GSK3β-SCFFBXW7α mediated phosphorylation and ubiquitination of IRF1 are required for its transcription-dependent turnover.

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

doi:10.1093/nar/gkz163

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 GSK3β (Glycogen Synthase Kinase 3β) via phosphorylation of the T181 residue which generates a phosphodegron for the SCF (Skp-Cul-Fbox) ubiquitin E3-ligase receptor protein Fbxw7α (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.

Highlights

IRF1 is a transcription factor essential for regulating a number of cellular responses including, immunity, apoptosis and DNA repair [1,2,3,4,5]
In a search for potential IRF1 phosphorylation sites we focused on a sequence within the IRF1 transactivation domain (TAD) that shares similarity to a subset of consensus GSK3 target sites
In cells transfected with FLAGIRF1 only, we were able to detect phosphorylation of IRF1 suggesting that endogenous GSK3 kinases or other kinases can target this residue

Summary

Introduction

IRF1 is a transcription factor essential for regulating a number of cellular responses including, immunity, apoptosis and DNA repair [1,2,3,4,5]. Phosphorylation of a cluster of residues in the C terminus by casein kinase II may be required for activity as mutation of these residues reduces reporter activity [6]. IRF1 is phosphorylated on Y109 in the DBD (DNA binding domain). This modification plays a role in dimerization with IRF8 and transcriptional activity [8]. Our understanding of how these modifications regulate IRF1 activity is still poorly understood

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Conclusion