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

The Journal of biological chemistry (2021-09-26)
Zachary J Waldrip, Lyle Burdine, David K Harrison, Ana Clara Azevedo-Pouly, Aaron J Storey, Olivia G Moffett, Samuel G Mackintosh, Marie Schluterman Burdine
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.

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InSolution Cycloheximide - CAS 66-81-9 - Calbiochem, InSolution Cycloheximide, CAS 66-81-9, is a 100 mg/ml, sterile-filtered solution of Cycloheximide (Cat. No. 239763) in DMSO. An antifungal antibiotic that inhibits protein synthesis in eukaryotes.