Boyer J, Asselin J, Bellé R, Ozon R
Dev Biol. 1986 Feb;113(2):420-8. doi: 10.1016/0012-1606(86)90176-4.
The [32P]phosphoproteins and [35S]thiophosphoproteins were analyzed by electrophoresis and autoradiography after microinjection of [gamma-32P]ATP or of [35S]ATP-gamma-S into living Xenopus oocytes. The level of 32P 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 [35S]ATP-gamma-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-gamma-S (1 mM) 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 phosphorylation of a 32-kDa phosphoprotein. An attractive hypothesis would be that the 20-kDa phosphoprotein is an inhibitor of protein phosphatase-1.
将[γ-32P]ATP或[35S]ATP-γ-S显微注射到非洲爪蟾卵母细胞后，通过电泳和放射自显影分析[32P]磷蛋白和[35S]硫代磷蛋白。孕酮处理（1至2小时之间）后，20-kDa蛋白的32P掺入水平降低。该20-kDa蛋白在体内被[35S]ATP-γ-S部分硫代磷酸化。此外，发现该磷蛋白通过TCA（1%）提取和热处理进行部分纯化。显微注射cAMP依赖性蛋白激酶的C亚基（0.6至1.2皮摩尔）可抑制成熟，并导致20-kDa蛋白和32-kDa蛋白的磷酸化水平升高，表明这两种蛋白在体内是cAMP依赖性蛋白激酶的底物（直接或间接）。当显微注射蛋白磷酸酶-1的抑制剂-1（每个卵母细胞5至10皮摩尔）时，减数分裂成熟受到抑制，32-kDa蛋白的磷酸化水平升高；注射ATP-γ-S（1 mM）后也得到相同结果。这些结果共同表明，一种20-kDa磷蛋白，其磷酸化水平因孕酮而降低，可能通过降低32-kDa磷蛋白的磷酸化水平参与成熟调节。一个有吸引力的假设是，20-kDa磷蛋白是蛋白磷酸酶-1的抑制剂。
