Activation of PARP-1 in response to bleomycin depends on the Ku antigen and protein phosphatase 5

Abstract

Poly (ADP-ribose) polymerase-1 (PARP-1) has an important role in the cellular response to a broad spectrum of DNA lesions. PARP-1 is strongly activated in response to double-stranded DNA breaks (DSBs), yet its contribution to the DSB response is poorly understood. Here we used bleomycin, a radiomimetic that generates DSBs with high specificity to focus on the response of PARP-1 to DSBs. We report that the induction of PARP-1 activity by bleomycin depends on the Ku antigen, a nonhomologous-DNA-End-Joining factor and protein phosphatase 5 (PP5). PARP-1 activation in response to bleomycin was reduced over 10-fold in Ku-deficient cells, whereas its activation in response to U.V. was unaffected. PARP-1 activation was rescued by reexpression of Ku, but was refractory to manipulation of DNA-dependent protein kinase or ATM. Similarly, PARP-1 activation subsequent to bleomycin was reduced 2-fold on ablation of PP5 and was increased 5-fold when PP5 was overexpressed. PP5 seemed to act directly on PARP-1, as its basal phosphorylation was reduced on overexpression of PP5, and PP5 dephosphorylated PARP-1 in vitro. These results highlight the functional importance of Ku antigen and PP5 for PARP-1 activity subsequent to DSBs.

Functional Aspects of PARP1 in DNA Repair and Transcription

“…Similarly, phosphorylation by activated calcium-dependent protein kinase (CaMKII) during neuronal development activates PARP1 enzyme and promotes the nuclear export of its negative regulator KIF4 [51]. However, overexpression of protein phosphatase 5 (PP5) increases PARP1 enzymatic activity towards dsDB [52], suggesting that the kinases and PP5 act on different residues to bring about contrasting effects on PARP1 enzymatic activity. Intricate cross-talks between PARP1 acetylation and sumoylation have also been observed, where modification by SUMO1 and SUMO3 prevents p300-mediated acetylation of PARP1 [53].…”

“…Perhaps acting as a convenient source of substrate, nuclear NMNAT catalyzes the final step of NAD + synthesis and associates with PAR to enhance PARP1 enzyme activity [54]. Other proteins regulating PARP1 activity include Ku [52], histone variant mH2A1.1 [55] and KIF4 [56]. Another important strategy often used in cell death pathways to control PARP1 activity is cleavage by proteases.…”

“…As V(D)J recombination for antibody class switching utilizes the NHEJ machinery, the unusual antibody profiles of PARP1 knockout mice point to its involvement in the process [94]. Instead of directly binding dsDB, PARP1 ADP-ribosylation activity is strongly enhanced by interaction with Ku, forming a functional complex with DNA-PK [52]. The kinase activity of DNA-PK is significantly increased by ADP-ribosylation [106].…”

Alternative splicing of FBP-interacting repressor coordinates c-Myc, P27Kip1/cyclinE and Ku86/XRCC5 expression as a molecular sensor for bleomycin-induced DNA damage pathway

“…Because FIR/FIRΔexon2/SAP155 interaction connects c-myc and cell-cycle regulation by integrating the expression of P89/FIR/FIRΔexon2 or P27/cdk2/cyclinE [14], FIR potentially plays some role in DNA-damage responses [14,15]. Bleomycin (BLM) produces much higher levels of DNA double strand breaks (DSBs) with relatively uniform and simple DNA ends [16,17]. Single-strand DNA breaks (SSDs) lead to DSBs that occur in close proximity and are produced with higher concentrations of BLM [18-20].…”

Pulmonary Hypertension Is a Probable NO/ONOO−Cycle Disease: A Review

“…e drug bleomycin has been shown to initiate some cases of PAH [162,181]. It is known to increase several mechanisms involved in the NO/ONOO − cycle including stimulating poly (ADP-ribose) polymerase (PARP), oxidative stress, superoxide generation, in�ammatory cytokines, oxidative stress, partial uncoupling of the NOSs (which is presumably caused by BH4 depletion), mitochondrial dysfunction, and NF-B elevation [181][182][183][184][185]. Superoxide is speci�cally implicated in having a causal role in bleomycininitiated PAH because overexpression of superoxide dismutase in a mouse model lessens subsequent pulmonary hypertension, �brosis, and vascular remodeling following bleomycin treatment [185].…”