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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 DAG and  InsP production in COS cells coexpressing PLCγ1. Cells were transfected  with the indicated expression vectors  and labeled with either [3H]arachidonic  acid (A) or [3H]inositol (B) and processed as described under Materials  and Methods. (A) DAG content was  measured as cpm incorporated in DAG  versus 103 cpm incorporated in total lipids. (B) InsPs content was measured as  cpm incorporated into InsPs per mg  protein 24 h after transfection. The  data represent the mean ± SD of three  separate experiments, each performed  in triplicate. (C) Pellets obtained after  TCA precipitation of representative  samples analyzed for InsP production  were resuspended in SDS-PAGE sample buffer and analyzed in Western blot  (50 μg in each lane) by using either  anti-PLCγ1bd or anti-kit antibodies.
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Figure 5: Tr-kit stimulates DAG and InsP production in COS cells coexpressing PLCγ1. Cells were transfected with the indicated expression vectors and labeled with either [3H]arachidonic acid (A) or [3H]inositol (B) and processed as described under Materials and Methods. (A) DAG content was measured as cpm incorporated in DAG versus 103 cpm incorporated in total lipids. (B) InsPs content was measured as cpm incorporated into InsPs per mg protein 24 h after transfection. The data represent the mean ± SD of three separate experiments, each performed in triplicate. (C) Pellets obtained after TCA precipitation of representative samples analyzed for InsP production were resuspended in SDS-PAGE sample buffer and analyzed in Western blot (50 μg in each lane) by using either anti-PLCγ1bd or anti-kit antibodies.

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 stimulates DAG and  InsP production in COS cells coexpressing PLCγ1. Cells were transfected  with the indicated expression vectors  and labeled with either [3H]arachidonic  acid (A) or [3H]inositol (B) and processed as described under Materials  and Methods. (A) DAG content was  measured as cpm incorporated in DAG  versus 103 cpm incorporated in total lipids. (B) InsPs content was measured as  cpm incorporated into InsPs per mg  protein 24 h after transfection. The  data represent the mean ± SD of three  separate experiments, each performed  in triplicate. (C) Pellets obtained after  TCA precipitation of representative  samples analyzed for InsP production  were resuspended in SDS-PAGE sample buffer and analyzed in Western blot  (50 μg in each lane) by using either  anti-PLCγ1bd or anti-kit antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132866&req=5

Figure 5: Tr-kit stimulates DAG and InsP production in COS cells coexpressing PLCγ1. Cells were transfected with the indicated expression vectors and labeled with either [3H]arachidonic acid (A) or [3H]inositol (B) and processed as described under Materials and Methods. (A) DAG content was measured as cpm incorporated in DAG versus 103 cpm incorporated in total lipids. (B) InsPs content was measured as cpm incorporated into InsPs per mg protein 24 h after transfection. The data represent the mean ± SD of three separate experiments, each performed in triplicate. (C) Pellets obtained after TCA precipitation of representative samples analyzed for InsP production were resuspended in SDS-PAGE sample buffer and analyzed in Western blot (50 μg in each lane) by using either anti-PLCγ1bd or anti-kit antibodies.
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