title: Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response.
abstract: 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.
authors.affiliation: Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Dong-Hyun
authors.initials: DH
authors.lastname: Lee
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Goodarzi
authors.forename: Guillaume O
authors.initials: GO
authors.lastname: Adelmant
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Pan
authors.forename: Penelope A
authors.initials: PA
authors.lastname: Jeggo
authors.forename: Jarrod A
authors.initials: JA
authors.lastname: Marto
authors.forename: Dipanjan
authors.initials: D
authors.lastname: Chowdhury
mesh_terms: D002455:Cell Division; D004247:DNA / Q000528:radiation effects; D004249:DNA Damage; D016195:G2 Phase; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011839:Radiation, Ionizing; D012097:Repressor Proteins / Q000378:metabolism*; D000076183:Tripartite Motif-Containing Protein 28
chemical_list: D012097:Repressor Proteins; D004247:DNA; C101116:TRIM28 protein, human; D000076183:Tripartite Motif-Containing Protein 28; D010749:Phosphoprotein Phosphatases; C080406:protein phosphatase 4