Involvement of phospholipase Cgamma1 in mouse egg activation induced by a truncated form of the C-kit tyrosine kinase present in spermatozoa.
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.
Sette
C
C
Dipartimento di Sanitá Pubblica e Biologia Cellulare, Sezione di Anatomia, Universitá di Roma Tor Vergata, Rome, Italy.
Bevilacqua
A
A
Geremia
R
R
Rossi
P
P
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D019556:COS Cells; D002453:Cell Cycle / Q000502:physiology; D004075:Diglycerides / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry* / Q000502:physiology; D008297:Male; D051379:Mice; D008845:Microinjections; D009865:Oocytes / Q000378:metabolism*; D010312:Parthenogenesis / Q000502:physiology; D051966:Phospholipase C gamma; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000494:pharmacology*; D011506:Proteins / Q000737:chemistry; D019009:Proto-Oncogene Proteins c-kit / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013094:Spermatozoa / Q000201:enzymology*; D014162:Transfection / Q000235:genetics; D010738:Type C Phospholipases / Q000737:chemistry* / Q000502:physiology; D018909:src Homology Domains / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies; D004075:Diglycerides; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D007295:Inositol Phosphates; D007527:Isoenzymes; D011506:Proteins; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; D019009:Proto-Oncogene Proteins c-kit; D010738:Type C Phospholipases; D051966:Phospholipase C gamma