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.
Gunther
K E
KE
Department of Zoology, Erindale College, University of Toronto, Mississauga, Ontario, Canada.
Ramkissoon
S
S
Lydan
M A
MA
O'Day
D H
DH
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D053119:Benzophenanthridines; D004023:Dictyostelium / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005306:Fertilization; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology; D010617:Phenanthridines / Q000494:pharmacology; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D000470:Alkaloids; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D010617:Phenanthridines; D010750:Phosphoproteins; C016299:chelerythrine; D011493:Protein Kinase C; D019311:Staurosporine; C088060:bisindolylmaleimide; D013755:Tetradecanoylphorbol Acetate