FERTILIZATION IN DICTYOSTELIUM - PHARMACOLOGICAL ANALYSES AND THE PRESENCE OF A SUBSTRATE PROTEIN SUGGEST PROTEIN-KINASE-C IS ESSENTIAL FORGAMETE FUSION

The role of protein kinase C (PKC) during fertilization in the model e ukaryote Dictyostelium discoideum was studied. Inhibition of PKC activ ity using staurosporine, chelerythrine, and bisindoylmaleimide resulte d in a dose-dependent decrease in gamete fusion without any detectable effect on cell morphology or growth. At 1.0 mu M, staurosporine led t o a greater than 90% inhibition of gamete fusion. In support of this, chelerythrine and bisindoylmaleimide at 10 mu M inhibited gamete cell fusion by 98 and 99%, respectively. In all cases, subsequent removal o f 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-tet radecanoylphorbol-13-acetate at 5 nM resulted in a 56% enhancement of cell fusion. In order to identify PKC substrates that may regulate fer tilization in D. discoideum, in vitro phosphorylation was carried out followed by SDS-PAGE, A number of proteins were phosphorylated, only o ne of which, a protein of about 50,000 M(r), appears to be a PKC subst rate. In total, these results coupled with earlier work suggest that P KC functions as part of a calcium-mediated signaling pathway that regu lates fertilization in D. discoideum, suggesting that the dual singali ng pathway that regulates fertilization in higher eukaryotes may have evolved very early. (C) 1995 Academic Press, Inc.