAG production with respect to both unstimulated cells and cells transfected with PLCγ1 alone (Fig. 5 A), even though PLCγ1 associates with c-kit after SCF stimulation (see below, Fig. 7 A). The inability of autophosphorylated c-kit to activate PLCγ1 is in agreement with our previous observation that SCF treatment fails to activate MII-arrested oocytes (Sette et al., 1997), which express the c-kit receptor (Manova et al., 1990; Horie et al., 1991; Yoshinaga et al., 1991). As shown for the closely related PDGFβ receptor (Valius et al., 1995), the simultaneous binding to the c-kit receptor to a particular blend of other signal transduction molecules might interfere with PLCγ1 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
Related in: MedlinePlus