Toulany Mahmoud, Maier Julia, Iida Mari, Rebholz Simone, Holler Marina, Grottke Astrid, Jüker Manfred, Wheeler Deric L, Rothbauer Ulrich, Rodemann H Peter
Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Tuebingen, Germany.
German Cancer Consortium (DKTK), Partner Site Tuebingen, and German Cancer Research Center (DKFZ), Heidelberg, Germany.
Cell Death Discov. 2017 Oct 30;3:17072. doi: 10.1038/cddiscovery.2017.72. eCollection 2017.
Akt1 through the C-terminal domain interacts with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and stimulates the repair of DNA double-strand breaks (DSBs) in K-RAS-mutated (K-RASmut) cells. We investigated the interactions of distinct domain(s) of DNA-PKcs in binding to full-length Akt1. Similarly, we analyzed potential interactions of DNA-PKcs with Akt2 and Akt3. Finally the effect of Akt isoforms in cell proliferation and tumor growth was tested. We demonstrated that Akt1 preferentially binds to the N-terminal domain of DNA-PKcs using pull-down studies with distinct eGFP-tagged DNA-PKcs fragments that were expressed by plasmids in combination with mCherry-tagged full-length Akt isoforms. These binding studies also indicated an interaction with the intermediate and C-terminal domains of DNA-PKcs. In contrast, Akt3 interacted with all four DNA-PKcs fragments without a marked preference for any specific domain. Notably, we could not see binding of Akt2 to any of the tested DNA-PKcs fragments. In subsequent studies, we demonstrated that Akt inhibition interferes with binding of Akt1 to the N-terminal domain of DNA-PKcs. This indicated a correlation between Akt1 activity and the Akt1/DNA-PKcs complex formation. Finally, knockdown studies revealed that the depletion of endogenous Akt1 and Akt3, but not Akt2, inhibit clonogenic activity and repair of ionizing radiation (IR)-induced DNA DSBs, leading to radiosensitization. Furthermore, in a xenograft study the expression of shAkt1 or shAkt3, but not shAkt2 in K-RASmut breast cancer cell line MDA-MB-231 showed major tumor growth delay. Together, these data indicate that Akt1 and Akt3, but not Akt2, physically interact with DNA-PKcs, thus stimulating the repair of DSBs and therefore protecting K-RASmut cells against IR. Likewise, interaction of Akt isoforms with DNA-PKcs could be crucial for their role in regulating tumor growth.
Akt1通过其C末端结构域与DNA依赖性蛋白激酶催化亚基（DNA-PKcs）相互作用，并刺激K-RAS突变（K-RASmut）细胞中DNA双链断裂（DSB）的修复。我们研究了DNA-PKcs不同结构域与全长Akt1结合的相互作用。同样，我们分析了DNA-PKcs与Akt2和Akt3之间的潜在相互作用。最后测试了Akt亚型对细胞增殖和肿瘤生长的影响。我们通过使用由质粒表达的不同eGFP标记的DNA-PKcs片段与mCherry标记的全长Akt亚型进行下拉研究，证明Akt1优先与DNA-PKcs的N末端结构域结合。这些结合研究还表明与DNA-PKcs的中间和C末端结构域存在相互作用。相比之下，Akt3与所有四个DNA-PKcs片段相互作用，对任何特定结构域没有明显偏好。值得注意的是，我们没有观察到Akt2与任何测试的DNA-PKcs片段结合。在随后的研究中，我们证明Akt抑制会干扰Akt1与DNA-PKcs N末端结构域的结合。这表明Akt1活性与Akt1/DNA-PKcs复合物形成之间存在相关性。最后，敲低研究表明，内源性Akt1和Akt3的缺失而非Akt2的缺失会抑制克隆形成活性和电离辐射（IR）诱导的DNA DSB的修复，导致放射增敏。此外，在异种移植研究中，K-RASmut乳腺癌细胞系MDA-MB-231中shAkt1或shAkt3而非shAkt2的表达显示出主要的肿瘤生长延迟。总之，这些数据表明Akt1和Akt3而非Akt2与DNA-PKcs发生物理相互作用，从而刺激DSB的修复，因此保护K-RASmut细胞免受IR损伤。同样，Akt亚型与DNA-PKcs的相互作用对于它们在调节肿瘤生长中的作用可能至关重要。
