Yosef Shaul

Interaction of c-Abl and p73α and their collaboration to induce apoptosis

c-Abl, a non-receptor tyrosine kinase, is activated by agents that damage DNA. This activation results in either arrest of the cell cycle in phase G1 or apoptotic cell death, both of which are dependent on the kinase activity of c-Abl. p73, a member of the p53 family of tumour-suppressor proteins,, can also induce apoptosis. Here we show that the apoptotic activity of p73α requires the presence of functional, kinase-competent c-Abl. Furthermore, p73 and c-Abl can associate with each other, and this binding is mediated by a PxxP motif in p73 and the SH3 domain of c-Abl. We find that p73 is a substrate of the c-Abl kinase and that the ability of c-Abl tophosphorylate p73 is markedly increased by γ-irradiation. Moreover, p73 is phosphorylated in vivo in response to ionizing radiation. These findings define a pro-apoptotic signalling pathway involving p73 and c-Abl.

Physical and Functional Interaction between p53 Mutants and Different Isoforms of p73

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 andin vivo with p73α, β, γ, and δ. 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.

NQO1 stabilizes p53 through a distinct pathway

Wild-type p53 is a tumor-suppressor gene that encodes a short-lived protein that, upon accumulation, induces growth arrest or apoptosis. Accumulation of p53 occurs mainly by posttranslational events that inhibit its proteosomal degradation. We have reported previously that inhibition of NAD(P)H: quinone oxidoreductase 1 (NQO1) activity by dicoumarol induces degradation of p53, indicating that NQO1 plays a role in p53 stabilization. We now have found that wild-type NQO1, but not the inactive polymorphic NQO1, can stabilize endogenous as well as transfected wild-type p53. NQO1-mediated p53 stabilization was especially prominent under induction of oxidative stress. NQO1 also partially inhibited p53 degradation mediated by the human papilloma virus E6 protein, but not when mediated by Mdm-2. Inhibitors of heat shock protein 90 (hsp90), radicicol and geldanamycin, induced degradation of p53 and suppressed p53-induced apoptosis in normal thymocytes and myeloid leukemic cells. Differences in the effectiveness of dicoumarol and hsp90 inhibitors to induce p53 degradation and suppress apoptosis in these cell types indicate that NQO1 and hsp90 stabilize p53 through different mechanisms. Our results indicate that NQO1 has a distinct role in the regulation of p53 stability, especially in response to oxidative stress. The present data on the genetic and pharmacologic regulation of the level of p53 have clinical implications for tumor development and therapy.

Yap1 Phosphorylation by c-Abl Is a Critical Step in Selective Activation of Proapoptotic Genes in Response to DNA Damage

Cells undergo apoptosis upon exposure to severe DNA damage stress. Under this condition, p73 is phosphorylated and activated by c-Abl. The transcription coactivator Yap1 binds p73 to generate a complex that escapes p73 proteasomal degradation and recruits p300 to support transcription of proapoptotic genes. However, the mechanism of selective activation of proapoptotic genes by Yap1 remained unclear. In this study, we show that c-Abl directly phosphorylates Yap1 at position Y357 in response to DNA damage. Tyrosine-phosphorylated Yap1 is a more stable protein that displays higher affinity to p73 and selectively coactivates p73 proapoptotic target genes. Furthermore, we show that Yap1 switches between p73-mediated proapoptotic and growth arrest target genes based on its phosphorylation state. Thus, our data demonstrate that modification of a transcription coactivator, namely the DNA damage-induced phosphorylation of Yap1 by c-Abl, influences the specificity of target gene activation.

Mdm-2 and ubiquitin-independent p53 proteasomal degradation regulated by NQO1

The tumor suppressor p53 is a labile protein whose level is known to be regulated by the Mdm-2–ubiquitin–proteasome degradation pathway. We have found another pathway for p53 proteasomal degradation regulated by NAD(P)H quinone oxidoreductase 1 (NQO1). Inhibition of NQO1 activity by dicoumarol induces p53 and p73 proteasomal degradation. A mutant p53 (p53[22,23]), which is resistant to Mdm-2-mediated degradation, was susceptible to dicoumarol-induced degradation. This finding indicates that the NQO1-regulated proteasomal p53 degradation is Mdm-2-independent. The tumor suppressor p14ARF and the viral oncogenes SV40 LT and adenovirus E1A that are known to stabilize p53 inhibited dicoumarol-induced p53 degradation. Unlike Mdm-2-mediated degradation, the NQO1-regulated p53 degradation pathway was not associated with accumulation of ubiquitin-conjugated p53. In vitro studies indicate that dicoumarol-induced p53 degradation was ubiquitin-independent and ATP-dependent. Inhibition of NQO1 activity in cells with a temperature-sensitive E1 ubiquitin-activating enzyme induced p53 degradation and inhibited apoptosis at the restrictive temperature without ubiquitination. Mdm-2 failed to induce p53 degradation under these conditions. Our results establish a Mdm-2- and ubiquitin-independent mechanism for proteasomal degradation of p53 that is regulated by NQO1. The lack of NQO1 activity that stabilizes a tumor suppressor such as p53 can explain why humans carrying a polymorphic inactive NQO1 are more susceptible to tumor development.

A human hepatitis B viral enhancer element.

Fragments of the cloned hepatitis B virus (HBV) genome were assayed in vivo for the presence of a transcriptional enhancer element. We demonstrate that sequences positioned approximately 450 bp upstream from the HBcAg gene promoter are required for its efficient activity. These HBV stimulatory sequences activate transcription when inserted upstream to a heterologous SV40 early promoter. Like other known enhancer elements, this HBV sequence acts in an orientation‐independent manner. Furthermore, the HBV enhancer element exhibits a preferred activity in a human hepatoma cell line.

The Yes-associated protein 1 stabilizes p73 by preventing Itch-mediated ubiquitination of p73

Upon DNA damage signaling, p73, a member of the p53 tumor suppressor family, accumulates to support transcription of downstream apoptotic genes. p73 interacts with Yes-associated protein 1 (Yap1) through its PPPY motif, and increases p73 transactivation of apoptotic genes. The ubiquitin E3 ligase Itch, like Yap1, interacts with p73. Given the fact that both Itch and Yap1 bind p73 via the PPPY motif, we hypothesized that Yap may also function to stabilize p73 by displacing Itch binding to p73. We show that the interaction of Yap1 and p73 was necessary for p73 stabilization. Yap1 competed with Itch for binding to p73, and prevented Itch-mediated ubiquitination of p73. Treatment of cells with cisplatin leads to an increase in p73 accumulation and induction of apoptosis, but both were dramatically reduced in the presence of Yap1 siRNA. Altogether, our findings attribute a central role to Yap1 in regulating p73 accumulation and function under DNA damage signaling.

Hepatitis B Virus pX Targets TFIIB in Transcription Coactivation

ABSTRACT pX, the hepatitis B virus (HBV)-encoded regulator, coactivates transcription through an unknown mechanism. pX interacts with several components of the transcription machinery, including certain activators, TFIIB, TFIIH, and the RNA polymerase II (POLII) enzyme. We show that pX localizes in the nucleus and coimmunoprecipitates with TFIIB from nuclear extracts. We used TFIIB mutants inactive in binding either POLII or TATA binding protein to study the role of TFIIB-pX interaction in transcription coactivation. pX was able to bind the former type of TFIIB mutant and not the latter. Neither of these sets of TFIIB mutants supports transcription. Remarkably, the latter TFIIB mutants fully block pX activity, suggesting the role of TFIIB in pX-mediated coactivation. By contrast, in the presence of pX, TFIIB mutants with disrupted POLII binding acquire the wild-type phenotype, both in vivo and in vitro. These results suggest that pX may establish the otherwise inefficient TFIIB mutant-POLII interaction, by acting as a molecular bridge. Collectively, our results demonstrate that TFIIB is the in vivo target of pX.

c-Abl: activation and nuclear targets

The c-Abl tyrosine kinase and its transforming variants have been implicated in tumorigenesis and in many important cellular processes. c-Abl is localized in the nucleus and the cytoplasm, where it plays distinct roles. The effects of c-Abl are mediated by multiple protein-protein and protein-DNA interactions and its tyrosine kinase domain. At the biochemical level, the mechanism of c-Abl kinase activation and the identification of its target proteins and cellular machineries have in part been solved. However, the phenotypic outcomes of these molecular events remained in large elusive. c-Abl has been shown to regulate the cell cycle and to induce under certain conditions cell growth arrest and apoptosis. In this respect the interaction of c-Abl with p53 and p73 has attracted particular attention. Recent findings have implicated c-Abl in an ionizing irradiation signaling pathway that elicits apoptosis. In this pathway p73 is an important immediate downstream effector. Here I review the current knowledge about these nuclear processes in which c-Abl is engaged and discuss some of their possible implications on cell physiology.

Cytokine induction by the hepatitis B virus capsid in macrophages is facilitated by membrane heparan sulfate and involves TLR2.

The hepatitis B virus (HBV) core Ag (HBcAg) serves as the structural subunit of the highly immunogenic capsid shell. HBcAg harbors a unique arginine-rich C terminus that was implicated in immune responses induced by the capsid. In this study, we examined the capacity of the HBV capsid to induce proinflammatory and regulatory cytokines in human THP-1 macrophages and the possible underlying mechanism. Full-length HBc capsids, but not ΗΒc-144 capsids lacking the arginine-rich domain of HBcAg, efficiently bound differentiated THP-1 macrophages and strongly induced TNF-α, IL-6, and IL-12p40. Capsid binding to macrophages and cytokine induction were independent of the RNA associated with the arginine-rich domain. Soluble heparin and heparan sulfate but not chondroitin sulfates greatly diminished cytokine induction through inhibition of capsid binding to THP-1 macrophages. Furthermore, serine phosphorylation in the arginine-rich domain modulates capsid binding to macrophages and the cytokine response. Induction of cytokines by the capsid involved activation of NF-κB, ERK-1/2, and p38 MAPK and did not require endosomal acidification. Finally, NF-κB activation by the capsid in HEK 293 cells specifically required expression of TLR2 and was compromised by soluble heparin. Thus, cytokine induction by the HBV capsid in macrophages is facilitated by interaction of its arginine-rich domain with membrane heparan sulfate and involves signaling through TLR2.