Involvement of phospholipase Cγ1 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 F. Rossi. 1997. Development [Camb.]. 124:2267-2274). Coinjection of glutathione-S-transferase (GST) fusion proteins containing the src-homology (SH) domains of the gamma 1 isoform of PLC (PLC gamma 1) competitively inhibits tr-kit-induced egg activation. A GST fusion protein containing the SH3 domain of PLC gamma 1 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 PLC gamma 1 is specific. Tr-kit-induced egg activation is also suppressed by co-injection of antibodies raised against the PLC gamma 1 SH domains, but not against the PLCy1 COOH-terminal region. In transfected COS cells, coexpression of PLC gamma 1 and tr-kit increases diacylglycerol and inositol phosphate production, and the phosphotyrosine content of PLC gamma 1 with respect to cells expressing PLC gamma 1 alone. These data indicate that tr-kit activates PLCy1, and that the SH3 domain of PLC gamma 1 is essential for tr-kit-induced egg activation.