Misra Uma Kant, Pizzo Salvatore Vincent
Department of Pathology, Duke University Medical Center, Durham, North Carolina, United States of America.
PLoS One. 2014 Feb 6;9(2):e88373. doi: 10.1371/journal.pone.0088373. eCollection 2014.
PDK1 phosphorylates multiple substrates including Akt by PIP3-dependent mechanisms. In this report we provide evidence that in prostate cancer cells stimulated with activated α2-macroglobulin (α2M*) PDK1 phosphorylates Akt in the T-loop at Thr(308) by using Raptor in the mTORC1 complex as a scaffold protein. First we demonstrate that PDK1, Raptor, and mTOR co-immunoprecipitate. Silencing the expression, not only of PDK1, but also Raptor by RNAi nearly abolished Akt phosphorylation at Akt(Thr308) in Raptor-immunoprecipitates of α2M*-stimulated prostate cancer cells. Immunodepleting Raptor or PDK from cell lysates of cells treated with α2M* drastically reduced Akt phosphorylation at Thr(308), which was recovered by adding the supernatant of Raptor- or PDK1-depleted cell lysates, respectively. Studies of insulin binding to its receptor on prostate cancer cells yielded similar results. We thus demonstrate that phosphorylating the T-loop Akt residue Thr(308) by PDK1 requires Raptor of the mTORC1 complex as a platform or scaffold protein.
PDK1通过依赖PIP3的机制磷酸化包括Akt在内的多种底物。在本报告中，我们提供证据表明，在活化的α2-巨球蛋白（α2M*）刺激的前列腺癌细胞中，PDK1通过使用mTORC1复合物中的Raptor作为支架蛋白，使Akt的T环中苏氨酸（Thr）308位点发生磷酸化。首先，我们证明PDK1、Raptor和mTOR能共同免疫沉淀。通过RNA干扰沉默PDK1以及Raptor的表达，几乎消除了α2M刺激的前列腺癌细胞的Raptor免疫沉淀产物中Akt（Thr308）的磷酸化。从用α2M处理的细胞裂解物中免疫去除Raptor或PDK，可显著降低苏氨酸（Thr）308位点的Akt磷酸化，分别添加Raptor或PDK1去除的细胞裂解物上清液可恢复该磷酸化。对胰岛素与其在前列腺癌细胞上的受体结合的研究也得到了类似结果。因此，我们证明PDK1使Akt的T环残基苏氨酸（Thr）308磷酸化需要mTORC1复合物的Raptor作为平台或支架蛋白。
