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EMBO Rep. Jun 2008; 9(6): 576–581.
Published online Apr 4, 2008. doi:  10.1038/embor.2008.48
PMCID: PMC2427386
Scientific Report

NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of the tumour suppressor p33ING1b

Abstract

The tumour suppressor p33ING1b (ING1b for inhibitor of growth family, member 1b) is important in cellular stress responses, including cell-cycle arrest, apoptosis, chromatin remodelling and DNA repair; however, its degradation pathway is still unknown. Recently, we showed that genotoxic stress induces p33ING1b phosphorylation at Ser 126, and abolishment of Ser 126 phosphorylation markedly shortened its half-life. Therefore, we suggest that Ser 126 phosphorylation modulates the interaction of p33ING1b with its degradation machinery, stabilizing this protein. Combining the use of inhibitors of the main degradation pathways in the nucleus (proteasome and calpains), partial isolation of the proteasome complex, and in vitro interaction and degradation assays, we set out to determine the degradation mechanism of p33ING1b. We found that p33ING1b is degraded in the 20S proteasome and that NAD(P)H quinone oxidoreductase 1 (NQO1), an oxidoreductase previously shown to modulate the degradation of p53 in the 20S proteasome, inhibits the degradation of p33ING1b. Furthermore, ultraviolet irradiation induces p33ING1b phosphorylation at Ser 126, which, in turn, facilitates its interaction with NQO1.

Keywords: tumour suppressor p33ING1b, NQO1, protein degradation, proteasome, phosphorylation

Introduction

Several studies have shown that p33ING1b (ING1b for inhibitor of growth family, member 1b) is a tumour suppressor (Feng et al, 2002; Kataoka et al, 2003), and it has been reported that p33ING1b is downregulated in several carcinomas. Overexpression of this protein inhibits cell growth, whereas its antisense expression promotes cell transformation (Garkavtsev et al, 1996). p33ING1b is important in cellular stress responses, including cell-cycle arrest, apoptosis, chromatin remodelling and DNA repair (Cheung & Li, 2001; Scott et al, 2001; Campos et al, 2004). This protein can also sensitize cells to stress agents such as etoposide and γ-irradiation in wild-type but not in p53-deficient cell lines (for a review see Campos et al, 2004). We found that p33ING1b is induced by ultraviolet radiation in human keratinocytes and melanoma cells (Cheung et al, 2000, 2001). In addition, we showed that p33ING1b markedly enhances nucleotide excision repair of ultraviolet-damaged DNA and promotes UVB-induced apoptosis in melanoma cells (Cheung et al, 2001; Cheung & Li, 2002). Recently, we discovered that p33ING1b is phosphorylated at Ser 126, and abolishment of Ser 126 phosphorylation markedly shortened its half-life. Furthermore, phosphorylation at the Ser 126 residue is important in the regulation of cyclin B1 expression and the proliferation of melanoma cells (Garate et al, 2007). These results indicate that the degradation of p33ING1b is modulated by phosphorylation at the Ser 126 residue; however, the exact degradation pathway of p33ING1b is unclear. In this work, we examined the effect of specific protease inhibitors on the half-life of p33ING1b and further confirm our results through the detection of this tumour suppressor in purified fractions of proteasome. Here, we provide evidence that p33ING1b is degraded in the 20S proteasome. Our results indicate that the NAD(P)H quinone oxidoreductase 1 (NQO1) inhibits the degradation of p33ING1b and that ultraviolet irradiation stabilizes p33ING1b by inducing phosphorylation at Ser 126, which enhances the p33ING1b interaction with NQO1.

Results And Discussion

To determine the degradation pathway of p33ING1b, MMRU cells were treated for 16 h with different inhibitors. MG132 was the first choice, as p33ING1b is localized in the nucleus where proteasome is the main degradation pathway. Calpain inhibitor I (CiI) was used as an alternative, as calpains are important proteases in the nucleus. Determination of p33ING1b protein levels in nuclear extracts (Fig 1A) showed that MG132 caused an increase of 55±7% in the expression of p33ING1b compared with cells degraded in the absence of inhibitor, whereas CiI had no significant effect. To confirm the role of proteasome in the degradation of p33ING1b, we determined the half-life of this protein by treating the cells with cycloheximide (CHX) in the presence or absence of lactacystin, an irreversible and specific inhibitor of proteasome. Consistent with our previous report (Garate et al, 2007), we estimated a half-life of 15.1 h for endogenous p33ING1b (Fig 1B), and lactacystin notably extended its half-life by approximately threefold. We confirmed the accuracy of the method used in this work by determining the turnover rate of two proteins—checkpoint kinase 1 (Chk1) and cyclin-dependent kinase 1 (Cdk1)—of known half-life (supplementary Fig S1 online). Furthermore, through immunolocalization, we eliminated the possibility that MG132 might induce relocalization of p33ING1b (supplementary Fig S2 online), suggesting that p33ING1b is degraded in proteasomes.

Figure 1
p33ING1b is degraded in the proteasome. (A) p33ING1b protein levels were determined by western blot in nuclear extracts of cells treated for 16 h with CHX or CHX plus MG132 or CiI. The density of the bands was determined and plotted as the relative fold ...

As ubiquitination triggers most proteins to be degraded in the proteasome (Grossman et al, 2003; Zhang et al, 2005), we evaluated, by pull-down assays, whether p33ING1b is ubiquitinated. Immunoprecipitation assays in Fig 2A show that there is a marked increase in the expression of both p53 (left panel) and p33ING1b (centre panel) when the cells are incubated in the presence of MG132. Conversely, isolation of ubiquitinated proteins (as described in the supplementary information online) showed only a single band of ubiquitinated p33ING1b, which did not increase in the presence of MG132 (right panel). Therefore, inhibition of proteasomal function did not affect the recovery of ubiquitinated p33ING1b but, not surprisingly, it increased the recovery of ubiquitinated p53, a known substrate of ubiquitin-dependent and ubiquitin-independent proteasomal pathways (Asher & Shaul, 2005), where several bands were detected as a sign of polyubiquitination (Fig 2A, right panel). These results indicate that p33ING1b is monoubiquitinated and degrades independently of ubiquitin. Furthermore, through ammonium sulphate fractionation of nuclear extracts (supplementary Fig S3 online), we found that p33ING1b is present in the 20S proteasome-containing fraction (38–70% ammonium sulphate-soluble fraction) only when the cells were incubated in the presence of MG132, suggesting that p33ING1b is degraded in the core of the proteasome. Further isolation of the 20S proteasome found that p33ING1b co-fractionate with this complex in every step. To show conclusively that p33ING1b is degraded in the 20S proteasome, we incubated affinity-isolated recombinant glutathione S-transferase (GST)-ING1b with purified mammalian 20S proteasome in vitro for different time points (Fig 2B, control). We found that 20S proteasome is able to degrade p33ING1b but not GST alone (supplementary Fig S4 online). It has been shown previously that NQO1 modulates the degradation of proteins such as p53 and p73 (Asher et al, 2005a) or ornitine decarboxylase (Asher et al, 2005b) in the 20S proteasome. Therefore, we assessed the possibility that the degradation of p33ING1b might be modulated by NQO1. NQO1 inhibited the degradation of p33ING1b by approximately 40%, and the degradation was inhibited by more than 80% in the presence of its substrate (NADH). To determine whether NQO1 modulates the degradation of p33ING1b in situ, NQO1 was knocked down. The results showed that knocking down NQO1 induced the degradation of p33ING1b (Fig 2C). As NQO1 is stabilized by interacting with its partner proteins, we investigated, by co-immunoprecipitation, whether NQO1 interacts with p33ING1b (Fig 2D). We were able to pull down p33ING1b using antibodies specific for NQO1 in cells expressing control short hairpin RNA (shRNA) but not in cells expressing NQO1 shRNA, confirming the interaction p33ING1b and NQO1.

Figure 2
p33ING1b is degraded in the 20S proteasome complex. (A) Nuclear extracts from cells treated for 16 h in the presence (+) or absence (−) of MG132 were immunoprecipitated (IP) with p53 and p33ING1 antibodies. Nuclear extracts expressing ...

It has been shown that phosphorylation targets various proteins for degradation, for example, Chk1 (Zhang et al, 2005) and p27 (Hasan et al, 2007). But other studies have clearly established that phosphorylation could also stabilize proteins such as p53 (Chehab et al, 1999) and p73 (Tsai & Yuan, 2003). In our recent work, we have shown that abolishment of Ser 126 phosphorylation shortens the half life of p33ING1b (Garate et al, 2007). To investigate whether p33ING1b phosphorylation at Ser 126 determines the degradation pathway of this protein, we assessed the effect of MG132 and CiI on the degradation of ectopic wild-type or unphosphorylatable S126A mutant p33ING1b. Consistent with the effect shown on the degradation of endogenous p33ING1b (Fig 1A), MG132 inhibited the degradation of both wild-type and S126A mutant p33ING1b (Fig 3A), causing an increase of 65±6% and 117±7%, respectively, in their expression compared with untreated cells. This suggests that p33ING1b is degraded in the proteasome complex independently of Ser 126 phosphorylation. Nevertheless, the fact that p33ING1b has a discrete signal for chaperone-mediated autophagy (CMA), 45QEILK49, persuaded us to assess the possibility that unphosphorylatable p33ING1b could be misfolded and degraded by autophagy. Our results show that neither form of p33ING1b is degraded by autophagy, as it is not possible to detect them in isolated lysosomes even in the presence of inhibitor (supplementary Fig S5 online). Therefore, we assessed whether phosphorylation at Ser 126 could affect the interaction of p33ING1b and NQO1. Through immunoprecipitation of this oxidoreductase from cells expressing wild-type, S126A or S126E (phosphomimetic) mutant p33ING1b, we found that the phosphomimetic form co-immunoprecipitated to a larger extent with NQO1 than the partly phosphorylated wild-type p33ING1b, whereas the unphosphorylatable S126A mutant did not co-precipitate with NQO1 (Fig 3B). The interaction of phosphorylated p33ING1b and NQO1 was confirmed by an independent pull-down assay (supplementary Fig S6 online). We found that NQO1 present in the nuclear extracts of MMRU cells binds more readily to phosphorylated GST-ING1b than to the unphosphorylated form, whereas GST is not able to bind to this oxidoreductase. To determine whether p33ING1b interacts directly with NQO1, we incubated GST-ING1b and isolated NQO1 in vitro in the absence or presence of NADH (Fig 3C). We found that NQO1 binds more readily to GST-ING1b in the presence of its substrate (NADH), but NQO1 does not bind to GST either in the presence or absence of NADH. To determine whether NQO1 differentially modulates the degradation of phosphorylatable and non-phosphorylatable p33ING1b in situ, we knocked down this enzyme. As shown in Fig 3D, knocking down NQO1 accelerated the degradation of ectopic wild-type p33ING1b, but not the S126A mutant compared with control cells, suggesting that phosphorylatable p33ING1b is stabilized preferentially by this oxidoreductase. Consistent with our previous results (Garate et al, 2007), the S126A mutant is degraded faster than the wild-type p33ING1b. The addition of MG132 ameliorates the degradation of both forms of p33ING1b, confirming that they share the same degradation pathway. Furthermore, these results confirm that NQO1 interacts preferentially with phosphorylated p33ING1b.

Figure 3
NQO1 preferentially binds to phosphorylated p33ING1b. (A) Phosphorylation at Ser 126 does not alter the degradation pathway of p33ING1b. Nuclear extracts obtained from cells transfected with pCI-NeoING1b (WT) or pCI-NeoS126A (S126A), and treated for 16 ...

We have shown previously that ultraviolet irradiation induces p33ING1b phosphorylation at Ser 126 (Garate et al, 2007). To explain the role of ultraviolet irradiation in p33ING1b degradation, we irradiated cells with different dosages of UVB and determined its effect on p33ING1b expression, phosphorylation and its interaction with NQO1. We found that ultraviolet irradiation induced both the expression and phosphorylation of p33ING1b, reaching a maximum at a dose of 10 mJ/cm2 (Fig 4A, left panel). By immunoprecipitation of NQO1 from control or ultraviolet-irradiated cells, we found that ultraviolet irradiation enhanced the interaction of p33ING1b with NQO1 (Fig 4A, right panel). The induction of p33ING1b phosphorylation also correlates with markedly increased stabilization of this protein. A half-life of 15.4 h is estimated for p33ING1b under control conditions, whereas it is extrapolated to be 57.6 h after ultraviolet irradiation (Fig 4B).

Figure 4
UVB irradiation induces binding of p33ING1b to NQO1 through phosphorylation at Ser 126. (A) p33ING1b expression and phosphorylation at Ser 126 were determined in nuclear extracts of cells irradiated with different dosages of UVB (mJ/cm2) and treated with ...

Conclusion

Our data provide evidence that under normal conditions poorly phosphorylated p33ING1b is degraded in the 20S proteasome (Fig 5A). This pathway is mediated by NQO1, which interacts preferentially with phosphorylated p33ING1b and partly inhibits its degradation in the 20S proteasome. Ultraviolet irradiation induces p33ING1b phosphorylation at Ser 126, inducing further stabilization of this protein through interaction with NQO1 (Fig 5B).

Figure 5
Proposed model for the effect of UVB irradiation on the mechanism of p33ING1b degradation. NQO1 preferentially binds to p33ING1b phosphorylated at Ser 126. (A) Under the conditions of non-ultraviolet stress, p33ING1b shows a low degree of phosphorylation ...

Methods

Cell culture, inhibitors and treatments. The human melanoma cell line MMRU (a kind gift from Dr R. Byers, Boston University School of Medicine) was maintained as described previously (Garate et al, 2007). Transfection of different DNA vectors was performed using Effectene reagent (Qiagen Inc., Mississauga, ON, Canada), according to the manufacturer's protocol, followed by a round of selection in media containing 1 mg/ml G418. Under these conditions, a transfection efficiency of more than 95% was achieved.

The different inhibitors and DNA vectors used in this work, as well as their respective concentrations, are summarized in the supplementary information online.

Western blot analysis. Nuclear pellets were obtained as described previously (Garate et al, 2007) and proteins were solubilized in nuclear extract buffer (20 mM Hepes, pH 7.9, 0.35 M NaCl, 1 mM EDTA, 20% glycerol, 1 mM DTT) plus protease and phosphatase inhibitors. The concentration of proteins was determined by using the DC Protein Assay (Bio-Rad, Hercules, CA, USA) and western blot analysis was performed using the Odyssey Infrared Imaging System as described previously (Razidlo et al, 2004). The intensity of the signal of interest was corrected by detecting β-actin as the input control. Immunoprecipitation was performed as described previously (Doyon et al, 2004).

Determination of p33ING1b half-life. MMRU cells treated with CHX were collected at different time points and whole-cell proteins were extracted as described previously (Wang & Li, 2006) for western blotting using a p33ING1b antibody. The half-life was estimated from the band density as described previously (Cuervo et al, 1998).

In vitro degradation of p33ING1b in the 20S proteasome. Recombinant GST-ING1b and NQO1 were isolated as described in the supplementary information online. Purified mammalian 20S proteasomes were purchased from Sigma-Aldrich (Oakville, ON, Canada).

A 250 ng portion of isolated GST-ING1b was mixed with similar amounts of bovine serum albumin, 100 ng of 20S proteasomes, 50 ng of NQO1 and 1 mM NADH in different combinations in degradation buffer (100 mmol/l Tris–HCl (pH 7.5), containing 150 mmol/l NaCl, 5 mmol/l MgCl2 and 2 mmol/l dithiothreitol) and incubated for different time points at 37°C. All samples were boiled in Laemmli denaturation buffer and analysed by western blot.

Statistical analysis. Student's t-test was used to perform statistical analyses. A P-value <0.05 was considered significant.

Supplementary information is available at EMBO reports online (http://www.emboreports.org).

Supplementary Material

supplementary Figs S1–S6

Acknowledgments

We thank A.M. Cuervo for the CMA motif search in the p33ING1b sequence and for helpful discussion. This work was funded by the Canadian Institutes of Health Research and the Canadian Dermatology Foundation.

Footnotes

The authors declare that they have no conflict of interest.

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