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UV induces nucleolar translocation of ING1 through two distinct nucleolar targeting sequences
M Scott1, F M Boisvert, D Vieyra
1Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada.
Abstract:
The ING1 candidate tumor suppressor is downregulated in a variety of primary tumors and established cancer cell lines. Blocking its expression experimentally promotes unregulated growth in vitro and in vivo, using cell and animal models. Alternative splicing products encode proteins that localize to the nucleus, inhibit cell cycle progression and affect apoptosis in different model systems. Here we show that ING1 proteins translocate to the nucleolus 12-48 h after UV-induced DNA damage. When a small 50 amino acid portion of ING1 was fused to green fluorescent protein, the fusion protein was efficiently targeted to the nucleolus, indicating that ING1 possesses an intrinsic nucleolar targeting sequence (NTS). We mapped this activity to two distinct 4 amino acid regions, which individually direct fused heterologous proteins to the nucleolus. Overexpression of ING1 induced apoptosis of primary fibroblasts in the presence and absence of UV exposure. In contrast, NTS mutants of ING1 that were not targeted to the nucleolus did not efficiently induce apoptosis when overexpressed and instead protected cells from UV-induced apoptosis. Taken together, these results indicate that UV induces ING1 to translocate to the nucleolus and that this translocation may facilitate apoptosis.
Insights
The ING1 tumor suppressor protein moves to the nucleolus after UV DNA damage. This translocation is crucial for ING1 to induce apoptosis and suppress cancer growth.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- The ING1 gene functions as a tumor suppressor, with its downregulation observed in various cancers.
- ING1 proteins are known to inhibit cell cycle progression and influence apoptosis.
- Previous studies indicate ING1's role in regulating cell growth and its nuclear localization.
Purpose of the Study:
- To investigate the subcellular localization of ING1 proteins in response to UV-induced DNA damage.
- To identify the specific regions within ING1 responsible for nucleolar targeting.
- To elucidate the role of ING1 nucleolar translocation in apoptosis and its tumor suppressor function.
Main Methods:
- Utilizing cell and animal models to study ING1 expression and function.
- Employing green fluorescent protein (GFP) fusion proteins to track ING1 localization.
- Site-directed mutagenesis to identify the nucleolar targeting sequence (NTS) within ING1.
- Assessing apoptosis induction via ING1 overexpression in primary fibroblasts with and without UV exposure.
Main Results:
- ING1 proteins were observed to translocate to the nucleolus 12-48 hours post-UV irradiation.
- A 50-amino acid fragment of ING1 was sufficient for nucleolar targeting, indicating an intrinsic NTS.
- Two distinct 4-amino acid regions within ING1 were identified as critical for nucleolar localization.
- Overexpression of wild-type ING1 induced apoptosis, while NTS mutants failed to induce apoptosis and protected cells from UV-induced apoptosis.
Conclusions:
- UV-induced DNA damage triggers the translocation of ING1 to the nucleolus.
- The nucleolar targeting of ING1 is essential for its pro-apoptotic function.
- ING1's translocation to the nucleolus is a key mechanism underlying its role as a tumor suppressor.