Fertilization in Dictyostelium: pharmacological analyses and the presence of a substrate protein suggest protein kinase C is essential for gamete fusion.

The role of protein kinase C (PKC) during fertilization in the model eukaryote Dictyostelium discoideum was studied. Inhibition of PKC activity using staurosporine, chelerythrine, and bisindoylmaleimide resulted in a dose-dependent decrease in gamete fusion without any detectable effect on cell morphology or growth. At 1.0 microM, staurosporine led to a greater than 90% inhibition of gamete fusion. In support of this, chelerythrine and bisindoylmaleimide at 10 microM inhibited gamete cell fusion by 98 and 99%, respectively. In all cases, subsequent removal of the inhibitor allowed for the completion of sexual development in a manner indistinguishable from untreated, control cultures. In contrast, the stimulation of PKC by the addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate at 5 nM resulted in a 56% enhancement of cell fusion. In order to identify PKC substrates that may regulate fertilization in D. discoideum, in vitro phosphorylation was carried out followed by SDS-PAGE. A number of proteins were phosphorylated, only one of which, a protein of about 50,000 M(r), appears to be a PKC substrate. In total, these results coupled with earlier work suggest that PKC functions as part of a calcium-mediated signaling pathway that regulates fertilization in D. discoideum, suggesting that the dual signaling pathway that regulates fertilization in higher eukaryotes may have evolved very early.