The 19 S Proteasomal Subunit POH1 Contributes to the Regulation of c-Jun Ubiquitination, Stability, and Subcellular Localization

Abstract

The AP1 (activator protein 1) transcription factor, c-Jun, is an important regulator of cell proliferation, differentiation, survival, and death. Its activity is regulated both at the level of transcription and post-translationally through phosphorylation, sumoylation, and targeted degradation. The degradation of c-Jun by the ubiquitin proteasome pathway has been well established. Here, we report that POH1, a subunit of the 19 S proteasome lid with a recently described deubiquitinase activity, is a regulator of c-Jun. Ectopic expression of POH1 in HEK293 cells decreased the level of c-Jun ubiquitination, leading to significant accumulation of the protein and a corresponding increase in AP1-mediated gene expression. The stabilization also correlated with a redistribution of c-Jun in the nucleus. These effects were reduced by mutation of a cysteine residue in the Mpr1 pad1 N-terminal plus motif of POH1 (Cys-120) and appeared to be selective for c-Jun, because POH1 had no effect on other proteasomal substrates. Our results identify a novel mechanism of c-Jun regulation in mammalian cells.The 26 S proteasome is the principal site of controlled protein degradation in eukaryotic cells. The 2-to 2.5-MDa proteasome complex consists of a central core (20 S proteasome) and one or two multiprotein regulatory particles (RPs 2 ; 19 S proteasome), which are further organized into base and lid regions (1). The 19 S RP mediates the binding, deubiquitination, and unfolding of substrates into the 20 S core, where proteolysis takes place (2). One of the many cellular proteins targeted for degradation by the proteasome is the AP1 transcription factor, c-Jun (3). In addition to being the most potent transcriptional activator of its group, c-Jun is involved in a myriad of cellular activities, including tumorigenesis (4). Tight control of intracellular concentrations of active c-Jun is therefore required, a process that is achieved through rapid turnover by ubiquitination and degradation. Novel mechanisms for targeted ubiquitination of c-Jun were recently shown to be carried out by c-Jun-specific ubiquitin ligases, such as Itch (5) and SCF Fbw7 (6). Whereas much continues to be learned about mechanisms of c-Jun ubiquitination, little is known about the process of deubiquitination and its contribution to the overall regulation of c-Jun. Cellular deubiquitinases (DUBs) play an important role in controlled protein degradation (7); when bound to the proteasome they mediate the removal of ubiquitin chains as the substrate is being degraded, a step that facilitates substrate translocation into the 20 S chamber and allows for recycling of ubiquitin in the cell. DUBs also provide ubiquitin-editing activity, which can rescue particular substrates from being degraded by removing or trimming the degradation signal. A wide range of DUBs has been identified in eukaryotic cells, including enzymes with specificities for particular ubiquitinated substrates (8) but no specific c-Jun deubiquitinase activity has yet been reported.A number of studies hav...

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