Multimodal control of Cdc25A by nitrosative stress

Abstract

Cdc25A propels cell cycle progression, is overexpressed in numerous human cancers, and possesses oncogenic and antiapoptotic activities. Reactive oxygen species, such as hydrogen peroxide, regulate Cdc25A, but the physiologic and pathologic effects of nitric oxide (*NO) and *NO-derived reactive species are not well defined. Herein, we report novel independent mechanisms governing Cdc25A in response to nitrosative insult. We observed direct and rapid inhibition of Cdc25A phosphatase activity after in vitro treatment with the low molecular mass cell-permeable S-nitrosothiol S-nitrosocysteine ethyl ester (SNCEE). In addition, treatment of cancer cells with SNCEE induced nitrosative stress and decreased Cdc25A protein levels in a time-dependent and concentration-dependent manner. Similarly, iNOS-derived *NO was sufficient to suppress Cdc25A expression, consistent with its role in mediating nitrosative stress. Whereas a decrease in Cdc25A half-life was not observed in response to SNCEE, we found the translational regulator eukaryotic initiation factor 2alpha (eIF2alpha) was hyperphosphorylated and total protein translation was decreased with kinetics consistent with Cdc25A loss. Inhibition of eIF2alpha decreased Cdc25A levels, supporting the hypothesis that SNCEE suppressed Cdc25A translation through inhibition of eIF2alpha. Nitrosative stress decreased the Cdc25A-bound fraction of apoptosis signal-regulating kinase-1 (ASK-1) and sensitized cells to apoptosis induced by the ASK-1-activating chemotherapeutic cis-diaminedichloroplatinum (II), suggesting that nitrosative stress-induced suppression of Cdc25A primed cells for ASK-1-dependent apoptosis. Together these data reveal novel *NO-dependent enzymatic and translational mechanisms controlling Cdc25A, and implicate Cdc25A as a mediator of *NO-dependent apoptotic signaling.

Figure 1

Low molecular mass RSNOs inhibited Cdc25A phosphatase activity in a redox-dependent manner. A, rHis-Cdc25A (500 ng) was pretreated for 30 minutes with the indicated concentrations of SNCEE before assay of OMFP phosphatase activity at 25°C over one hour. Results are expressed as percent of vehicle-pretreated Cdc25A activity. B, rHis-Cdc25A (500 ng) was pretreated for 30 minutes with 1 mM DTT, 100 μM Na₃VO₄, or the indicated concentrations of CEE and SNCEE at which time it was incubated with 250 μg cyclin B₁ immunoprecipitate for 60 minutes at 37°C. Levels of phospho-Tyr15 Cdk1, total Cdk1, and Cdc25A were then determined by Western blotting. C, rHis-Cdc25A was treated with 100 μM decomposed SNCEE or SNCEE and then with β-mercaptoethanol or not before SDS-PAGE. Migration of Cdc25A was then assessed by Western blotting with Cdc25A antibodies. D, rHis-Cdc25A was treated with 100 μM SNCEE or not as in Figure 1B, at which time 20 mM DTT was added where indicated immediately before assay of OMFP phosphatase activity. *, p < 0.001.

Figure 2

Concentration- and time-dependent decrease in Cdc25A following SNCEE treatment. A, HCT116 cells were treated with 100 μM of the indicated compounds and harvested two hours later for Western blotting. B, HCT116 cells were treated with 100 μM SNCEE and samples were harvested at the indicated time points for Western blotting. C and D, HCT116 cells (C) and HeLa cells (D) were treated with the indicated concentrations of SNCEE and harvested two hours post-treatment for Western blotting.

Figure 3

Regulation of Cdc25A expression by iNOS. A, The concentration of nitrite and nitrate in the medium (from B) was determined using a colorimetric detection kit from Cayman Chemical according to the manufacturer’s instructions. B, HCT116 cells were infected with 10 MOI of adenoviruses encoding the β-galactosidase gene (LacZ) or human the iNOS cDNA in the presence or absence of 1 mM l-NMMA. Twenty-four hours later, cells were harvested for Western blotting.

Figure 4

Cdc25A protein stability was not decreased following SNCEE treatment. A, HCT116 cells were co-treated with 25 μg/mL CHX and either 100 μM decomposed SNCEE, 100 μM SNCEE, or 60 J/m² UV. Cells were harvested at the indicated timepoints for Western blotting. Western blots were then densitometrically scanned, and remaining Cdc25A levels were expressed as fraction Cdc25A at time = 0 after normalization to β-tubulin. Black, gray, and white bars represent Cdc25A levels from decomposed SNCEE-, SNCEE-, and UV-treated cells, respectively. B, representative Western blots used to generate A. C, HCT116 cells were treated simultaneously with DMSO or 20 μM LLnL and with 100 μM of the indicated compounds or 60 J/m² UV irradiation as described in Materials and Methods. Cells were harvested two hours later for Western blotting.

Figure 5

eIF2α-mediated translational Cdc25A suppression following SNCEE treatment. A, HCT116 cells were transfected with plasmids encoding HA-tagged Cdc25A as described in Material and Methods. After 24 hours, cells were treated with 100 μM of the indicated compounds. Two hours later, cells were harvested for Western blotting. B, HCT116 cells were incubated for 1 hour in medium lacking l-Cys and l-Met, and then treated with 100 μM decomposed SNCEE, or 100 μM SNCEE for 2 hours. We added 300 μCi/mL [³⁵S]-l-Cys and [³⁵S]-l-Met to the medium at the start of the indicated hour post-SNCEE treatment and cells were harvested for autoradiography and Western blotting 60 minutes later. Non-adjacent lanes are shown from the same gel. C, HCT116 cells were treated for the indicated times with 100 μM SNCEE and harvested for Western blotting. D, HCT116 cells were treated for 24 hours with DMSO or with 75 μM salubrinal (Sal) and were then harvested for Western blotting.

Figure 6

Nitrosative stress attenuated Cdc25A-binding to ASK-1 and sensitized cells to apoptosis. A, HCT116 cells expressing Cdc25A and HA-ASK-1 were treated for the indicated times with 100 μM SNCEE. Cells were harvested, and HA-ASK-1 was immunoprecipitated as described in Materials and Methods. Immunoprecipitates were subjected to Western blotting with the indicated antibodies. B, HCT116 cells were pretreated with 100 μM decomposedSNCEE or fresh SNCEE for one hour before exposure to 10 μM CDDP. Fourty-eight hours after CDDP treatment, cells were fixed and nuclei were stained, and apoptotic nuclei were counted. N.S., not significant; *, p < 0.0001. C, HCT116 cells were treated as described in B and harvested after 24 hours for Western blotting with the indicated antibodies.