ABSTRACT
Previous studies have shown that UV-induced binding of p21(WAF1) to PCNA through the PCNA-interacting protein (PIP) domain in p21(WAF1) promotes a switch from DNA replication to DNA repair by altering the PCNA protein complex. Here we show that the p33(ING1b) isoform of the ING1 candidate tumour suppressor contains a PIP domain. UV rapidly induces p33(ING1b) to bind PCNA competitively through this domain, a motif also found in DNA ligase, the DNA repair-associated FEN1 and XPG exo/endonucleases, and DNA methyltransferase. Interaction of p33(ING1b) with PCNA occurs between a significant proportion of ING1 and PCNA, increases more than tenfold in response to UV and is specifically inhibited by overexpression of p21(WAF1), but not by p16(MTS1), which has no PIP sequence. In contrast to wild-type p33(ING1b), ING1 PIP mutants that do not bind PCNA do not induce apoptosis, but protect cells from UV-induced apoptosis, suggesting a role for this PCNA-p33(ING1b) interaction in eliminating UV-damaged cells through programmed cell death. These data indicate that ING1 competitively binds PCNA through a site used by growth regulatory and DNA damage proteins, and may contribute to regulating the switch from DNA replication to DNA repair by altering the composition of the PCNA protein complex.
Subject(s)
Apoptosis/physiology , Proliferating Cell Nuclear Antigen/metabolism , Proteins/genetics , Proteins/metabolism , Binding Sites/physiology , Cell Cycle Proteins , Cell Line , Cell Nucleus/metabolism , DNA Repair/physiology , DNA Replication/physiology , DNA-Binding Proteins , Fibroblasts/cytology , Gene Expression , Genes, Tumor Suppressor , Humans , Inhibitor of Growth Protein 1 , Intracellular Signaling Peptides and Proteins , Nuclear Proteins , Protein Binding/physiology , Protein Binding/radiation effects , Protein Structure, Tertiary , Proteins/chemistry , RNA Splicing , Tumor Suppressor Proteins , Ultraviolet RaysABSTRACT
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.
Subject(s)
Cell Nucleolus/metabolism , Cell Nucleolus/radiation effects , Protein Sorting Signals/physiology , Proteins/chemistry , Proteins/metabolism , Amino Acid Sequence , Apoptosis/radiation effects , Cell Cycle Proteins , Cells, Cultured , Consensus Sequence , DNA Damage/radiation effects , DNA-Binding Proteins , Fibroblasts , Fluorescent Antibody Technique, Indirect , Gene Products, tat/genetics , Gene Products, tat/metabolism , Genes, Tumor Suppressor/genetics , Humans , Inhibitor of Growth Protein 1 , Intracellular Signaling Peptides and Proteins , Kinetics , Molecular Sequence Data , Mutation/genetics , Nuclear Localization Signals/genetics , Nuclear Localization Signals/physiology , Nuclear Proteins , Protein Binding , Protein Sorting Signals/genetics , Protein Transport/radiation effects , Proteins/genetics , RNA Polymerase I/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription, Genetic/radiation effects , Transfection , Tumor Suppressor Proteins , Ultraviolet RaysABSTRACT
Nine monoclonal antibodies (MAbs) against human and rodent ING1 protein have been generated using an IL6-secreting mouse myeloma line. These antibodies are all effective in recognizing ING1 protein in ELISAs, Western blot assays, and by indirect immunofluorescence. Combining different CAb monoclonal antibodies in a Western blot assay also allows detection of the very low levels of endogenous ING1 found in fibroblast cells in culture and the identification of at least two isoforms of ING1 in normal human diploid fibroblasts and established brain cancer cell lines.
