Involvement of Phospholipase C{gamma}1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

doi:10.1083/jcb.142.4.1063

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© The Rockefeller University Press, 0021-9525/1998//1063 $5.00
The Journal of Cell Biology, Volume 142, Number 4, , 1998 1063-1074

Articles

Involvement of Phospholipase C ${gamma}$ 1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

Claudio Sette^*, Arturo Bevilacqua, Raffaele Geremia^*, and Pellegrino Rossi^*

^* Dipartimento di Sanitá Pubblica e Biologia Cellulare, Sezione di Anatomia, Universitá di Roma Tor Vergata, Rome, Italy; and Dipartimento di Istologia ed Embriologia Medica and Dipartimento di Psicologia, Universitá di Roma La Sapienza, Rome, Italy

Microinjection of a truncated form of the c-kit tyrosine kinasepresent in mouse spermatozoa (tr-kit) activates mouse eggsparthenogenetically, and tr-kit– induced egg activationis inhibited by preincubation with an inhibitor of phospholipaseC (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 proteinscontaining the src-homology (SH) domains of the ${gamma}$ 1 isoform ofPLC (PLC ${gamma}$ 1) competitively inhibits tr-kit– induced eggactivation. A GST fusion protein containing the SH3 domainof PLC ${gamma}$ 1 inhibits egg activation as efficiently as the wholeSH region, while a GST fusion protein containing the two SH2domains is much less effective. A GST fusion protein containingthe SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit–induced egg activation, showing that the effectof the SH3 domain of PLC ${gamma}$ 1 is specific. Tr-kit–inducedegg activation is also suppressed by co-injection of antibodies raised against the PLC ${gamma}$ 1 SH domains, but not against the PLC ${gamma}$ 1COOH-terminal region. In transfected COS cells, coexpressionof PLC ${gamma}$ 1 and tr-kit increases diacylglycerol and inositol phosphateproduction, and the phosphotyrosine content of PLC ${gamma}$ 1 with respectto cells expressing PLC ${gamma}$ 1 alone. These data indicate that tr-kit activates PLC ${gamma}$ 1, and that the SH3 domain of PLC ${gamma}$ 1 is essentialfor tr-kit–induced egg activation.

Key Words: truncated-c-kit • spermatozoa • egg-activation • phospholipase-C- ${gamma}$ 1 • src-homology-domains

Abbreviations used in this paper: DAG, diacylglycerol; GST, glutathione-S-transferase; InsP, inositol phosphate; InsP₃, inositol 1,4,5-trisphosphate; MII, metaphase II; NRTK, non-receptor tyrosine kinase; PIP₂, phosphatidylinositol-4,5-bisphosphate; PIP₃, phosphatidylinositol-3,4,5-trisphosphate; PKC, protein kinase C; PLC, phospholipase C; RTK, receptor tyrosine kinase; SCF, stem cell factor; SH, src-homology; SH2, src-homology 2; SH3, src-homology 3; tr-kit, truncated c-kit.

Address all correspondence to P. Rossi, Dipartimento di SanitáPubblica e Biologia Cellulare, Sezione di Anatomia, Universitádi Roma Tor Vergata, via O. Raimondo 8, 00173, Rome, Italy.Tel.: 39-6-72596272. Fax: 39-6-72596268. E-mail: pellegrino.rossi{at}med.uniroma2.it

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