Ook-Joon Yoo

Downregulation of p53 by phosphatase of regenerating liver 3 is mediated by MDM2 and PIRH2.

AIMS The phosphatase of regenerating liver (PRL) family is related to tumorigenesis and metastasis in various cancer types. Its overexpression increases cell motility and proliferation via the downregulation of p21 expression. In a previous study, we reported that PRL-1 downregulates p53 and is a target gene of p53. In this study, we investigated whether a member of the PRL family, PRL-3, could regulate p53 like PRL-1 in cancer cells. MAIN METHODS To elucidate the role of PRL-3 in regulating p53 in cancer cells, we used a cell culture system to measure protein level, transcriptional level, apoptosis or localization. KEY FINDINGS We determined that PRL-3 overexpression reduced the activity of the p21 and p53 reporters. Additionally, the levels of endogenous and exogenous p53 protein were reduced in cells transiently expressing PRL-3, whereas the ablation of PRL-3 by siRNA increased levels of the p53 protein. The downregulation of p53 by PRL-3 inhibited p53-mediated apoptosis. However, the phosphatase-dead mutant C104S, prenylated-site mutant C170S, and C104S/C170S PRL-3 evidenced minimal effects on the downregulation of p53 protein as compared with wild-type PRL-3. Further examinations revealed that PRL-3 expression reduced the stability of p53 by inducing the transcription of p53 induced protein with a RING-H2 domain (PIRH2) through early growth response (EGR) and by increasing the phosphorylation of mouse double minute 2 (MDM2), and then both negatively regulated p53. SIGNIFICANCE These findings demonstrated that PRL-3, like PRL-1, can negatively regulate p53 via the activation of PIRH2 and MDM2 in cancer cells.

A genetic screen for modifiers of Drosophila caspase Dcp-1 reveals caspase involvement in autophagy and novel caspase-related genes

BackgroundCaspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. Recent reports have described non-apoptotic functions of caspases, including autophagy. In this report, we searched for novel modifiers of the phenotype of Dcp-1 gain-of-function (GF) animals by screening promoter element- inserted Drosophila melanogaster lines (EP lines).ResultsWe screened ~15,000 EP lines and identified 72 Dcp-1-interacting genes that were classified into 10 groups based on their functions and pathways: 4 apoptosis signaling genes, 10 autophagy genes, 5 insulin/IGF and TOR signaling pathway genes, 6 MAP kinase and JNK signaling pathway genes, 4 ecdysone signaling genes, 6 ubiquitination genes, 11 various developmental signaling genes, 12 transcription factors, 3 translation factors, and 11 other unclassified genes including 5 functionally undefined genes. Among them, insulin/IGF and TOR signaling pathway, MAP kinase and JNK signaling pathway, and ecdysone signaling are known to be involved in autophagy. Together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts Dcp-1-induced apoptosis. Consistent with this idea, we show that expression of eGFP-Atg5 rescued the eye phenotype caused by Dcp-1 GF. Paradoxically, we found that over-expression of full-length Dcp-1 induced autophagy, as Atg8b-GFP, an indicator of autophagy, was increased in the eye imaginal discs and in the S2 cell line. Taken together, these data suggest that autophagy suppresses Dcp-1-mediated apoptotic cell death, whereas Dcp-1 positively regulates autophagy, possibly through feedback regulation.ConclusionsWe identified a number of Dcp-1 modifiers that genetically interact with Dcp-1-induced cell death. Our results showing that Dcp-1 and autophagy-related genes influence each other will aid future investigations of the complicated relationships between apoptosis and autophagy.

The essential role of FKBP38 in regulating phosphatase of regenerating liver 3 (PRL-3) protein stability.

The phosphatase of regenerating liver-3 (PRL-3) is a member of protein tyrosine phosphatases and whose deregulation is implicated in tumorigenesis and metastasis of many cancers. However, the underlying mechanism by which PRL-3 is regulated is not known. In this study, we identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as an interacting protein of PRL-3 using a yeast two-hybrid system. FKBP38 specifically binds to PRL-3 in vivo, and that the N-terminal region of FKBP38 is crucial for binding with PRL-3. FKBP38 overexpression reduces endogenous PRL-3 expression levels, whereas the depletion of FKBP38 by siRNA increases the level of PRL-3 protein. Moreover, FKBP38 promotes degradation of endogenous PRL-3 protein via protein-proteasome pathway. Furthermore, FKBP38 suppresses PRL-3-mediated p53 activity and cell proliferation. These results demonstrate that FKBP38 is a novel regulator of the oncogenic protein PRL-3 abundance and that alteration in the stability of PRL-3 can have a dramatic impact on cell proliferation. Thus, FKBP38 may play a critical role in tumorigenesis.

Sex determination in single mouse blastomeres by polymerase chain reaction

In sex determination of mammalian preimplantation embryos, viability of biopsied embryos and accuracy of sexing are together important. In consideration of this point, single blastomeres were mechanically isolated from mouse embryos using a micromanipulator and then sexed by polymerase chain reaction (PCR) using mouse Y chromosome-specific primers. All of 260 embryos biopsied at the four-cell and morula stage survived. Developmental rate of the embryos to normal blastocysts was 93 and 94%, respectively. Sex determination of single blastomeres was performed by amplification of a mouse Y chromosome-specific DNA sequence using PCR technique. The ratio of male to female embryos was 53 and 47%, respectively. The sex-determined embryos were transferred to the uteri of pseudopregnant recipients to test the consistency of the assay system. The sex of 27 of 29 mice developed from male and female embryos agreed with the predicted sex. The method developed for embryo biopsy and sexing could be used for diagnosis of defective genes at the stage of the preimplantation embryos of human and other domestic animals.

Molecular cloning of gaf2, A schizosaccharomyces pombe GATA factor, which has two zinc‐finger sequences

By low stringency screening of a λ‐Shizosaccharomyces pombe genomic library, we have cloned a GATA factor homologous gene, gaf2+, within a 3.1‐kb EcoRI fragment. The gaf2 ORF predicts a protein of Mr 61 kDa consisting of intronless 564 amino acids corresponding to 1,692 bp. Gaf2 has two zinc‐fingers as Urbs1 of Ustilago maydis, whereas most of fungal GATA factors have only one zinc‐finger. The separation between two zinc‐fingers of Gaf2 is rather long. In addition to gaf2, the sequence analysis revealed a Val‐tRNA gene in the 3′‐flanking region of gaf2. Northern blot analysis indicated that the gaf2 gene is transcribed constitutively irrespective of the nitrogen source in a medium.

Molecular cloning of Gaf1, a Schizosaccharomyces pombe GATA factor, which can function as a transcriptional activator

As a first step to elucidate the functions of Schizosaccharomyces pombe (S. pombe) GATA factors, we have isolated the gaf1+ gene (GATA-factor like gene) in S. pombe. The predicted amino acid (aa) sequence of Gaf1 reveals a single zinc finger domain typical of fungal GATA factors, and the zinc finger exhibits 60% aa identity to that of human GATA-1. The open reading frame of Gaf1 predicts a protein of Mr 32 kDa consisting of 290 intronless amino acids. Disruption of this gene has no effect on cell viability and growth rate. The GST-Gaf1 fusion protein binds specifically to GATA motifs of its own promoter as well as DAL7 UAS, a canonical GATA motif of Saccharomyces cerevisiae (S. cerevisiae) The specific DNA-binding activity resides within the N-terminal half of Gaf1 (Gaf1N; aa 1-120) containing the zinc finger, whereas the C-terminal half (Gaf1C; aa 121-290) contains transactivation sequences that induce the expression of the lacZ reporter when fused to the GAL4 DNA binding domain. These results demonstrate that Gaf1 may function as a transcriptional activator consisting of DNA-binding and transactivation domains.

Identification of an enhancer region in the mouse ephA8 locus directing expression to the anterior region of the dorsal mesencephalon

Eph receptors and ephrins are dynamically expressed in a wide range of regions of the vertebrate during embryogenesis. The dorsal mesencephalon appears to be segmented into two broad regions demarcated by the mutually exclusive expression of EphA receptors and ephrinA ligands. It is of considerable interest to elucidate how these expression domains are established in the development of the mesencephalon. In this study, we used a transgenic approach to define the cis‐acting DNA regulatory elements involved in the anterior mesencephalon‐specific expression of the mouse ephA8 gene. Our analyses of the temporal and spatial expression patterns of various ephA8/lacZ gene fusions in transgenic mice revealed that the 10‐kb genomic DNA 5′ immediately upstream of the ephA8 coding sequence is capable of directing lacZ expression in an ephA8‐specific manner. Further deletion analyses of the ephA8 genomic region led to the identification of a 1‐kb enhancer region, which directs expression in the embryo to the anterior region of the developing midbrain. This ephA8‐specific regulatory DNA sequences can now be used in biochemical analyses to identify proteins modulating the anterior differentiation of the optic tectum, and in functional analyses to direct the expression of other developmentally important genes to this region. Developmental Dynamics 226:596–603, 2003.

The BTB/POZ-ZF transcription factor dPLZF is involved in Ras/ERK signaling during Drosophila wing development.

In Drosophila, broad complex, tramtrack, bric à brac (BTB)/poxvirus and zinc finger (POZ) transcription factors are essential regulators of development. We searched the Drosophila genome for BTB/POZ-ZF domains and discovered an unknown Drosophila gene, dPLZF, which encodes an orthologue of human PLZF. We then characterized the biological function of the dPLZF via genetic interaction analysis. Ectopic expression of dPLZF in the wing induced extra vein formation during wing development in Drosophila. Genetic interactions between dPLZF and Ras or extracellular signal-regulated kinase (ERK) significantly enhanced the formation of vein cells. On the other hand, loss-of-function mutations in dPLZF resulted in a dramatic suppression of the extra and ectopic vein formation induced by elevated Ras/ERK signaling. Moreover, dPLZF activity upregulated the expression of rhomboid (rho) and spitz, which perform crucial functions in vein cell formation in the developing wing. These results indicate that dPLZF is a transcription factor controlled by the Ras/ERK signaling pathway, which is a prominent regulator of vein cell formation during wing development in Drosophila.

Altered expression of phosphatase of regenerating liver gene family in non-small cell lung cancer

Protein tyrosine phophatases (PTPs) are implicated in the tumorigenesis and metastasis of human cancer. The phosphatase of regenerating liver (PRL) gene family, a subgroup of PTPs is also linked to these processes. In many solid cancers, high levels of PRL-3 expression are related with metastasis and poor prognosis. However, the expression patterns of PRL-1 and -2 have not been explored in lung cancer yet. Thus, we investigated the expression levels of PRL-1, -2 and -3 in the tissues of primary lung cancer patients. The protein expression levels of PRL-2, but not PRL-1 and -3 were increased in cancer tissues. However, there was no correlation between mRNA and protein expression levels of the PRLs. Reporter assays showed that PRLs suppressed the activity of the p21 promoter but promoted AP-1 activity. Furthermore, transfection of PRLs showed significantly increased cell proliferation. Therefore, these results suggest that PRL-2 plays an important role in lung cancer and can be a biomarker of lung cancer, substituting for the function of other PRLs.