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

U E Lang1, P Kocabayoglu, G Z Cheng

  • 1Division of Liver Diseases, Mount Sinai School of Medicine, New York, NY, USA.

Oncogene
|October 23, 2012
PubMed

Insights

Glycogen synthase kinase 3β (GSK3β) directly phosphorylates Krüppel-like factor 6 (KLF6), enhancing its tumor suppressor activity by increasing p21 expression. This mechanism explains how KLF6 mutations promote cancer growth.

Area of Science:

  • Molecular Biology
  • Cancer Biology
  • Biochemistry

Background:

  • Krüppel-like factor 6 (KLF6) is a tumor suppressor frequently inactivated in human cancers.
  • KLF6 suppresses tumor growth partly by activating p21 expression in a p53-independent manner.
  • Cancer-associated KLF6 mutations often affect a glycogen synthase kinase 3β (GSK3β) phosphorylation site, suggesting a role for GSK3β.

Purpose of the Study:

  • To investigate the role of GSK3β in regulating KLF6 function and its tumor suppressor activity.
  • To determine if GSK3β directly interacts with and phosphorylates KLF6.
  • To elucidate the functional consequences of GSK3β-mediated KLF6 phosphorylation on p21 expression and cell growth.

Main Methods:

  • Transient transfection assays to assess KLF6-mediated p21 promoter activity.
  • Co-immunoprecipitation to validate KLF6-GSK3β interaction.
  • In vitro and in vivo phosphorylation assays (32P incorporation) to confirm KLF6 phosphorylation by GSK3β.
  • Site-directed mutagenesis to create a non-phosphorylatable KLF6 mutant (KLF6-4A).
  • Western blotting to detect KLF6 isoforms and p21 protein levels.
  • Cell growth assays to evaluate the functional impact of KLF6 phosphorylation.

Main Results:

  • GSK3β enhances KLF6's ability to activate the p21 promoter.
  • KLF6 and GSK3β physically interact.
  • GSK3β promotes KLF6 phosphorylation, evidenced by increased 32P incorporation and the loss of a higher molecular weight isoform in the KLF6-4A mutant.
  • GSK3β-mediated KLF6 phosphorylation is required for robust p21 induction and KLF6's growth-suppressive function.
  • The KLF6-4A mutant fails to be augmented by GSK3β, indicating the importance of phosphorylation at these sites.

Conclusions:

  • GSK3β directly phosphorylates KLF6 at specific sites.
  • This phosphorylation event augments KLF6's transcriptional activity towards p21, leading to enhanced growth suppression.
  • The findings reveal a novel regulatory mechanism for KLF6 function and suggest that impaired GSK3β-mediated phosphorylation of KLF6 may contribute to cancer development.

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