p73, a member of the p53 family of transcription factors, is upregulated in response to DNA damage, inducing cell cycle arrest and apoptosis. Besides indications that this p73 response is post‐transcriptional, little is known about the underlying molecular mechanisms of p73 protein degradation. Ubiquitination and proteasomal‐dependent degradation of p53 are regulated by its transcriptional target MDM2. However, unlike p53, p73 binds to, but is not degraded by, MDM2. Here we describe the binding of p73 to Itch, a Hect ubiquitin–protein ligase. Itch selectively binds and ubiquitinates p73 but not p53; this results in the rapid proteasome‐dependent degradation of p73. Upon DNA damage Itch itself is downregulated, allowing p73 protein levels to rise and thus interfere with p73 function. In conclusion, we have identified a key mechanism in the control of p73 protein levels both in normal as well as in stress conditions.

BCL6 is the product of a proto-oncogene implicated in the pathogenesis of human B-cell lymphomas1,2. By binding specific DNA sequences, BCL6 controls the transcription of a variety of genes involved in B-cell development, differentiation and activation. BCL6 is overexpressed in the majority of patients with aggressive diffuse large B-cell lymphoma (DLBCL), the most common lymphoma in adulthood, and transgenic mice constitutively expressing BCL6 in B cells develop DLBCLs similar to the human disease3,4. In many DLBCL patients, BCL6 overexpression is achieved through translocation ( 40%) or hypermutation of its promoter ( 15%). However, many other DLBCLs overexpress BCL6 through an unknown mechanism. Here we show that BCL6 is targeted for ubiquitylation and proteasomal degradation by a SKP1–CUL1–F-box protein (SCF) ubiquitin ligase complex that contains the orphan F-box protein FBXO11 (refs 5, 6). The gene encoding FBXO11 was found to be deleted or mutated in multiple DLBCL cell lines, and this inactivation of FBXO11 correlated with increased levels and stability of BCL6. Similarly, FBXO11 was either deleted or mutated in primary DLBCLs. Notably, tumour-derived FBXO11 mutants displayed an impaired ability to induce BCL6 degradation. Reconstitution of FBXO11 expression in FBXO11-deleted DLBCL cells promoted BCL6 ubiquitylation and degradation, inhibited cell proliferation, and induced cell death. FBXO11-deleted DLBCL cells generated tumours in immunodeficient mice, and the tumorigenicity was suppressed by FBXO11 reconstitution. We reveal a molecular mechanism controlling BCL6 stability and propose that mutations and deletions in FBXO11 contribute to lymphomagenesis through BCL6 stabilization. The deletions/mutations found in DLBCLs are largely monoallelic, indicating that FBXO11 is a haplo-insufficient tumour suppressor gene.

Specific protein-protein interactions are involved in a large number of cellular processes and are mainly mediated by structurally and functionally defined domains. Here we report that the nuclear phosphoprotein p73 can engage in a physical association with the Yesassociated protein (YAP). This association occurs under physiological conditions as shown by reciprocal co-immunoprecipitation of complexes from lysates of P19 cells. The WW domain of YAP and the PPPPY motif of p73 are directly involved in the association. Furthermore, as required for ligands to group I WW domains, the terminal tyrosine (Y) of the PPPPY motif of p73 was shown to be essential for the association with YAP. Unlike p73␣, p73␤, and p63␣, which bind to YAP, the endogenous as well as exogenously expressed wild-type p53 (wt-p53) and the p73␥ isoform do not interact with YAP. Indeed, we documented that YAP interacts only with those members of the p53 family that have a well conserved PPXY motif, a target sequence for WW domains. Overexpression of YAP causes an increase of p73␣ transcriptional activity. Differential interaction of YAP with members of the p53 family may provide a molecular explanation for their functional divergence in signaling.

p53 is the most frequently inactivated tumor suppressor gene in human cancer, whereas its homologue, p73, is rarely mutated. Similarly to p53, p73 can promote growth arrest or apoptosis when overexpressed in certain p53-null tumor cells. It has previously been shown that some human tumor-derived p53 mutants can exert gain of function activity. The molecular mechanism underlying this activity remains to be elucidated. We show here that human tumor-derived p53 mutants (p53His175 and p53Gly281) associate in vitro and in vivo with p73␣, ␤, ␥, and ␦. This association occurs under physiological conditions, as verified in T47D and SKBR3 breast cancer cell lines. The core domain of mutant p53 is sufficient for the association with p73, whereas both the specific DNA binding and the oligomerization domains of p73 are required for the association with mutant p53. Furthermore, p53His175 and p53Gly281 mutants markedly reduce the transcriptional activity of the various isoforms of p73. Thus, human tumor-derived p53 mutants can associate with p73 not only physically but also functionally. These findings define a network involving mutant p53 and the various spliced isoforms of p73 that may confer upon tumor cells a selective survival advantage.

p63, a member of the p53 family of transcription factors, plays an important role in epithelial development, regulating both cell cycle and apoptosis. Even though p63 activity is regulated mainly at the posttranslational level, the control of p63 protein stability is far from being fully understood. Here, we show that the Hect (homologous to the E6-associated protein C terminus)-containing Nedd4-like ubiquitin protein ligase Itch binds, ubiquitylates, and promotes the degradation of p63. The physical interaction occurs at the border between the PY and the SAM (sterile ␣ motif) domains; a single Y504F mutation significantly affects p63 degradation. Itch and p63 are coexpressed in the epidermis and in primary keratinocytes where Itch controls the p63 protein steadystate level. Accordingly, p63 protein levels are significantly increased in Itch knockout keratinocytes. These data suggest that Itch has a fundamental role in the mechanism that controls endogenous p63 protein levels and therefore contributes to regulation of p63 in physiological conditions. keratinocytes ͉ ubiquitination ͉ p73 ͉ AIP4

3). In particular, the U7 small nuclear ribonucleoproteins and other factors involved in histone precursor mRNA processing are known to accumulate within CBs (1, 4). Notably, CBs also associate with the major histone gene clusters in a variety of organisms, including mammals, amphibians, and dipterans (5, 6). In addition to participating in various RNA-processing activities, CBs have also been implicated in transcriptional regulation of the cell-cycledependent histone genes. Phosphorylation of a CB component p220͞nuclear protein, ataxia-telangiectasia (NPAT) by cyclin E͞Cdk2 is required for activation of histone transcription, exit from G 1 , and progression through S phase (7-12). Taken together, these observations suggest that CBs are intimately involved in histone gene expression.In this study, we identify FADD-like IL-1␤-converting enzyme (FLICE) associated huge protein (FLASH) (13) as a component of the histone gene expression machinery. Although FLASH was originally identified as a component of the apoptotic signaling complex known as the death-inducing signaling complex (DISC) that is assembled in response to Fas ligand binding (13, 14), we have recently shown that FLASH is an essential component of CBs and is required for maintenance of their structure (15). We show that FLASH colocalizes with the histone transcriptional activator, NPAT, in CBs and is required for efficient expression of histone genes. Results FLASH Down-Regulation Results in S-Phase Block.One of the hallmarks of proteins that are involved in expression of the cell-cycle-dependent histone genes is that perturbation of their function results in an accumulation of cells in S phase. Accordingly, we found that treatment of cells with short hairpin RNAs (shRNAs) targeting FLASH (shFLASH) resulted in a dramatic block of cells within S-phase of the cell cycle (Fig. 1a). Such a block was observed in all cell lines tested (HEK293, HeLa, MCF-7, SAOS2, 3T3 and MEFs) reaching up to 70% after 72 h (see Fig. 5, which is published as supporting information on the PNAS web site). These findings were confirmed through use of a colony-forming assay, revealing that down-regulation of FLASH resulted in a remarkable reduction in growth of the shFLASH-treated cells (Fig. 1b). Western blot in Fig. 1c confirms FLASH protein levels downregulation after shRNA treatment.Another hallmark of genes involved in histone gene expression is that their protein levels are up-regulated during S phase. Endogenous FLASH expression showed a clear cell-cycledependence, peaking during S-phase, when cells were synchronized by thymidine block and deoxycytidine release (Fig. 1d). Consistent with these observations, we found that the number of FLASH-positive bodies was correlated with the cell cycle. Primary (IMR90) cells were used for this analysis, as they are diploid. As shown in Fig. 1e, the number of FLASH bodies in BrdU-positive (S-phase cells) was typically four, whereas in BrdU-negative cells, the number was typically two. FLASH Interacts with NPAT and Is Bound to Histone Ge...

The HECT-type E3 ubiquitin ligase (E3) Itch is absent in the non-agouti-lethal 18H or Itchy mice, which develop a severe immunological disease, including lung and stomach inflammation and hyperplasia of lymphoid and hematopoietic cells. The involvement of Itch in multiple signaling pathways and pathological conditions is presently an area of extensive scientific interest. This review aims to bring together a growing body of work exploring Itch-regulated biological processes, and to highlight recent discoveries on the regulatory mechanisms modulating its catalytic activity and substrate recognition capability. Our contribution is also an endeavor to correlate Itch substrate specificity with the pathological defects manifested by the mutant Itchy mice.

Inhibition of distinct ubiquitin E3 ligases might represent a powerful therapeutic tool. ITCH is a HECT domain-containing E3 ligase that promotes the ubiquitylation and degradation of several proteins, including p73, p63, c-Jun, JunB, Notch and c-FLIP, thus affecting cell fate. Accordingly, ITCH depletion potentiates the effect of chemotherapeutic drugs, revealing ITCH as a potential pharmacological target in cancer therapy. Using high throughput screening of ITCH auto-ubiquitylation, we identified several putative ITCH inhibitors, one of which is clomipramine—a clinically useful antidepressant drug. Previously, we have shown that clomipramine inhibits autophagy by blocking autophagolysosomal fluxes and thus could potentiate chemotherapy in vitro. Here, we found that clomipramine specifically blocks ITCH auto-ubiquitylation, as well as p73 ubiquitylation. By screening structural homologs of clomipramine, we identified several ITCH inhibitors and putative molecular moieties that are essential for ITCH inhibition. Treating a panel of breast, prostate and bladder cancer cell lines with clomipramine, or its homologs, we found that they reduce cancer cell growth, and synergize with gemcitabine or mitomycin in killing cancer cells by blocking autophagy. We also discuss a potential mechanism of inhibition. Together, our study (i) demonstrates the feasibility of using high throughput screening to identify E3 ligase inhibitors and (ii) provides insight into how clomipramine and its structural homologs might interfere with ITCH and other HECT E3 ligase catalytic activity in (iii) potentiating chemotherapy by regulating autophagic fluxes. These results may have direct clinical applications.