Mutual regulation between Polo-like kinase 3 and SIAH2 E3 ubiquitin ligase defines a regulatory network that fine-tunes the cellular response to hypoxia and nickel

Cen Li,Soyoung Park,Xiaowen Zhang,Wei Dai,Dazhong Xu

doi:10.1074/jbc.m116.767178

Cen Li, Soyoung Park + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m116.767178

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Abstract

Elevated cellular response to hypoxia, which contributes to cell transformation and tumor progression, is a prominent feature of malignant cells in solid tumors. Polo-like kinase 3 (Plk3) is a serine/threonine protein kinase known to inhibit the cellular response to hypoxia and tumorigenesis. Nickel compounds are well-established human carcinogens that induce tumorigenesis partly through their hypoxia-mimicking effects. Despite previous research efforts, the role of Plk3 in the hypoxic response induced by hypoxia or nickel is not completely understood. Here, we show that NiCl2 (Ni(II)) or hypoxia reduces the protein level and shortens the half-life of cytoplasmic Plk3 in a ubiquitin-proteasome-dependent manner. We identify SIAH2, a RING finger E3 ubiquitin ligase associated with the cellular hypoxic response, to be the ubiquitin E3 ligase that mediates the degradation of Plk3. We show that SIAH2 binds to Plk3 and mediates its ubiquitination primarily through its polo-box domain. We report that USP28, a deubiquitinase known to be inhibitable by Ni(II) or hypoxia, may also contribute to the suppression of the Plk3 protein by Ni(II). We also show that Plk3 in turn suppresses the SIAH2 protein level in a kinase activity-dependent manner. Our study revealed an interesting mutual regulation between Plk3 and SIAH2 and uncovered a regulatory network that functions to fine-tune the cellular hypoxic response. We propose that suppression of Plk3 expression contributes to carcinogenesis and tumor progression induced by nickel compounds.

Highlights

Elevated cellular response to hypoxia, which contributes to cell transformation and tumor progression, is a prominent feature of malignant cells in solid tumors
We identify Seven in Absentia Homologue 2 (SIAH2), a RING finger E3 ubiquitin ligase associated with the cellular hypoxic response, to be the ubiquitin E3 ligase that mediates the degradation of Plk[3]
We show that SIAH2 binds to Plk[3] and mediates its ubiquitination primarily through its polo-box domain

Summary

Results

Given the involvement of Plk[3] in the hypoxic response (10), we examined whether Plk[3] expression is regulated by Ni(II) in A549 lung carcinoma and BEAS-2B lung branchial epithelial cells. Note that the HIF-1␣ protein levels were elevated as a result of ectopic expression of wildtype SIAH2 but not mutant SIAH2 and that inhibition of the proteasome increased the endogenous HIF-1␣ protein levels in both wild-type and mutant SIAH2 groups (Fig. 5C) These results are consistent with the previous findings that SIAH2 up-regulates the HIF-1␣ protein level by promoting ubiquitination and degradation of the prolyl hydroxylase (22). During the analysis of our co-transfection experiments investigating the regulation of Plk[3] by SIAH2, we observed that ectopic co-expression of full-length Plk[3] or the KD but not the PBD of Plk[3] dramatically inhibited the protein levels of both wild-type and mutant SIAH2 (Fig. 5, A–C, HA signals). These results indicate that while showing some similarities, the mechanisms underlying the regulation of Plk[3] by SIAH2 in response to Ni(II) and hypoxia are not identical

Discussion

Cell lines and cell culture

RNA interference

Western blotting

Cell fractionation assay

Statistical analysis