PML isoforms IV and V contribute to adenovirus-mediated oncogenic transformation by functionally inhibiting the tumor-suppressor p53.

Although modulation of the cellular tumor-suppressor p53 is considered to have the major role in E1A/E1B-55K-mediated tumorigenesis, other promyelocytic leukemia nuclear body (PML-NB)/PML oncogenic domain (POD)-associated factors including SUMO, Mre11, Daxx, as well as the integrity of these nuclear bodies contribute to the transformation process. However, the biochemical consequences and oncogenic alterations of PML-associated E1B-55K by SUMO-dependent PML-IV and PML-V interaction have so far remained elusive. We performed mutational analysis to define a PML interaction motif within the E1B-55K polypeptide. Our results showed that E1B-55K/PML binding is not required for p53, Mre11 and Daxx interaction. We also observed that E1B-55K lacking subnuclear PML localization because of either PML-IV or PML-V-binding deficiency was no longer capable of mediating E1B-55K-dependent SUMOylation of p53, inhibition of p53-mediated transactivation or efficiently transforming primary rodent cells. These results together with the observation that E1B-55K-dependent SUMOylation of p53 is required for efficient cell transformation, provides evidence for the idea that the SUMO ligase activity of the E1B-55K viral oncoprotein is intimately linked to its growth-promoting oncogenic activities.

Cross-talk between phosphorylation and SUMOylation regulates transforming activities of an adenoviral oncoprotein.

Since the discovery of post-translational modification (PTM) by the small ubiquitin-related modifiers (SUMOs), a multitude of proteins have been described to be reversibly modified, resulting in the alteration of several cellular pathways. Interestingly, various pathogens gain access to this modification system, although the molecular mechanisms and functional consequences are barely understood. We show here that the adenoviral oncoprotein E1B-55K is a substrate of the SUMO conjugation system, which is directly linked to its C-terminal phosphorylation. This regulative connection is indispensable for modulation of the tumor suppressor p53/chromatin-remodeling factor Daxx by E1B-55K and, consequently, its oncogenic potential in primary mammalian cells. In virus infection, E1B-55K PTMs are necessary for localization to viral transcription/replication sites. Furthermore, we identify the E2 enzyme Ubc9 as an interaction partner of E1B-55K, providing a possible molecular explanation for SUMO-dependent modulation of cellular target proteins. In conclusion, these results for the first time provide evidence how E1B-55K PTMs are regulated and subsequently facilitate exploitation of the host cell SUMOylation machinery.

The adenovirus E4orf4 protein induces growth arrest and mitotic catastrophe in H1299 human lung carcinoma cells.

The human adenovirus E4orf4 protein, when expressed alone, induces p53-independent death in a wide range of cancer cells. Earlier studies by our groups suggested that although in some cases cell death can be associated with some hallmarks of apoptosis, it is not always affected by caspase inhibitors. Thus it is unlikely that E4orf4-induced cell death occurs uniquely through apoptosis. In the present studies using H1299 human lung carcinoma cells as a model system we found that death is induced in the absence of activation of any of the caspases tested, accumulation of reactive oxygen species, or release of cytochrome c from mitochondria. E4orf4 caused a substantial change in cell morphology, including vigorous membrane blebbing, multiple nuclei in many cells and increased cell volume. Most of these characteristics are not typical of apoptosis, but they are of necrosis. FACS analysis and western blotting for cell cycle markers showed that E4orf4-expressing cells became arrested in G(2)/M and also accumulated high levels of cyclin E. The presence of significant numbers of tetraploid and polyploid cells and some cells with micronuclei suggested that E4orf4 appears to induce death in these cells through a process resulting from mitotic catastrophe.

SIRT1 negatively regulates HDAC1-dependent transcriptional repression by the RBP1 family of proteins.

Both RBP1 and the highly related protein BCAA play a role in the induction of growth arrest and cellular senescence via mechanisms involving transcriptional repression. While investigating the transcriptional repression activities of RBP1, we observed a genetic link between RBP1 and SIR2. Further work uncovered an interaction between RBP1 family proteins and the mammalian homologue of SIR2, SIRT1. Interestingly, the HDAC-dependent transcriptional repression domain of RBP1 proteins, termed R2, is necessary and sufficient for the interaction with SIRT1. In vitro and in vivo binding studies indicated that the p33(ING1b) and p33(ING2) subunits of the mSIN3A/HDAC1 complex are responsible for the recruitment of SIRT1 to the R2 domain. To investigate the biological relevance of this interaction, we used the sirtuin activator resveratrol and the sirtuin inhibitor sirtinol in transcriptional repression assays and demonstrated that SIRT1 activity negatively regulates R2-mediated transcriptional repression activity. We therefore propose a novel mechanism of class I HDAC regulation by a class III HDAC. Explicitly, SIRT1 is recruited by ING proteins and inhibits R2-associated mSIN3A/HDAC1 transcriptional repression activity.

Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs via a novel mechanism involving a Cullin-containing complex.

Although MDM2 plays a major role in regulating the stability of the p53 tumor suppressor protein, other poorly understood MDM2-independent pathways also exist. Human adenoviruses have evolved strategies to regulate p53 function and stability to permit efficient viral replication. One mechanism involves adenovirus E1B55K and E4orf6 proteins, which collaborate to target p53 for degradation. To determine the mechanism of this process, a multiprotein E4orf6-associated complex was purified and shown to contain a novel Cullin-containing E3 ubiquitin ligase that is (1) composed of Cullin family member Cul5, Elongins B and C, and the RING-H2 finger protein Rbx1(ROC1); (2) remarkably similar to the von Hippel-Lindau tumor suppressor and SCF (Skp1-Cul1/Cdc53-F-box) E3 ubiquitin ligase complexes; and (3) capable of stimulating ubiquitination of p53 in vitro in the presence of E1/E2 ubiquitin-activating and -conjugating enzymes. Cullins are activated by NEDD8 modification; therefore, to determine whether Cullin complexes are required for adenovirus-induced p53 degradation, studies were conducted in ts41 Chinese hamster ovary cells that are temperature sensitive for the NEDD8 pathway. E4orf6/E1B55K failed to induce the degradation of p53 at the nonpermissive temperature. Thus, our results identify a novel role for the Cullin-based machinery in regulation of p53.

The role of adenovirus E4orf4 protein in viral replication and cell killing.

It has only been within the last few years that insights have been gained into the remarkable diversity of functions of the adenovirus early transcription region 4 (E4) products. The polypeptide encoded by E4 open reading frame 4 (E4orf4) has emerged as an enigmatic product. Although it accomplishes certain functions that propel viral replication, it has also been shown to be highly toxic, an effect that could dampen the infectious cycle, but that also might serve to facilitate release of viral progeny. When expressed alone, E4orf4 induces a novel form of p53-independent apoptosis in cancer cells but not in normal human cells, thus making it of potential use in cancer gene therapy. In addition, knowledge of its mechanism of action, especially with regard to its interaction with protein phosphatase 2A (PP2A), could provide insights to develop new small molecule anti-cancer drugs. Thus future studies on E4orf4 should be both informative and potentially valuable therapeutically. In this study we review the current status of knowledge on E4orf4.

Toxicity of human adenovirus E4orf4 protein in Saccharomyces cerevisiae results from interactions with the Cdc55 regulatory B subunit of PP2A.

The E4orf4 protein of human adenovirus induces p53-independent apoptosis, a process that may promote cell death and viral spread. When expressed alone, E4orf4 kills transformed cells but not normal human cells. The only clear target of E4orf4 in mammalian cells is the Balpha (B55) subunit of protein phosphatase 2A (PP2A), a member of one of three classes of regulatory B subunits. Here we report the effects of E4orf4 in Saccharomyces cerevisiae, which encodes two PP2A regulatory B subunits, CDC55 and RTS1, that share homology with mammalian B and B' subunits, respectively. E4orf4 expression was found to be toxic in yeast, resulting in the accumulation of cells in G2/M phase that failed to grow upon removal of E4orf4. E4orf4-expressing yeast also displayed an elongated cell morphology similar to cdc55 deletion strains. E4orf4 required CDC55 to elicit its effect, whereas RTS1 was dispensable. The recruitment of the PP2A holoenzyme by E4orf4 was entirely dependent on Cdc55. These studies indicate that E4orf4-induced apoptosis in mammalian cells and cell death in yeast require functional interactions with B-type subunits of PP2A. However, some inhibition of growth by E4orf4 was observed in the cdc55 strain and with an E4orf4 mutant that fails to interact with Cdc55, indicating that E4orf4 may possess a second Cdc55-independent function affecting cell growth.

Muf1, a novel Elongin BC-interacting leucine-rich repeat protein that can assemble with Cul5 and Rbx1 to reconstitute a ubiquitin ligase.

The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.

RBP1 recruits the mSIN3-histone deacetylase complex to the pocket of retinoblastoma tumor suppressor family proteins found in limited discrete regions of the nucleus at growth arrest.

Retinoblastoma (RB) tumor suppressor family pocket proteins induce cell cycle arrest by repressing transcription of E2F-regulated genes through both histone deacetylase (HDAC)-dependent and -independent mechanisms. In this study we have identified a stable complex that accounts for the recruitment of both repression activities to the pocket. One component of this complex is RBP1, a known pocket-binding protein that exhibits both HDAC-dependent and -independent repression functions. RB family proteins were shown to associate via the pocket with previously identified mSIN3-SAP30-HDAC complexes containing exclusively class I HDACs. Such enzymes do not interact directly with RB family proteins but rather utilize RBP1 to target the pocket. This mechanism was shown to account for the majority of RB-associated HDAC activity. We also show that in quiescent normal human cells this entire RBP1-mSIN3-SAP30-HDAC complex colocalizes with both RB family members and E2F4 in a limited number of discrete regions of the nucleus that in other studies have been shown to represent the initial origins of DNA replication following growth stimulation. These results suggest that RB family members, at least in part, drive exit from the cell cycle by recruitment of this HDAC complex via RBP1 to repress transcription from E2F-dependent promoters and possibly to alter chromatin structure at DNA origins.

Identification of three functions of the adenovirus e4orf6 protein that mediate p53 degradation by the E4orf6-E1B55K complex.

Complexes containing adenovirus E4orf6 and E1B55K proteins play critical roles in productive infection. Both proteins interact directly with the cellular tumor suppressor p53, and in combination they promote its rapid degradation. To examine the mechanism of this process, degradation of exogenously expressed p53 was analyzed in p53-null human cells infected with adenovirus vectors encoding E4orf6 and/or E1B55K. Coexpression of E4orf6 and E1B55K greatly reduced both the level and the half-life of wild-type p53. No effect was observed with the p53-related p73 proteins, which did not appear to interact with E4orf6 or E1B55K. Mutant forms of p53 were not degraded if they could not efficiently bind E1B55K, suggesting that direct interaction between p53 and E1B55K may be required. Degradation of p53 was independent of both MDM2 and p19ARF, regulators of p53 stability in mammalian cells, but required an extended region of E4orf6 from residues 44 to 274, which appeared to possess three separate biological functions. First, residues 39 to 107 were necessary to interact with E1B55K. Second, an overlapping region from about residues 44 to 218 corresponded to the ability of E4orf6 to form complexes with cellular proteins of 19 and 14 kDa. Third, the nuclear retention signal/amphipathic arginine-rich alpha-helical region from residues 239 to 253 was required. Interestingly, neither the E4orf6 nuclear localization signal nor the nuclear export signal was essential. These results suggested that if nuclear-cytoplasmic shuttling is involved in this process, it must involve another export signal. Degradation was significantly blocked by the 26S proteasome inhibitor MG132, but unlike the HPV E6 protein, E4orf6 and E1B55K were unable to induce p53 degradation in vitro in reticulocyte lysates. Thus, this study implies that the E4orf6-E1B55K complex may direct p53 for degradation by a novel mechanism.

Identification and elimination of an aberrant splice product from cDNAs encoding the human adenovirus type 5 E4orf6 protein.

Growing awareness of the central role of the E4orf6 protein in regulating the infectious cycle of human adenoviruses has led to greatly intensified efforts to define its functions and mechanisms of action. Many workers employ cDNAs to generate plasmid or viral vectors to express E4orf6 in the absence of other viral products. In addition to the normal 34-kDa product, such vectors consistently produce a polypeptide of about 8 kDa. In the present report we show that this protein is produced by an aberrant mRNA utilizing the 5' splice donor site used normally by the virus to produce the E4orf6/7 product, which shares 58 residues with E4orf6. This amino terminal coding sequence is linked to a 3' sequence via a novel splice acceptor site in an alternative reading frame of the E4orf6 cDNA. The 5' donor site was altered by PCR-directed mutagenesis to yield a construct that produces high levels of E4orf6 in the absence of the 8-kDa polypeptide. This construct should eliminate some of the problems encountered previously using the wild-type E4orf6 coding sequence.

Induction of p53-independent apoptosis by the adenovirus E4orf4 protein requires binding to the Balpha subunit of protein phosphatase 2A.

Previous studies have indicated that the E4orf4 protein of human adenovirus type 2 (Ad2) induces p53-independent apoptosis. We believe that this process may play a role in cell death and viral spread at the final stages of productive infection. E4orf4 may also be of therapeutic value in treating some diseases, including cancer, through its ability to induce apoptosis when expressed individually. The only previously identified biochemical function of E4orf4 is its ability to associate with the Balpha subunit of protein phosphatase 2A (PP2A). We have used a genetic approach to determine the role of such interactions in E4orf4-induced cell death. E4orf4 deletion mutants were of only limited value, as all were highly defective. We found that E4orf4 proteins from most if not all adenovirus serotypes induced cell death, and thus point mutations were introduced that converted the majority of highly conserved residues to alanines. Such mutants were used to correlate Balpha-subunit binding, association with PP2A activity, and cell killing following the transfection of appropriate cDNAs into p53-null H1299 or C33A cells. The results indicated that binding of the Balpha subunit is essential for induction of cell death, as every mutant that failed to bind efficiently was totally defective for cell killing. This class of mutations (class I) largely involved residues between amino acids 51 and 89. Almost all E4orf4 mutant proteins that associated with PP2A killed cancer cells at high levels; however, several mutants that associated with significant levels of PP2A were defective for killing (class II). Thus, binding of E4orf4 to PP2A is essential for induction of p53-independent apoptosis, but E4orf4 may possess one or more additional functions required for cell killing.

Human immunodeficiency virus type 1 Vpr-mediated G(2) cell cycle arrest: Vpr interferes with cell cycle signaling cascades by interacting with the B subunit of serine/threonine protein phosphatase 2A.

The Vpr protein of primate lentiviruses arrests cell cycling at the G(2)/M phase through an inactivation of cyclin B-p34(cdc2) and its upstream regulator cdc25. We provide here biochemical and functional evidence demonstrating that human immunodeficiency virus type 1 (HIV-1) Vpr mediates G(2) arrest by forming a complex with protein phosphatase 2A (PP2A), an upstream regulator of cdc25. Vpr associates with PP2A through a specific interaction with the B55 regulatory subunit. This interaction is necessary but not sufficient for G(2) arrest. Interestingly, we found that Vpr association with B55-containing PP2A targets the enzymatic complex to the nucleus and, importantly, enhances the recruitment and dephosphorylation of the cdc25 substrate. Our data suggest that Vpr mediates G(2) arrest by enhancing the nuclear import of PP2A and by positively modulating its catalytic activity towards active phosphorylated nuclear cdc25.

Modulation of EWS/WT1 activity by the v-Src protein tyrosine kinase.

Desmoplastic small round cell tumor (DSRCT) is a malignant human cancer that is associated with a specific t(11;22) chromosome translocation, where 265 amino acids from the EWS amino-terminus are fused to the DNA binding domain of the WT1 tumor suppressor gene. We have noticed the presence of several SH3 interacting domains within the amino-terminus of EWS and have assessed the potential of EWS/WT1 to interact with such motifs. We find that EWS/WT1 can associate with the SH3 domain of several proteins, including v-Src. Ectopic expression of v-Src phosphorylates EWS/WT1 in vivo, as well as enhances the transactivation ability of the EWS amino-terminal domain. Structural alteration of the v-Src SH2 or SH3 domains produced mutants that could not interact with EWS/WT1 nor augment the transcriptional properties of EWS. Taken together, our results suggest the possibility that some transcriptional properties of EWS/WT1 may be regulated by a cytoplasmic signaling pathway.

Modification of EWS/WT1 functional properties by phosphorylation.

In many human cancers, tumor-specific chromosomal rearrangements are known to create chimeric products with the ability to transform cells. The EWS/WT1 protein is such a fusion product, resulting from a t(11;22) chromosomal translocation in desmoplastic small round cell tumors, where 265 aa from the EWS amino terminus are fused to the DNA binding domain of the WT1 tumor suppressor gene. Herein, we find that EWS/WT1 is phosphorylated in vivo on serine and tyrosine residues and that this affects DNA binding and homodimerization. We also show that EWS/WT1 can interact with, and is a substrate for, modification on tyrosine residues by c-Abl. Tyrosine phosphorylation of EWS/WT1 by c-Abl negatively regulates its DNA binding properties. These results indicate that the biological activity of EWS/WT1 is closely linked to its phosphorylation status.

Viruses and apoptosis.

Successful viral replication requires not only the efficient production and spread of progeny, but also evasion of host defense mechanisms that limit replication by killing infected cells. In addition to inducing immune and inflammatory responses, infection by most viruses triggers apoptosis or programmed cell death of the infected cell. This cell response often results as a compulsory or unavoidable by-product of the action of critical viral replicative functions. In addition, some viruses seem to use apoptosis as a mechanism of cell killing and virus spread. In both cases, successful replication relies on the ability of certain viral products to block or delay apoptosis until sufficient progeny have been produced. Such proteins target a variety of strategic points in the apoptotic pathway. In this review we summarize the great amount of recent information on viruses and apoptosis and offer insights into how this knowledge may be used for future research and novel therapies.

Promoter analysis of the murine T-cell protein tyrosine phosphatase gene.

The T-cell protein tyrosine phosphatase (TC PTP) is expressed ubiquitously at all stages of mammalian development. However, mRNA levels fluctuate in a cell-cycle-dependent manner, reaching peak levels in late G1, and rapidly decreasing in S phase. Furthermore, TC PTP being present in higher amounts in lymphoid tissues, we have recently shown that it is essential for proper maintenance of both the bone marrow micro-environment and B- and T-cell functions. In order to better understand the elements controlling the expression pattern of this gene, we have isolated and characterized approx. 4kb of the murine TC PTP promoter. DNA sequencing of the proximal 5' region revealed the absence of both TATAA and CAAT boxes. Primer extension analysis and S1 nuclease mapping techniques identified multiple transcription initiation sites. Functional promoter activity was determined using transfection experiments of promoter deletion constructs fused to a CAT reporter construct. Our results indicate that the minimal promoter sequence required for functional expression is contained within the first 147bp of the TC PTP promoter. In addition, consistent with the cell-cycle-dependent expression of TC PTP, we localized a domain between 492 and 1976bp from the transcription initiation site through which repression occurs. In conclusion, although initiator-driven transcription allows for ubiquitous expression of TC PTP, we define general transcription motifs present within the promoter that may mediate specific modulations of the TC PTP gene.

RBP1 recruits both histone deacetylase-dependent and -independent repression activities to retinoblastoma family proteins.

Retinoblastoma (RB) tumor suppressor family proteins block cell proliferation in part by repressing certain E2F-specific promoters. Both histone deacetylase (HDAC)-dependent and -independent repression activities are associated with the RB "pocket." The mechanism by which these two repression functions occupy the pocket is unknown. A known RB-binding protein, RBP1, was previously found by our group to be an active corepressor which, if overexpressed, represses E2F-mediated transcription via its association with the pocket. We show here that RBP1 contains two repression domains, one of which binds all three known HDACs and represses them in an HDAC-dependent manner while the other domain functions independently of the HDACs. Thus, RB family members repress transcription by recruiting RBP1 to the pocket. RBP1, in turn, serves as a bridging molecule to recruit HDACs and, in addition, provides a second HDAC-independent repression function.

RBP1 induces growth arrest by repression of E2F-dependent transcription.

Growth arrest and cell cycle progression are regulated by the retinoblastoma tumour suppressor pRB and related proteins p130 and p107 that bind to and inhibit the E2F family of transcription factors. Although the precise mechanism of this inhibition remains to be established, previous studies indicated the presence of transcriptional repression activity in the 'pocket' of RB family members. We show here that RBP1, a known pRB pocket-binding protein, possesses transcriptional repression activity and associates with p130-E2F and pRB-E2F complexes specifically during growth arrest. Overexpression of RBP1 both inhibited E2F-dependent gene expression and suppressed cell growth. Thus repression of E2F-dependent transcription by RBP1 via RB family members may play a central role in inducing growth arrest.

Analysis of synthesis, stability, phosphorylation, and interacting polypeptides of the 34-kilodalton product of open reading frame 6 of the early region 4 protein of human adenovirus type 5.

The 34-kDa early-region 4 open reading frame 6 (E4orf6) product of human adenovirus type 5 forms complexes with both the cellular tumor suppressor p53 and the viral E1B 55-kDa protein (E1B-55kDa). E4orf6 can inhibit p53 transactivation activity, as can E1B-55kDa, and in combination these viral proteins cause the rapid turnover of p53. In addition, E4orf6-55kDa complexes play a critical role at later times in the regulation of viral mRNA transport and shutoff of host cell protein synthesis. In the present study, we have further characterized some of the biological properties of E4orf6. Analysis of extracts from infected cells by Western blotting indicated that E4orf6, like E1A and E1B products, is present at high levels until very late times, suggesting that it is available to act throughout the infectious cycle. This pattern is similar to that of E4orf4 but differs markedly from that of another E4 product, E4orf6/7, which is present only transiently. Synthesis of E4orf6 is maximal at early stages but ceases completely with the onset of shutoff of host protein synthesis; however, it was found that unlike E4orf6/7, E4orf6 is very stable, thus allowing high levels to be maintained even at late times. E4orf6 was shown to be phosphorylated at low levels. Coimmunoprecipitation studies in cells lacking p53 indicated that E4orf6 interacts with a number of other proteins. Five of these were shown to be viral or virally induced proteins ranging in size from 102 to 27 kDa, including E1B-55kDa. One such species, of 72 kDa, was shown not to represent the E2 DNA-binding protein and thus remains to be identified. Another appeared to be the L4 100-kDa nonstructural adenovirus late product, but it appeared to be present nonspecifically and not as part of an E4orf6 complex. Apart from p53, three additional cellular proteins, of 84, 19, and 14 kDa were detected by using an adenovirus vector that expresses only E4orf6. The 19-kDa species and a 16-kDa cellular protein were also shown to interact with E4orf6/7. It is possible that complex formation with these viral and cellular proteins plays a role in one or more of the biological activities associated with E4orf6 and E4orf6/7.