Phorbol Myristate Acetate Induces the Phosphorylation of Plasma Membrane‐Associated Proteins in Sea Urchin Eggs

Immediately after fertilization sea urchin eggs undergo an increase in cytoplasmic pH from 6.8 to 7.2. This pH change occurs by activation of a Na+/H+ antiporter, and is a necessary signal for later steps in metabolic activation of development. Activators of protein kinase C such as phorbol myristate acetate (PMA) and diacylglycerol produce a similar pH increase in eggs. Phosphorylation of the antiporter or a regulatory protein may be a step in activating Na+/H+ exchange. Here we show that treatment of sea urchin eggs (S. purpuratus) with PMA results in increased phosphorylation of over a dozen proteins. Of these, three proteins of Mr=240, 92 and 80 kD are located in the egg cortex; under‐representation of these bands in isolated cortical granules suggests that they are plasma membrane‐associated. Phosphorylation of the 92 kD band is concentration‐dependent over a range of 10 to 1000 nM PMA and occurs over a time‐course of 1 to 3 min. Phosphoamino acid analysis indicates that phosphorylation is on serine residues. Phosphorylation appeares to be mediated by protein kinase C since the inactive PMA analogue, 4α‐phorbol 12, 13‐didecanoate, does not induce phosphorylation nor does experimental alkalinization of the egg cytoplasm.