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AKT-mediated phosphorylation enhances protein stability and transcription activity of ZNF322A to promote lung cancer progression

Abstract

ZNF322A is an oncogenic zinc-finger transcription factor. Our published results show that ZNF322A positively regulates transcription of alpha-adducin (ADD1) and cyclin D1 (CCND1) to promote tumorgenicity of lung cancer. However, the upstream regulatory mechanisms of ZNF322A protein function remain elusive. Here, we demonstrate that AKT could phosphorylate ZNF322A by in vitro kinase assay and cell-based mass spectrometry analysis. Overexpression of AKT promoted ZNF322A protein stability and transcriptional activity, whereas these effects were inhibited by knockdown of AKT or treating with AKT inhibitor. We studied AKT-mediated phosphorylation sites, viz. Thr-150, Ser-224, Thr-234, and Thr-262. ZNF322A phosphorylation at Thr-262 by AKT promoted ZNF322A protein stability thus increased ADD1 promoter activity. Interestingly, phosphorylation at Thr-150, Ser-224, and Thr-234 enhanced transcription activity without affecting protein stability of ZNF322A. Chromatin immunoprecipitation and DNA affinity precipitation assays showed that ZNF322A phosphorylation defective mutants Thr-150A, Ser-224A, and Thr-234A attenuated chromatin binding and DNA binding affinity to ADD1 and CCND1 promoters compared with wild-type ZNF322A. Furthermore, AKT-mediated Thr-150, Ser-224, Thr-234, and Thr-262 phosphorylation promoted lung cancer cell growth and metastasis in vitro and in vivo. Clinically, expression of phosphorylated ZNF322A (p-ZNF) correlated with actively phosphorylated AKT (p-AKT) in tumor specimens from 150 lung cancer patients. Multivariate Cox regression analysis indicated that combined p-AKT and p-ZNF expression profile was an independent factor to predict the clinical outcome in lung cancer patients. Our results reveal a new mechanism of AKT signaling in promoting ZNF322A protein stability and transcriptional activity in lung cancer cell, xenograft, and clinical models.

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Acknowledgements

We are grateful for the support from the Human Biobank, Research Center of Clinical Medicine, National Cheng Kung University Hospital. We thank Mass Spectrometry Facilities of National Cheng Kung University for MS analysis. This work is supported by the Taiwan Ministry of Science and Technology Grant MOST106-2320-B-006-071-MY3.

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YCW conceived the project. SYL, IYK and YCW designed the experiments. SYL, IYK, YTC, PCL, YFL and HYW conducted the experiments. WWL provided lung cancer patient specimens.

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Correspondence to Yi-Ching Wang.

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Liao, SY., Kuo, IY., Chen, YT. et al. AKT-mediated phosphorylation enhances protein stability and transcription activity of ZNF322A to promote lung cancer progression. Oncogene 38, 6723–6736 (2019). https://doi.org/10.1038/s41388-019-0928-x

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