Joel P B, Smith J, Sturgill T W, Fisher T L, Blenis J, Lannigan D A
Center for Cell Signaling and Department of Pharmacology, University of Virginia, Charlottesville 22908, USA.
Mol Cell Biol. 1998 Apr;18(4):1978-84. doi: 10.1128/MCB.18.4.1978.
The estrogen receptor alpha (ER), a member of the steroid receptor superfamily, contains an N-terminal hormone-independent transcriptional activation function (AF-1) and a C-terminal hormone-dependent transcriptional activation function (AF-2). Here, we used in-gel kinase assays to determine that pp90rsk1 activated by either epidermal growth factor (EGF) or phorbol myristate acetate specifically phosphorylates Ser-167 within AF-1. In vitro kinase assays demonstrated that pp90rsk1 phosphorylates the N terminus of the wild-type ER but not of a mutant ER in which Ser-167 was replaced by Ala. In vivo, EGF stimulated phosphorylation of Ser-167 as well as Ser-118. Ectopic expression of active pp90rsk1 increased the level of phosphorylation of Ser-167 compared to that of either a mutant pp90rsk1, which is catalytically inactive in the N-terminal kinase domain, or to that of vector control. The ER formed a stable complex with the mutant pp90rsk1 in vivo. Transfection of the mutant pp90rsk1 depressed ER-dependent transcription of both a wild-type ER and a mutant ER that had a defective AF-2 domain (ER TAF-1). Furthermore, replacing either Ser-118 or Ser-167 with Ala in ER TAF-1 showed similar decreases in transcription levels. A double mutant in which both Ser-118 and Ser-167 were replaced with Ala demonstrated a further decrease in transcription compared to either of the single mutations. Taken together, our results strongly suggest that pp90rsk1 phosphorylates Ser-167 of the human ER in vivo and that Ser-167 aids in regulating the transcriptional activity of AF-1 in the ER.
雌激素受体α（ER）是类固醇受体超家族的成员，包含一个N端激素非依赖性转录激活功能（AF-1）和一个C端激素依赖性转录激活功能（AF-2）。在此，我们使用凝胶内激酶分析来确定由表皮生长因子（EGF）或佛波酯肉豆蔻酸酯激活的pp90rsk1特异性磷酸化AF-1内的Ser-167。体外激酶分析表明，pp90rsk1磷酸化野生型ER的N端，但不磷酸化Ser-167被丙氨酸取代的突变型ER的N端。在体内，EGF刺激Ser-167以及Ser-118的磷酸化。与在N端激酶结构域无催化活性的突变型pp90rsk1或载体对照相比，活性pp90rsk1的异位表达增加了Ser-167的磷酸化水平。ER在体内与突变型pp90rsk1形成稳定复合物。突变型pp90rsk1的转染抑制了野生型ER和具有缺陷AF-2结构域的突变型ER（ER TAF-1）的ER依赖性转录。此外，在ER TAF-1中将Ser-118或Ser-167替换为丙氨酸显示转录水平有类似下降。与任何一个单突变相比，Ser-118和Ser-167都被替换为丙氨酸的双突变体显示转录进一步下降。综上所述，我们的结果强烈表明，pp90rsk1在体内磷酸化人ER的Ser-167，并且Ser-167有助于调节ER中AF-1的转录活性。
