Systematic Analysis Reveals Elongation Factor 2 and α-Enolase as Novel Interaction Partners of AKT2.
Systematic Analysis Reveals Elongation Factor 2 and α-Enolase as Novel Interaction Partners of AKT2.
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DOI：
10.1371/journal.pone.0066045
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Gödecke A
中科院分区：
文献类型：
--
作者：
Bottermann K;Reinartz M;Barsoum M;Kötter S;Gödecke A
AKT 2是参与细胞代谢调节的蛋白激酶AKT的三种同种型之一。由于蛋白质-蛋白质相互作用是传递信号转导特异性的一种可能性，我们进行了AKT 2-蛋白质相互作用分析，以阐明它们与AKT 2依赖性细胞功能的相关性。我们通过串联亲和纯化和质谱分析相结合的方法在HEK 293 T细胞中鉴定了热休克蛋白90 kDa（HSP 90）、Cdc 37、热休克蛋白70 kDa（HSP 70）、78 kDa葡萄糖调节蛋白（GRP 78）、微管蛋白、GAPDH、α-烯醇化酶和延伸因子2（EF 2）为AKT 2相互作用蛋白。使用细胞培养物中的氨基酸的稳定同位素标记的定量MS分析（SILAC）揭示，只有HSP 90和Cdc 37与AKT 2稳定地相互作用，而其他蛋白质与AKT 2以低亲和力相互作用。进一步分析AKT 2与α-烯醇化酶和EF 2的相互作用，以揭示这些新发现的结合伴侣的功能相关性。尽管AKT 2和α-烯醇化酶的相互作用（通过邻近连接试验（PLA）进行了额外验证），但在活性测量中未检测到AKT对α-烯醇化酶活性的显著影响。通过胰岛素刺激AKT和/或用ATP竞争性抑制剂CCT 128930抑制AKT不会改变α-烯醇化酶的酶活性。有趣的是，发现AKT 2和EF 2的直接相互作用在胚胎大鼠心肌细胞中受到动态调节。在用几种激素刺激之前用PI 3-激酶抑制剂LY 294002处理使复合物稳定，而单独刺激导致复合物解离，其用PLA原位分析。总之，这些发现指出了蛋白质合成和葡萄糖代谢中AKT 2介导的信号转导的新方面。
AKT2 is one of the three isoforms of the protein kinase AKT being involved in the modulation of cellular metabolism. Since protein-protein interactions are one possibility to convey specificity in signal transduction, we performed AKT2-protein interaction analysis to elucidate their relevance for AKT2-dependent cellular functions. We identified heat shock protein 90 kDa (HSP90), Cdc37, heat shock protein 70 kDa (HSP70), 78 kDa glucose regulated protein (GRP78), tubulin, GAPDH, α-enolase and elongation factor 2 (EF2) as AKT2-interacting proteins by a combination of tandem affinity purification and mass spectrometry in HEK293T cells. Quantitative MS-analysis using stable isotope labeling by amino acids in cell culture (SILAC) revealed that only HSP90 and Cdc37 interact stably with AKT2, whereas the other proteins interact with low affinity with AKT2. The interactions of AKT2 with α-enolase and EF2 were further analyzed in order to uncover the functional relevance of these newly discovered binding partners. Despite the interaction of AKT2 and α-enolase, which was additionally validated by proximity ligation assay (PLA), no significant impact of AKT on α-enolase activity was detected in activity measurements. AKT stimulation via insulin and/or inhibition with the ATP-competitive inhibitor CCT128930 did not alter enzymatic activity of α-enolase. Interestingly, the direct interaction of AKT2 and EF2 was found to be dynamically regulated in embryonic rat cardiomyocytes. Treatment with the PI3-kinase inhibitor LY294002 before stimulation with several hormones stabilized the complex, whereas stimulation alone led to complex dissociation which was analyzed in situ with PLA. Taken together, these findings point to new aspects of AKT2-mediated signal transduction in protein synthesis and glucose metabolism.
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