Critical role for Daxx in regulating Mdm2

The tumour suppressor p53 induces apoptosis or cell-cycle arrest in response to genotoxic and other stresses. In unstressed cells, the anti-proliferative effects of p53 are restrained by mouse double minute 2 (Mdm2), a ubiquitin ligase (E3) that promotes p53 ubiquitination and degradation. Mdm2 also mediates its own degradation through auto-ubiquitination. It is unclear how the cis- and trans-E3 activities of Mdm2, which have opposing effects on cell fate, are differentially regulated. Here, we show that death domain-associated protein (Daxx) is required for Mdm2 stability. Downregulation of Daxx decreases Mdm2 levels, whereas overexpression of Daxx strongly stabilizes Mdm2. Daxx simultaneously binds to Mdm2 and the deubiquitinase Hausp, and it mediates the stabilizing effect of Hausp on Mdm2. In addition, Daxx enhances the intrinsic E3 activity of Mdm2 towards p53. On DNA damage, Daxx dissociates from Mdm2, which correlates with Mdm2 self-degradation. These findings reveal that Daxx modulates the function of Mdm2 at multiple levels and suggest that the disruption of the Mdm2-Daxx interaction may be important for p53 activation in response to DNA damage.     

Multiple scaffolding functions of {beta}-arrestins in the degradation of G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 (GRK2) plays a fundamental role in the regulation of G protein-coupled receptors (GPCRs), and changes in GRK2 expression levels can have an important impact on cell functions. GRK2 is known to be degraded by the proteasome pathway. We have shown previously that β-arrestins participate in enhanced kinase turnover upon GPCR stimulation by facilitating GRK2 phosphorylation by c-Src or by MAPK or by recruiting the Mdm2 E3 ubiquitin ligase to the receptor complex. In this report, we have investigated how such diverse β-arrestin scaffold functions are integrated to modulate GRK2 degradation. Interestingly, we found that in the absence of GPCR activation, β-arrestins do not perform an adaptor role for GRK2/Mdm2 association, but rather compete with GRK2 for direct Mdm2 binding to regulate basal kinase turnover. Upon agonist stimulation, β-arrestins-mediated phosphorylation of GRK2 at serine 670 by MAPK facilitates Mdm2-mediated GRK2 degradation, whereas c-Src-dependent phosphorylation would support the action of an undetermined β-arrestin-recruited ligase in the absence of GPCR activation. The ability of β-arrestins to play different scaffold functions would allow coordination of both Mdm2-dependent and -independent processes aimed at the specific modulation of GRK2 turnover in different signaling contexts.     

Regulation of p53 in embryonic stem cells

Despite an increasing interest in the role of the p53 tumour suppressor protein in embryonic stem cells, not much is known about its regulation in this cell type.We show that the relatively high amount of p53 protein correlates with a higher amount of p53 RNA in ES cells compared to differentiated cells. Moreover, p53 RNA is more stable in embryonic stem cells and the p53 protein is more often transcribed. This is at least partly due to decreased expression of miRNA-125a and 125b in embryonic stem cells. Despite its cytoplasmic localisation, p53 is degraded in 26S proteasomes in embryonic stem cells. This process is controlled by Mdm2, the deubiquitinating enzyme Hausp and Ubc13. In contrast, the E3 ligase PirH2 appears to be less important for the control of p53 in embryonic stem cells. During differentiation, p53 protein and RNA levels are decreased which corresponds to increased expression of miRNA-125a and miRNA-125b.     

Effects of MdmX on Mdm2-mediated downregulation of pRB

Mdm2, a RING-finger type ubiquitin ligase, is overexpressed in a variety of human cancers. It promotes ubiquitination of the tumor suppressor p53 and can function as an oncogene by largely downregulating p53. Recently, we reported that Mdm2 degrades retinoblastoma tumor suppressor protein (pRB) via the ubiquitin-proteasome system. In the present study, we assessed the effects of MdmX, a structural homolog of Mdm2, on the Mdm2-mediated ubiquitination of pRB. MdmX is known to negatively regulate p53 function by enhancing the Mdm2-mediated ubiquitination and degradation of p53. Interestingly, MdmX inhibited the Mdm2-mediated pRB ubiquitination. Furthermore, an MdmX siRNA decreased the endogenous pRB level, while MdmX overexpression stimulated pRB functions in cultured cells. Therefore, MdmX may have different roles in the regulation of Mdm2 activity for ubiquitination of pRB and p53.     

Mechanistic studies of MDM2-mediated ubiquitination in p53 regulation

As a central regulator for cell cycle arrest, apoptosis, and cellular senescence, p53 requires multiple layers of regulatory control to ensure correct temporal and spatial functions. It is well accepted that Mdm2-mediated ubiquitination plays a crucial role in p53 regulation. In addition to proteasome-mediated degradation, ubiquitination of p53 by Mdm2 acts a key signal for its nuclear export. Nuclear export has previously been thought to require the disassociation of the p53 tetramer and exposure of the intrinsic nuclear export signal. To elucidate the molecular mechanism of degradation-independent repression on p53 by Mdm2, we have developed a two-step approach to purify ubiquitinated forms of p53 induced by Mdm2 from human cells. Surprisingly, however, we found that ubiquitination has no effect on the tetramerization/oligomerization of p53, arguing against this seemingly well accepted model. Moreover, nuclear export of p53 alone is not sufficient to completely abolish p53 activity. Ubiquitination-mediated repression of p53 by Mdm2 acts at least, in part, through inhibiting the sequence-specific DNA binding activity. Thus, our results have important implications regarding the mechanisms by which Mdm2 acts on p53.     

Regulation of Actinomycin D induced upregulation of Mdm2 in H1299 cells

Mdm2 is a critical negative regulator of the p53 tumor suppressor and also has many p53-independent functions. Deregulation of Mdm2 is closely associated with tumorigenesis. However, how Mdm2 is regulated in response to various stresses is not well understood. In this study, we found that Mdm2 was stabilized and upregulated upon Actinomycin D (ActD) treatment in the p53-deficient H1299 cell line. This Mdm2 upregulation was not dependent on the ribosomal protein L11, an essential player in ribosomal stress-induced p53 activation, but did require a NEDDylation-dependent mechanism. We further demonstrated that the ActD-induced Mdm2 stabilization may be modulated by the cell growth signaling, and that knockdown of Mdm2 enhanced ActD-induced cell death in H1299 cells. These results suggested a role of Mdm2 in the ribosomal stress response in the p53 deficient cells, which could be exploited in therapeutic use for treating cancers harboring p53 mutations.     

Ets-1-dependent expression of vascular endothelial growth factor receptors is activated by latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus through interaction with Daxx

Vascular endothelial growth factor (VEGF) and its receptors are highly expressed in Kaposi's sarcoma (KS) lesion and play a key role in angiogenesis. Latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) has multiple functions related to viral latency and KSHV-induced oncogenesis. In this report, we have identified Daxx as a LANA-binding protein by co-immunoprecipitation analysis of HeLa cells stably expressing LANA. LANA associated with Daxx in a PEL cell line infected with KSHV. LANA and Daxx also bound in vitro, suggesting direct interaction. From the results of binding assays, a region containing the Glu/Asp-rich domain within LANA, and a central region including the second paired amphipathic helix within Daxx contributed to the interaction. To address the physiological significance of this interaction, we focused on a Daxx-mediated VEGF receptor gene regulation. We found that Daxx repressed Ets-1-dependent Flt-1/VEGF receptor-1 gene expression, and that LANA inhibited the repression by Daxx in a reporter assay. Analyses of flow cytometry and real-time PCR revealed that expression of VEGF receptor-1 and -2 in LANA-expressing human umbilical vein endothelial cells (HUVECs) significantly increased. Co-immunoprecipitation and immunoblotting experiments suggested that LANA-bound Daxx to inhibit the interaction between Daxx and Ets-1. Chromatin immunoprecipitation assays showed that Daxx associated with VEGF receptor-1 promoter in HUVECs, and that LANA expression reduced this association. These results suggested that LANA contributes to a high expression of VEGF receptors in KS lesion by interfering with the interaction between Daxx and Ets-1.     

Phosphorylation-dependent ubiquitylation and degradation of androgen receptor by Akt require Mdm2 E3 ligase

The androgen receptor (AR) controls several biological functions including prostate cell growth and apoptosis. However, the mechanism by which AR maintains its stability to function properly remains largely unknown. Here we show that Akt and Mdm2 form a complex with AR and promote phosphorylation-dependent AR ubiquitylation, resulting in AR degradation by the proteasome. The effect of Akt on AR ubiquitylation and degradation is markedly impaired in a Mdm2-null cell line compared with the wild-type cell line, suggesting that Mdm2 is involved in Akt-mediated AR ubiquitylation and degradation. Furthermore, we demonstrate that the E3 ligase activity of Mdm2 and phosphorylation of Mdm2 by Akt are essential for Mdm2 to affect AR ubiquitylation and degradation. These results suggest that phosphorylation-dependent AR ubiquitylation and degradation by Akt require the involvement of Mdm2 E3 ligase activity, a novel mechanism that provides insight into how AR is targeted for degradation.     

Hetero-oligomerization with MdmX rescues the ubiquitin/Nedd8 ligase activity of RING finger mutants of Mdm2

Mdm2 is a member of the RING finger family of ubiquitin ligases and is best known for its role in targeting the tumor suppressor p53 for ubiquitination and degradation. Mdm2 can bind to itself and to the structurally related protein MdmX, and these interactions involve the RING finger domain of Mdm2 and MdmX, respectively. In this study, we performed a mutational analysis of the RING finger domain of Mdm2, and we identified several amino acid residues that are important for Mdm2 to exert its ubiquitin ligase function. Mutation of some of these residues interfered with the Mdm2-Mdm2 interaction indicating that a homomeric complex represents the active form of Mdm2. Mutation of other residues did not detectably affect the ability of Mdm2 to interact with itself but reduced the ability of Mdm2 to interact with UbcH5. Remarkably, MdmX efficiently rescued the ubiquitin ligase activity of the latter Mdm2 mutants in vitro and within cells. Because the interaction of Mdm2 with MdmX is more stable than the Mdm2-Mdm2 interaction, this suggests that Mdm2-MdmX complexes play a prominent role in p53 ubiquitination in vivo. Furthermore, we show that, similar to Mdm2, the Mdm2-MdmX complex has Nedd8 ligase activity and that all mutations that affect the ubiquitin ligase activity of Mdm2 also affect its Nedd8 ligase activity. From a mechanistic perspective, this suggests that the actual function of Mdm2 and Mdm2-MdmX, respectively, in p53 ubiquitination and in p53 neddylation is similar for both processes.     

Sumoylation of Daxx regulates IFN-induced growth suppression of B lymphocytes and the hormone receptor-mediated transactivation

Daxx has been shown to play an essential role in type I IFN-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the molecular mechanisms of how Daxx acts on growth suppression of B lymphocytes, we examined functions of a sumoylation-defective Daxx KA mutant (Daxx K630/631A), which substituted Lys 630 and Lys 631 to Ala. Importantly, Daxx KA localized in the cytoplasm, whereas wild-type Daxx localized in the nucleus. Murine pro-B cell line Ba/F3 expressing Daxx KA revealed a resistance to the IFN-induced growth suppression. It is noteworthy that treatment with an exportin inhibitor, leptomycin B, resulted in nuclear localization of Daxx KA and recovery of the IFN-induced growth suppression in Ba/F3 cells. Moreover, Daxx KA decreased the binding potential to promyelocytic leukemia protein (PML), and overexpression of PML recruited Daxx KA into PML oncogenic domains. Notably, a Daxx-small ubiquitin-related modifier fusion protein exhibited increased nuclear localization and ability to suppress cell growth in Ba/F3 cells. These results demonstrate that the IFN-induced growth suppression of B lymphocytes requires nuclear localization of Daxx through its sumoylation and proper interactions with PML.     

Oncolytic vesicular stomatitis virus induces apoptosis via signaling through PKR, Fas, and Daxx

Matrix (M) protein mutants of vesicular stomatitis virus (VSV) are promising oncolytic agents for cancer therapy. Previous research has implicated Fas and PKR in apoptosis induced by other viruses. Here, we show that dominant-negative mutants of Fas and PKR inhibit M protein mutant virus-induced apoptosis. Most previous research has focused on the adapter protein FADD as a necessary transducer of Fas-mediated apoptosis. However, the expression of dominant-negative FADD had little effect on the induction of apoptosis by M protein mutant VSV. Instead, virus-induced apoptosis was inhibited by the expression of a dominant-negative mutant of the adapter protein Daxx. These data indicate that Daxx is more important than FADD for apoptosis induced by M protein mutant VSV. These results show that PKR- and Fas-mediated signaling play important roles in cell death during M protein mutant VSV infection and that Daxx has novel functions in the host response to virus infection by mediating virus-induced apoptosis.