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

U. E. Lang; P. Kocabayoglu; G. Z. Cheng; Z. Ghiassi-Nejad; U. Muñoz; D. Vetter; D. A. Eckstein; R. A. Hannivoort; M. J. Walsh; S. L. Friedman

doi:10.1038/onc.2012.457

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

U. E. Lang, P. Kocabayoglu, G. Z. Cheng, Z. Ghiassi-Nejad, U. Muñoz, D. Vetter, D. A. Eckstein, R. A. Hannivoort, M. J. Walsh, S. L. Friedman

Research output: Contribution to journal › Article › peer-review

44 Scopus citations

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.

Original language	English
Pages (from-to)	4557-4564
Number of pages	8
Journal	Oncogene
Volume	32
Issue number	38
DOIs	https://doi.org/10.1038/onc.2012.457
State	Published - 19 Sep 2013

Keywords

GSK3β
KLF6
p21
phosphorylation

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title = "GSK3β phosphorylation of the KLF6 tumor suppressor promotes its transactivation of p21",

abstract = "KLF6, a ubiquitously expressed Kr{\"u}ppel-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.",

keywords = "GSK3β, KLF6, p21, phosphorylation",

author = "Lang, {U. E.} and P. Kocabayoglu and Cheng, {G. Z.} and Z. Ghiassi-Nejad and U. Mu{\~n}oz and D. Vetter and Eckstein, {D. A.} and Hannivoort, {R. A.} and Walsh, {M. J.} and Friedman, {S. L.}",

note = "Funding Information: 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 GSK3b 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).",

year = "2013",

month = sep,

day = "19",

doi = "10.1038/onc.2012.457",

language = "English",

volume = "32",

pages = "4557--4564",

journal = "Oncogene",

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publisher = "Springer Nature",

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TY - JOUR

T1 - GSK3β phosphorylation of the KLF6 tumor suppressor promotes its transactivation of p21

AU - Lang, U. E.

AU - Kocabayoglu, P.

AU - Cheng, G. Z.

AU - Ghiassi-Nejad, Z.

AU - Muñoz, U.

AU - Vetter, D.

AU - Eckstein, D. A.

AU - Hannivoort, R. A.

AU - Walsh, M. J.

AU - Friedman, S. L.

N1 - Funding Information: 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 GSK3b 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).

PY - 2013/9/19

Y1 - 2013/9/19

N2 - 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.

AB - 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.

KW - GSK3β

KW - KLF6

KW - p21

KW - phosphorylation

UR - http://www.scopus.com/inward/record.url?scp=84884596944&partnerID=8YFLogxK

U2 - 10.1038/onc.2012.457

DO - 10.1038/onc.2012.457

M3 - Article

C2 - 23085750

AN - SCOPUS:84884596944

SN - 0950-9232

VL - 32

SP - 4557

EP - 4564

JO - Oncogene

JF - Oncogene

IS - 38

ER -

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

Abstract

Keywords

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