Overlapping signals for protein degradation and nuclear localization define a role for intrinsic RAG-2 nuclear uptake in dividing cells.
Expression of the recombinase proteins RAG-1 and RAG-2 is discordant: while RAG-1 is relatively long lived, RAG-2 is degraded periodically at the G(1)-S transition. Destruction of RAG-2 is mediated by a conserved interval in the recombination-dispensable region. The need for RAG-2 to reaccumulate in the nucleus at each cell division ... suggested the existence of an intrinsic RAG-2 nuclear localization signal (NLS). RAG-1 or RAG-2, expressed individually, is a nuclear protein. A screen for proteins that bind the recombination-dispensable region of RAG-2 identified the nuclear transport protein Importin 5. Mutation of residues 499 to 508 in RAG-2 abolished Importin 5 binding, nuclear accumulation, and periodic degradation of RAG-2. The Importin 5 binding site overlaps an NLS, defined by mutagenesis. RAG-1 rescued the localization of degradation-defective, RAG-2 NLS mutants; this required an intact RAG-1 NLS. Mutations in RAG-2 that abolish intrinsic nuclear accumulation but spare periodic degradation impaired recombination in cycling cells; induction of quiescence restored recombination to wild-type levels. Recombination defects were correlated with a cell cycle-dependent defect in the ability of RAG-1 to rescue localization of the RAG-2 mutants. These results suggest that the intrinsic RAG-2 NLS functions in the nuclear uptake of RAG-2 following its reexpression in cycling cells.
Mesh Terms:
3T3 Cells, Active Transport, Cell Nucleus, Animals, Binding Sites, Cell Cycle, Cell Division, Cell Line, Cell Nucleus, DNA, DNA Nucleotidyltransferases, DNA-Binding Proteins, G1 Phase, Genes, RAG-1, Homeodomain Proteins, Humans, Mice, Microscopy, Confocal, Microscopy, Fluorescence, Mutagenesis, Site-Directed, Mutation, Nuclear Localization Signals, Nuclear Proteins, Plasmids, Protein Binding, Protein Structure, Tertiary, Recombination, Genetic, S Phase, Transfection, VDJ Recombinases, alpha Karyopherins
3T3 Cells, Active Transport, Cell Nucleus, Animals, Binding Sites, Cell Cycle, Cell Division, Cell Line, Cell Nucleus, DNA, DNA Nucleotidyltransferases, DNA-Binding Proteins, G1 Phase, Genes, RAG-1, Homeodomain Proteins, Humans, Mice, Microscopy, Confocal, Microscopy, Fluorescence, Mutagenesis, Site-Directed, Mutation, Nuclear Localization Signals, Nuclear Proteins, Plasmids, Protein Binding, Protein Structure, Tertiary, Recombination, Genetic, S Phase, Transfection, VDJ Recombinases, alpha Karyopherins
Mol. Cell. Biol.
Date: Aug. 01, 2003
PubMed ID: 12861017
View in: Pubmed Google Scholar
Download Curated Data For This Publication
12548
Switch View:
- Interactions 2