Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response

• Published in: The EMBO journal Vol. 31; no. 10; pp. 2403 - 2415
• Main Authors: Lee, Dong-Hyun, Goodarzi, Aaron A, Adelmant, Guillaume O, Pan, Yunfeng, Jeggo, Penelope A, Marto, Jarrod A, Chowdhury, Dipanjan
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 16-05-2012; Nature Publishing Group UK; Blackwell Publishing Ltd; Nature Publishing Group
• Subjects: Cell Division; checkpoint; Deoxyribonucleic acid; DNA; DNA - radiation effects; DNA Damage; DNA damage signalling; double-strand DNA break; EMBO13; EMBO31; Enzymes; G2 Phase; HeLa Cells; Humans; Ionizing radiation; Lesions; Models, Biological; Molecular biology; Mutants; phosphatase; Phosphoprotein Phosphatases - metabolism; Phosphorylation; Protein Processing, Post-Translational; Radiation, Ionizing; Repressor Proteins - metabolism; Signal transduction; Substrates; Tripartite Motif-Containing Protein 28; checkpoint; phosphatase; double‐strand DNA break; DNA damage signalling
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1038/emboj.2012.86

Protein phosphatase PP4C has been implicated in the DNA damage response (DDR), but its substrates in DDR remain largely unknown. We devised a novel proteomic strategy for systematic identification of proteins dephosphorylated by PP4C and identified KRAB‐domain‐associated protein 1 (KAP‐1) as a substrate. Ionizing radiation leads to phosphorylation of KAP‐1 at S824 (via ATM) and at S473 (via CHK2). A PP4C/R3β complex interacts with KAP‐1 and silencing this complex leads to persistence of phospho‐S824 and phospho‐S473. We identify a new role for KAP‐1 in DDR by showing that phosphorylation of S473 impacts the G2/M checkpoint. Depletion of PP4R3β or expression of the phosphomimetic KAP‐1 S473 mutant (S473D) leads to a prolonged G2/M checkpoint. Phosphorylation of S824 is necessary for repair of heterochromatic DNA lesions and similar to cells expressing phosphomimetic KAP‐1 S824 mutant (S824D), or PP4R3β‐silenced cells, display prolonged relaxation of chromatin with release of chromatin remodelling protein CHD3. Our results define a new role for PP4‐mediated dephosphorylation in the DDR, including the regulation of a previously undescribed function of KAP‐1 in checkpoint response.
ATM‐dependent phosphorylation of KAP1, which opens heterochromatin to allow DNA repair, is reverted by PP4, revealing a key phosphatase target in the DNA damage response.