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.