Progesterone and cAMP-dependent protein kinase regulate in vivo the level of phosphorylation of two proteins (Mr 20,000 and Mr 32,000) in Xenopus oocytes☆

Abstract The [ 32 P]phosphoproteins and [ 35 S]thiophosphoproteins were analyzed by electrophoresis and autoradiography after microinjection of [γ- 32 P]ATP or of [ 35 S]ATP-γ-S into living Xenopus oocytes. The level of 32 P incorporation into a 20-kDa protein was decreased following progesterone treatment (between 1 and 2 hr). This 20-kDa protein was partially thiophosphorylated in vivo by [ 35 S]ATP-γ-S. Furthermore it was found that this phosphoprotein was partially purified by TCA (1%) extraction and heat treatment. Microinjection of the C-subunit of cAMP-dependent protein kinase (0.6 to 1.2 pmole) inhibited maturation and provoked an increase in the level of phosphorylation of the 20-kDa protein and of a 32-kDa protein, indicating that both proteins were in vivo substrates (directly or indirectly) for cAMP-dependent protein kinase. When inhibitor-1 of protein phosphatase-1 was microinjected (5 to 10 pmole per oocyte) meiotic maturation was inhibited and the level of phosphorylation of the 32-kDa protein was increased; the same result was obtained following ATP-γ-S (1 m M ) microinjection. Altogether these results suggest that a 20-kDa phosphoprotein, whose level of phosphorylation is decreased by progesterone, could be involved in the regulation of maturation by lowering the level of phosphorylation of a 32-kDa phosphoprotein. An attractive hypothesis would be that the 20-kDa phosphoprotein is an inhibitor of protein phosphatase-1.