The Ataxia telangiectasia-mutated and Rad3-related protein kinase regulates cellular hydrogen sulfide concentrations

Abstract

The ataxia telangiectasia-mutated and Rad3-related (ATR) serine/threonine kinase plays a central role in the repair of replication-associated DNA damage, the maintenance of S and G2/M-phase genomic stability, and the promotion of faithful mitotic chromosomal segregation. A number of stimuli activate ATR, including persistent single-stranded DNA at stalled replication folks, R loop formation, hypoxia, ultraviolet light, and oxidative stress, leading to ATR-mediated protein phosphorylation. Recently, hydrogen sulfide (H₂S), an endogenous gasotransmitter, has been found to regulate multiple cellular processes through complex redox reactions under similar cell stress environments. Three enzymes synthesize H₂S: cystathionine-β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase. Since H₂S can under some conditions cause DNA damage, we hypothesized that ATR activity may regulate cellular H₂S concentrations and H₂S-syntheszing enzymes. Here we show that human colorectal cancer cells carrying biallelic knock-in hypomorphic ATR mutations have lower cellular H₂S concentrations than do syngeneic ATR wild-type cells, and all three H₂S-synthesizing enzymes show lower protein expression in the ATR hypomorphic mutant cells. Additionally, ATR serine 428 phosphorylation is altered by H₂S donor and H₂S synthesis enzyme inhibition, while the oxidative-stress induced phosphorylation of the ATR-regulated protein CHK1 on serine 345 is increased by H₂S synthesis enzyme inhibition. Lastly, inhibition of H₂S production potentiated oxidative stress-induced double-stranded DNA breaks in the ATR hypomorphic mutant compared to ATR wild-type cells. Our findings demonstrate that the ATR kinase regulates and is regulated by H₂S.

Keywords: 3-Mercaptopyruvate sulfurtransferase; ATR; CHK1; Cystathionine γ-lyase; Cystathionine-β-synthase; H(2)S; Hydrogen sulfide; Nicotinamide; phosphoribosyltransferase.

Figure 1.

The effects of t-BOOH and H₂S inhibitor and donor treatments in the colony-forming efficiency assay with ATR and ATR-H cells. Twelve hours after plating in appropriate media, exponentially growing ATR and ATR-H cells were treated for two hours with either an H₂S inhibitor (1 mM β-cyano-L-alanine, βCA) or an H₂S donor (20 μM diallyl trisulfide, DATS), and subjected to 15 minutes 50, 100, or 200 μM t-BOOH oxidative stress. After 11 days, the cells were fixed, stained, and the colonies counted. Data indicates survival as a percentage of untreated cells.

Figure 2.

Free cellular H₂S concentrations in the ATR wild-type cells compared to the ATR-H mutant cells. Figure 2A, H₂S concentrations were compared in ATR and ATR-H, with and without the ATR kinase inhibitor NU6027. The cells were treated for two hours with 12 μM NU6027 in standard media and harvested (2A). Figure 2B, H₂S concentrations were compared in ATR and ATR-H, with and without the CBS and CSE inhibitor β-cyanol-L-alanine. The cells were treated with 1 mM β-cyanol-L-alanine for two hours in standard media and harvested (2B). Figure 2C, H₂S concentrations were compared in ATR and ATR-H, with and without 20μM diallyl trisulfide for two hours in standard media and harvested (2C). “βCA” = 1 mM β-cyanol-L-alanine. Free H₂S is in nmol/mg protein.

Figure 3.

Representative western blots for CBS (3A), CSE (3B), 3-MST (3C), and Nampt (3D) comparing ATR and ATR -H cells protein expression with β-actin as a control protein. All western blots were performed at least in triplicate.

Figure 4.

Quantitative real-time polymerase chain reaction analyses of CBS, CSE, and 3-MST mRNA levels in the ATR and ATR-H cell lines. Statistical analyses revealed no significant differences between CBS, CSE, and 3-MST mRNA levels between the two cell lines. The primers used in the quantitative real-time polymerase chain reactions are given in Table 1. GADPH was used as a control.

Figure 5.

ATR protein phosphorylation on serine-435 with an H₂S donor and inhibitor, or t-BOOH treatments. ATR and ATR-H cells were treated with 1mM β-cyanol-L-alanine or 20 μM diallyl trisulfide for two hours and harvested (5A). In Figure 5B, ATR and ATR-H cells were treated with 100 μM t-BOOH for 15 minutes, incubated in standard media for 45 minutes, and harvested. In Figure 5C, ATR and ATR-H cells were treated with 15,000 μJ/cm² UV light and the cells were harvested 45 minutes later.

Figure 6.

CHK1 serine 345 phosphorylation in ATR and ATR-H cells with H₂S synthesis inhibition followed by t-BOOH treatment was examined. ATR and ATR-H cells were pretreated with 1mM β-cyanol-L-alanine for two hours, then 15 minutes with 10μM t-BOOH, and harvested following a 45-minute incubation in standard media.

Figure 7.

dsDNA break formation in ATR and ATR-H cells following H₂S synthesis inhibition. ATR and ATR-H cells were pretreated with 1mM β-cyanol-L-alanine for two hours, then 15 minutes with t-BOOH, cultured one hour in standard media, treated with colcemid for four hours, and harvested. dsDNA breaks in Giemsa stained, Colcemid-treated cells, were counted under oil immersion microscopy. The t-BOOH concentration was 10μM.

Figure 8.

A summary of the findings presented in this paper. The ATR kinase regulates intracellular H₂S concentrations (arrow 1) and the levels of the three H₂S-synthesizing enzymes (arrow 2). Increased in intracellular H₂S concentrations decreased ATR serine 435 phosphorylation, while decreased in intracellular H₂S concentrations increased this phosphorylation (arrow 3). Attenuation of intracellular H₂S synthesis also potentiates CHK1 serine 345 phosphorylation following ATR cell exposure to low t-BOOH concentration, an event not seen in ATR-H cells (arrow 4). Lastly, low cellular H₂S concentrations in the hypomorphic ATR-H cells increase genomic instability by itself, and when combined with a low dose of t-BOOH. Taken together, our data suggests that the ATR kinase regulates and is in turn regulated by H₂S.