The inhibitor ABIN-2 disrupts the interaction of receptor-interacting protein with the kinase subunit IKKgamma to block activation of the transcription factor NF-kappaB and potentiate apoptosis.

NF-kappaB (nuclear factor kappaB) proteins are key transcription factors that regulate gene expression in response to various extracellular stimuli. The pathway leading to the activation of NF-kappaB involves a complicated network that includes a number of signalling molecules. The recent identification of a wide range of negative regulators of NF-kappaB has given another layer of complexity in NF-kappaB activation. We and others have previously identified the protein ABIN-2 (A20 binding inhibitor of NF-kappaB 2) as an inhibitor of NF-kappaB activation. In the present paper, we demonstrate that ABIN-2 exerts its inhibitory function by blocking the interaction of RIP (receptor-interacting protein) with the downstream effector IKKgamma, a non-kinase component of the IkappaB (inhibitory kappaB) kinase complex. When overexpressed in cells, ABIN-2 bound to IKKgamma and prevented the association of IKKgamma with RIP. By a deletion mapping, a stretch of 50 amino acids on ABIN-2 is found to be essential for its interaction with IKKgamma. The ABIN-2 mutant that lacked these 50 amino acids did not interact with IKKgamma and, consequently, failed to inhibit NF-kappaB activation. Strikingly, a portion of RIP, which is similar to this 50-residue domain of ABIN-2, is also essential for RIP interaction with IKKgamma. The RIP mutant with deletion of this similar region did not associate with IKKgamma and had substantial reduction of its ability to mediate NF-kappaB activation. Taken together, these conserved 50 residues of ABIN-2 and RIP define a novel structural domain in mediating a key step in the NF-kappaB signalling pathway through the interaction with IKKgamma. Finally, the signalling pathway of NF-kappaB activation is known to promote survival in many cellular events. The mechanism for decision between cell death and survival is under fine regulation. In the present paper, we demonstrated further that the expression of ABIN-2 could promote the RIP-mediated apoptosis by presumably suppressing the anti-apoptotic effect of NF-kappaB.

The A20-binding protein ABIN-2 exerts unexpected function in mediating transcriptional coactivation.

The human ABIN-2 was originally identified as an A20-associating cytosolic protein to block NF-kappaB activation induced by various stimuli. Here we report that ABIN-2 has the potential to enter the nucleus and plays a role in mediating transcriptional activation in both yeast and mammalian cells. The Gal4BD-ABIN-2 fusion protein is able to drive the expression of the GAL4-responsive reporter gene in yeast efficiently without the need of the Gal4p activation domain, suggesting that ABIN-2 functions as a transcriptional coactivator and facilitates transcription in yeast. In contrast to the activity in yeast, however, only the C-terminal fragment of ABIN-2 exerts the transactivating activity in mammalian cells but not the full-length ABIN-2 protein. This observation has led to the identification of the N-terminal 195 amino acids of ABIN-2 as a regulatory domain, which retains the full-length ABIN-2 in the cytoplasm of mammalian cells and thus cannot transactivate. We have also found that BAF60a, a component of chromatin-remodeling complex, interacts with ABIN-2 by the yeast two-hybrid analysis. Together, our results suggest that the nuclear ABIN-2 defines a novel transcriptional coactivator and acts presumably by recruiting a chromatin-remodeling complex to the site of the target gene.

Ung1p-mediated uracil-base excision repair in mitochondria is responsible for the petite formation in thymidylate deficient yeast.

The budding yeast CDC21 gene, which encodes thymidylate synthase, is crucial in the thymidylate biosynthetic pathway. Early studies revealed that high frequency of petites were formed in heat-sensitive cdc21 mutants grown at the permissive temperature. However, the molecular mechanism involved in such petite formation is largely unknown. Here we used a yeast cdc21-1 mutant to demonstrate that the mutant cells accumulated dUMP in the mitochondrial genome. When UNG1 (encoding uracil-DNA glycosylase) was deleted from cdc21-1, we found that the ung1Delta cdc21-1 double mutant reduced frequency of petite formation to the level found in wild-type cells. We propose that the initiation of Ung1p-mediated base excision repair in the uracil-laden mitochondrial genome in a cdc21-1 mutant is responsible for the mitochondrial petite mutations.

The yeast Cdc8 exhibits both deoxythymidine monophosphate and diphosphate kinase activities.

The existence of multifunctional enzymes in the nucleotide biosynthesis pathways is believed to be one of the important mechanisms to facilitate the synthesis and the efficient supply of deoxyribonucleotides for DNA replication. Here, we used the bacterially expressed yeast thymidylate kinase (encoded by the CDC8 gene) to demonstrate that the purified Cdc8 protein possessed thymidylate-specific nucleoside diphosphate kinase activity in addition to thymidylate kinase activity. The yeast endogenous nucleoside diphosphate kinase is encoded by YNK1, which appears to be non-essential. Our results suggest that Cdc8 has dual enzyme activities and could duplicate the function of Ynk1 in thymidylate synthesis. We also discuss the importance of the coordinated expression of CDC8 during the cell cycle progression in yeast.

Integrative genomics based identification of potential human hepatocarcinogenesis-associated cell cycle regulators: RHAMM as an example.

DNA microarray has been widely used to examine gene expression profile of different human tumors. The information generated from microarray analysis usually represents the overall range of cancer-associated abnormality associated with gene regulation. In order to identify key regulatory genes involved in carcinogenesis of human cancer, hypothesis driven data mining of the microarray data plus experimental validation becomes a critical approach in the post-genome era. Here, we present an integrative genomic analysis of published microarray data and homolog gene database. Over 20,000 genes were examined to reveal 16 genes specific to vertebrates, cell cycle G2/M regulated, and overexpressed in human HCC. Using Affymetrix microarray analysis, we found that all 16 genes were up-regulated in human HCC. Among these 16 genes, we experimentally validated the up-regulation of receptor for hyaluronan-mediated motility (RHAMM) in different cell model systems. We first confirmed elevation of RHAMM in the G2/M phase of synchronized HeLa cells. We also found that RHAMM had an elevated level of expression in all the HCC samples we examined and it was induced during the G2/M phase of regenerating mouse hepatocytes after partial hepatectomy. Thus, the expression of RHAMM appears to be tightly regulated during mammalian cell cycle G2/M progression. The ectopic overexpression of RHAMM in 293T cells resulted in the accumulation of cells at G2/M phase. RHAMM-induced mitotic arrest of cells was predominantly in the prophase. Taken together, using an integrated functional genomic approach, we have uncovered a set of genes that may play specific roles in cell cycle progression and in HCC development. To elucidate the function of these genes in cell cycle regulation may shed light on the control mechanism of human HCC in the future.

An LKB1-interacting protein negatively regulates TNFalpha-induced NF-kappaB activation.

The Peutz-Jeghers syndrome (PJS) is a hereditary disorder that predisposes an individual to benign and malignant tumors in multiple organ systems. Recently, the locus responsible for PJS was mapped genetically to the LKB1 gene, with a subsequent investigation proving that it is responsible for most cases of PJS. LKB1 encodes a nuclear serine/threonine protein kinase, and potential tumor-suppressing activity has been attributed to LKB1 kinase. However, how LKB1 exerts its tumor-suppressing function remains to be determined. In this report, we describe the identification of a putative human LKB1-interacting protein, FLIP1, using the yeast two-hybrid system. Two regions of the LKB1 sequence have been determined to be crucial for the interaction with FLIP1. FLIP1 encodes a protein of 429 amino acids with a predicted molecular weight of 47 kd. In contrast to LKB1, which is mainly nuclear, FLIP1 is a cytoplasmic protein, and its expression is ubiquitous in all human tissues examined to date. Interestingly, deletion of the 195 N- terminal amino acids allows FLIP1 to enter the nucleus, suggesting the presence of a regulatory mechanism through its N-terminus for nuclear entry. In addition, we found that ectopic expression of FLIP1 selectively blocks cytokine-induced NF-kappaB activation. The involvement of FLIP1 in the regulation of NF-kappaB activity may shed new light on the role of LKB1 in tumor suppression.

Overexpression of a novel imprinted gene, PEG10, in human hepatocellular carcinoma and in regenerating mouse livers.

Many of the promising applications of the microarray technology are pertinent to identifying abnormalities in gene expression that contribute to malignant progression. We developed a bioinformatics tool to identify differentially expressed genes in human hepatocellular carcinoma (HCC). This involved the construction of a liver EST database (http://lestdb.nhri.org.tw) and in silico verification of differentially expressed genes with a human hepatoma microarray database. The stringency of the search was reinforced with a statistical analysis. A novel imprinted gene, paternally expressed 10(PEG10) was identified as having an elevated level of expression in the majority of the HCC samples and was also induced to express during G2/M phase of regenerating mouse liver. Ectopic expression of PEG10 in 293T cells affects cell cycle progression. PEG10 is distributed in the cytosol and associates with the nuclear membrane. This is the first time that an imprinted gene has been found to reexpress in both human HCC and in the regenerating mouse liver. This result indicates that the induction of the paternally imprinted gene may play an important role during liver regeneration or carcinogenesis of the human hepatocyte. Understanding the molecular basis of the abnormal imprinting of PEG10 will shed new light on the process that leads to liver disease.