Denner L A, Schrader W T, O'Malley B W, Weigel N L
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1990 Sep 25;265(27):16548-55.
The present studies examine the effects of in vivo and in situ progesterone treatment in the regulation of site-specific phosphorylation of the chicken oviduct progesterone receptor (PR). By gas-phase protein sequencing we have identified three hormonally regulated phosphorylation sites: Ser-211, Ser-260, and Ser-530. We determined phosphorylation stoichiometries by analyzing the amounts of phosphorylated and dephosphorylated serine at each site. Stoichiometries of sites 211 and 260 were about 20% under basal conditions and increased 1.5-2-fold by in situ progesterone treatment. Site 530 was virtually absent under basal conditions and induced to greater than 33% by in situ progesterone treatment. We tested several protein kinases for phosphorylation of the PR in vitro on these sites or peptides containing these sites. We found that the catalytic subunit of cAMP-dependent protein kinase mimicked the in vivo, hormone-induced altered mobility of PRs in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the in vivo and in vitro alterations were reversed by alkaline phosphatase. Finally, we showed that cAMP-dependent protein kinase phosphorylated Ser-528.
本研究检测了体内和原位孕酮处理对鸡输卵管孕酮受体（PR）位点特异性磷酸化调节的影响。通过气相蛋白质测序，我们确定了三个受激素调节的磷酸化位点：Ser-211、Ser-260和Ser-530。我们通过分析每个位点磷酸化和去磷酸化丝氨酸的量来确定磷酸化化学计量。在基础条件下，位点211和260的化学计量约为20%，原位孕酮处理使其增加了1.5至2倍。位点530在基础条件下几乎不存在，原位孕酮处理可使其诱导至超过33%。我们测试了几种蛋白激酶在体外对这些位点或含这些位点的肽段进行PR磷酸化的情况。我们发现，环磷酸腺苷（cAMP）依赖性蛋白激酶的催化亚基在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中模拟了体内激素诱导的PR迁移率改变。体内和体外的改变均可被碱性磷酸酶逆转。最后，我们表明cAMP依赖性蛋白激酶使Ser-528磷酸化。
