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The xeroderma pigmentosum group E gene product DDB2 is a specific target of cullin 4A in mammalian cells
1Department of Biochemistry and Molecular Biology (M/C 536), University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Abstract:
The damaged-DNA binding protein DDB consists of two subunits, DDB1 (127 kDa) and DDB2 (48 kDa). Mutations in the DDB2 subunit have been detected in patients suffering from the repair deficiency disease xeroderma pigmentosum (group E). In addition, recent studies suggested a role for DDB2 in global genomic repair. DDB2 also exhibits transcriptional activity. We showed that expression of DDB1 and DDB2 stimulated the activity of the cell cycle regulatory transcription factor E2F1. Here we show that DDB2 is a cell cycle-regulated protein. It is present at a low level in growth-arrested primary fibroblasts, and after release the level peaks at the G(1)/S boundary. The cell cycle regulation of DDB2 involves posttranscriptional mechanisms. Moreover, we find that an inhibitor of 26S proteasome increases the level of DDB2, suggesting that it is regulated by the ubiquitin-proteasome pathway. Our previous study indicated that the cullin family protein Cul-4A associates with the DDB2 subunit. Because cullins are involved in the ubiquitin-proteasome pathway, we investigated the role of Cul-4A in regulating DDB2. Here we show that DDB2 is a specific target of Cul-4A. Coexpression of Cul-4A, but not Cul-1 or other highly related cullins, increases the ubiquitination and the decay rate of DDB2. A naturally occurring mutant of DDB2 (2RO), which does not bind Cul-4A, is not affected by coexpression of Cul-4A. Studies presented here identify a specific function of the Cul-4A gene, which is amplified and overexpressed in breast cancers.
Insights
The damaged-DNA binding protein 2 (DDB2) is regulated by the ubiquitin-proteasome pathway and specifically targeted by Cul-4A. This finding is crucial for understanding DNA repair and cell cycle control.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- The damaged-DNA binding protein (DDB) comprises DDB1 and DDB2 subunits.
- Mutations in DDB2 are linked to xeroderma pigmentosum (group E) and it plays a role in DNA repair and transcription.
- DDB2 expression stimulates the E2F1 transcription factor, suggesting cell cycle involvement.
Purpose of the Study:
- To investigate the cell cycle regulation of DDB2.
- To determine the role of the ubiquitin-proteasome pathway in DDB2 regulation.
- To elucidate the specific function of Cul-4A in DDB2 degradation.
Main Methods:
- Analysis of DDB2 protein levels during the cell cycle in primary fibroblasts.
- Treatment with a 26S proteasome inhibitor to assess degradation pathways.
- Coexpression studies with Cul-4A and related cullins to examine ubiquitination and decay rates of DDB2.
- Assessment of a DDB2 mutant (2RO) that does not bind Cul-4A.
Main Results:
- DDB2 is a cell cycle-regulated protein, with levels peaking at the G(1)/S boundary.
- DDB2 levels increase upon proteasome inhibition, indicating ubiquitin-proteasome pathway regulation.
- Cul-4A specifically targets DDB2 for ubiquitination and increased decay, unlike other cullins.
- A DDB2 mutant unable to bind Cul-4A is resistant to Cul-4A-mediated degradation.
Conclusions:
- DDB2 is regulated post-transcriptionally via the ubiquitin-proteasome pathway.
- Cul-4A acts as a specific E3 ubiquitin ligase for DDB2.
- This Cul-4A-DDB2 interaction is important for regulating DDB2 levels and potentially its function in DNA repair and cancer, given Cul-4A's amplification in breast cancer.