DNA-PKcs kinase activity stabilizes the transcription factor Egr1 in activated immune cells

Zachary J Waldrip,Lyle Burdine,David K Harrison,Ana Clara Azevedo-Pouly,Aaron J Storey,Olivia G Moffett,Samuel G Mackintosh,Marie Schluterman Burdine

doi:10.1016/j.jbc.2021.101209

Abstract

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is known primarily for its function in DNA double-stranded break repair and nonhomologous end joining (NHEJ). However, DNA-PKcs also has a critical yet undefined role in immunity impacting both myeloid and lymphoid cell lineages spurring interest in targeting DNA-PKcs for therapeutic strategies in immune-related diseases. To gain insight into the function of DNA-PKcs within immune cells, we performed a quantitative phosphoproteomic screen in T cells to identify phosphorylation targets of DNA-PKcs. Our results indicate that DNA-PKcs phosphorylates the transcription factor Egr1 (early growth response protein 1) at serine 301. Expression of Egr1 is induced early upon T cell activation and dictates T cell response by modulating expression of cytokines and key costimulatory molecules such as IL (interleukin) 2, IL6, IFNγ, and NFκB. Inhibition of DNA-PKcs by treatment with a DNA-PKcs specific inhibitor NU7441 or shRNA knockdown increased proteasomal degradation of Egr1. Mutation of serine 301 to alanine via CRISPR-Cas9 reduced EGR1 protein expression and decreased Egr1-dependent transcription of IL2 in activated T cells. Our findings identify DNA-PKcs as a critical intermediary link between T cell activation and T cell fate and a novel phosphosite involved in regulating Egr1 activity.

Highlights

This emphasizes a function for DNA-PKcs in the mature immune system that is yet to be clearly defined
immediate early response gene (IEG) genes such as early growth response protein 1 (Egr1) are transcribed within minutes of T cell receptor (TCR) stimulation to rapidly turn on transcription of genes needed for immune cell function [17, 18]
We analyzed for differentially phosphorylated proteins between stimulated cells and stimulated cells pretreated with NU7441

Summary

Introduction

This emphasizes a function for DNA-PKcs in the mature immune system that is yet to be clearly defined. Ferguson et al determined that following viral DNA detection, DNA-PKcs drives activation of the innate immune response by directly binding the transcription factor interferon (IFN) regulator factor-3 (Irf-3), and promoting its translocation into the nucleus to induce cytokine gene expression [13]. IEG genes such as Egr are transcribed within minutes of TCR stimulation to rapidly turn on transcription of genes needed for immune cell function [17, 18] This includes genes such as NFKB, ELK, and NFAT, which can be activated quickly through degradation of inhibitors or through posttranslational modifications via MAP kinase cascades [19,20,21]. Regulation of early signaling effectors such as Egr suggests that DNA-PKcs functions as a critical link between TCR stimulation and subsequent gene transcription capable of guiding T cell signaling toward specific outcomes

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Conclusion