Genetic code expansion and live cell imaging reveal that Thr-308 phosphorylation is irreplaceable and sufficient for Akt1 activity
Genetic code expansion and live cell imaging reveal that Thr-308 phosphorylation is irreplaceable and sufficient for Akt1 activity
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DOI：
10.1074/jbc.ra118.002357
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发表时间：
2018-07-06
影响因子：
4.8
通讯作者：
O'Donoghue, Patrick
中科院分区：
文献类型：
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作者：
Balasuriya, Nileeka;Kunkel, Maya T.;O'Donoghue, Patrick
原癌基因Akt/蛋白激酶B （PKB）是生长和生存的关键信号传感器。生长因子刺激导致Akt在两个调控位点（Thr-308和Ser-473）磷酸化，剧烈激活Akt信号。然而，描述每个调控位点的确切作用在技术上具有挑战性，并且仍然难以捉摸。在这里，我们使用遗传密码扩增来产生位点特异性磷酸化的Akt1，以剖析每个调控位点对Akt1活性的贡献。我们首次实现了含有位点特异性pThr和pSer残基的全长Akt1的重组生产。我们对Akt1位点特异性磷酸化的分析显示，相对于未磷酸化的Akt1，两个位点的磷酸化使表观催化速率增加了1500倍，这主要归因于Thr-308位点的磷酸化。COS-7细胞的实时成像证实，Akt的细胞活化需要Thr-308的磷酸化，而不是Ser-473。我们在体外和细胞中发现，酸性残基不能模仿pThr-308的功能，Ala突变也不能模仿未磷酸化的Thr-308。具有pSer-308的Akt1变体仅具有部分酶和细胞信号活性，揭示了pThr-308的-甲基与Akt1活性位点的Cys-310之间的关键相互作用。因此，pThr-308是刺激细胞中Akt信号传导的必要和充分条件，通常使用的拟磷物不适合用于Akt信号传导的生物学研究。我们的数据还表明，pThr-308应被视为Akt活性的主要诊断标志物。
The proto-oncogene Akt/protein kinase B (PKB) is a pivotal signal transducer for growth and survival. Growth factor stimulation leads to Akt phosphorylation at two regulatory sites (Thr-308 and Ser-473), acutely activating Akt signaling. Delineating the exact role of each regulatory site is, however, technically challenging and has remained elusive. Here, we used genetic code expansion to produce site-specifically phosphorylated Akt1 to dissect the contribution of each regulatory site to Akt1 activity. We achieved recombinant production of full-length Akt1 containing site-specific pThr and pSer residues for the first time. Our analysis of Akt1 site-specifically phosphorylated at either or both sites revealed that phosphorylation at both sites increases the apparent catalytic rate 1500-fold relative to unphosphorylated Akt1, an increase attributable primarily to phosphorylation at Thr-308. Live imaging of COS-7 cells confirmed that phosphorylation of Thr-308, but not Ser-473, is required for cellular activation of Akt. We found in vitro and in the cell that pThr-308 function cannot be mimicked with acidic residues, nor could unphosphorylated Thr-308 be mimicked by an Ala mutation. An Akt1 variant with pSer-308 achieved only partial enzymatic and cellular signaling activity, revealing a critical interaction between the -methyl group of pThr-308 and Cys-310 in the Akt1 active site. Thus, pThr-308 is necessary and sufficient to stimulate Akt signaling in cells, and the common use of phosphomimetics is not appropriate for studying the biology of Akt signaling. Our data also indicate that pThr-308 should be regarded as the primary diagnostic marker of Akt activity.