PP6 regulatory subunit R1 is bidentate anchor for targeting protein phosphatase-6 to DNA-dependent protein kinase.
DNA-dependent protein kinase (DNA-PK) becomes activated in response to DNA double strand breaks, initiating repair by the non-homologous end joining pathway. DNA·PK complexes with the regulatory subunit SAPSR1 (R1) of protein phosphatase-6 (PP6). Knockdown of either R1 or PP6c prevents DNA-PK activation in response to ionizing radiation-induced DNA damage and radiosensitizes glioblastoma cells. Here, we demonstrate that R1 is necessary for and bridges the interaction between DNA-PK and PP6c. Using R1 deletion mutants, DNA-PK binding was mapped to two distinct regions of R1 spanning residues 1-326 and 522-700. Either region expressed alone was sufficient to bind DNA-PK, but only deletion of residues 1-326, not 522-700, eliminated interaction of R1 with DNA-PK. We assign 1-326 as the dominant domain and 522-700 as the supporting region. These results demonstrate that R1 acts as a bidentate anchor to DNA-PK and recruits PP6c. Targeting the dominant interface with small molecule or peptidomimetic inhibitors could specifically prevent activation of DNA-PK and thereby sensitize cells to ionizing radiation and other genotoxic agents.
Department of Radiation Oncology, University of Virginia Health System, Charlottesville, Virginia 22908, USA.
Amol S
AS
Hosing
Nicholas C K
NC
Valerie
Jaroslaw
J
Dziegielewski
David L
DL
Brautigan
James M
JM
Larner
D020816:Amino Acid Motifs; D002460:Cell Line; D051747:DNA-Activated Protein Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
D051747:DNA-Activated Protein Kinase; D010749:Phosphoprotein Phosphatases; C106079:protein phosphatase 6; C572633:protein phosphatase-6 SAPSP1 subunit, human