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

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. 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. The canonical function for DNA-dependent protein kinase catalytic subunit or DNA-PKcs is in the sensing and repair of DNA double-strand breaks (DSB) through nonhomologous end joining (NHEJ). However, all vertebrates harboring kinase loss of function mutations in DNA-PKcs present with a severe immunodeficient phenotype with defects in antibody production and impaired B and T cell maturation (1Davis A.J. Chen B.P. Chen D.J. DNA-PK: A dynamic enzyme in a versatile DSB repair pathway.DNA Repair (Amst). 2014; 17: 21-29Crossref PubMed Scopus (224) Google Scholar, 2Pannunzio N.R. Watanabe G. Lieber M.R. Nonhomologous DNA end-joining for repair of DNA double-strand breaks.J. Biol. Chem. 2018; 293: 10512-10523Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 3Taccioli G.E. Amatucci A.G. Beamish H.J. Gell D. Xiang X.H. Torres Arzayus M.I. Priestley A. Jackson S.P. Marshak Rothstein A. Jeggo P.A. Herrera V.L. Targeted disruption of the catalytic subunit of the DNA-PK gene in mice confers severe combined immunodeficiency and radiosensitivity.Immunity. 1998; 9: 355-366Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 4van der Burg M. van Dongen J.J. van Gent D.C. DNA-PKcs deficiency in human: Long predicted, finally found.Curr. Opin. Allergy Clin. Immunol. 2009; 9: 503-509Crossref PubMed Scopus (46) Google Scholar). These defects have been primarily attributed to the function of DNA-PKcs in V(D)J recombination, which is required for antibody and receptor diversity in adaptive immune cells (5Smider V. Chu G. The end-joining reaction in V(D)J recombination.Semin. Immunol. 1997; 9: 189-197Crossref PubMed Scopus (53) Google Scholar). Interestingly, this enzyme is robustly expressed in mature lymphocytes and consistently activated by various lymphocyte stimulants (6Kim Wiese A. Schluterman Burdine M. Turnage R.H. Tackett A.J. Burdine L.J. DNA-PKcs controls calcineurin mediated IL-2 production in T lymphocytes.PLoS One. 2017; 12e0181608Crossref PubMed Scopus (2) Google Scholar, 7Ghonim M.A. Pyakurel K. Ju J. Rodriguez P.C. Lammi M.R. Davis C. Abughazleh M.Q. Mansy M.S. Naura A.S. Boulares A.H. DNA-dependent protein kinase inhibition blocks asthma in mice and modulates human endothelial and CD4+ T-cell function without causing severe combined immunodeficiency.J. Allergy Clin. Immunol. 2015; 135: 425-440Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). This emphasizes a function for DNA-PKcs in the mature immune system that is yet to be clearly defined.T cells are a key component of the adaptive immune response providing long-term protection against evading pathogens. Uncontrolled or defective T cell activity, however, can have deleterious effects including transplant graft rejection, graft versus host disease, and a plethora of autoimmune diseases (8Dornmair K. Goebels N. Weltzien H.U. Wekerle H. Hohlfeld R. T-cell-mediated autoimmunity: Novel techniques to characterize autoreactive T-cell receptors.Am. J. Pathol. 2003; 163: 1215-1226Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 9Issa F. Schiopu A. Wood K.J. Role of T cells in graft rejection and transplantation tolerance.Expert Rev. Clin. Immunol. 2010; 6: 155-169Crossref PubMed Scopus (78) Google Scholar). Therefore, understanding molecular mechanisms that regulate T cell activity is critical for the development of novel therapeutics to prevent/treat T cell-mediated disorders. T cell receptor (TCR) activation induces signaling cascades that regulate T cell proliferation, survival, and differentiation. The end result is widely dependent on the activation of transcription factors that promote expression of cytokines and chemokines that, depending on the level and combination, can have varying effects on T cell response. For instance, graded expression of the T-box transcription factor T-bet in naïve CD4+ T cells coordinates helper (Th) 1 or T follicular helper (Tfh) cell differentiation with higher levels driving a Th1 cell fate (10Yu F. Sharma S. Edwards J. Feigenbaum L. Zhu J. Dynamic expression of transcription factors T-bet and GATA-3 by regulatory T cells maintains immunotolerance.Nat. Immunol. 2015; 16: 197-206Crossref PubMed Scopus (165) Google Scholar). It is becoming clear that DNA-PKcs strongly influences T cell activity, as well as other immune cells, through regulation of transcription factor expression. In CD4+ T cells, following TCR activation, DNA-PKcs regulates expression of both T-bet and Gata3 highlighting it as a master regulator of Th1 and Th2 differentiation (7Ghonim M.A. Pyakurel K. Ju J. Rodriguez P.C. Lammi M.R. Davis C. Abughazleh M.Q. Mansy M.S. Naura A.S. Boulares A.H. DNA-dependent protein kinase inhibition blocks asthma in mice and modulates human endothelial and CD4+ T-cell function without causing severe combined immunodeficiency.J. Allergy Clin. Immunol. 2015; 135: 425-440Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 11Mishra A. Brown A.L. Yao X. Yang S. Park S.J. Liu C. Dagur P.K. McCoy J.P. Keeran K.J. Nugent G.Z. Jeffries K.R. Qu X. Yu Z.X. Levine S.J. Chung J.H. Dendritic cells induce Th2-mediated airway inflammatory responses to house dust mite via DNA-dependent protein kinase.Nat. Commun. 2015; 6: 6224Crossref PubMed Scopus (24) Google Scholar). Our laboratory recently reported that DNA-PKcs also controls expression of the p65 subunit of NF-κB in activated T cells, and loss of DNA-PKcs activity significantly reduces expression of NF-κB target genes including Interleukin (IL)-6 (12Harrison D.K. Waldrip Z.J. Burdine L. Shalin S.C. Burdine M.S. DNA-PKcs Inhibition Extends Allogeneic Skin Graft Survival.Transplantation. 2020; 105: 540-549Crossref Scopus (2) Google Scholar). 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 (13Ferguson B.J. Mansur D.S. Peters N.E. Ren H. Smith G.L. DNA-PK is a DNA sensor for IRF-3-dependent innate immunity.Elife. 2012; 1e00047Crossref PubMed Scopus (266) Google Scholar). Similarly, our studies indicate that DNA-PKcs plays a pivotal role in the calcineurin-mediated translocation of NFAT to the nucleus. Inhibition of DNA-PKcs blocked calcineurin activity, thereby preventing the translocation of NFAT to the nucleus and expression of cytokine IL2 (6Kim Wiese A. Schluterman Burdine M. Turnage R.H. Tackett A.J. Burdine L.J. DNA-PKcs controls calcineurin mediated IL-2 production in T lymphocytes.PLoS One. 2017; 12e0181608Crossref PubMed Scopus (2) Google Scholar). Herein, we report that DNA-PKcs also regulates expression of the immediate early response gene (IEG) Egr1 (early growth response 1), a transcription factor critical for cytokine production (14Shin H.J. Lee J.B. Park S.H. Chang J. Lee C.W. T-bet expression is regulated by EGR1-mediated signaling in activated T cells.Clin. Immunol. 2009; 131: 385-394Crossref PubMed Scopus (31) Google Scholar, 15Li B. Power M.R. Lin T.J. De novo synthesis of early growth response factor-1 is required for the full responsiveness of mast cells to produce TNF and IL-13 by IgE and antigen stimulation.Blood. 2006; 107: 2814-2820Crossref PubMed Scopus (37) Google Scholar, 16Pang Z. Raudonis R. McCormick C. Cheng Z. Early growth response 1 deficiency protects the host against pseudomonas aeruginosa lung infection.Infect. Immun. 2019; 88e00678-19Crossref PubMed Scopus (8) Google Scholar). IEG genes such as Egr1 are transcribed within minutes of TCR stimulation to rapidly turn on transcription of genes needed for immune cell function (17Mortlock S.A. Wei J. Williamson P. T-cell activation and early gene response in dogs.PLoS One. 2015; 10e0121169Crossref PubMed Scopus (7) Google Scholar, 18Lee S.M. Vasishtha M. Prywes R. Activation and repression of cellular immediate early genes by serum response factor cofactors.J. Biol. Chem. 2010; 285: 22036-22049Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). 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 (19Oeckinghaus A. Ghosh S. The NF-kappaB family of transcription factors and its regulation.Cold Spring Harb. Perspect. Biol. 2009; 1a000034Crossref PubMed Scopus (1607) Google Scholar, 20Besnard A. Galan-Rodriguez B. Vanhoutte P. Caboche J. Elk-1 a transcription factor with multiple facets in the brain.Front. Neurosci. 2011; 5: 35Crossref PubMed Scopus (129) Google Scholar, 21Lee J.U. Kim L.K. Choi J.M. Revisiting the concept of targeting NFAT to control T cell immunity and autoimmune diseases.Front. Immunol. 2018; 9: 2747Crossref PubMed Scopus (79) Google Scholar). IEGs, therefore, are responsible for coaxing T cells down specific response pathways predetermined by the type of immunogenic stimulus encountered. We identified Egr1 to be a phosphorylation target of DNA-PKcs. Inhibition of DNA-PKcs or mutation of serine 301 of Egr1 resulted in significant downregulation of Egr1 protein leading to reduced secretion of IL2. Regulation of early signaling effectors such as Egr1 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.ResultsEgr1 is phosphorylated by DNA-PKcs following stimulation of T cellsGiven the diversity of signaling events in which DNA-PKcs is involved, we sought to identify potential DNA-PKcs phosphorylation targets involved in T cell activation. To accomplish this goal, we performed a quantitative proteomic mass spectrometry screen for phosphoproteins utilizing TMT (tandem mass tag, Thermo) technology. This was accomplished using human T cells (Jurkat) stimulated with phorbol myristate acetate and phytohemagglutinin (PMA and PHA, respectively) and treated with or without NU7441, a specific small-molecule kinase inhibitor of DNA-PKcs (22Zhao Y. Thomas H.D. Batey M.A. Cowell I.G. Richardson C.J. Griffin R.J. Calvert A.H. Newell D.R. Smith G.C. Curtin N.J. Preclinical evaluation of a potent novel DNA-dependent protein kinase inhibitor NU7441.Cancer Res. 2006; 66: 5354-5362Crossref PubMed Scopus (335) Google Scholar, 23Dong J. Ren Y. Zhang T. Wang Z. Ling C.C. Li G.C. He F. Wang C. Wen B. Inactivation of DNA-PK by knockdown DNA-PKcs or NU7441 impairs non-homologous end-joining of radiation-induced double strand break repair.Oncol. Rep. 2018; 39: 912-920PubMed Google Scholar). A phospho-TMT analysis combines affinity enrichment of phosphorylated proteins/peptides with quantitative TMT isobaric reagents; thus, allowing for a highly quantitative and extensive analysis of phosphorylation events in a cell. We analyzed for differentially phosphorylated proteins between stimulated cells and stimulated cells pretreated with NU7441. Reporter ion intensities of identified phosphosites were normalized against reporter ion intensities of the corresponding protein to account for changes in overall protein abundance. Figure 1 contains a list of all phosphoproteins with a fold change of >10. We identified phosphorylation of DNA-PKcs to be downregulated providing validity to our screen given that DNA-PKcs is known to autophosphorylate itself at numerous sites (24Uematsu N. Weterings E. Yano K. Morotomi-Yano K. Jakob B. Taucher-Scholz G. Mari P.O. van Gent D.C. Chen B.P. Chen D.J. Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks.J. Cell Biol. 2007; 177: 219-229Crossref PubMed Scopus (308) Google Scholar). One of the more prominent phosphopeptides mapped to the IEG transcription factor Egr1. The phosphopeptide shown in Figure 1 was approximately 30-fold more prevalent in the noninhibitor-treated sample. Analysis of this phosphorylation site revealed that it falls within the DNA-PKcs kinase recognition motif, SQD/E (25Bednarski J.J. Sleckman B.P. At the intersection of DNA damage and immune responses.Nat. Rev. Immunol. 2019; 19: 231-242Crossref PubMed Scopus (52) Google Scholar). To further substantiate the potential biological significance of this site, a sequence alignment of this peptide in vertebrates revealed a very high degree of conservation from zebrafish to humans (Fig. 2 and Table 1). As far as we know, specific phosphorylation of this serine residue (S301) has not previously been described.Figure 2Schematic of Egr1 protein highlighting the position of S301 and known domains. The S301 phosphorylation sites lies within the identified repressor domain of Egr1 (35Magee N. Zhang Y. Role of early growth response 1 in liver metabolism and liver cancer.Hepatoma Res. 2017; 3: 268-277Crossref PubMed Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Egr1 S301 phosphorylation site detected by mass spectrometry is highly conserved in vertebrate animalsSpeciesTryptic peptideH. sapiensAFATQSGSQDLKP. troglodytesAFATQSGSQDLKM. mulattaAFATQSGSQDLKC. lupus familiarisAFATQSGSQDLKB. taurusAFATQSGSQDLKM. musculusAFATQSGSQDLKR. norvegicusAFATQSGSQDLKG. gallusAFATQTGSQELKX. tropicalisAFATQT- SQDLKD. rerioAFATQTGSQDLKhttps://www.ncbi.nlm.nih.gov/homologene/56394 Open table in a new tab To further confirm that DNA-PKcs phosphorylates Egr1, we performed an in vitro DNA-PKcs luminescent kinase assay using an Egr1 fragment containing the S301 amino acid. Significant kinase activity was detected when DNA-PKcs was incubated with the Egr1 fragment (Fig. 3). Additionally, we analyzed the phosphorylated Egr1 peptide by mass spectrometry following the in vitro kinase assay to verify DNA-PKcs phosphorylation of S301 within the Egr1 peptide. Figure S1 contains the annotated spectrum of the phosphorylated Egr1 peptide. Figure 4 highlights the MaxQuant intensity ratio of phosphorylated S301 to unphosphorylated S301 with or without DNA-PKcs. No phosphorylated S301 was detected in the absence of DNA-PKcs but was found highly phosphorylated in the presence of DNA-PKcs.Figure 3DNA-PKcs phosphorylates Egr1 in vitro. A luminescent in vitro kinase assay was performed to measure DNA-PKcs activity. A significant increase in kinase activity was detected when recombinant DNA-PKcs was incubated with an Egr1 peptide. Error bars = s.d. of the mean of technical replicates. ∗p < 0.001 no substrate vs. Egr1 substrate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Serine 301 of Egr1 is phosphorylated by DNA-PKcs in vitro. Phosphorylation of the Egr1 peptide following an in vitro kinase assay was analyzed by mass spectrometry. Intensities are presented as a ratio of phosphorylated S301/unphosphorylated S301. pS301 was not detected in the absence of DNA-PKcs. Error bars = S.D. p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Inhibition of DNA-PKcs kinase activity reduces Egr1 protein expressionWestern blotting revealed that Egr1 was highly induced upon stimulation (Fig. 5). However, Egr1 levels were markedly lower in NU7441-treated human T cells suggesting that DNA-PKcs kinase activity is required for Egr1 protein expression (Fig. 5A). To confirm inhibition of DNA-PKcs activity by NU7441 in this assay, we probed for phospho-AKT at S473, a target of DNA-PKcs (23Dong J. Ren Y. Zhang T. Wang Z. Ling C.C. Li G.C. He F. Wang C. Wen B. Inactivation of DNA-PK by knockdown DNA-PKcs or NU7441 impairs non-homologous end-joining of radiation-induced double strand break repair.Oncol. Rep. 2018; 39: 912-920PubMed Google Scholar). Phosphorylation of S473 was significantly lower in NU7441 treated samples. To validate that this result is due to specific downregulation of DNA-PKcs activity and not off target effects of the drug, we analyzed Egr1 expression patterns in total mouse splenocytes from wild-type (WT) or PRKDC (gene for DNA-PKcs) knockout mice (KO) treated with or without NU7441 (Fig. 5B) and in the embryonic kidney cell line HEK293 treated with shRNA to specifically knock down DNA-PKcs expression (Fig. 5C). Inhibition of DNA-PKcs kinase activity significantly reduced Egr1 protein expression in both additional cell lines indicating the robustness of this finding and confirming that this mechanism of regulation occurs in other cell lines that induce Egr1 expression to rapidly respond to cellular stimuli.Figure 5Protein and RNA expression patterns associated with Egr1 in multiple cell types. Egr1 expression pattern was analyzed by Western blotting in (A) Jurkat T cells, (B) total mouse splenocytes (WT indicates WT mouse and KO indicates PRKDC functional knockout mouse), and (C) HEK293 kidney cells, which were chemically stimulated for 3 h and treated with NU7441 as indicated. D, real-time qPCR analysis of EGR1 transcripts in Jurkat cells. Error bars represent standard error of the mean. NS indicates no significant difference. E, Jurkat cells were treated as in (A) with the addition of the proteasome inhibitor MG132 and Egr1 was detected by Western blot. Error bars = s.d. of the mean of biological replicates.View Large Image Figure ViewerDownload Hi-res image Download (PPT)EGR1 is known to be tightly regulated at the transcriptional level and, like many other T cell-responsive transcription factors, the gene is highly induced upon T cell receptor or phorbol ester stimulation. To determine whether an effect of NU7441 on EGR1 transcript levels explains the drop in Egr1 protein, qPCR was carried out in the presence or absence of NU7441. Transcript levels were unaffected (Fig. 5D) by the loss of DNA-PKcs kinase activity. To assay whether the reduction in Egr1 protein was a result of proteasomal degradation, we analyzed Egr1 levels in NU7441-treated T cells in the presence of the proteasome inhibitor MG132. Our results indicate that proteasomal inhibition is able to restore the drop in Egr1 levels observed in the presence of NU7441 (Fig. 5E).Phosphorylation of serine 301 of Egr1 is required for protein stabilityTo further analyze the functional relevance of S301 phosphorylation, plasmid-based FLAG-tagged Egr1 S301 mutants were expressed under the control of a constitutive promoter in HEK293 cells. An alanine mutant (S301A) as well as two phospho-mimetic mutants (S301D and S301E) were generated. Egr1 S301A protein levels were significantly decreased in contrast to both the WT protein (S301S) and the phospho-mimetic mutants S301D and S301E (Fig. 6A). In fact, the phospho-mimetic mutations appeared to enhance protein stability. Next, we generated an EGR1 knockout (EGR1Δ) as well as an endogenous S301A mutant in Jurkat T cells using CRISPR genome editing. This yielded a similar result where a single amino acid mutation to S301A to block phosphorylation resulted in a significantly reduced level of Egr1 protein despite normal transcription (Fig. 6B). Transcript levels of the S301A mutant gene were similar to WT EGR1 levels (Fig. S2). Additionally, we performed a cycloheximide chase assay to analyze the degradation of the Egr1 S301A mutant over time. This study indicated that the S301A degraded at a much faster rate than WT further validating the importance of this site to Egr1 protein stability (Fig. 6C).Figure 6Effect of Egr1 phosphorylation on protein stability. A, four plasmid-based variants of Egr1-3xFlag at amino acid 301, as indicated by the single-letter amino acid abbreviation (A, D, and E), were expressed in stimulated HEK293 cells. B, endogenous EGR1 S301A and knockout mutants along with the wild type S301S strain were generated in Jurkat cells using CRISPR. C, degradation of Egr1 or one of the Egr1 S301A variants over time following cycloheximide treatment and quantification of relative EGR1 protein levels from three independent experiments normalized to GAPDH. Δ = EGR1 CRISPR knockout, A = mutation generating S301A mutant (three separate clones are represented), S = WT EGR1. Error bars = standard error.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Loss of S301 phosphorylation abrogates production of IL2Egr1 is a transcriptional regulator of the cytokine IL2. Therefore, to validate the relevance of this phosphorylation site to immune cell function, we analyzed expression of IL2. We used our CRISPR-generated EGR1Δ cell line to determine if the reintroduction of the Egr1 S301 variants (S301S, S301A, S301D, S301E) via electroporated plasmids would rescue IL2 expression. We hypothesized that the unstable S301A mutant would not fully restore or rescue IL2 levels. EGR1Δ cells were electroporated with one of five plasmids and stimulated 48 h later using PMA and ionomycin for 6 h. IL2 levels in the media were determined by ELISA. EGR1Δ samples transfected with plasmids expressing WT Egr1 S301S or the mutant S301D or S301E more than doubled their IL2 production relative to samples receiving control plasmid (Fig. 7). In line with our findings, transfection of the S301A variant generated the lowest amount of IL2 rescue. IL2 levels from the cells expressing this variant were approximately 2.3-fold over the control, compared with 2.9–3.3 fold from cells expressing Egr1 S301S, S301D, or S301E.Figure 7Effect of Egr1 phosphorylation on IL2 expression. IL2 concentrations were measured by ELISA in Jurkat EGR1Δ cells transfected by electroporation with plasmids expressing the indicated variant of Egr1. Control indicates transfection with a plasmid containing GFP in place of EGR1. Variability is represented by standard deviation of four replicates. ∗ indicates p < 0.01 evaluated by t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionWhile DNA-PKcs is a well-known mediator of double-stranded DNA damage through promotion of NHEJ, it is becoming increasing clear that it is also a critical regulator of the immune system. This is not a characteristic unique to DNA-PKcs. Other DDR kinases such as ATR and ATM have been linked to multiple processes in both the innate and adaptive responses (26Sun L.L. Yang R.Y. Li C.W. Chen M.K. Shao B. Hsu J.M. Chan L.C. Yang Y. Hsu J.L. Lai Y.J. Hung M.C. Inhibition of ATR downregulates PD-L1 and sensitizes tumor cells to T cell-mediated killing.Am. J. 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Paillard A. et al.ATM activity in T cells is critical for immune surveillance of lymphoma in vivo.Leukemia. 2020; 34: 771-786Crossref PubMed Scopus (6) Google Scholar). These functions are largely separate from their roles in NHEJ and HR (homologous recombination), which highlight a clear, yet largely undefined area of immune regulation. The goal of our study was to further understand mechanisms used by DNA-PKcs to govern T cell activation by uncovering novel target proteins. A mass spectrometry phosphoproteomic screen determined that the IEG transcription factor Egr1 is a robust phospho-target of DNA-PKcs. Phosphorylation of Egr1 at S301 by DNA-PKcs is required for protein stability and prevention of proteasomal degradation. Interestingly, the region within Egr1 containing this site has previously been reported to be critical for regulation of Egr1 transcriptional activity. Jain et al., demonstrate in mouse NIH 3T3 fibroblasts that casein kinase II (CKII) phosphorylates Egr1 within this region, which reduces Egr1 binding to DNA and transcription (30Jain N. Mahendran R. Philp R. Guy G.R. Tan Y.H. Cao X. Casein kinase II associates with Egr-1 and acts as a negative modulator of its DNA binding and transcription activities in NIH 3T3 cells.J. Biol. Chem. 1996; 271: 13530-13536Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Our data further validates the importance of this region, specifically S301, and the regulation of Egr1 through phosphorylation.IEGs are activated within 30 minutes of TCR activation and are decisive factors in mediating T cell responses to immunogenic stimuli (17Mortlock S.A. Wei J. Williamson P. T-cell activation and early gene response in dogs.PLoS One. 2015; 10e0121169Crossref PubMed Scopus (7) Google Scholar, 31Bahrami S. Drabløs F. Gene regulation in the immediate-early response process.Adv. Biol. Regul. 2016; 62: 37-49Crossref PubMed Scopus (198) Google Scholar). Identifying a role for DNA-PKcs in regulating expression and thus activity of Egr1 suggests that it has a much greater influence on immune response outcome than previously understood and could become a novel therapeutic target for a number of immune-related disorders. For instance, our previous study indicates that DNA-PKcs is a critical regulator of the T cell response to allogeneic antigens (12Harrison D.K. Waldrip Z.J. Burdine L. Shalin S.C. Burdine M.S. DNA-PKcs Inhibition Extends Allogeneic Skin Graft Survival.Transplantation. 2020; 105: 540-549Crossref Scopus (2) Google Scholar). Loss of DNA-PKcs activity prevented T cells from producing a host of inflammatory cytokines in response to alloantigen recognition resulting in reduced graft rejection in vivo. This suggests it may be a prime therapeutic target for the prevention of transplant rejection. While we show in those studies that the reduction in cytokine production from DNA-PKcs inhibition was partly due to a drop in protein expression of the NFκB subunit p65, the loss of Egr1 expression most likely was also involved in this outcome given that Egr1 promotes cytokine expression including IL2, a