Biomedical subjects

Aart G Jochemsen

Publications and source records attributed to Aart G Jochemsen.

15 recordsLinked to original sources

PubMed2006

Inactivation of the p53 pathway in retinoblastoma.

Most human tumours have genetic mutations in their Rb and p53 pathways, but retinoblastoma is thought to be an exception. Studies suggest that retinoblastomas, which initiate with mutations in the gene retinoblastoma 1 (RB1), bypass the p53 pathway because they arise from intrinsically death-resistant cells during retinal development. In contrast to this prevailing theory, here we show that the tumour surveillance pathway mediated by Arf, MDM2, MDMX and p53 is activated after loss of RB1 during retinogenesis. RB1-deficient retinoblasts undergo p53-mediated apoptosis and exit the cell cycle. Subsequently, amplification of the MDMX gene and increased expression of MDMX protein are strongly selected for during tumour progression as a mechanism to suppress the p53 response in RB1-deficient retinal cells. Our data provide evidence that the p53 pathway is inactivated in retinoblastoma and that this cancer does not originate from intrinsically death-resistant cells as previously thought. In addition, they support the idea that MDMX is a specific chemotherapeutic target for treating retinoblastoma.

Animals↗

PubMed2006

Differential roles of ATM- and Chk2-mediated phosphorylations of Hdmx in response to DNA damage.

The p53 tumor suppressor plays a major role in maintaining genomic stability. Its activation and stabilization in response to double strand breaks (DSBs) in DNA are regulated primarily by the ATM protein kinase. ATM mediates several posttranslational modifications on p53 itself, as well as phosphorylation of p53's essential inhibitors, Hdm2 and Hdmx. Recently we showed that ATM- and Hdm2-dependent ubiquitination and subsequent degradation of Hdmx following DSB induction are mediated by phosphorylation of Hdmx on S403, S367, and S342, with S403 being targeted directly by ATM. Here we show that S367 phosphorylation is mediated by the Chk2 protein kinase, a downstream kinase of ATM. This phosphorylation, which is important for subsequent Hdmx ubiquitination and degradation, creates a binding site for 14-3-3 proteins which controls nuclear accumulation of Hdmx following DSBs. Phosphorylation of S342 also contributed to optimal 14-3-3 interaction and nuclear accumulation of Hdmx, but phosphorylation of S403 did not. Our data indicate that binding of a 14-3-3 dimer and subsequent nuclear accumulation are essential steps toward degradation of p53's inhibitor, Hdmx, in response to DNA damage. These results demonstrate a sophisticated control by ATM of a target protein, Hdmx, which itself is one of several ATM targets in the ATM-p53 axis of the DNA damage response.

14-3-3 Proteins↗

PubMed2005

ATM-mediated phosphorylations inhibit Mdmx/Mdm2 stabilization by HAUSP in favor of p53 activation.

The p53 tumor suppressor protein has a major role in protecting genome integrity. Under normal circumstances Mdmx and Mdm2 control the activity of p53. Both proteins inhibit the transcriptional regulation by p53, while Mdm2 also functions as an E3 ubiquitin ligase to target both p53 and Mdmx for proteasomal degradation. HAUSP counteracts the destabilizing effect of Mdm2 by direct deubiquitination of p53. Subsequently, HAUSP was shown to deubiquitinate Mdm2 and Mdmx, thereby stabilizing these proteins. The ATM protein kinase is a key regulator of the p53 pathway in response to double strand breaks (DSBs) in the DNA. ATM fine-tunes p53's response to DNA damage by directly phosphorylating it, by regulating additional post-translational modifications of this protein, and by affecting two p53 regulators: Mdm2 and Mdmx. ATM directly and indirectly induces Mdm2 and Mdmx phosphorylation, resulting in decreased activity and stability of these proteins. We recently provided a mechanism for the reduced stability of Mdm2 and Mdmx by showing that ATM-dependent phosphorylation lowers their affinity for the deubiquitinating enzyme HAUSP. Altogether, the emerging picture portrays an elaborate, but fine-tuned, ATM-mediated control of p53 activation and stabilization following DNA damage. Further insight into the mechanism by which ATM switches the interactions between HAUSP, Mdmx, Mdm2 and p53, to favor p53 activation may offer new tools for therapeutic intervention in the p53 pathway for cancer treatment.

Adenosine Triphosphate↗

PubMed2005

Mdmx as an essential regulator of p53 activity.

The murine double minute 2 (Mdm2) is a critical negative regulator of the p53 tumor suppressor. Almost 10 years ago, a search for new p53-interactors revealed the existence of an Mdm2-structurally related protein, Mdmx (or Mdm4). Since then a large body of biochemical data has accumulated on the functions of Mdmx, often leading to conflicting molecular models. Nevertheless, virtually all these data pointed toward a critical role for Mdmx in the regulation of the p53-Mdm2 network. A view that was recently confirmed by genetic studies. This review is a summary of our current understanding of this molecule, its structure and biological functions, as well as its relationship to its known binding partners.

Amino Acid Motifs↗

PubMed2005

Loss of HAUSP-mediated deubiquitination contributes to DNA damage-induced destabilization of Hdmx and Hdm2.

The p53 tumor suppressor protein has a major role in protecting the integrity of the genome. In unstressed cells, p53 is maintained at low levels by the ubiquitin-proteasome pathway. A balance between ubiquitin ligase activity (Hdm2, COP1, and Pirh2) and the ubiquitin protease activity of the Herpes virus-associated ubiquitin-specific protease (HAUSP) determines the half-life of p53. HAUSP also modulates p53 stability indirectly by deubiquitination and stabilization of Hdm2. The Hdmx protein affects p53 stability as well through its interaction with and regulation of Hdm2. Vice versa, Hdmx is a target for Hdm2-mediated ubiquitination and degradation. Here, we show that HAUSP also interacts with Hdmx, resulting in its direct deubiquitination and stabilization. HAUSP activity is required to maintain normal Hdmx protein levels. Therefore, the balance between HAUSP and Hdm2 activity determines Hdmx protein stability. Importantly, impaired deubiquitination of Hdmx/Hdm2 by HAUSP contributes to the DNA damage-induced degradation of Hdmx and transient instability of Hdm2.

Animals↗

PubMed2005

Phosphorylation of Hdmx mediates its Hdm2- and ATM-dependent degradation in response to DNA damage.

Maintenance of genomic stability depends on the DNA damage response, an extensive signaling network that is activated by DNA lesions such as double-strand breaks (DSBs). The primary activator of the mammalian DSB response is the nuclear protein kinase ataxia-telangiectasia, mutated (ATM), which phosphorylates key players in various arms of this network. The activation and stabilization of the p53 protein play a major role in the DNA damage response and are mediated by ATM-dependent posttranslational modifications of p53 and Mdm2, a ubiquitin ligase of p53. p53's response to DNA damage also depends on Mdm2-dependent proteolysis of Mdmx, a homologue of Mdm2 that represses p53's transactivation function. Here we show that efficient damage-induced degradation of human Hdmx depends on functional ATM and at least three sites on the Hdmx that are phosphorylated in response to DSBs. One of these sites, S403, is a direct ATM target. Accordingly, each of these sites is important for Hdm2-mediated ubiquitination of Hdmx after DSB induction. These results demonstrate a sophisticated mechanism whereby ATM fine-tunes the optimal activation of p53 by simultaneously modifying each player in the process.

Ataxia Telangiectasia↗

PubMed2005

DNA damage-induced phosphorylation of MdmX at serine 367 activates p53 by targeting MdmX for Mdm2-dependent degradation.

Understanding how p53 activity is regulated is crucial in elucidating mechanisms of cellular defense against cancer. Genetic data indicate that Mdmx as well as Mdm2 plays a major role in maintaining p53 activity at low levels in nonstressed cells. However, biochemical mechanisms of how Mdmx regulates p53 activity are not well understood. Through identification of Mdmx-binding proteins, we found that 14-3-3 proteins are associated with Mdmx. Mdmx harbors a consensus sequence for binding of 14-3-3. Serine 367 (S367) is located within the putative binding sequence for 14-3-3, and its substitution with alanine (S367A) abolishes binding of Mdmx to 14-3-3. Transfection assays indicated that the S367A mutation, in cooperation with Mdm2, enhances the ability of Mdmx to repress the transcriptional activity of p53. The S367A mutant is more resistant to Mdm2-dependent ubiquitination and degradation than wild-type Mdmx, and Mdmx phosphorylated at S367 is preferentially degraded by Mdm2. Several types of DNA damage markedly enhance S367 phosphorylation, coinciding with increased binding of Mdmx to 14-3-3 and accelerated Mdmx degradation. Furthermore, promotion of growth of normal human fibroblasts after introduction of Mdmx is enhanced by the S367 mutation. We propose that Mdmx phosphorylation at S367 plays an important role in p53 activation after DNA damage by triggering Mdm2-dependent degradation of Mdmx.

14-3-3 Proteins↗

PubMed2004

Mdmx and Mdm2: brothers in arms?

The p53 tumor suppressor pathway is inactivated in most if not all human tumors. In about 50% of the cases this is accomplished directly by gene mutations. The tumors that retain wild type p53 frequently show defects either in effector target genes, or in the expression of p53 regulatory proteins. The Mdm2 protein is generally considered THE master regulator of the p53 tumor suppressor activity. Recently, however, the Mdm2-related protein Mdmx is taking the stage in the p53-Mdm2-Mdmx play. We summarize here observations unambiguously assigning a critical role for the Mdmx protein in the regulation of p53 function during development and tumor formation.

Animals↗

PubMed2004

Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity.

Human tumors are believed to harbor a disabled p53 tumor suppressor pathway, either through direct mutation of the p53 gene or through aberrant expression of proteins acting in the p53 pathway, such as p14(ARF) or Mdm2. A role for Mdmx (or Mdm4) as a key negative regulator of p53 function in vivo has been established. However, a direct contribution of Mdmx to tumor formation remains to be demonstrated. Here we show that retrovirus-mediated Mdmx overexpression allows primary mouse embryonic fibroblast immortalization and leads to neoplastic transformation in combination with HRas(V12). Furthermore, the human Mdmx ortholog, Hdmx, was found to be overexpressed in a significant percentage of various human tumors and amplified in 5% of primary breast tumors, all of which retained wild-type p53. Hdmx was also amplified and highly expressed in MCF-7, a breast cancer cell line harboring wild-type p53, and interfering RNA-mediated reduction of Hdmx markedly inhibited the growth potential of these cells in a p53-dependent manner. Together, these results make Hdmx a new putative drug target for cancer therapy.

Animals↗

PubMed2003

Hdmx protein stability is regulated by the ubiquitin ligase activity of Mdm2.

The stability of the p53 tumor suppressor protein is critically regulated by the Hdm2 and Hdmx proteins. Hdm2 protein levels are auto-regulated by the self-ubiquitination activity of Hdm2 and on the transcriptional level by p53-activated transcription of the hdm2 gene. Little is known about the regulation of Hdmx expression levels, apart from the observation that the Mdmx protein can be cleaved by caspase-3 in a p53-inducible manner. In the functional analysis of two mutant Hdmx proteins, products of two alternatively spliced mRNAs, it was found that Hdmx proteins are targets for ubiquitination by Mdm2. The stability of the Hdmx protein is partly dependent on the presence of its internal acidic domain. Mdm2 appears only to require an intact RING domain to be able to ubiquitinate Hdmx and target it for proteasomal degradation. These findings highlight the intricate functional relationships between p53, Mdm2, and Hdmx.

Alternative Splicing↗

PubMed2003

Critical role for a central part of Mdm2 in the ubiquitylation of p53.

The stability of the p53 protein is regulated by Mdm2. By acting as an E3 ubiquitin ligase, Mdm2 directs the ubiquitylation of p53 and its subsequent degradation by the 26S proteasome. In contrast, the Mdmx protein, although structurally similar to Mdm2, cannot ubiquitylate or degrade p53 in vivo. To ascertain which domains determine this functional difference between Mdm2 and Mdmx and consequently are essential for p53 ubiquitylation and degradation, we generated Mdm2-Mdmx chimeric constructs. Here we show that, in addition to a fully functional Mdm2 RING finger, an internal domain of Mdm2 (residues 202 to 302) is essential for p53 ubiquitylation. Strikingly, the function of this domain can be fulfilled in trans, indicating that the RING domain and this internal region perform distinct activities in the ubiquitylation of p53.

Binding Sites↗

PubMed2002

Mutual dependence of MDM2 and MDMX in their functional inactivation of p53.

MDMX, an MDM2-related protein, has emerged as yet another essential negative regulator of p53 tumor suppressor, since loss of MDMX expression results in p53-dependent embryonic lethality in mice. However, it remains unknown why neither homologue can compensate for the loss of the other. In addition, results of biochemical studies have suggested that MDMX inhibits MDM2-mediated p53 degradation, thus contradicting its role as defined in gene knockout experiments. Using cells deficient in either MDM2 or MDMX, we demonstrated that these two p53 inhibitors are in fact functionally dependent on each other. In the absence of MDMX, MDM2 is largely ineffective in down-regulating p53 because of its extremely short half-life. MDMX renders MDM2 protein sufficiently stable to function at its full potential for p53 degradation. On the other hand, MDMX, which is a cytoplasmic protein, depends on MDM2 to redistribute into the nucleus and be able to inactivate p53. We also showed that MDMX, when exceedingly overexpressed, inhibits MDM2-mediated p53 degradation by competing with MDM2 for p53 binding. Our findings therefore provide a molecular basis for the nonoverlapping activities of these two p53 inhibitors previously revealed in genetic studies.

Animals↗

PubMed2002

MDMX stability is regulated by p53-induced caspase cleavage in NIH3T3 mouse fibroblasts.

MDMX is a p53 binding protein, which shares a high degree of homology with MDM2, a negative regulator of the tumor suppressor p53. MDMX has been shown to counteract MDM2-dependent p53 degradation and to stabilize p53 in its inactive form. In this study: we identify two MDMX proteolytic pathways that control its intracellular levels, and show that MDMX post-translational processing may be regulated by p53. Mouse MDMX is cleaved in vitro and in vivo by caspase activity, between aminoacids 358 and 361, producing a p54 minor form. In addition, MDMX is subjected to proteasome-mediated degradation, which concurs to MDMX proteolysis mainly through degradation of p54. A D361A-MDMX mutant, resistant to caspase cleavage, exhibits prolonged intracellular lifetime in comparison to wild-type protein, indicating that caspase cleavage affects stability of MDMX protein probably by modulating its further degradation. Overexpression of exogenous p53 increases the intracellular levels of p54 product. Similarly, activation of endogenous p53 by adriamycin enhances MDMX cleavage and produces a marked decrease of its intracellular levels, while not affecting the D361A-MDMX mutant. In addition, the D361A-MDMX mutant lacks the ability to inhibit p53 transactivation in respect to wild-type MDMX, suggesting that MDMX caspase cleavage play an important functional role. In conclusion, our results demonstrate that, in analogy to MDM2, MDMX may be subjected to proteolytic modifications that regulate its intracellular levels. Moreover, decrease of MDMX protein levels following p53 activation suggests a p53-dependent regulatory feedback of MDMX function.

3T3 Cells↗

PubMed2002

p63.

The p63 gene, a member of the p53 gene family, is expressed into at least six protein isoforms which are divided into two groups, those containing the transcription activation domain (TA isoforms) and those that do not (Delta N isoforms). The TA isoforms are similar to p53 in that they are able to activate transcription of specific target genes and induce cell cycle arrest and apoptosis. The Delta N isoforms are unable to activate transcription, and act in a dominant negative manner, inhibiting transcription activation by both p53 and TA isoforms. p63 knock-out studies in mice have shown that p63 plays an important role in development rather than in tumour suppression. In humans, mutations in the p63 gene have been linked with several developmental abnormalities. Studies on human tumours suggest an oncogenic function for Delta N isoforms rather than a tumour suppressor function for the TA forms.

Animals↗

PubMed2002

Induction of p21 mRNA synthesis after short-wavelength UV light visualized in individual cells by RNA FISH.

Expression of the cyclin-dependent kinase inhibitor gene p21 is induced after DNA damage and plays a role in cell survival. The exact mechanism of induction is not known, but enhancement of mRNA stability has recently been implicated as an important factor. To obtain further insight into the dynamics of p21 gene expression at the individual cell level, normal fibroblasts, GM1492 fibroblasts from a Bloom's syndrome patient, and U2OS osteosarcoma cells were UVC irradiated, fixed at different time points, and subjected to mRNA fluorescence in situ hybridization (FISH) and immunocytochemical staining. In mock-irradiated normal fibroblasts, a subfraction of cells revealed low levels of p21 mRNA synthesis. After UVC treatment, p21 transcripts accumulated over time in nuclear locations other than transcription foci. At 6 hr after irradiation, almost 50% of the cells displayed p21 mRNA in three different distribution patterns within the nuclei. The highest frequency of cells with cytoplasmic accumulation of p21 mRNA was seen at 17 hr after UVC treatment. We conclude that increased p21 gene transcription and possibly stabilization of newly synthesized p21 mRNA contribute to elevated levels of p21 protein after UVC irradiation. (J Histochem Cytochem 50:81-89, 2002)

Cell Line↗