Phosphoproteomics reveals novel modes of function and inter‐relationships among PIKKs in response to genotoxic stress. (20th November 2020)

• Record Type: Journal Article
• Authors: Schlam‐Babayov, Sapir; Bensimon, Ariel; Harel, Michal; Geiger, Tamar; Aebersold, Ruedi; Ziv, Yael; Shiloh, Yosef
• Is Part Of: EMBO journal. Volume 40:Number 2(2021)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2020-11-20
• Subjects: ataxia‐telangiectasia -- ATM -- DNA damage response -- phosphoproteomics -- PIKKs; Molecular biology -- Periodicals; 572.805
• DOI: 10.15252/embj.2020104400
• Languages: English
• ISSNs: 0261-4189

Abstract

Abstract: The DNA damage response (DDR) is a complex signaling network that relies on cascades of protein phosphorylation, which are initiated by three protein kinases of the family of PI3‐kinase‐related protein kinases (PIKKs): ATM, ATR, and DNA‐PK. ATM is missing or inactivated in the genome instability syndrome, ataxia‐telangiectasia (A‐T). The relative shares of these PIKKs in the response to genotoxic stress and the functional relationships among them are central questions in the genome stability field. We conducted a comprehensive phosphoproteomic analysis in human wild‐type and A‐T cells treated with the double‐strand break‐inducing chemical, neocarzinostatin, and validated the results with the targeted proteomic technique, selected reaction monitoring. We also matched our results with 34 published screens for DDR factors, creating a valuable resource for identifying strong candidates for novel DDR players. We uncovered fine‐tuned dynamics between the PIKKs following genotoxic stress, such as DNA‐PK‐dependent attenuation of ATM. In A‐T cells, partial compensation for ATM absence was provided by ATR and DNA‐PK, with distinct roles and kinetics. The results highlight intricate relationships between these PIKKs in the DDR. Synopsis: PI3K‐related kinases (PIKKs) ATM, ATR and DNA‐PK are master regulators of DNA damage response (DDR) signaling. Here, quantitative phosphoproteomics uncover complex relationships among them in the cellular response to DNA double‐strand breaks both in wild‐type cells and cells from patients with the genome instability disorder, ataxia‐telangiectasia (A‐T). A multitude of new phosphorylation and dephosphorylation events in the DDR are identified and matched with 34 previous proteomic and functional screens for DDR factors. Shared and individual targets of ATM, ATR and DNA‐PK reveal their concerted function in DDR regulation, and cross‐talk among PIKKs is exemplified by DNA‐PK‐dependent attenuation of ATM. Most of the compensation for lack of ATM in A‐T cells occurs during the late phase of the response and is ATR‐dependent, while early phase compensation is less prominent and is largely DNA‐PK‐dependent. DSB‐induced, ATR‐dependent compensation for lack or inhibition of ATM involves substrates that this PIKK normally targets following replication stress. Abstract : Targeted proteomics via selected reaction monitoring defines the relative contributions of the PI3K‐related master kinases ATM, ATR and DNA‐PK to DNA damage response signaling in wild‐type and ataxia‐telangiectasia patient cells.