CRM1-mediated nuclear export is required for 26 S proteasome-dependent degradation of the TRIP-Br2 proto-oncoprotein.
Overexpression of the proto-oncogene TRIP-Br2 (SERTAD2) has been shown to induce E2F activity and promote tumorigenesis, whereas ablation of TRIP-Br2 arrests cell proliferation. Timely degradation of many cell cycle regulators is fundamental to the maintenance of proper cell cycle progression. Here we report novel mechanism(s) that govern the tight regulation of TRIP-Br2 levels during cell cycle progression. TRIP-Br2 was observed to be a short-lived protein in which the expression level peaks at the G(1)/S boundary. TRIP-Br2 accumulated in cells treated with 26 S proteasome inhibitors. Co-immunoprecipitation studies revealed that TRIP-Br2 forms ubiquitin conjugates. In silico analysis identified a putative leucine-rich nuclear export signal (NES) motif that overlaps with the PHD-Bromo interaction domain in the acidic C-terminal transactivation domain (TAD) of TRIP-Br2. This NES motif is highly conserved in widely divergent species and in all TRIP-Br family members. TRIP-Br2 was shown to be stabilized in G(2)/M phase cells through nuclear entrapment, either by deletion of the acidic C-terminal TAD, which includes the NES motif, or by leptomycin B-mediated inhibition of the CRM1-dependent nuclear export machinery. Mutation of leucine residue 238 of this NES motif abolished the interaction between CRM1 and TRIP-Br2, as well as the nuclear export of TRIP-Br2 and its subsequent 26 S proteasome-dependent degradation. These data suggest that CRM1-mediated nuclear export may be required for the proper execution of ubiquitin-proteasome-dependent degradation of TRIP-Br2.
Cheong
Jit Kong
JK
Renal Division and Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Gunaratnam
Lakshman
L
Hsu
Stephen I-Hong
SI
D021581:Active Transport, Cell Nucleus; D000818:Animals; D019556:COS Cells; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D028884:Karyopherins / Q000737:chemistry / Q000502:physiology*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D000090063:Proto-Oncogene Mas; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000502:physiology*; D014157:Transcription Factors / Q000737:chemistry*; D015533:Transcriptional Activation; D025801:Ubiquitin / Q000737:chemistry; D000097606:Exportin 1 Protein; D000092468:Bromodomain Containing Proteins
D028884:Karyopherins; D046988:Proteasome Endopeptidase Complex; D000090063:Proto-Oncogene Mas; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D025801:Ubiquitin; D000097606:Exportin 1 Protein; C000715470:MAS1 protein, human; C426827:SERTAD2 protein, human; D000092468:Bromodomain Containing Proteins; C061553:ATP dependent 26S protease