Phosphoprotein inhibition of calcium-stimulated exocytosis in sea urchin eggs.
We have investigated the role of protein phosphorylation in the control of exocytosis in sea urchin eggs by treating eggs with a thio-analogue of ATP. ATP gamma S (adenosine 5'-O-3-thiotriphosphate) is a compound which can be used as a phosphoryl donor by protein kinases, leading to irreversible protein thiophosphorylation (Gratecos, D., and E.H. Fischer. 1974. Biochem. Biophys. Res. Commun. 58:960-967). Microinjection of ATP gamma S inhibits cortical granule exocytosis, but has no effect on the sperm-egg signal transduction mechanisms which normally cause exocytosis by generating an increase in [Ca2+]i. ATP gamma S requires cytosolic factors for its inhibition of cortical granule exocytosis: it does not affect exocytosis when applied directly to the isolated exocytotic apparatus. Our data suggest that ATP gamma S irreversibly inhibits exocytosis via thiophosphorylation of proteins associated with the egg cortex. We have identified two thiophosphorylated proteins (33 and 27 kD) that are associated with the isolated exocytotic apparatus. They may mediate the inhibition of exocytosis by ATP gamma S. In addition, we show that okadaic acid, an inhibitor of phosphoprotein phosphatases, prevents cortical granule exocytosis at fertilization without affecting calcium mobilization. Like ATP gamma S, okadaic acid has no effect on exocytosis in vitro. Our results suggest that an inhibitory phosphoprotein can obstruct calcium-stimulated exocytosis in sea urchin eggs; on the other hand, they do not readily support the idea that a protein phosphatase is an essential component of the mechanism controlling exocytosis.
Whalley
T
T
Department of Physiology, University College London, United Kingdom.
Crossley
I
I
Whitaker
M
M
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology* / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000502:physiology; D004988:Ethers, Cyclic / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects*; D005306:Fertilization / Q000187:drug effects; D007425:Intracellular Membranes / Q000378:metabolism; D008561:Membrane Fusion; D019319:Okadaic Acid; D010063:Ovum / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology*; D010766:Phosphorylation; D012617:Sea Urchins; D015398:Signal Transduction; D013997:Time Factors
D004988:Ethers, Cyclic; D010750:Phosphoproteins; D019319:Okadaic Acid; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D002118:Calcium