Structural and functional characterization of the USP11 deubiquitinating enzyme, which interacts with the RanGTP-associated protein RanBPM.
RanBPM is a RanGTP-binding protein required for correct nucleation of microtubules. To characterize the mechanism, we searched for RanBPM-binding proteins by using a yeast two-hybrid method and isolated a cDNA encoding the ubiquitin-specific protease USP11. The full-length cDNA of USP11 was cloned from a Jurkat cell library. Sequencing revealed that ... USP11 possesses Cys box, His box, Asp and KRF domains, which are highly conserved in many ubiquitin-specific proteases. By immunoblotting using HeLa cells, we concluded that 921-residue version of USP11 was the predominant form, and USP11 may be a ubiquitous protein in various human tissues. By immunofluorescence assay, USP11 primarily was localized in the nucleus of non-dividing cells, suggesting an association between USP11 and RanBPM in the nucleus. Furthermore, the association between USP11 and RanBPM in vivo was confirmed not only by yeast two-hybrid assay but also by co-immunoprecipitation assays using exogenously expressed USP11 and RanBPM. We next revealed proteasome-dependent degradation of RanBPM by pulse-chase analysis using proteasome inhibitors. In fact, ubiquitinated RanBPM was detected by both in vivo and in vitro ubiquitination assays. Finally, ubiquitin conjugation to RanBPM was inhibited in a dose-dependent manner by the addition of recombinant USP11. We conclude that RanBPM was the enzymic substrate for USP11 and was deubiquitinated specifically.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Baculoviridae, COS Cells, Carbon-Nitrogen Lyases, Cell Line, Cell Nucleus, Centrifugation, Density Gradient, Cloning, Molecular, Cysteine Endopeptidases, Cytoskeletal Proteins, DNA, Complementary, Dose-Response Relationship, Drug, Escherichia coli, Gene Library, Hela Cells, Humans, Immunoblotting, Jurkat Cells, Mice, Molecular Sequence Data, Multienzyme Complexes, Nuclear Proteins, Plasmids, Proteasome Endopeptidase Complex, Protein Binding, Protein Structure, Tertiary, Recombinant Proteins, Structure-Activity Relationship, Substrate Specificity, Sucrose, Thiolester Hydrolases, Time Factors, Transfection, Two-Hybrid System Techniques, Ubiquitin, ran GTP-Binding Protein
Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Baculoviridae, COS Cells, Carbon-Nitrogen Lyases, Cell Line, Cell Nucleus, Centrifugation, Density Gradient, Cloning, Molecular, Cysteine Endopeptidases, Cytoskeletal Proteins, DNA, Complementary, Dose-Response Relationship, Drug, Escherichia coli, Gene Library, Hela Cells, Humans, Immunoblotting, Jurkat Cells, Mice, Molecular Sequence Data, Multienzyme Complexes, Nuclear Proteins, Plasmids, Proteasome Endopeptidase Complex, Protein Binding, Protein Structure, Tertiary, Recombinant Proteins, Structure-Activity Relationship, Substrate Specificity, Sucrose, Thiolester Hydrolases, Time Factors, Transfection, Two-Hybrid System Techniques, Ubiquitin, ran GTP-Binding Protein
Biochem. J.
Date: Oct. 01, 2002
PubMed ID: 12084015
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