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GSK3β phosphorylation of the KLF6 tumor suppressor promotes its transactivation of p21

Abstract

KLF6, a ubiquitously expressed Krüppel-like transcription factor, is frequently inactivated in human cancer and has significant roles in cellular proliferation, apoptosis, differentiation and development. A key mechanism of KLF6-mediated growth suppression is through p53-independent transactivation of p21. Several cancer-derived KLF6 mutants lead to the loss of p21-mediated growth suppression through an unknown mechanism. Because several colorectal cancer and hepatocellular carcinoma-derived KLF6 mutations affect a glycogen synthase kinase 3β (GSK3β) phosphorylation consensus site, we investigated the role of GSK3β in the regulation of KLF6 function. Based on transient transfection, GSK3β augments the transactivation of a p21 promoter luciferase by KLF6. Reciprocal co-immunoprecipitation of hemagglutinin (HA)-GSK3β and Flag-KLF6 validated the interaction between these two proteins. KLF6 phosphorylation is augmented in the presence of GSK3β based on in vitro and in vivo 32P incorporation assays. Site-directed mutagenesis of the candidate phosphorylation sites to alanines (‘KLF6-4A’ phosphomutant) eliminated a higher molecular weight phosphorylated isoform of KLF6 based on western blot. GSK3β augmented the transactivation by wild-type KLF6, but not KLF6-4A, towards the p21 promoter, and increased p21 protein. Functionally, GSK3β enhanced KLF6-mediated growth suppression, which was abrogated by the KLF6-4A phosphomutant. These data establish that GSK3β directly phosphorylates KLF6, which augments its induction of p21 and resultant growth suppression. This interaction may account for the growth-promoting effects of cancer-derived KLF6 mutants that lack tumor suppressor activity.

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Acknowledgements

We thank Dr Ming-Ming Zhou at the Mount Sinai School of Medicine for his expert advice in protein purification of GST-KLF6. Dr Jin Q Cheng at H Lee Moffitt Cancer Center shared all GSK3β plasmids and contributed technical support for the in vivo kinase assays. We acknowledge funding support from the NIH (to SLF: RO1DK37340, RO1DK 56621, 1K05 AA018408, 1P20 AA017067; to MJW: 1RO1CA154809-01; to UEL and ZGN: T32GM00728), Deutsche Forschungsgemeinschaft (DFG) (to PK) KO 4086/1-1, Graduate School for Drug Exploration (GUIDE) and the Stichting Nicholaas Muleriusfonds, Groningen, the Netherlands (to RAH), the Ellison Medical Foundation Senior Scholar Award in Aging (SS-AG2482-1) (to MJW), the Swiss National Fund (SNF) (to DV) and the Alonso Martin Escudero foundation (to UM).

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Correspondence to S L Friedman.

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Lang, U., Kocabayoglu, P., Cheng, G. et al. GSK3β phosphorylation of the KLF6 tumor suppressor promotes its transactivation of p21. Oncogene 32, 4557–4564 (2013). https://doi.org/10.1038/onc.2012.457

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