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.The retinoblastoma (RB) tumor suppressor protein pRB plays a critical role in the control of cell proliferation. In addition, loss of the Rb gene is known to contribute to the establishment of a variety of cancers. pRB and the related proteins p130 and p107 control cell cycle progression through interactions with the E2F family of transcription factors (reviewed in reference 11). Such interactions regulate transcription by mechanisms requiring the "pocket" domain of RB family members (1,3,5,18,32,39,40,46). One role of the pocket is to interact with and mask the transcriptional activation domain of E2F; however, this mechanism does not explain the repression of E2F-dependent promoters in which E2F binding sites act as negative elements that, if deleted, result in relief from repression (7,11,20,28). The pocket can interact simultaneously with both E2F and certain cellular and viral proteins that bind by utilizing a conserved Leu-X-Cys-X-Glu FIG. 1. Endogenous interactions of pRB (A) and p107 (B) with HDACs. (A) Immunoprecipitations were done in lysates from either H1299 or 293T cells. Plates (100 mm) of both cell types were lysed in low-stringency buffer. Cell extracts were incubated with 1 g of the indicated antisera and 30 l of a 50% slurry of protein G-Sepharose for at least 12 h. Immunoprecipitated complexes were washed six times with the 150 mM low-stringency buffer and eluted by boiling with 2ϫ sample buffer. Eluted proteins were subjected to SDS-PAGE with a 6% polyacrylamide gel. The presence of pRB was detected with monoclonal antibody (G3-245) against pRB (PharMingen). (B) Binding studies similar to those described for panel A were done, but coimmunoprecipitation of p107 was detected with a rabbit polyclonal antibody (C-18) against p107 (␣-107) (Santa Cruz).

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 Ba (B55) subunit of protein phosphatase 2A (PP2A), a member of one of three classes of regulatory B subunits. Here we report the eects 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 eect, 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 aecting cell growth. Oncogene (2001) 20, 5279 ± 5290.

Ribavirin is a guanosine ribonucleoside analog that displays broad-spectrum anti-viral activity and is currently used for the treatment of some viral infections. Ribavirin has recently been proposed to also be a mimic of the 7-methyl guanosine cap found at the 5 0 end of mRNAs. To obtain supporting functional data for this hypothesis, we assessed the ability of ribavirin triphosphate to interfere with the interaction between eIF4E and 7-methyl guanosine capped mRNA. In chemical cross-linking assays, cap-affinity chromatography, and cap-dependent translation assays, ribavirin was unable to function as a cap analog.

In many human cancers, tumor-specific chromosomal rearrangements are known to create chimeric products with the ability to transform cells. The EWS͞WT1 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 EWS͞WT1 is phosphorylated in vivo on serine and tyrosine residues and that this affects DNA binding and homodimerization. We also show that EWS͞WT1 can interact with, and is a substrate for, modification on tyrosine residues by c-Abl. Tyrosine phosphorylation of EWS͞WT1 by c-Abl negatively regulates its DNA binding properties. These results indicate that the biological activity of EWS͞WT1 is closely linked to its phosphorylation status.desmoplastic small round cell tumor ͉ DNA binding ͉ homodimerization ͉ c-Abl D esmoplastic small round cell tumor is an aggressive rare tumor that generally occurs in adolescence and is located to the peritoneal surfaces of the abdomen. This malignancy is associated with a recurrent translocation t(11;22)(p13;q22), which fuses the amino-terminal domain (NTD) of the EWS gene in-frame to three of the four carboxyl-terminal zinc fingers of the WT1 tumor suppressor gene (1, 2). The EWS gene is involved in several tumor-related chromosomal translocations that produce fusions with genes postulated to function as transcription factors (for a review, see ref.3). In each case, the translocation produces chimeric molecules containing the EWS NTD fused to the DNA domain of the partner.There are two isoforms of EWS͞WT1 generated as a result of an alternative splicing event between the third and last WT1 zinc finger and resulting in the insertion or removal of three amino acids (ϮKTS). This event produces EWS͞WT1 isoforms with distinct DNA binding properties (4). The EWS͞WT1(ϪKTS) isoform recognizes GC-rich WT1 binding sites [5Ј-GCGGGGGCG-3Ј] with Ϸ10-fold higher affinity than WT1 and can transform NIH 3T3 cells (4, 5). On the other hand, EWS͞WT1(ϩKTS) does not bind GC-rich WT1 sites and has no transforming activity. The NTD (amino acids 1-265) of EWS͞WT1 is composed almost exclusively of tyrosine, glutamine, alanine, serine, threonine, glycine, and proline residues, of which some are organized in a repeated and degenerate polypeptide motif having the consensus NSYGQQS. This domain shares distant homology with the carboxyl-terminal domain (CTD) of eukaryotic RNA polymerase II (6) and is a potent transcriptional activator (4,7,8). One model to account for the transforming properties of EWS͞WT1(ϪKTS) is that some of the genes normally under WT1 control are deregulated by EWS͞WT1.During our characterization of the EWS͞WT1 oncogene we discovered that this product can self-associate and that the responsible region maps to the chimeric portion of the molecule, requiring both EWS and WT1 domains (see below). Herein, we report that phosphorylation of the EWS͞WT1 chimeric product dramatically alters...

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 aminoterminal 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.z 2000 Federation of European Biochemical Societies.

Tie2 is an endothelium-specific receptor tyrosine kinase required for normal blood vessel maturation. We report that Tie2 mRNA translation is maintained under hypoxic conditions. To identify the mechanism responsible for this, we undertook structure/function analysis of the Tie2 5-untranslated region (UTR). Transcription start site mapping indicates the existence of a several mRNA isoforms containing unusually long 5-UTRs (>350 nucleotides) with five upstream open reading frames. We find internal ribosome binding activity that allows the Tie2 mRNA to initiate in a cap-independent fashion. Our data provide a framework for understanding how Tie2 mRNA is translated despite a cumbersome structured 5-UTR and how its production is secured under unfavorable environmental conditions.

Intralaryngeal extension should be considered when there is hoarseness, dysphagia, or dyspnea associated with a thyroglossal duct cyst. Office laryngoscopy and computed tomography make the diagnosis. Care must be taken with airway management and intraoperative dissection for good outcomes.

Tie2 is an endothelium-specific receptor tyrosine kinase required for normal blood vessel maturation, remodeling, and stability. Tie2 expression is also upregulated in various cancers implicating a role in tumor angiogenesis. Its mRNA transcript contains an unusually long (372 nucleotides) 5 0 untranslated region (UTR) with five upstream open reading frames (uORFs) and an internal ribosome entry site (IRES) that allows this mRNA to be translated under hypoxic conditions. This sets up an alternative initiation pathway with the potential to clash with 5 0 end-mediated initiation from the same template. Herein, we define experimental conditions under which the Tie2 IRES is not active, allowing us to assess the contribution of the 5 0 UTR to cap-dependent translation on the Tie2 transcript. We find that the Tie2 5 0 UTR is inhibitory to translation initiation with ribosome flow decreasing following encounters with each uORF. No single uORF was found to harbor significant cisacting inhibitory activity. Our results suggest that the uORFs within the Tie2 5 0 UTR serve to decrease the percent of ribosomes competent for reinitiation as these traverse the mRNA 5 0 UTR, thus minimizing interference with the IRES.