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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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Expression of PLCγ1 in MII-arrested  mouse eggs, and schematic representation of the  bacterial fusion proteins containing different  PLCγ1 domains. In the schematic representation  of PLCγ1 on the top of this figure, brackets identify the regions of PLCγ1 recognized by the two  antibodies used for microinjection experiments  shown in Table II. PH, pleckstrin homology domain; Ca2+, EF-hand domain (calcium binding  motif); X and Y, split catalytic domain; P and H,  split pleckstrin homology domain; SH2 and SH3,  src-homology domains; C2, Ca2+-dependent lipid  binding domain. (A) Western blot from extracts  of COS cells transfected with a PLCγ1 expression vector (50 μg), and from extracts of 50  mouse eggs, probed with the anti-PLCγ1bd antibody, described in the Materials and Methods  section, and used for the microinjection experiments shown in Table II. (B) Bacterial fusion  proteins containing different PLCγ1 domains used for the experiments shown in Table I. The Coomassie blue staining of a 10% SDS-PAGE gel loaded with affinity-purified GST fusion proteins is shown on the right: lane 1, GST; lane 2, GST-PLCγ1-SH2SH2SH3; lane  3, GST-PLCγ1-SH2SH2; lane 4, GST-PLCγ1-SH3.
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Figure 3: Expression of PLCγ1 in MII-arrested mouse eggs, and schematic representation of the bacterial fusion proteins containing different PLCγ1 domains. In the schematic representation of PLCγ1 on the top of this figure, brackets identify the regions of PLCγ1 recognized by the two antibodies used for microinjection experiments shown in Table II. PH, pleckstrin homology domain; Ca2+, EF-hand domain (calcium binding motif); X and Y, split catalytic domain; P and H, split pleckstrin homology domain; SH2 and SH3, src-homology domains; C2, Ca2+-dependent lipid binding domain. (A) Western blot from extracts of COS cells transfected with a PLCγ1 expression vector (50 μg), and from extracts of 50 mouse eggs, probed with the anti-PLCγ1bd antibody, described in the Materials and Methods section, and used for the microinjection experiments shown in Table II. (B) Bacterial fusion proteins containing different PLCγ1 domains used for the experiments shown in Table I. The Coomassie blue staining of a 10% SDS-PAGE gel loaded with affinity-purified GST fusion proteins is shown on the right: lane 1, GST; lane 2, GST-PLCγ1-SH2SH2SH3; lane 3, GST-PLCγ1-SH2SH2; lane 4, GST-PLCγ1-SH3.

Mentions: The glutathione-S-transferase (GST)-encoding plasmid pGEX3X was obtained from Pharmacia Biotech, Inc. (Piscataway, NJ). Plasmid DNAs encoding for GST fusion proteins of bovine PLCγ1 SH2–SH2 and human PLCγ1 SH3 (see Fig. 3) in pGEX2T′6 were a kind gift from S. Courtneidge (Sugen, Inc., Redwood City, CA).


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)

Expression of PLCγ1 in MII-arrested  mouse eggs, and schematic representation of the  bacterial fusion proteins containing different  PLCγ1 domains. In the schematic representation  of PLCγ1 on the top of this figure, brackets identify the regions of PLCγ1 recognized by the two  antibodies used for microinjection experiments  shown in Table II. PH, pleckstrin homology domain; Ca2+, EF-hand domain (calcium binding  motif); X and Y, split catalytic domain; P and H,  split pleckstrin homology domain; SH2 and SH3,  src-homology domains; C2, Ca2+-dependent lipid  binding domain. (A) Western blot from extracts  of COS cells transfected with a PLCγ1 expression vector (50 μg), and from extracts of 50  mouse eggs, probed with the anti-PLCγ1bd antibody, described in the Materials and Methods  section, and used for the microinjection experiments shown in Table II. (B) Bacterial fusion  proteins containing different PLCγ1 domains used for the experiments shown in Table I. The Coomassie blue staining of a 10% SDS-PAGE gel loaded with affinity-purified GST fusion proteins is shown on the right: lane 1, GST; lane 2, GST-PLCγ1-SH2SH2SH3; lane  3, GST-PLCγ1-SH2SH2; lane 4, GST-PLCγ1-SH3.
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Figure 3: Expression of PLCγ1 in MII-arrested mouse eggs, and schematic representation of the bacterial fusion proteins containing different PLCγ1 domains. In the schematic representation of PLCγ1 on the top of this figure, brackets identify the regions of PLCγ1 recognized by the two antibodies used for microinjection experiments shown in Table II. PH, pleckstrin homology domain; Ca2+, EF-hand domain (calcium binding motif); X and Y, split catalytic domain; P and H, split pleckstrin homology domain; SH2 and SH3, src-homology domains; C2, Ca2+-dependent lipid binding domain. (A) Western blot from extracts of COS cells transfected with a PLCγ1 expression vector (50 μg), and from extracts of 50 mouse eggs, probed with the anti-PLCγ1bd antibody, described in the Materials and Methods section, and used for the microinjection experiments shown in Table II. (B) Bacterial fusion proteins containing different PLCγ1 domains used for the experiments shown in Table I. The Coomassie blue staining of a 10% SDS-PAGE gel loaded with affinity-purified GST fusion proteins is shown on the right: lane 1, GST; lane 2, GST-PLCγ1-SH2SH2SH3; lane 3, GST-PLCγ1-SH2SH2; lane 4, GST-PLCγ1-SH3.
Mentions: The glutathione-S-transferase (GST)-encoding plasmid pGEX3X was obtained from Pharmacia Biotech, Inc. (Piscataway, NJ). Plasmid DNAs encoding for GST fusion proteins of bovine PLCγ1 SH2–SH2 and human PLCγ1 SH3 (see Fig. 3) in pGEX2T′6 were a kind gift from S. Courtneidge (Sugen, Inc., Redwood City, CA).

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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