Abstract
Daxx is a multifunctional protein that regulates a variety of cellular processes, including transcription, cell cycle, and apoptosis. SPOP is a BTB (Bric-a-brac/Tramtrack/Broad complex) protein that constitutes Cul3-based ubiquitin ligases. Here we show that SPOP serves as an adaptor of Daxx for the ubiquitination by Cul3-based ubiquitin ligase and subsequent degradation by the proteasome. Expression of SPOP with Cul3 markedly reduced Daxx level, and this degradation was blocked by SPOP-specific short hairpin RNAs. Inhibition of the proteasome by MG132 caused the prevention of Daxx degradation in parallel with the accumulation of ubiquitinated Daxx. Expression of SPOP with Cul3 reversed Daxx-mediated repression of ETS1- and p53-dependent transcription, and short hairpin RNA-mediated knock down of SPOP blocked the recovery of their transcriptional activation. Furthermore, Daxx degradation led to the cleavage of poly(ADP-ribose) polymerase and the increase in the number of terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end-labeling-positive apoptotic cells. These results suggest that SPOP/Cul3-ubiquitin ligase plays an essential role in the control of Daxx level and, thus, in the regulation of Daxx-mediated cellular processes, including transcriptional regulation and apoptosis.
Highlights
Which Cul1 serves as a scaffold molecule that interacts with Skp1 and a small RING-finger protein Roc1, known as Hrt1 and Rbx1 [7,8,9]
The present studies have demonstrated that SPOP BTB protein serves as an adaptor of Daxx for the ubiquitination by Cul3-based Ub ligase and subsequent degradation by the proteasome
In addition to BTB domain that mediates the interaction with Cul3, SPOP has a MATH domain that functions in protein-protein interaction [12, 17]
Summary
Plasmids and Cells—pGL3-MMP1-Luc and various vectors expressing Daxx, p53, ETS1, and wild-type and mutant forms of human SPOP were prepared as described [35]. Bound proteins were eluted by boiling and subjected to SDS-PAGE followed by immunoblot with mouse anti-HA antibody (Roche Applied Science), rabbit anti-HA-antibody (Upstate Biotechnology, Inc.) , mouse anti-FLAG M2 monoclonal antibody (Sigma), anti-Xpress antibody (Invitrogen), rabbit anti-Daxx-antibody (Calbiochem or Upstate), rabbit anti-Cul3-antibody (Zymed Laboratories Inc.), or anti-SPOP antiserum. Transcription Assay—For assaying the transcription of ETS1-dependent luciferase reporter gene, 293T cells were cultured on 24-well plates and transiently transfected with pGL3-MMP1-Luc and various combinations of expression vectors for HA-Daxx, FLAG-ETS1, FLAG-SPOP, FLAGCul, and FLAG-Roc. Terminal Deoxynucleotidyltransferase-mediated dUTP-fluorescein Nick End-labeling (TUNEL) Assay—HeLa cells cultured on cover-glasses were transfected with various combinations of expression vectors for SPOP, Cul, Roc, and Daxx. After incubation for 24 h, cells were fixed with 3.7% paraformaldehyde for 10 min and subjected to TUNEL assay by following the manufacturer’s instruction (Roche Applied Science). The nuclei were counterstained with 4,6-diamidino-2-phenylindole for counting total cell numbers
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