Protein phosphorylation during activation of surf clam oocytes.
We have investigated the increase of phosphorylated proteins upon activation of surf clam (Spisula solidissima) oocytes, by measuring the cumulative incorporation of 32P in proteins and by performing an SDS-PAGE and autoradiographic analysis of 32P-labeled proteins, from oocytes initially radiolabeled with 32P-orthophosphate. The phosphorylation inhibitor 6-dimethylaminopurine (6-DMAP) inhibits both germinal vesicle breakdown (GVBD) and the normal increase in phosphorylated proteins observed upon activation by KCl, in a reversible and dose-dependent manner. Using different artificial seawaters (normal, Ca(2+)-free, Na(+)-free), we observed that the increase of phosphorylated proteins, upon K+ stimulation, occurs only when GVBD is allowed to proceed along with an increased Ca2+ influx, in normal or Na(+)-free seawater. Stimulation of oocytes by ammonia, which directly raises intracellular pH (pHi) but does not trigger GVBD, is without effect on the level or pattern of phosphorylated proteins. The link between the Ca2+ influx and the level of phosphorylated proteins was further investigated using conditions altering the duration or the level of Ca2+ influx upon K+ stimulation. In all conditions tested, both GVBD and the level of phosphorylated proteins were similarly affected by alterations of the Ca2+ influx, indicating that these processes are tightly coupled one with another. Upon activation of oocytes, six major proteins of estimated molecular weights of 31, 41, 48, 56, 80 and 86 kDa undergo an increased phosphorylation that is reversibly sensitive to 6-DMAP. Our results suggest that increased protein phosphorylation, sensitive to 6-DMAP, is necessary for GVBD and that it is indirectly linked to the increased Ca2+ influx that stands as an upstream trigger for activation, while an elevated pHi alone has no effect on these processes.
Dubé
F
F
Département d'Océanographie, Université du Québec à Rimouski, Canada.
Dufresne
L
L
Coutu
L
L
Clotteau
G
G
D000818:Animals; D049872:Bivalvia / Q000378:metabolism* / Q000502:physiology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D004527:Egg Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D009866:Oogenesis; D010766:Phosphorylation; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism
D015220:Calcium Channels; D004527:Egg Proteins; D015221:Potassium Channels; D015222:Sodium Channels