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RING3 kinase transactivates promoters of cell cycle regulatory genes through E2F
1Cancer Research Center, Boston University School of Medicine, Massachusetts 02118, USA. gdenis@bu.edu
Abstract:
RING3 is a novel, nuclear-localized, serine-threonine kinase that has elevated activity in human leukemias. RING3 transforms NIH/3T3 cells and is activated by mitogenic signals, all of which suggest that it may play a role in cell cycle-responsive transcription. We tested this hypothesis with transient transfection of RING3 into fibroblasts and assayed transactivation of the promoters of cyclin D11 cyclin A, cyclin E, and dihydrofolate reductase (dhfr) genes. RING3 transactivates these promoters in a manner dependent on ras signaling. A kinase-deficient point mutant of RING3 does not transactivate. Mutational analysis of the dhfr promoter reveals that transactivation also depends on the presence of a functional E2F binding site. Furthermore, ectopic expression of Rb protein, a negative regulator of E2F activity, suppresses the RING3-dependent transactivation of this promoter. Consistent with a potential role of E2F in RING3-dependent transcription, anti-RING3 immunoaffinity chromatography or recombinant RING3 protein affinity chromatography of nuclear extracts copurified a protein complex that contains E2F-1 and E2F-2. These data suggest that RING3 is a potentially important regulator of E2F-dependent cell cycle genes.
Insights
RING3, a novel kinase, regulates genes essential for cell cycle progression. It activates transcription of cyclin and dihydrofolate reductase genes, suggesting a role in cell proliferation and leukemia.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- RING3 is a nuclear serine-threonine kinase with heightened activity in human leukemias.
- RING3 has transforming capabilities and is activated by mitogenic signals, indicating a potential role in cell cycle-responsive transcription.
Purpose of the Study:
- To investigate the role of RING3 in cell cycle-responsive transcription.
- To determine if RING3 regulates the promoters of key cell cycle genes, including cyclin D1, cyclin A, cyclin E, and dihydrofolate reductase (dhfr).
Main Methods:
- Transient transfection of RING3 into fibroblasts.
- Assaying transactivation of target gene promoters.
- Mutational analysis of the dhfr promoter.
- Ectopic expression of Retinoblastoma (Rb) protein.
- Immunoaffinity chromatography using anti-RING3 or recombinant RING3.
Main Results:
- RING3 transactivates cyclin D1, cyclin A, cyclin E, and dhfr promoters in a manner dependent on ras signaling.
- A kinase-deficient RING3 mutant failed to transactivate these promoters.
- Transactivation of the dhfr promoter by RING3 requires a functional E2F binding site.
- Ectopic expression of Rb protein suppressed RING3-dependent transactivation.
- Nuclear extracts from cells treated with RING3 contained E2F-1 and E2F-2.
Conclusions:
- RING3 is a novel regulator of E2F-dependent cell cycle genes.
- RING3's kinase activity and interaction with E2F suggest a significant role in regulating cell cycle progression.
- These findings highlight RING3 as a potential therapeutic target in leukemias characterized by elevated RING3 activity.