USP13 Enzyme Regulates Siah2 Ligase Stability and Activity via Noncatalytic Ubiquitin-binding Domains

Abstract

The RING finger E3 ubiquitin ligase Siah2 is implicated in control of diverse cellular biological events, including MAPK signaling and hypoxia. Here we demonstrate that Siah2 is subject to regulation by the deubiquitinating enzyme USP13. Overexpression of USP13 increases Siah2 stability by attenuating its autodegradation. Consequently, the ability of Siah2 to target its substrates prolyl hydroxylase 3 and Spry2 (Sprouty2) for ubiquitin-mediated proteasomal degradation is attenuated. Conversely, inhibition of USP13 expression with corresponding shRNA decreases the stability of both Siah2 and its substrate Spry2. Thus, USP13 limits Siah2 autodegradation and its ubiquitin ligase activity against its target substrates. Strikingly, the effect of USP13 on Siah2 is not mediated by its isopeptidase activity: mutations in its ubiquitin-binding sequences positioned within the ubiquitin-specific processing protease and ubiquitin-binding domains, but not within putative catalytic sites, abolish USP13 binding to and effect on Siah2 autodegradation and targeted ubiquitination. Notably, USP13 expression is attenuated in melanoma cells maintained under hypoxia, thereby relieving Siah2 inhibition and increasing its activity under low oxygen levels. Significantly, on melanoma tissue microarray, high nuclear expression of USP13 coincided with high nuclear expression of Siah2. Overall, this study identifies a new layer of Siah2 regulation mediated by USP13 binding to ubiquitinated Siah2 protein with a concomitant inhibitory effect on its activity under normoxia.Siah proteins are RING finger E3 ubiquitin ligases implicated in the ubiquitination and proteasome-dependent degradation of substrate molecules, which also limit their own availability through self-ubiquitination and degradation (1-3). Siah was first identified in Drosophila melanogaster as seven in absentia (sina), which regulates formation of the R7 photoreceptor through control of tramtrack stability (4, 5). Three murine Siah genes (Siah1a, Siah1b, and Siah2) share significant homology with the two human (SIAH1 and SIAH2) orthologues (6, 7).Siah2 is an important regulator of pathways activated under stress and hypoxia. Among substrates reportedly regulated by Siah under these conditions are TRAF2, ␣-ketoglutarate dehydrogenase, Spry2 (Sprouty2), and two of the three prolyl hydroxylases (8 -10). Given the role of PHD proteins as oxygen sensors and regulators of HIF1␣, the master transcription factor responding to hypoxia, Siah is implicated in the control of hypoxia, particularly within the range of 2-6% oxygen at which PHD proteins retain sufficient activity (11,12). The role of Siah2 in tumor development has been extensively studied. Inhibition of Siah reportedly attenuates breast, pancreatic, lung, prostate, and melanoma tumors (13-16). Mechanistically, Siah2 contributes to tumor development and metastasis via its control of diverse substrates, as shown in a melanoma model (14).A search for novel Siah2 interacting factors led to identification of the isopeptidase USP13 ...

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“…Siah proteins induce ubiquitination and subsequent degradation of several substrates and thus regulate numerous signaling pathways and biological processes (10). Like other ubiquitin ligases (11), Siah can also self-ubiquitinate and promote its own degradation through the ubiquitin-proteasome pathway (12,13). Thus, Siah proteins are generally present at very low levels in cells.…”

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