• The Korean Journal of Physiology and Pharmacology
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• v.4 no.6
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• pp.497-506
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• 2000

In this work, GLUTs phosphorylations by a downstream effector of PI3-kinase, $PKC-{\zeta},$ were studied, and GLUT4 phosphorylation was compared with GLUT2 phosphorylation in relation to the translocation mechanism. Prior to phosphorylation experiment, $PKC-{\zeta}$ kinase activity was determined as $20.76{\pm}4.09$ pmoles Pi/min/25 ng enzymes. GLUT4 was phosphorylated by $PKC-{\zeta}$ and the phosphorylation was increased on the vesicles immunoadsorpted from LDM and on GLUT4 immunoprecipitated from GLUT4- contianing vesicles of adipocytes treated with insulin. However, GLUT2 in hepatocytes was neither phosphorylated by $PKC-{\zeta}$ nor changed in response to insulin treatment. It was confirmed by measuring the subcellular distribution of GLUT2 based on GLUT2 immunoblot density among the four membrane fractions before and after insulin treatment. Total GLUT2 distributions at PM, LYSO, HDM and LDM were $37.7{\pm}12.0%,\;42.4{\pm}12.1%,\;19.2{\pm}5.0%\;and\;0.7{\pm}1.2%$ in the absence of insulin. Total GLUT2 distribution in the presence of insulin was almost same as that in the absence of insulin. Present data with previous findings suggest that GLUT4 translocation may be attributed to GLUT4 phosphorylation by $PKC-{\zeta}$ but GLUT2 does not translocate because GLUT2 is not phosphorylated by the kinase. Therefore, GLUT phosphorylation may be required in GLUT translocation mechanism.

• Journal of Embryo Transfer
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• v.28 no.1
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• pp.57-61
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• 2013

Microtubule-associated protein 1B (MAP1B), a member of MAP1 family, plays a key role in neuronal development. MAP1B binds to many kinds of proteins directly or indirectly. This study was performed to investigate whether MAP1B interacts with GAPDH in bovine follicles using immunoprecipitation (IP) with Western blot analysis and immunohistochemisty. The mRNA expressions of MAP1B and glyceraldehydes 3-phosphate dehydrogenase (GAPDH) were down-regulated in bovine follicular cystic follicles (FCF). In parallel with the mRNA levels, their protein levels were also down-regulated in FCFs. In addition, MAP1B and GAPDH were co-localized at the cytoplasm of follicles. IP with Western blot analysis showed that MAP1B bound to GAPDH in normal follicles, but their binding was absent in FCFs, suggesting a low level of MAP1B and/or GAPDH expressions in FCFs. Taken together, these results suggest that MAP1B interacted with GAPDH may play a role in bovine follicle development, and that GAPDH does not function always as a loading control in bovine follicles.

• Molecules and Cells
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• v.26 no.3
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• pp.291-298
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• 2008

Human karyopherin ${\beta}3$, highly homologous to a yeast protein secretion enhancer (PSE1), has often been reported to be associated with a mediator of a nucleocytoplasmic transport pathway. Previously, we showed that karyopherin ${\beta}3$ complemented the PSE1 and KAP123 double mutant. Our research suggested that karyopherin beta has an evolutionary function similar to that of yeast PSE1 and/or KAP 123. In this study, we performed yeast two-hybrid screening to find a protein which would interact with karyopherin ${\beta}3$ and identified apolipoprotein A-I (apo A-I), a secretion protein with a primary function in cholesterol transport. By using in vitro binding assay, co-immunoprecipitation, and colocalization studies, we defined an interaction between karyopherin ${\beta}3$ and apo A-I. In addition, overexpression of karyopherin ${\beta}3$ significantly increased apo A-I secretion. These results suggest that karyopherin ${\beta}3$ plays a crucial role in apo A-I secretion. These findings may be relevant to the study of a novel function of karyopherin ${\beta}3$ and coronary artery diseases associated with apo A-I.

• Development and Reproduction
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• v.24 no.2
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• pp.101-111
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• 2020

Coiled-coil domain containing 110 (CCDC110, KM-HN-1) is a protein containing C-terminal coiled-coil domain (CCD) which was previously discovered as a member of the human cancer/testis antigen (CTA). In addition, CCDC110 has both nuclear localization signal sequence and the leucine zipper motif. Although the functional role of CCDC110 has yet to be fully identified, the mRNA expression levels of CCDC110 are known to be highly elevated in various cancer types including testis, implying its relevance to cancer pathogenesis. In this study, we first developed several monoclonal antibody (mAb) hybridoma clones targeting CCDC110 and further isolated clone by characterizing for its specificity using immunoblotting and immunoprecipitation approaches with basal parenchymal sperm cells in testis tissue. Next, using these mAbs, we showed that the Tet-inducible overexpression of CCDC110 protein delayed the entry of G2/M phase in U2-OS osteosarcoma cells. Based on these results, we propose that CCDC110 plays a crucial role in cell cycle progression.

• The Korean Journal of Food And Nutrition
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• v.24 no.2
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• pp.258-267
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• 2011

14-3-3 is a highly conserved, ubiquitously expressed protein family. It associates with diverse cellular proteins through its specific phosphoserine/phosphothreonine-binding activity and thus contributes to the regulation of crucial cellular processes such as metabolism, signal transduction, cell-cycle control, apoptosis, protein trafficking, transcription and stress responses. This study aims to determine changes in levels of 14-3-3 isoforms and 14-3-3 - associated proteins in Helicobacter pylori(H. pylori)-infected gastric epithelial AGS cells. AGS cells were stimulated with H. pylori(NCTC 11637) at the ratio of 300:1(bacterium:cell). Western blot analysis revealed that 14-3-3 $\sigma$ was elevated at 3 hr after H. pylori treatment. Other isoforms were not significantly affected by H. pylori infection. Using immunoprecipitation to 14-3-3 $\sigma$, followed by proteomic analysis, we found that S phase kinase associated protein isoform 2 bound to 14-3-3 $\sigma$ has increased. In contrast, three proteins (DEAD-box polypeptide 3, heterogeneous nuclear ribonucleoprotein H2 and WD repeat-containing protein isoform 1) bound to 14-3-3 decreased by H. pylori infection. Our results suggest that 14-3-3 may play an important regulatory role in H. pylori-induced signal transduction in gastric epithelial cells.

• The Korean Journal of Zoology
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• v.35 no.4
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• pp.456-465
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• 1992

The 94 KDa glucose-resulsted Protein (SH 94), one of stress Proteins, is a Ca2+-binding protein in the endoplasmic reticulum (ER). In this study, the possible effect of Ca2+ on the native conformation of grp 94 was examined. When the purified grp 94 was analyzed by Sel filtration in the presence of either EGTA or CaCl2, it was eluted with apparent molecular weight (MW) of 100 KDa in both cases. When similarly analyzed with microtome or cell Ivsate, however, srp 94 was eluted with apparent IW of 200 KDa in the presence of E6TA, while with apparent MW of 100 KDa in the presence of CaCl2, indicating possible association of grp 94 with one or more other proteins in the absence of CaCl2. Consequently, immunoprecipitation with anti-grp 94 was carried out to determine which proteins specifically interact with grp 94. It is sho%un that srp 94 may interact, in a Ca2+_dependent manner. with other proteins including BiP (grp 78) which is also a stress protein in the ER.

• Journal of Plant Biology
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• v.32 no.1
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• pp.51-65
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• 1989

Glycinin is the major storage protein in soybean. It has been known that a molecule of glycinin is composed of 6 subunits, each of which consists of two different kinds of polypeptides, acidic (A) and basic (B) one (NW 39K and 19K, respectively). To study the molecular origin and the relationship of glycinin subunit polypeptides, antibodies against A-and B-polypeptide were obtained by immunizing rabbits with either of the antigens purified by gel filtration and preparative electrophoresis. Each antibody was not only specific for its own antigen polypeptide in soybeans but also recoginzed the precursor which was synthesized in vivo and in vitro. The polyadenylated mRNAs were isolated from immature seeds and leaves and were translated in vitro using wheat germ extract. One of the seed-specific translation products. MW 60K, was identified to be the precursor of glycinin subunit by immunoprecipitation with antibodies against glycinin A- and B-polypeptide. Mature A- and B-polypeptides were not detected in the translte in vitro. These results suggest that the precursor polypeptide is synthesized from the mRNA and is cleaved to yield A- and B-polypeptides which from a glycinin subunit in the cell. Glycinin genes were expressed with the maturation of soybean seeds in a tissue-specific and developmental stage-specific manner.

• Animal cells and systems
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• v.4 no.2
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• pp.165-171
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• 2000

Ubiquitinated proteins in lysosomes were characterized by using two monoclonal antibodies (mAbs): LYS8-1, a mAb to lysosomal proteins, and NYA124, a mAb to ubiquitin. LYS8-1 stained lysosome-like vesicles in immunofluorescence microscopy of Amoeba proteus and Acanthamoeba castellanii. In immunoblotting, LYS8-1's antigens (LYS proteins) were detected as 68-kDa and 77-kDa proteins in A. proteus, and as 30-kDa and 39-kDa proteins in A. castellanii. In immunoprecipitation of A. castellanii, at least four distinct LYS proteins, LVS35p, LyS39p, LyS42p, and LYS46p, were detected and accumulated upon inhibition of lysosome functions but not upon that of 26S proteasome functions. They were all found to be ubiquitinated, and were recovered in the lysosome fractions in subcellular fractionation experiments. In chemical fractionation analyses, LYS35p and LYS39p were demonstrated to be peripherally associated with lysosome membrane, while LYS42p and LYS46p tightly bound to the membrane. These results suggest that the LYS proteins become associated to lysosomal membrane upon ubiquitination.

• Molecules and Cells
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• v.41 no.7
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• pp.665-675
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• 2018

Rice is a facultative short-day (SD) plant in which flowering is induced under SD conditions or by other environmental factors and internal genetic programs. Overexpression of Histone Deacetylase 701 (HDT701) accelerates flowering in hybrid rice. In this study, mutants defective in HDT701 flowered late under both SD and long-day conditions. Expression levels of florigens Heading date 3a (Hd3a) and Rice Flowering Locus T1 (RFT1), and their immediate upstream floral activator Early heading date 1 (Ehd1), were significantly decreased in the hdt701 mutants, indicating that HDT701 functions upstream of Ehd1 in controlling flowering time. Transcript levels of OsINDETERMINATE SPIKELET 1 (OsIDS1), an upstream repressor of Ehd1, were significantly increased in the mutants while those of OsGI and Hd1 were reduced. Chromatin-immunoprecipitation assays revealed that HDT701 directly binds to the promoter region of OsIDS1. These results suggest that HDT701 induces flowering by suppressing OsIDS1.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.5
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• pp.515-523
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• 2016

Adhesion events of monocytes represent an important step in inflammatory responses induced by chemokines. The ${\beta}1$-integrin CD29 is a major adhesion molecule regulating leukocyte migration and extravasation. Although several adhesion molecules have been known as regulators of CD29, the molecular interactions between CD29 and its regulatory adhesion molecules (such as CD98 and CD147) have not been fully elucidated. Therefore, in this study, we examined whether these molecules are functionally, biochemically, and cell-biologically associated using monocytic U937 cells treated with aggregation-stimulating and blocking antibodies, as well as enzyme inhibitors. The surface levels of CD29, CD98, and CD147 (but not CD43, CD44, and CD82) were increased. The activation of CD29, CD98, and CD147 by ligation of them with aggregation-activating antibodies triggered the induction of cell-cell adhesion, and sensitivity to various enzyme inhibitors and aggregation-blocking antibodies was similar for CD29-, CD98-, and CD147-induced U937 cell aggregation. Molecular association between these molecules and the actin cytoskeleton was confirmed by confocal microscopy and immunoprecipitation. These results strongly suggest that CD29 might be modulated by its biochemical and cellular regulators, including CD98 and CD147, via the actin cytoskeleton.