Myung-sun Kim

Stability and Function of Mammalian Lethal Giant Larvae-1 Oncoprotein Are Regulated by the Scaffolding Protein RanBPM

The evolutionarily conserved lethal giant larvae (Lgl) tumor suppressor gene has an essential role in establishing apical-basal cell polarity, cell proliferation, differentiation, and tissue organization. However, the precise molecular mechanism by which the Lgl carries out its function remains obscure. In the current study, we have identified Ran-binding protein M (RanBPM) as a novel binding partner of Mgl-1, a mammalian homolog of Drosophila tumor suppressor protein lethal (2) giant larvae (L(2)gl) by yeast two-hybrid screening. RanBPM seems to act as a scaffolding protein with a modulatory function with respect to Mgl-1. The Mgl-1 and RanBPM association was confirmed by co-immunoprecipitation and GST pull-down experiments. Additionally, expression of RanBPM resulted in inhibition of Mgl-1 degradation, and thereby extended the half-life of Mgl-1. Furthermore, the ability of Mgl-1 activity in cell migration and colony formation assay was enhanced by RanBPM. Taken together, our findings reveal that RanBPM plays a novel role in regulating Mgl-1 stability and contributes to its biological function as a tumor suppressor.

HAUSP, a deubiquitinating enzyme for p53, is polyubiquitinated, polyneddylated, and dimerized

The tumor suppressor protein p53 is ubiquitinated and neddylated by MDM2 and then degraded by 26S proteasome. However, p53 is stabilized by the HAUSP (Herpes‐virus‐associated ubiquitin‐specific protease) deubiquitinating enzyme. In this study, we discovered that rat HAUSP (rHAUSP) is polyubiquitinated, polyneddylated, and dimerized using co‐immunoprecipitation assays. This suggests that rHAUSP may function as a dimer or multimer and is also degraded through the proteasome‐mediated degradation. Transfection of rHAUSP into RGC‐Lac‐Z cell line with the integrated p53 response element revealed that rHAUSP contributed to p53 stabilization, and a rHAUSP (C224S) mutant contributed to p53 destabilization in a dose‐dependent manner.

14-3-3γ Is Stimulated by IL-3 and Promotes Cell Proliferation

IL-3 plays important roles in the growth and survival of hematopoietic progenitor cells, processes modeled in studies of the IL-3-dependent cell line Ba/F3. To gain insights into molecular mechanisms governing cell fate, we examined the patterns of proteins up-regulated following stimulation of Ba/F3 cells with IL-3. Through two-dimensional electrophoresis and proteomics-based approaches, we identified 11 proteins. Of these, expression of 14-3-3γ was significantly increased by IL-3 stimulation at both the transcriptional and translational levels. 14-3-3γ overexpression in Ba/F3 cells abrogated dependence on IL-3 and was associated with activation of PI3K and MAPK signaling cascades, suggesting that the functions of 14-3-3γ in normal hematopoietic progenitors are to promote survival and growth through the activation of distinct signaling pathways. Additionally, the up-regulation of Bax and Bad was seen with the ablation of 14-3-3γ, resulting in cell death. These results indicate that deregulated expression of 14-3-3γ may contribute to malignant transformation, possibly providing a new target for therapeutic intervention in hematopoietic neoplasms.

Hyaluronan- and RNA-binding deubiquitinating enzymes of USP17 family members associated with cell viability

BackgroundProtein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. Deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway.ResultsWhile identifying putative ubiquitin specific protease (USP) enzymes that contain a conserved Asp (I) domain in humans, 4 USP17 subfamily members, highly homologous to DUB-3, have been found (USP17K, USP17L, USP17M, and USP17N), from human chorionic villi. Expression analysis showed that USP17 transcripts are highly expressed in the heart, liver, and pancreas and are expressed moderately in various human cancerous cell lines. Amino acid sequence analysis revealed that they contain the highly conserved Cys, His, and Asp domains which are responsible for the deubiquitinating activity. Biochemical enzyme assays indicated that they have deubiquitinating activity. Interestingly, the sequence analysis showed that these proteins, with exception of USP17N, contain the putative hyaluronan/RNA binding motifs, and cetylpyridinium chloride (CPC)-precipitation analysis confirmed the association between these proteins and intracellular hyaluronan and RNA.ConclusionHere, we report that the overexpression of these proteins, with exception of USP17N, leads to apoptosis, suggesting that the hyaluronan and RNA binding motifs in these enzymes play an important role in regulating signal transduction involved in cell death.

Protein stability of mitochondrial superoxide dismutase SOD2 is regulated by USP36

SOD2 is a key mitochondrial antioxidant enzyme and its perturbation leads to oxidative cell death, which results in various disorders. In this study, we identified a deubiquitinating enzyme USP36 that regulates the protein stability of SOD2. The regulatory effect of USP36 on SOD2 was initially identified by 2‐DE and MALDI‐TOF/MS analyses. In addition, endogenous USP36 and SOD2 were shown to interact in an immunoprecipitation assay, which was verified using the yeast two‐hybrid system. Furthermore, we demonstrated that SOD2 binds with ubiquitin molecules to form polyubiquitination chains and undergoes degradation through the ubiquitin‐proteasomal pathway. Finally, USP36 was shown to be a specific deubiquitinating enzyme that reduces the ubiquitination level of SOD2 and was involved in SOD2 protein stability by extending its half‐life. J. Cell. Biochem. 112: 498–508, 2011.

DUB-1, a fate determinant of dynein heavy chain in B-lymphocytes, is regulated by the ubiquitin–proteasome pathway

Ubiquitinaiton and deubiquitination of post‐translational modification play counter roles in determining the fate of protein function in eukaryotic system for maintaining the cellular homeostasis. Even though novel family members of growth‐regulating deubiquitinating enzymes (DUB‐1 and DUB‐2) have been identified, their target proteins and functions are poorly understood. Dub genes encoding DUB‐1 and DUB‐2 are immediate‐early genes and are induced in response to cytokine stimuli rapidly and transiently. In order to explore the possible proteins regulated by DUB‐1, we performed the matrix assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) analysis followed by immunoprecipitation. We confirmed that DUB‐1 interacts with dynein heavy chain, which is known to regulate the movement of organelles and microtubule binding ability. In addition, structural and immunoprecipitation analyses revealed that DUB‐1 contains a putative PEST motif and is polyubiquitinated, indicating that DUB‐1 is also regulated by the ubiquitin–proteasome pathway. J. Cell. Biochem. 105: 1420–1429, 2008.

Protein chip analysis of pluripotency-associated proteins in NIH3T3 fibroblast

Specific transcription factors regulate the totipotent and pluripotent capability of embryonic stem cells. Amongst these regulatory transcription factors in embryonic stem cells, Oct4 and Nanog are master factors that also have unique characteristic ability of cell‐specific pluripotency and self‐renewal. The expression of Nanog in fibroblasts confirms increased cell proliferation and transformation of foci‐forming phenotype indicative of its oncogenic potential. The expression of Oct4, interestingly, leads to transformation of non‐tumorgenic mouse into tumorigenic mouse. Our current investigation ascertains that the resultant increase in DNA synthesis and cell proliferation is the consequence of transforming the phenotype into foci formation. We used a manually curetted ProteoChip to carry out the signaling protein microarray analysis, which revealed up‐regulated expression of various proteins including FAK1, MEK1 and Raf1. Some of the proteins explain the mechanism by which Oct4 and Nanog transform the phenotype. In NIH3T3 cells expressed with mouse Oct4 (mOct4), mouse Nanog (mNanog) separately as well as together, the specific knockdown of mFAK1 inhibited morphological transformation of the cells, and their invasion activity. The mFAK1 overexpression leads to morphological transformation as shown with mOct4 and mNanog. Additionally, we showed that the ERK1/2 pathway is involved in the up‐regulation of c‐myc and cyclin D1 expression mediated by mFAK1. Our results signify that the combinatorial signaling protein‐array using biomolecular approach may possibly provide us with a new tool to understand cellular homeostasis.

Murine Asb-17 expression during mouse testis development and spermatogenesis

In this study we isolated a murine mAsb-17 from mouse testis by RT-PCR using primers designed based on the sequences from the GenBank database. The sequence analysis showed that mAsb-17 encodes a 295 amino acid polypeptide with a molecular weight of approximately 34 kDa containing two ankyrin repeats and one SOCS box. The amino acid sequence of mASB-17 showed 87.5%, 98.3% and 92.9% identity with that of human, rat and dog, respectively. Interestingly, northern blot analysis showed that mAsb-17 was expressed only in the testis. The expression analysis by RT-PCR for mAsb-17 in mouse indicates that mAsb-17 is expressed from the fourth week after birth to adult, with the highest expression in round spermatids. Both northern blot and RT-PCR analyses suggest that mASB-17 may play essential roles in testis development and spermatogenesis.

Hardware-in-the-loop Simulation for CPU/GPU Heterogeneous Platforms

Multi-core CPU/GPU heterogeneous platforms became popular in embedded systems. A full system simulator is typically used to observe the internal system behavior by running complete software stacks without modification on simulation models of CPUs and other devices in the system. However, there are few known full system simulators for CPU/GPU heterogeneous platforms and existent GPU simulators are prohibitively slow for running application software. In this paper, we propose a hardware-in-the-loop simulation technique that integrates GPU hardware into a full system simulator. A novel interfacing mechanism between CPU simulator and the development board, where GPU hardware is integrated, is devised. In the experiments, we took Exynos 4412 as a case study, where gem5 simulator is used to simulate mainly a quad-core ARM CPU in the platform and an Exynos development board is used to run the Mali GPU hardware. We could successfully run Android apps on the proposed hardware-in-the-loop simulation framework with up to 1.5 M cycles per second performance.

Impact of BARC on SEM shrinkage of ArF resist

The shrinkage of resist pattern during in-line SEM measurement has been argued and studied as one of the problems unsettled for manufacturing with ArF photolithography. Many of attempts to solve this problem have focused their attentions on the improvement of resist and inspection equipment. We bring up BARC (bottom anti-reflective coating) as a new impact factor on SEM shrinkage of resist. Practically, although the same resist was employed, our shrinkage tests gave the results depending on the kind of BARC. Feature size and depth of focus also affect SEM shrinkage of resist. Effect of reflectivity on SEM shrinkage was evaluated by changing thickness of BARCs and resultantly was somewhat significant. In this paper, the BARC-dependent results of SEM shrinkage are analyzed and discussed to provide a possibility that BARC may have another function of reducing SEM shrinkage.