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The adenovirus E1A protein has been shown to be involved in the potentiation of apoptosis induced by chemotherapeutic agents, yet the molecular events of E1A-mediated apoptosis are not very clear. A recent report has suggested that deamidation of the Bcl-X(L) protein inhibits its antiapoptotic ability and leads to apoptosis induced by alkylating agents in Rb-deficient tumor cells. Since Rb is known to interact with E1A, which interrupts Rb's normal function, we examined Bcl-X(L) deamidation and cell death induced by cisplatin in E1A transfectants. We found that the E1A transfectants became sensitive to cisplatin-induced apoptosis compared to the parental cells, SKOV3.ip1. Our data show that cisplatin treatment induced the modification of Bcl-X(L) in the E1A transfectants in dosage and time-dependent manner. Furthermore, phosphatase treatment had no effect on the level of Bcl-X(L) modification, whereas alkaline lysis buffer appeared to induce the same modification of Bcl-X(L). Ectopic expression of the deamidated forms of Bcl- X(L) in SKOV3.ip1 cells revealed that the modification to the Bcl- X(L) protein molecules was deamidation. Expression of the E1A mutant (dl1108) which contains deletion at CR2 domain suppressed Bcl-X(L) deamidation and apoptosis induced by cisplatin. We also found that expression of the nondeamidated Bcl-X(L) protected E1A transfectants from apoptosis. These findings suggest that Bcl-X(L) deamidation contributes to E1A-mediated cisplatin sensitization in SKOV3.ip1 cells.

Introduction Toll-like receptors (TLRs) play important roles in innate immunity by eliciting inflammation. Upon receptor engagement, activation of IKKs and MAPKs control the production of cytokines. It has been shown that TAK1 is the essential mediator that transmits signal from receptor to downstream effectors. Meanwhile, in our previous study [1] , we implied that IKKs activation in TLR4 is independent to TAK1 phosphorylation. Therefore, the molecular mechanisms underlying how MAPKs and IKKs are differentially activated by TAK1 in TLR4 need for further investigation. Methods In order to examine the role of TAK1 in MAPKs and IKKs activation, TAK1-silencing cells were stable expressed with TAK1 mutants and stimulated with LPS. Additionally, LC-MS analysis was used to identify novel post-translation modification and binding partners of TAK1. Results As previous reports, we found that the production of cytokines in response to LPS was diminished in TAK1-silencing cells. Reconstituting cells with various mutated TAK1, mutants in either activation loop (T184 and T187) or kinase domain (D156) leaded to inhibition of MAPKs. The activation of IKKs was slightly decreased in D156A mutant, but not T (184; 187) A. Furthermore, mutation in phosphorylation sites of TAK1 enhanced the K63-linked ubiquitination. By in vivo ubiquitination assay, we showed that mutation in either K158 or K209 of TAK1 was unable to abolish polyubiquitination completely, but we confirmed that K158 of TAK1 as the critical site in TLR4 pathway for IKK activation and TAK1 phosphorylation that, thereby, activates of MAPKs. The proteomic analysis for binding partners of TAK1 in different modification status was in line with activity assay. Furthermore, we also identified K562 of TAK1 as a novel ubiquitination site. Conclusion We identified that ubiquitination in K209 of TAK1 regulates IKKs activation and the phosphorylation of TAK1 itself. The activation of TAK1 is able to transmit signal to activate MAPKs, and, in turn, block the K63-linked ubiquitination of TAK1 in feedback regulation. Our data suggested that TAK1-mediated activation of downstream effectors in TLR4 is carefully regulated by the crosstalk of phosphorylation and ubiquitination.

Exon sizes in vertebrate genes are, with a few exceptions, limited to less than 300 bases. It has been proposed that this limitation may derive from the exon definition model of splice site recognition. In this model, a downstream donor site enhances splicing at the upstream acceptor site of the same exon. This enhancement may require contact between factors bound to each end of the exon; an exon size limitation would promote such contact. To test the idea that proximity was required for exon definition, we inserted random DNA fragments from Escherichia coli into a central exon in a three-exon dihydrofolate reductase minigene and tested whether the expanded exons were efficiently spliced. DNA from a plasmid library of expanded minigenes was used to transfect a CHO cell deletion mutant lacking the dhfr locus. PCR analysis of DNA isolated from the pooled stable cotransfectant populations displayed a range of DNA insert sizes from 50 to 1,500 nucleotides. A parallel analysis of the RNA from this population by reverse transcription followed by PCR showed a similar size distribution. Central exons as large as 1,400 bases could be spliced into mRNA. We also tested individual plasmid clones containing exon inserts of defined sizes. The largest exon included in mRNA was 1,200 bases in length, well above the 300-base limit implied by the survey of naturally occurring exons. We conclude that a limitation in exon size is not part of the exon definition mechanism.

We describe a Drosophila DNA clone of tandemly duplicated genes encoding an amino acid sequence nearly identical to human ribosomal protein S14 and yeast rp59. Despite their remarkably similar exons, the locations and sizes of introns differ radically among the Drosophila, human, and yeast (Saccharomyces cerevisiae) ribosomal protein genes. Transcripts of both Drosophila RPS14 genes were detected in embryonic and adult tissues and are the same length as mammalian S14 message. Drosophila RPS14 was mapped to region 7C5-9 on the X chromosome. This interval also encodes a previously characterized Minute locus, M(1)7C.

We describe sequences of two human ribosomal proteins, S14 and S17, and messenger RNAs that encode them. cDNAs were used as molecular hybridization probes to recognize complementary genes in rodent, Drosophila, and yeast chromosomal DNAs. Human ribosomal protein sequences are compared to analogous Chinese hamster, yeast, and bacterial genes. Our observations suggest that some ribosomal protein genes have been conserved stringently in the several phylogenetic lines examined. These genes apparently were established early in evolution and encode products that are fundamental to the translational apparatus. Other ribosomal protein genes examined, although similar enough to heterologous DNA sequences to indicate their structural relationships, appear to have diverged substantially during evolution, probably reflecting adaptations to different genetic environments.