title: 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 (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. 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.
authors.affiliation: Division of Liver Diseases, Mount Sinai School of Medicine, New York, NY, USA.
authors.forename: U E
authors.initials: UE
authors.lastname: Lang
authors.forename: P
authors.initials: P
authors.lastname: Kocabayoglu
authors.forename: G Z
authors.initials: GZ
authors.lastname: Cheng
authors.forename: Z
authors.initials: Z
authors.lastname: Ghiassi-Nejad
authors.forename: U
authors.initials: U
authors.lastname: Muñoz
authors.forename: D
authors.initials: D
authors.lastname: Vetter
authors.forename: D A
authors.initials: DA
authors.lastname: Eckstein
authors.forename: R A
authors.initials: RA
authors.lastname: Hannivoort
authors.forename: M J
authors.initials: MJ
authors.lastname: Walsh
authors.forename: S L
authors.initials: SL
authors.lastname: Friedman
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D016384:Consensus Sequence; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics* / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D055550:Protein Stability; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; C105465:KLF6 protein, human; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3