The IL-2 Induced De-Ubiquitinating Enzyme, DUB-2, Is a Nuclear-Cytoplasmic Shuttle Which Requires Catalytic Activity for Nuclear Localization.

DUB-2 is a T-cell specific de-ubiquitinating enzyme whose expression is induced by IL-2. Expression of DUB-2 results in prolonged STAT5 phosphorylation, enhanced IL-2 induced gene expression, and resistance to apoptosis resulting from cytokine withdrawal. The molecular basis of DUB-2s effects, however, is not understood. In order to better understand the role of DUB-2 in the IL-2 response, we performed a systematic analysis of DUB-2 catalytic activity, subcellular localization, and domain organization. Subcellular localization of DUB-2 was examined using a C-terminal DUB-2-Cherry (enhanced red fluorescence) fusion and live-cell imaging. When expressed in HeLa cells, wild-type DUB-2-Cherry is predominantly localized to the cytoplasm, although 8–10% of cells have a strong nuclear accumulation. Strikingly, the nuclear localization is entirely eliminated by a mutation in the catalytic cysteine (c60s). This effect on DUB-2s localization is not due to changes in cell cycle progression, as we observe identical cell cycle profiles (DNA content) in wt or c60s expressing cells. Conversely, enhanced DUB-2 nuclear localization is seen following nocodazole-induced cell cycle synchronization and with mutant DUB-2-Cherry fusions lacking a duplicated 8-residue sequence within the C-terminal domain.

We next analyzed the catalytic activity of DUB-2 toward ubiquitin and other ubiquitin-like (UbL) molecules using a series of semi-synthetic activity-based probes. These probes consist of full-length Ub or UbL molecules equipped with a C-terminal electrophilic group. These probes act as suicide substrates, forming a covalent bond with the active site cysteine of catalytically active (but not inactive) Ub(L) isopeptidases. Unexpectedly, DUB-2 forms adducts not only with the Ub-based probe, but also the probe derivative of the UbL molecule ISG15, which suggests that it has dual-specificity. The activity towards Ub and ISG15 is abrogated by a mutation in the catalytic cysteine (c60s) of DUB-2, but is not affected by deletions of the poly-lysine or hypervariable domains.

Taken together, these results suggest that DUB-2 is a bi-specific (Ub and ISG15) isopeptidase which shuttles between the cytoplasm and the nucleus in a manner dependent on its catalytic activity. This work, and the ultimate elucidation of the molecular target of DUB-2, will further increase our understanding of DUB-2’s role in IL-2-dependent T-cell proliferation and immunologic tolerance.