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DUB-1, a deubiquitinating enzyme with growth-suppressing activity
1Division of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
Cytokines regulate cell growth by inducing the expression of specific target genes. Using the differential display method, we have cloned a cytokine-inducible immediate early gene, DUB-1 (for deubiquitinating enzyme). DUB-1 is related to members of the UBP superfamily of deubiquitinating enzymes, which includes the oncoprotein Tre-2. A glutathione S-transferase-DUB-1 fusion protein cleaved ubiquitin from a ubiquitin-beta-galactosidase protein. When a conserved cysteine residue of DUB-1, required for ubiquitin-specific thiol protease activity, was mutated to serine (C60S), deubiquitinating activity was abolished. Continuous expression of DUB-1 from a steroid-inducible promoter induced growth arrest in the G1 phase of the cell cycle. Cells arrested by DUB-1 expression remained viable and resumed proliferation upon steroid withdrawal. Our results suggest that DUB-1 regulates cellular growth by modulating either the ubiquitin-dependent proteolysis or the ubiquitination state of an unknown growth regulatory factor(s).
Insights
A newly discovered gene, deubiquitinating enzyme-1 (DUB-1), regulates cell growth. Its continuous expression causes cell cycle arrest, suggesting a role in controlling cell proliferation via ubiquitin pathways.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Cytokines are crucial regulators of cell growth, mediating their effects through the induction of specific target genes.
- The identification of novel genes involved in cytokine signaling pathways is essential for understanding cell growth regulation.
Purpose of the Study:
- To clone and characterize a novel cytokine-inducible immediate early gene.
- To investigate the function of the cloned gene, DUB-1, in cell cycle regulation.
Main Methods:
- Differential display method was used to identify cytokine-inducible genes.
- Cloning and expression of the DUB-1 gene.
- Biochemical assays using glutathione S-transferase-DUB-1 fusion protein to assess deubiquitinating activity.
- Site-directed mutagenesis (C60S) to abolish enzymatic activity.
- Induction of DUB-1 expression using a steroid-inducible promoter to study cell cycle effects.
Main Results:
- A novel cytokine-inducible immediate early gene, DUB-1 (deubiquitinating enzyme-1), was cloned.
- DUB-1 demonstrated deubiquitinating activity, cleaving ubiquitin from a model substrate.
- Mutation of a conserved cysteine residue (C60S) abolished DUB-1's enzymatic activity.
- Continuous DUB-1 expression induced G1 phase cell cycle arrest, with cells remaining viable.
- Cessation of DUB-1 expression allowed cells to resume proliferation.
Conclusions:
- DUB-1 is a functional deubiquitinating enzyme involved in cell cycle regulation.
- DUB-1 plays a role in controlling cellular growth, likely by modulating ubiquitin-dependent proteolysis or the ubiquitination status of growth regulatory factors.
- DUB-1 represents a potential target for understanding and manipulating cell proliferation.