Laetitia K. Linares

HdmX stimulates Hdm2-mediated ubiquitination and degradation of p53

The RING finger proteins HdmX and Hdm2 share significant structural and functional similarity. Hdm2 is a member of the RING finger family of ubiquitin-protein ligases E3 and targets the tumor suppressor protein p53 for degradation. Although HdmX also binds to p53, HdmX does not induce p53 degradation. Moreover, HdmX has been reported to interfere with p53 degradation in overexpression experiments. To obtain insight into the mechanism by which HdmX interferes with p53 degradation, we studied the effect of HdmX on the E3 activity of Hdm2 in vitro. Surprisingly, this revealed that HdmX stimulates Hdm2-mediated ubiquitination of p53 and that HdmX facilitates ubiquitination of Hdm2 and vice versa. In addition, down-regulation of HdmX expression within cells results in the accumulation of both p53 and Hdm2. Because HdmX alone does not have appreciable E3 activity, these data indicate that HdmX acts as a stimulator, rather than as an inhibitor, of the E3 activity of Hdm2 and that, at least under certain conditions, HdmX is actively involved in the degradation of both p53 and Hdm2.

E4F1 is an atypical ubiquitin ligase that modulates p53 effector functions independently of degradation.

p53 is regulated by multiple posttranslational modifications, including Hdm2-mediated ubiquitylation that drives its proteasomal degradation. Here, we identify the p53-associated factor E4F1, a ubiquitously expressed zinc-finger protein first identified as a cellular target of the viral oncoprotein E1A, as an atypical ubiquitin E3 ligase for p53 that modulates its effector functions without promoting proteolysis. E4F1 stimulates oligo-ubiquitylation in the hinge region of p53 on lysine residues distinct from those targeted by Hdm2 and previously described to be acetylated by the acetyltransferase PCAF. E4F1 and PCAF mediate mutually exclusive posttranslational modifications of p53. E4F1-dependent Ub-p53 conjugates are associated with chromatin, and their stimulation coincides with the induction of a p53-dependent transcriptional program specifically involved in cell cycle arrest, and not apoptosis. Collectively, our data reveal that E4F1 is a key posttranslational regulator of p53, which modulates its effector functions involved in alternative cell fates: growth arrest or apoptosis.

A non-proteolytic role for ubiquitin in Tat-mediated transactivation of the HIV-1 promoter.

The human immunodeficiency virus type 1 (HIV-1) encodes a potent transactivator, Tat, which functions through binding to a short leader RNA, called transactivation responsive element (TAR). Recent studies suggest that Tat activates the HIV-1 long terminal repeat (LTR), mainly by adapting co-activator complexes, such as p300, PCAF and the positive transcription elongation factor P-TEFb, to the promoter. Here, we show that the proto-oncoprotein Hdm2 interacts with Tat and mediates its ubiquitination in vitro and in vivo. In addition, Hdm2 is a positive regulator of Tat-mediated transactivation, indicating that the transcriptional properties of Tat are stimulated by ubiquitination. Fusion of ubiquitin to Tat bypasses the requirement of Hdm2 for efficient transactivation, supporting the notion that ubiquitin has a non-proteolytic function in Tat-mediated transactivation.

Intrinsic ubiquitination activity of PCAF controls the stability of the oncoprotein Hdm2

The p300–CBP-associated factor (PCAF) is a histone acetyltransferase (HAT) involved in the reversible acetylation of various transcriptional regulators, including the tumour suppressor p53. It is implicated in many cellular processes, such as transcription, differentiation, proliferation and apoptosis. We observed that knockdown of PCAF expression in HeLa or U2OS cell lines induces stabilization of the oncoprotein Hdm2, a RING finger E3 ligase primarily known for its role in controlling p53 stability. To investigate the molecular basis of this effect, we examined whether PCAF is involved in Hdm2 ubiquitination. Here, we show that PCAF, in addition to its acetyltransferase activity, possesses an intrinsic ubiquitination activity that is critical for controlling Hdm2 expression levels, and thus p53 functions. Our data highlight a regulatory crosstalk between PCAF and Hdm2 activities, which is likely to have a central role in the subtle control of p53 activity after DNA damage.

Tip60 is targeted to proteasome-mediated degradation by Mdm2 and accumulates after UV irradiation.

Acetylation is a prominent post‐translational modification of nucleosomal histone N‐terminal tails, which regulates chromatin accessibility. Accordingly, histone acetyltransferases (HATs) play major roles in processes such as transcription. Here, we show that the HAT Tip60, which is involved in DNA repair and apoptosis following γ irradiation, is subjected to proteasome‐dependent proteolysis. Furthermore, we provide evidence that Mdm2, the ubiquitin ligase of the p53 tumour suppressor, interacts physically with Tip60 and induces its ubiquitylation and proteasome‐dependent degradation. Moreover, a ubiquitin ligase‐defective mutant of Mdm2 had no effect on Tip60 stability. Our results indicate that Mdm2 targets both p53 and Tip60, suggesting that these two proteins could be co‐regulated with respect to protein stability. Consistent with this hypothesis, Tip60 levels increased significantly upon UV irradiation of Jurkat cells. Collectively, our results suggest that degradation of Tip60 could be part of the mechanism leading to cell transformation by Mdm2.

BAT3 modulates p300-dependent acetylation of p53 and autophagy-related protein 7 (ATG7) during autophagy

Significance Autophagy allows the lysosomal degradation of intracellular material. It is a tightly regulated process controlled by the tumor-suppressor gene p53, among others. Here we report a unique regulator of autophagy, BAT3, which modulates the intracellular localization of the enzyme p300, an enzyme that adds some acetyl residues on targets proteins (acetylation) to modulate their activity. To stimulate autophagy, BAT3 allows the acetylation of p53 by p300 in the nucleus, but limits the p300-dependent acetylation of ATG7, a protein specific for autophagy, in the cytosol. Thus, BAT3 acts on both the cytosol and the nucleus to tightly modulate autophagy. Autophagy is regulated by posttranslational modifications, including acetylation. Here we show that HLA-B–associated transcript 3 (BAT3) is essential for basal and starvation-induced autophagy in embryonic day 18.5 BAT3−/− mouse embryos and in mouse embryonic fibroblasts (MEFs) through the modulation of p300-dependent acetylation of p53 and ATG7. Specifically, BAT3 increases p53 acetylation and proautophagic p53 target gene expression, while limiting p300-dependent acetylation of ATG7, a mechanism known to inhibit autophagy. In the absence of BAT3 or when BAT3 is located exclusively in the cytosol, autophagy is abrogated, ATG7 is hyperacetylated, p53 acetylation is abolished, and p300 accumulates in the cytosol, indicating that BAT3 regulates the nuclear localization of p300. In addition, the interaction between BAT3 and p300 is stronger in the cytosol than in the nucleus and, during starvation, the level of p300 decreases in the cytosol but increases in the nucleus only in the presence of BAT3. We conclude that BAT3 tightly controls autophagy by modulating p300 intracellular localization, affecting the accessibility of p300 to its substrates, p53 and ATG7.

Transcription factor E4F1 is essential for epidermal stem cell maintenance and skin homeostasis

A growing body of evidence suggests that the multifunctional protein E4F1 is involved in signaling pathways that play essential roles during normal development and tumorigenesis. We generated E4F1 conditional knockout mice to address E4F1 functions in vivo in newborn and adult skin. E4F1 inactivation in the entire skin or in the basal compartment of the epidermis induces skin homeostasis defects, as evidenced by transient hyperplasia in the interfollicular epithelium and alteration of keratinocyte differentiation, followed by loss of cellularity in the epidermis and severe skin ulcerations. E4F1 depletion alters clonogenic activity of epidermal stem cells (ESCs) ex vivo and ends in exhaustion of the ESC pool in vivo, indicating that the lesions observed in the E4F1 mutant skin result, at least in part, from cell-autonomous alterations in ESC maintenance. The clonogenic potential of E4F1 KO ESCs is rescued by Bmi1 overexpression or by Ink4a/Arf or p53 depletion. Skin phenotype of E4F1 KO mice is also delayed in animals with Ink4a/Arf and E4F1 compound gene deficiencies. Our data identify a regulatory axis essential for ESC-dependent skin homeostasis implicating E4F1 and the Bmi1–Arf–p53 pathway.

Chromatin-Bound MDM2 Regulates Serine Metabolism and Redox Homeostasis Independently of p53

The mouse double minute 2 (MDM2) oncoprotein is recognized as a major negative regulator of the p53 tumor suppressor, but growing evidence indicates that its oncogenic activities extend beyond p53. Here, we show that MDM2 is recruited to chromatin independently of p53 to regulate a transcriptional program implicated in amino acid metabolism and redox homeostasis. Identification of MDM2 target genes at the whole-genome level highlights an important role for ATF3/4 transcription factors in tethering MDM2 to chromatin. MDM2 recruitment to chromatin is a tightly regulated process that occurs during oxidative stress and serine/glycine deprivation and is modulated by the pyruvate kinase M2 (PKM2) metabolic enzyme. Depletion of endogenous MDM2 in p53-deficient cells impairs serine/glycine metabolism, the NAD(+)/NADH ratio, and glutathione (GSH) recycling, impacting their redox state and tumorigenic potential. Collectively, our data illustrate a previously unsuspected function of chromatin-bound MDM2 in cancer cell metabolism.

The ubiquitin-protein ligase activity of Hdm2 is inhibited by nucleic acids

The proto‐oncoprotein Hdm2 is a member of the RING finger‐type family of ubiquitin–protein ligases E3. The RING finger domain is assumed to mediate the specific interaction of an E3 with its cognate ubiquitin‐conjugating enzyme E2, which catalyzes the covalent attachment of ubiquitin to substrate proteins. In addition, the RING finger domain of Hdm2 is involved in Hdm2 homooligomer formation and has the capacity to bind to RNA in a sequence‐specific manner. Here we report that interaction with nucleic acids interferes with both Hdm2/Hdm2 complex formation and auto‐ubiquitination of Hdm2 in vitro. Furthermore, although binding of Hdm2 to the tumor suppressor p53 is not inhibited by nucleic acids, Hdm2‐mediated ubiquitination of p53 is significantly decreased. Taken together, these results provide the first example of an E3 whose activity can be regulated by direct interaction with nucleic acids.

E4F1 deficiency results in oxidative stress–mediated cell death of leukemic cells

Deletion of E4F1 inflicts mitochondrial damage and oxidative stress on murine and human myeloid leukemia cells but not healthy macrophages.