GSK3-SCFFBXW7 mediated phosphorylation and ubiquitination of IRF1 are required for its transcription-dependent turnover

Alexander J. Garvin; Ahmed H.A. Khalaf; Alessandro Rettino; Jerome Xicluna; Laura Butler; Joanna R. Morris; David M. Heery; Nicole M. Clarke

doi:10.1093/nar/gkz163

GSK3-SCF^FBXW7 mediated phosphorylation and ubiquitination of IRF1 are required for its transcription-dependent turnover

Alexander J. Garvin, Ahmed H.A. Khalaf, Alessandro Rettino, Jerome Xicluna, Laura Butler, Joanna R. Morris, David M. Heery^*, Nicole M. Clarke

^*Corresponding author for this work

Cancer and Genomic Sciences

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

11 Citations (Scopus)

Abstract

IRF1 (Interferon Regulatory Factor-1) is the prototype of the IRF family of DNA binding transcription factors. IRF1 protein expression is regulated by transient up-regulation in response to external stimuli followed by rapid degradation via the ubiquitin-proteasome system. Here we report that DNA bound IRF1 turnover is promoted by GSK3 (Glycogen Synthase Kinase 3) via phosphorylation of the T181 residue which generates a phosphodegron for the SCF (Skp-Cul-Fbox) ubiquitin E3-ligase receptor protein Fbxw7 (F-box/WD40 7). This regulated turnover is essential for IRF1 activity, as mutation of T181 results in an improperly stabilized protein that accumulates at target promoters but fails to induce RNA-Pol-II elongation and subsequent transcription of target genes. Consequently, the anti-proliferative activity of IRF1 is lost in cell lines expressing T181A mutant. Further, cell lines with dysfunctional Fbxw7 are less sensitive to IRF1 overexpression, suggesting an important co-activator function for this ligase complex. As T181 phosphorylation requires both DNA binding and RNA-Pol-II elongation, we propose that this event acts to clear 'spent' molecules of IRF1 from transcriptionally engaged target promoters.

Original language	English
Pages (from-to)	4476-4494
Number of pages	19
Journal	Nucleic Acids Research
Volume	47
Issue number	9
DOIs	https://doi.org/10.1093/nar/gkz163
Publication status	Published - 21 May 2019

Bibliographical note

Funding Information:
N.M.C, A.R. and J.X. were supported by grants from Cancer Research UK [grant number C24478/A9004] and Biotechnology and Biological Sciences Research Council [grant number BB/F000340/1] awarded to N.M.C.;A.J.G., L.B. and J.R.M. were supported by grants from Breast Cancer Campaign [grant number 2006NovPHD13] Cancer Research UK [grant number C8820/A19062] and the Wellcome Trust [grant number Z06343/Z/17/Z]; D.M.H. was supported by Cancer Research UK [grant number C1506/A11643]. A.J.G. was supported by a PhD stipend from the School of Pharmacy; A.H.A.K. was supported by the School of Pharmacy and a Qalaa Holdings (Egypt) Scholarship; Funding for open access charge: School of Pharmacy, University of Nottingham. Conflict of interest statement. None declared.

Publisher Copyright:
© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

ASJC Scopus subject areas

Genetics

Access to Document

10.1093/nar/gkz163

Cite this

@article{9a133355f84544f3bdec355e586ea974,

title = "GSK3-SCFFBXW7 mediated phosphorylation and ubiquitination of IRF1 are required for its transcription-dependent turnover",

abstract = "IRF1 (Interferon Regulatory Factor-1) is the prototype of the IRF family of DNA binding transcription factors. IRF1 protein expression is regulated by transient up-regulation in response to external stimuli followed by rapid degradation via the ubiquitin-proteasome system. Here we report that DNA bound IRF1 turnover is promoted by GSK3 (Glycogen Synthase Kinase 3) via phosphorylation of the T181 residue which generates a phosphodegron for the SCF (Skp-Cul-Fbox) ubiquitin E3-ligase receptor protein Fbxw7 (F-box/WD40 7). This regulated turnover is essential for IRF1 activity, as mutation of T181 results in an improperly stabilized protein that accumulates at target promoters but fails to induce RNA-Pol-II elongation and subsequent transcription of target genes. Consequently, the anti-proliferative activity of IRF1 is lost in cell lines expressing T181A mutant. Further, cell lines with dysfunctional Fbxw7 are less sensitive to IRF1 overexpression, suggesting an important co-activator function for this ligase complex. As T181 phosphorylation requires both DNA binding and RNA-Pol-II elongation, we propose that this event acts to clear 'spent' molecules of IRF1 from transcriptionally engaged target promoters.",

author = "Garvin, {Alexander J.} and Khalaf, {Ahmed H.A.} and Alessandro Rettino and Jerome Xicluna and Laura Butler and Morris, {Joanna R.} and Heery, {David M.} and Clarke, {Nicole M.}",

note = "Funding Information: N.M.C, A.R. and J.X. were supported by grants from Cancer Research UK [grant number C24478/A9004] and Biotechnology and Biological Sciences Research Council [grant number BB/F000340/1] awarded to N.M.C.;A.J.G., L.B. and J.R.M. were supported by grants from Breast Cancer Campaign [grant number 2006NovPHD13] Cancer Research UK [grant number C8820/A19062] and the Wellcome Trust [grant number Z06343/Z/17/Z]; D.M.H. was supported by Cancer Research UK [grant number C1506/A11643]. A.J.G. was supported by a PhD stipend from the School of Pharmacy; A.H.A.K. was supported by the School of Pharmacy and a Qalaa Holdings (Egypt) Scholarship; Funding for open access charge: School of Pharmacy, University of Nottingham. Conflict of interest statement. None declared. Publisher Copyright: {\textcopyright} The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.",

year = "2019",

month = may,

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doi = "10.1093/nar/gkz163",

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T1 - GSK3-SCFFBXW7 mediated phosphorylation and ubiquitination of IRF1 are required for its transcription-dependent turnover

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AU - Xicluna, Jerome

AU - Butler, Laura

AU - Morris, Joanna R.

AU - Heery, David M.

AU - Clarke, Nicole M.

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PY - 2019/5/21

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N2 - IRF1 (Interferon Regulatory Factor-1) is the prototype of the IRF family of DNA binding transcription factors. IRF1 protein expression is regulated by transient up-regulation in response to external stimuli followed by rapid degradation via the ubiquitin-proteasome system. Here we report that DNA bound IRF1 turnover is promoted by GSK3 (Glycogen Synthase Kinase 3) via phosphorylation of the T181 residue which generates a phosphodegron for the SCF (Skp-Cul-Fbox) ubiquitin E3-ligase receptor protein Fbxw7 (F-box/WD40 7). This regulated turnover is essential for IRF1 activity, as mutation of T181 results in an improperly stabilized protein that accumulates at target promoters but fails to induce RNA-Pol-II elongation and subsequent transcription of target genes. Consequently, the anti-proliferative activity of IRF1 is lost in cell lines expressing T181A mutant. Further, cell lines with dysfunctional Fbxw7 are less sensitive to IRF1 overexpression, suggesting an important co-activator function for this ligase complex. As T181 phosphorylation requires both DNA binding and RNA-Pol-II elongation, we propose that this event acts to clear 'spent' molecules of IRF1 from transcriptionally engaged target promoters.

AB - IRF1 (Interferon Regulatory Factor-1) is the prototype of the IRF family of DNA binding transcription factors. IRF1 protein expression is regulated by transient up-regulation in response to external stimuli followed by rapid degradation via the ubiquitin-proteasome system. Here we report that DNA bound IRF1 turnover is promoted by GSK3 (Glycogen Synthase Kinase 3) via phosphorylation of the T181 residue which generates a phosphodegron for the SCF (Skp-Cul-Fbox) ubiquitin E3-ligase receptor protein Fbxw7 (F-box/WD40 7). This regulated turnover is essential for IRF1 activity, as mutation of T181 results in an improperly stabilized protein that accumulates at target promoters but fails to induce RNA-Pol-II elongation and subsequent transcription of target genes. Consequently, the anti-proliferative activity of IRF1 is lost in cell lines expressing T181A mutant. Further, cell lines with dysfunctional Fbxw7 are less sensitive to IRF1 overexpression, suggesting an important co-activator function for this ligase complex. As T181 phosphorylation requires both DNA binding and RNA-Pol-II elongation, we propose that this event acts to clear 'spent' molecules of IRF1 from transcriptionally engaged target promoters.

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