Activation of DNA-PK by Ionizing Radiation Is Mediated by Protein Phosphatase 6

Abstract

DNA-dependent protein kinase (DNA-PK) plays a critical role in DNA damage repair, especially in non-homologous end-joining repair of double-strand breaks such as those formed by ionizing radiation (IR) in the course of radiation therapy. Regulation of DNA-PK involves multisite phosphorylation but this is incompletely understood and little is known about protein phosphatases relative to DNA-PK. Mass spectrometry analysis revealed that DNA-PK interacts with the protein phosphatase-6 (PP6) SAPS subunit PP6R1. PP6 is a heterotrimeric enzyme that consists of a catalytic subunit, plus one of three PP6 SAPS regulatory subunits and one of three ankyrin repeat subunits. Endogenous PP6R1 co-immunoprecipitated DNA-PK, and IR enhanced the amount of complex and promoted its import into the nucleus. In addition, siRNA knockdown of either PP6R1 or PP6 significantly decreased IR activation of DNA-PK, suggesting that PP6 activates DNA-PK by association and dephosphorylation. Knockdown of other phosphatases PP5 or PP1γ1 and subunits PP6R3 or ARS-A did not reduce IR activation of DNA-PK, demonstrating specificity for PP6R1. Finally, siRNA knockdown of PP6R1 or PP6 but not other phosphatases increased the sensitivity of glioblastoma cells to radiation-induced cell death to a level similar to DNA-PK deficient cells. Our data demonstrate that PP6 associates with and activates DNA-PK in response to ionizing radiation. Therefore, the PP6/PP6R1 phosphatase is a potential molecular target for radiation sensitization by chemical inhibition.

Serine/threonine phosphatases in the DNA damage response and cancer

“…Due to space limitations, this review focuses only on phosphatase-dependent regulation of g-H2AX, ATM, Chk1, Chk2 and p53, factors critically involved in checkpoint signaling. However, other important DDR factors, particularly those in DNA repair pathways, including DNA-PK, RPA2, BRCA1 and so on, are also regulated by Ser/Thr phosphatases (Douglas et al, 2001;Liu et al, 2002;Wechsler et al, 2004;Feng et al, 2009;Mi et al, 2009a;Lee et al, 2010). In most cases, PPs negatively regulate the DNA damage signaling, thus controlling the threshold of DDR activation and promoting DNA damage recovery, consistent with the essential role of protein kinases in activation of the response.…”

“…In most cases, PPs negatively regulate the DNA damage signaling, thus controlling the threshold of DDR activation and promoting DNA damage recovery, consistent with the essential role of protein kinases in activation of the response. However, PP5 positively regulates ATM/ ATR-dependent signal transduction (Ali et al, 2004;Zhang et al, 2005), PP6 mediates DNA-PK activation (Mi et al, 2009a), and PP2A is required for activation of the ATR/Chk1 pathway in the G2/M checkpoint response (Yan et al, 2010). These studies suggest dephosphorylation of inhibitory sites in DDR factors is also important, and future identification of these sites will shed light on the underlying mechanisms.…”

Phosphorylation of the Cryptochrome 1 C-terminal Tail Regulates Circadian Period Length

“…It is possible that DNA-PK phosphorylates and activates a protein phosphatase that then dephosphorylates CRY1. DNA-PK (either DNA-PKcs itself or the DNA-PK complex) has been shown to interact with several protein phosphatases, including PP6, PP2A, PP5, and PP1c (53)(54)(55), and PP5 and PP1c have previously been shown to interact with cryptochromes and other clock components (56 -58). Alternatively, DNA-PK may phosphorylate and inhibit some other kinase that directly targets Ser-588 of CRY1.…”

Pro-tumorigenic Effects of miR-31 Loss in Mesothelioma

“…Validation of Protein Phosphatase 6 as an miR-31 Target and a Diagnostic Marker for MM-Among the predicted miR-31 targets, protein phosphatase PPP6C is one of the most intriguing due to its ability to trigger stress-related response and establish links with cell cycle regulation (30), apoptosis, and chemoresistance (31), resistance to radiation (32), and regulation of chromosome segregation (33). Because all these PPP6C properties suggest a principal role for the gene in progression of MM, a cancer distinguished with multiple chromosomal instability (10) and resistance to common therapies, we asked if PPP6C is indeed a direct miR-31 target.…”