Glycogen synthase kinase-3 protects estrogen receptor α from proteasomal degradation and is required for full transcriptional activity of the receptor
Glycogen synthase kinase-3 protects estrogen receptor α from proteasomal degradation and is required for full transcriptional activity of the receptor
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DOI：
10.1210/me.2007-0129
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发表时间：
2007-10-01
影响因子：
--
通讯作者：
Mayer, Doris
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文献类型：
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作者：
Grisouard, Jean;Medunjanin, Senad;Mayer, Doris
糖原合成酶-3(GSK-3)在包括类固醇受体在内的转录因子的调控中起着关键作用。在确定雌激素受体α(ERα)是GSK-3的底物后，GSK-3对ERα功能和活性对17β-雌二醇(E_2)依赖的激活的影响尚不清楚。在这里，我们通过使用小干扰技术结合免疫印迹、基因表达分析和荧光素酶报告分析表明，沉默GSK-3α或GSK-3β导致ERα阳性乳腺癌细胞中ERα水平和转录活性的降低。利用MCF-7细胞，我们证明了GSK-3沉默后ERα水平的降低是由于ERα蛋白酶体降解增加而不是抑制ERα蛋白合成。事实上，在这种情况下，在蛋白质合成抑制剂放线菌酮存在的情况下，使用蛋白酶体抑制剂MG132拯救了ERα蛋白。此外，在MG132存在下，GSK-3沉默GSK-3后，泛素化的ERα有较强的积累。我们的结论是，GSK-3保护ERα不受蛋白酶体降解的影响，在ERα蛋白的稳定和转换中起着关键作用。此外，体外激酶分析表明，GSK-3β在Ser-118处使ERα磷酸化。GSK-3沉默导致E2诱导的ERα在Ser-118处的磷酸化水平降低，以及E2诱导的雌激素反应元件依赖的荧光素酶报告基因表达降低。MG132对Ser-118磷酸化和荧光素酶活性均无恢复作用。此外，雌激素反应基因(PS2和孕激素受体)的表达在GSK-3沉默后降低。这些发现表明，GSK-3对于E2诱导的ERα在Ser118的磷酸化和在E2刺激下受体的完全转录活性是必需的。
Glycogen synthase kinase- 3 ( GSK- 3) plays a key role in the regulation of transcription factors including steroid receptors. Having identified estrogen receptor-alpha ( ER alpha) as substrate for GSK- 3, the impact of GSK- 3 on ER alpha function and activity upon 17 beta- estradiol ( E2)- dependent activation remains to be clarified. Here we show by using small interfering technology in combination with immunoblot, gene expression analysis, and luciferase reporter assays that silencing of GSK- 3 alpha or GSK- 3 beta results in the reduction of ER alpha levels and transcriptional activity in ER alpha- positive breast cancer cells. Using MCF- 7 cells we demonstrate that reduction of ER alpha levels upon GSK- 3 silencing was due to increased proteasomal degradation of ER alpha rather than inhibition of ER alpha protein synthesis. Indeed, under this condition, ER alpha protein was rescued using the proteasome inhibitor MG132 in presence of the protein synthesis inhibitor cycloheximide. In addition, strong accumulation of ubiquitinated ER alpha was obtained after GSK- 3 silencing in the presence of MG132. We conclude that GSK- 3 protects ER alpha from proteasomal degradation and plays a crucial role in ER alpha protein stabilization and turnover. Furthermore, in vitro kinase assay depicted that GSK- 3 beta phosphorylates ER alpha at Ser- 118. GSK- 3 silencing resulted in decrease of E2- induced nuclear ER alpha phosphorylation at Ser- 118 and E2- induced estrogen response element- dependent luciferase reporter gene expression. Neither Ser- 118 phosphorylation nor luciferase activity was restored by use of MG132. Moreover, the expression of estrogenresponsive genes ( pS2 and progesterone receptor) was decreased upon GSK- 3 silencing. These findings demonstrated that GSK- 3 is required for E2- induced ER alpha phosphorylation at Ser118 and full transcriptional activity of the receptor upon E2 stimulation.