Context-dependent expression of a conditionally-inducible form of active Akt.
Context-dependent expression of a conditionally-inducible form of active Akt.
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DOI：
10.1371/journal.pone.0197899
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Greene LA
中科院分区：
文献类型：
--
作者：
Park S;Burke RE;Kareva T;Kholodilov N;Aimé P;Franke TF;Levy O;Greene LA
Akt激酶是增殖活性细胞和有丝分裂后细胞中的关键信号组分。在这里，我们试图创造一种条件诱导形式的活性Akt，用于体外和体内应用。我们融合了一个来自E.大肠杆菌二氢叶酸还原酶转化为Akt 1的组成型活性突变体形式Akt（E40 K）。先前的工作表明，这样的融合蛋白可以被稳定和诱导的配体，抗生素甲氧苄啶（TMP）。我们观察到TMP在培养的几种细胞背景（包括神经元）中对总DD-Akt和磷酸化/活性DD-Akt（E40 K）的剂量依赖性可逆诱导。Akt底物FoxO 4的磷酸化在DD-Akt（E40 K）诱导后显著升高，表明诱导的蛋白具有功能活性。诱导的Akt（E40 K）保护细胞免于由血清剥夺诱发的凋亡，并且在帕金森病的两种细胞模型（6-OHDA和MPP+暴露）中具有神经保护作用。没有诱导没有显著的保护作用。我们还评估了通过AAV 1腺相关病毒载体递送神经元后，TMP在小鼠黑质和纹状体中对Akt（E40 K）的诱导。虽然在纹状体中有显著的诱导，但在黑质中没有明显的诱导。为了探索这种差异的可能基础，我们检查了培养的腹侧中脑神经元中DD-Akt（E40 K）的诱导。TMP处理后，培养物中的多巴胺能和非多巴胺能神经元均显示DD-Akt（E40 K）诱导。然而，多巴胺能神经元的基础DD-Akt（E40 K）表达高3倍，导致该群体中TMP的诱导显著降低。这些发现表明，多巴胺能神经元可能是相对低效的蛋白质降解，一个属性，可能涉及到他们缺乏明显的DD-Akt（E40 K）诱导在体内和他们的选择性脆弱性帕金森氏病。总之，我们产生了Akt的可诱导的生物活性形式。诱导的程度似乎反映了细胞环境，这将为该试剂和相关试剂的最适当应用提供信息。
Akt kinases are key signaling components in proliferation-competent and post-mitotic cells. Here, we sought to create a conditionally-inducible form of active Akt for both in vitro and in vivo applications. We fused a ligand-responsive Destabilizing Domain (DD) derived from E. coli dihydrofolate reductase to a constitutively active mutant form of Akt1, Akt(E40K). Prior work indicated that such fusion proteins may be stabilized and induced by a ligand, the antibiotic Trimethoprim (TMP). We observed dose-dependent, reversible induction of both total and phosphorylated/active DD-Akt(E40K) by TMP across several cellular backgrounds in culture, including neurons. Phosphorylation of FoxO4, an Akt substrate, was significantly elevated after DD-Akt(E40K) induction, indicating the induced protein was functionally active. The induced Akt(E40K) protected cells from apoptosis evoked by serum deprivation and was neuroprotective in two cellular models of Parkinson's disease (6-OHDA and MPP+ exposure). There was no significant protection without induction. We also evaluated Akt(E40K) induction by TMP in mouse substantia nigra and striatum after neuronal delivery via an AAV1 adeno-associated viral vector. While there was significant induction in striatum, there was no apparent induction in substantia nigra. To explore the possible basis for this difference, we examined DD-Akt(E40K) induction in cultured ventral midbrain neurons. Both dopaminergic and non-dopaminergic neurons in the cultures showed DD-Akt(E40K) induction after TMP treatment. However, basal DD-Akt(E40K) expression was 3-fold higher for dopaminergic neurons, resulting in a significantly lower induction by TMP in this population. Such findings suggest that dopaminergic neurons may be relatively inefficient in protein degradation, a property that could relate to their lack of apparent DD-Akt(E40K) induction in vivo and to their selective vulnerability in Parkinson's disease. In summary, we generated an inducible, biologically active form of Akt. The degree of inducibility appears to reflect cellular context that will inform the most appropriate applications for this and related reagents.
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影响因子：
3.7
作者：
Aas A;Isakson P;Bindesbøll C;Alemu EA;Klungland A;Simonsen A
通讯作者：
Simonsen A
影响因子：
5.3
作者：
Liu J;Pasini S;Shelanski ML;Greene LA
通讯作者：
Greene LA
DOI：
10.1007/978-1-61779-536-7_18
发表时间：
2012
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Grau, Cristina Malagelada;Greene, Lloyd A
通讯作者：
Greene, Lloyd A
影响因子：
3.7
作者：
Deneka AY;Haber L;Kopp MC;Gaponova AV;Nikonova AS;Golemis EA
通讯作者：
Golemis EA
影响因子：
4
作者：
Greene, Lloyd A.;Levy, Oren;Malagelada, Cristina
通讯作者：
Malagelada, Cristina