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.
Ideguchi
Haruko
H
First Department of Internal Medicine, Yokohama City University School of Medicine, 3-9 Fukuura, Yokohama 236-0004, Japan.
Ueda
Atsuhisa
A
Tanaka
Masatsugu
M
Yang
Jun
J
Tsuji
Takashi
T
Ohno
Shigeru
S
Hagiwara
Eri
E
Aoki
Akiko
A
Ishigatsubo
Yoshiaki
Y
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D019556:COS Cells; D019759:Carbon-Nitrogen Lyases / Q000737:chemistry; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013395:Sucrose / Q000494:pharmacology; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; C116005:Ran binding protein 9; D011994:Recombinant Proteins; C101359:USP11 protein, human; D025801:Ubiquitin; D013395:Sucrose; D013869:Thiolester Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D020931:ran GTP-Binding Protein; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase