GSK3? 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 3? (GSK3?) phosphorylation consensus site, we investigated the role of GSK3? in the regulation of KLF6 function. Based on transient transfection, GSK3? augments the transactivation of a p21 promoter luciferase by KLF6. Reciprocal co-immunoprecipitation of hemagglutinin (HA)-GSK3? and Flag-KLF6 validated the interaction between these two proteins. KLF6 phosphorylation is augmented in the presence of GSK3? based on in vitro and in vivo (32)P 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. GSK3? augmented the transactivation by wild-type KLF6, but not KLF6-4A, towards the p21 promoter, and increased p21 protein. Functionally, GSK3? enhanced KLF6-mediated growth suppression, which was abrogated by the KLF6-4A phosphomutant. These data establish that GSK3? 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.

SUBMITTER: Lang UE 

PROVIDER: S-EPMC3892988 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature