Visualization of inositol phosphate-dependent mobility of Ku: depletion of the DNA-PK cofactor InsP6 inhibits Ku mobility

Abstract

Repair of DNA double-strand breaks (DSBs) in mammalian cells by nonhomologous end-joining (NHEJ) is initiated by the DNA-PK protein complex. Recent studies have shown inositol hexakisphosphate (InsP6) is a potent cofactor for DNA-PK activity in NHEJ. Specifically, InsP6 binds to the Ku component of DNA-PK, where it induces a conformational change and a corresponding increase in DNA end-joining activity. However, the effect of InsP6 on the dynamics of Ku, such as its mobility in the nucleus, is unknown. Importantly, these dynamics reflect the character of Ku's interactions with other molecules. To address this question, the diffusion of Ku was measured by fluorescence photobleaching experiments using cells expressing green fluorescent protein (GFP)-labeled Ku. InsP6 was depleted by treating cells with calmodulin inhibitors, which included the compounds W7 and chlorpromazine. These treatments caused a 50% reduction in the mobile fraction of Ku-GFP, and this could be reversed by replenishing cells with InsP6. By expressing deletion mutants of Ku-GFP, it was determined that its W7-sensitive region occurred at the N-terminus of the dimerization domain of Ku70. These results therefore show that InsP6 enhances Ku mobility through a discrete region of Ku70, and modulation of InsP6 levels in cells represents a potential avenue for regulating NHEJ by affecting the dynamics of Ku and hence its interaction with other nuclear proteins.

Protocols for Regulation and Study of Diphosphoinositol Polyphosphates

“…4). Similar data were obtained with chlorpromazine (Byrum et al, 2004), whereas KN-93 (500 M) and K-252a (50 M) calmodulin kinase inhibitors had no effect. Because it has been proposed that W-7 is cytotoxic (Jan et al, 2000), its effects were more systematically assessed.…”

“…Nevertheless, an increasing number of inositol phosphates are being identified that have varied and diverse functions. In addition to calcium signaling, they have been implicated in nuclear mRNA export (York et al, 1999), chromatin remodelling (Shen et al, 2003;Steger et al, 2003), DNA repair (Hanakahi et al, 2000;Byrum et al, 2004), membrane trafficking (Ye et al, 1995;Saiardi et al, 2002), and control of cell proliferation (Orchiston et al, 2004).…”

The antipsychotic drug trifluoperazine inhibits DNA repair and sensitizes non–small cell lung carcinoma cells to DNA double-strand break–induced cell death

“…Recent studies have shown that inositol phosphates serve as cofactors for DNA-PK cs during NHEJ (28 -30). Notably, inositol hexakisphosphate (InsP 6 ) has been shown to regulate the mobility of Ku proteins and depletion of InsP 6 by the TFP-related compound chlorpromazine causes a reduction in the mobile fraction of Ku (31). Therefore, one might speculate that the inhibitory action of TFP on DNA DSB repair shown in this study could be a consequence of calmodulin antagonism.…”

DNA-dependent Protein Kinase-mediated Phosphorylation of Protein Kinase B Requires a Specific Recognition Sequence in the C-terminal Hydrophobic Motif

“…Inositol hexakisphosphate (IP6) is a novel factor that is bound by DNA-PK and stimulates DNA end joining in vitro (41,42). Therefore, we used IP6 to stimulate DNA-PK activity in vitro in the presence/absence of Ku70/Ku86 subunits with FSYtide as substrate.…”