Department of Medical Biochemistry and Microbiology, The Biomedical Center, Uppsala University, Uppsala, Sweden.
PLoS One. 2012;7(2):e32081. doi: 10.1371/journal.pone.0032081. Epub 2012 Feb 22.
A tight control over AKT/PKB activation is essential for cells, and they realise this in part by regulating the phosphorylation of Ser473 in the "hydrophobic motif" of the AKT carboxy-terminal region. The RICTOR-mTOR complex (TORC2) is a major kinase for AKT Ser473 phosphorylation after stimulation by several growth factors, in a reaction proposed to require p21-activated kinase (PAK) as a scaffold. However, other kinases may catalyse this reaction in stimuli-specific manners. Here we characterised the requirement of RICTOR, ILK, and PAK for AKT Ser473 phosphorylation downstream of selected family members of integrins, G protein-coupled receptors, and tyrosine-kinase receptors and analysed the importance of this phosphorylation site for adhesion-mediated survival. siRNA-mediated knockdown in HeLa and MCF7 cells showed that RICTOR-mTOR was required for phosphorylation of AKT Ser473, and for efficient phosphorylation of the downstream AKT targets FOXO1 Thr24 and BAD Ser136, in response to β1 integrin-stimulation. ILK and PAK1/2 were dispensable for these reactions. RICTOR knockdown increased the number of apoptotic MCF7 cells on β1 integrin ligands up to 2-fold after 24 h in serum-free conditions. β1 integrin-stimulation induced phosphorylation of both AKT1 and AKT2 but markedly preferred AKT2. RICTOR-mTOR was required also for LPA-induced AKT Ser473 phosphorylation in MCF7 cells, but, interestingly, not in HeLa cells. PAK was needed for the AKT Ser473 phosphorylation in response to LPA and PDGF, but not to EGF. These results demonstrate that different receptors utilise different enzyme complexes to phosphorylate AKT at Ser473, and that AKT Ser473 phosphorylation significantly contributes to β1 integrin-mediated anchorage-dependent survival of cells.
AKT/PKB 的激活受到严格的调控，这对细胞至关重要，细胞通过调节 AKT 羧基末端区域“疏水区”中丝氨酸 473 的磷酸化来实现这一点。在受到多种生长因子刺激后，RICTOR-mTOR 复合物（TORC2）是 AKT 丝氨酸 473 磷酸化的主要激酶，该反应需要 p21 激活激酶（PAK）作为支架。然而，其他激酶可能以刺激特异性的方式催化此反应。在这里，我们研究了 RICTOR、ILK 和 PAK 在整合素、G 蛋白偶联受体和酪氨酸激酶受体家族成员刺激后 AKT 丝氨酸 473 磷酸化中的需求，并分析了该磷酸化位点对黏附介导的存活的重要性。HeLa 和 MCF7 细胞中的 siRNA 介导的敲低表明，RICTOR-mTOR 对于 AKT 丝氨酸 473 的磷酸化以及对于下游 AKT 靶标 FOXO1 苏氨酸 24 和 BAD 丝氨酸 136 的有效磷酸化是必需的，这是对β1 整合素刺激的反应。ILK 和 PAK1/2 对于这些反应是可有可无的。在无血清条件下，RICTOR 敲低使 MCF7 细胞在β1 整合素配体上的凋亡细胞数量增加了 2 倍以上，时间长达 24 小时。β1 整合素刺激诱导 AKT1 和 AKT2 的磷酸化，但明显偏向 AKT2。RICTOR-mTOR 对于 MCF7 细胞中 LPA 诱导的 AKT 丝氨酸 473 磷酸化也是必需的，但在 HeLa 细胞中却不是必需的。PAK 对于 LPA 和 PDGF 刺激的 AKT 丝氨酸 473 磷酸化是必需的，但对于 EGF 则不是必需的。这些结果表明，不同的受体利用不同的酶复合物来磷酸化 AKT 的丝氨酸 473，并且 AKT 丝氨酸 473 磷酸化显著有助于β1 整合素介导的细胞锚定依赖性存活。
