Abstract
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.
Highlights
The major mechanisms that lead to a constitutive activation of the PI3K/Akt pathway are mutations and overexpression of upstream receptor tyrosine kinases such as erbB family members, activating mutations of PIK3CA or RAS and the loss of tumor suppressor protein phosphatase and tensin homolog (PTEN).[1]
We demonstrated that Akt[1] 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
K-RAS mutated in codon as well as in codon stimulates autocrine production of EGFR ligands and enhances basal activation of the PI3K/Akt pathway.[5,6]
Summary
The major mechanisms that lead to a constitutive activation of the PI3K/Akt pathway are mutations and overexpression of upstream receptor tyrosine kinases such as erbB family members, activating mutations of PIK3CA or RAS and the loss of tumor suppressor protein phosphatase and tensin homolog (PTEN).[1]. Akt isoforms have a N-terminal PH (pleckstrin homology) domain and a kinase domain, which are separated by a 39-amino-acid hinge region.[2] The PH domains are approx. 60% identical and the kinase domains are more than 85% identical.[3] Catalytically active Akt regulates the function of numerous substrates involved in cell survival, growth, proliferation, metabolism and protein synthesis (reviewed in Manning, Cantley[4]). K-RAS mutated in codon as well as in codon stimulates autocrine production of EGFR ligands and enhances basal activation of the PI3K/Akt pathway.[5,6] Likewise, K-RAS mutation leads to enhanced cell proliferation and tumor cell clonogenicity.[6]
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