Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors.

Small ubiquitin-like modifier (SUMO) modification has emerged as an important posttranslational control of protein functions. Daxx, a transcriptional corepressor, was reported to repress the transcriptional potential of several transcription factors and target to PML oncogenic domains (PODs) via SUMO-dependent interactions. The mechanism by which Daxx binds to sumoylated factors mediating transcriptional and subnuclear compartmental regulation remains unclear. Here, we define a SUMO-interacting motif (SIM) within Daxx and show it to be crucial for targeting Daxx to PODs and for transrepression of several sumoylated transcription factors, including glucocorticoid receptor (GR). In addition, the capability of Daxx SIM to bind SUMO also controls Daxx sumoylation. We further demonstrate that arsenic trioxide-induced sumoylation of PML correlates with a change of endogenous Daxx partitioning from GR-regulated gene promoter to PODs and a relief of Daxx repression on GR target gene expression. Our results provide mechanistic insights into Daxx in SUMO-dependent transcriptional control and subnuclear compartmentalization.

Daxx mediates the small ubiquitin-like modifier-dependent transcriptional repression of Smad4.

Daxx has been shown to function as an apoptosis regulator and transcriptional repressor via its interaction with various cytoplasmic and nuclear proteins. Here, we showed that Daxx interacts with Smad4 and represses its transcriptional activity via the C-terminal domain of Daxx. In vitro and in vivo interaction studies indicated that the binding of Smad4 to Daxx depends on Smad4 sumoylation. Substitution of Smad4 SUMO conjugation residue lysine 159, but not 113, to arginine not only disrupted Smad4-Daxx interaction but also relieved Daxx-elicited repression of Smad4 transcriptional activity. Furthermore, chromatin immunoprecipitation analyses revealed the recruitment of Daxx to an endogenous, Smad4-targeted promoter in a Lys(159) sumoylation-dependent manner. Finally, down-regulation of Daxx expression by RNA interference enhanced transforming growth factor beta-induced transcription of reporter and endogenous genes through a Smad4-dependent, but not K159R-Smad4-dependent, manner. Together, these results indicate that Daxx suppresses Smad4-mediated transcriptional activity by direct interaction with the sumoylated Smad4 and identify a novel role of Daxx in regulating transforming growth factor beta signaling.

Negative modulation of androgen receptor transcriptional activity by Daxx.

The transcriptional activity of the androgen receptor (AR) modulated by positive or negative regulators plays a critical role in controlling the growth and survival of prostate cancer cells. Although numerous positive regulators have been identified, negative regulators of AR are less well understood. We report here that Daxx functions as a negative AR coregulator through direct protein-protein interactions. Overexpression of Daxx suppressed AR-mediated promoter activity in COS-1 and LNCaP cells and AR-mediated prostate-specific antigen expression in LNCaP cells. Conversely, downregulation of endogenous Daxx expression by RNA interference enhances androgen-induced prostate-specific antigen expression in LNCaP cells. In vitro and in vivo interaction studies revealed that Daxx binds to both the amino-terminal and the DNA-binding domain of the AR. Daxx proteins interfere with the AR DNA-binding activity both in vitro and in vivo. Moreover, sumoylation of AR at its amino-terminal domain is involved in Daxx interaction and trans-repression. Together, these findings not only provide a novel role of Daxx in controlling AR transactivation activity but also uncover the mechanism underlying sumoylation-dependent transcriptional repression of the AR.

Caspase activation of mammalian sterile 20-like kinase 3 (Mst3). Nuclear translocation and induction of apoptosis.

Mammalian Sterile 20-like kinase 3 (Mst3), the physiological functions of which are unknown, is a member of the germinal center kinase-III family. It contains a conserved kinase domain at its NH(2) terminus, whereas there is a regulatory domain at its COOH terminus. In this study we demonstrate that endogenous Mst3 is specifically cleaved when Jurkat cells were treated with anti-Fas antibody or staurosporine and that this cleavage is inhibited by the caspase inhibitor, Ac-DEVD-CHO. Using apoptotic Jurkat cell extracts and recombinant caspases, we mapped the caspase cleavage site, AETD(313), which is at the junction of the NH(2)-terminal kinase domain and the COOH-terminal regulatory domain. Caspase-mediated cleavage of Mst3 activates its intrinsic kinase activity, suggesting that the COOH-terminal domain of Mst3 negatively regulates the kinase domain. Furthermore, proteolytic removal of the Mst3 COOH-terminal domain by caspases promotes nuclear translocation. Ectopic expression of either wild-type or COOH-terminal truncated Mst3 in cells results in DNA fragmentation and morphological changes characteristic of apoptosis. By contrast, no such changes were exhibited for catalytically inactive Mst3, implicating the involvement of Mst3 kinase activity for mediation of these effects. Collectively, these results support the notion that caspase-mediated proteolytic activation of Mst3 contributes to apoptosis.