• Animal cells and systems
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• v.11 no.1
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• pp.39-50
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• 2007

Tau plays a role in numerous neuronal processes, such as vesicle transport, microtubule-plasma membrane interaction and intracellular localization of proteins. SUMO (Small Ubiquitin-like Modifier) modification (SUMOylation) appears to regulate diverse cellular processes including nuclear transport, signal transduction, apoptosis, autophagy, cell cycle control, ubiquitin-dependent degradation, as well as gene transcription. We noticed that putative SUMOylation site is localized at $^{340}K$ of $Tau(^{339}VKSE^{342})$ with the consensus sequence information (${\Phi}KxE$ ; where ${\Phi}$ represents L, I, V or F and x is any amino acid). In this report, we demonstrated that $^{340}K$ of Tau is the SUMOylation site and that a point mutant of Tau S214E (an analog of the phospho $^{214}S$ Tau) promotes its SUMOylation at $^{340}K$ and its nuclear or nuclear vicinity localization, by co-immunoprecipitation and confocal microscopy analysis. Further, we demonstrate that the Tau S214E (neither Tau S214A nor Tau K340R) mutant increases its protein stability. However, the SUMOylation at $^{340}K$ of Tau did not influence cell survival, as determined by FACS analysis. Therefore, our results suggested that the phosphorylation of Tau on $^{214}S$ residue promotes its SUMOylation on $^{340}K$ residue and nuclear vicinity localization, and increases its stability, without influencing cell survival.

• Reproductive and Developmental Biology
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• v.31 no.4
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• pp.221-226
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• 2007

Embryonic stem (ES) cells are known to have an infinite proliferation and pluripotency that are associated with complex processes. The objective of this study was to examine expression of genes differentially regulated during differentiation of human ES cells by suppression subtractive hybridization (SSH). Human ES cells were induced to differentiate into neural precursor cells via embryoid body. Neural precursor cells were isolated physically based on morphological criteria. Immunocytochemical analysis showed expression of pax6 in neural precursor cells, confirming that the isolated cells were neural precursor cells. Undifferentiated human ES cells and neural precursor cells were subject to the SSH. TPX2 (Targeting Protein for Xklp2 (Xenopus centrosomal kinesin-like protein 2)) was identified, cloned and analyzed during differentiation of human ES cells into neural lineages. Expression of TPX2 was gradually down-regulated in embryoid bodies and neural precursor cells relative to undifferentiated ES cells. Targeting Protein for Xklp2 has been shown to be involved in cell division by interaction with microtubule development in cancer cells. Taken together, result of this study suggests that TPX2 may be involved in proliferation and differentiation of human ES cells.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.2
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• pp.155-161
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• 2014

Overexpression of amyloid precursor protein with the Swedish mutation causes abnormal hyperphosphorylation of the microtubule-associated protein tau. Hyperphosphorylated isoforms of tau are major components of neurofibrillary tangles, which are histopathological hallmarks of Alzheimer's disease. Protein phosphatase 2A (PP2A), a major tau protein phosphatase, consists of a structural A subunit, catalytic C subunit, and a variety of regulatory B subunits. The B subunits have been reported to modulate function of the PP2A holoenzyme by regulating substrate binding, enzyme activity, and subcellular localization. In the current study, we characterized regulatory B subunit-specific regulation of tau protein phosphorylation. We showed that the PP2A B subunit PPP2R2A mediated dephosphorylation of tau protein at Ser-199, Ser-202/Thr-205, Thr-231, Ser-262, and Ser-422. Down-regulation of PPP2R5D expression decreased tau phosphorylation at Ser-202/Thr-205, Thr-231, and Ser-422, which indicates activation of the tau kinase glycogen synthase kinase 3 beta ($GSK3{\beta}$) by PP2A with PPP2R5D subunit. The level of activating phosphorylation of the $GSK3{\beta}$ kinase Akt at Thr-308 and Ser-473 were both increased by PPP2R5D knockdown. We also characterized B subunit-specific phosphorylation sites in tau using mass spectrometric analysis. Liquid chromatography-mass spectrometry revealed that the phosphorylation status of the tau protein may be affected by PP2A, depending on the specific B subunits. These studies further our understanding of the function of various B subunits in mediating site-specific regulation of tau protein phosphorylation.

• Journal of Life Science
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• v.29 no.3
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• pp.369-375
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• 2019

Microtubules form through the polymerization of ${\alpha}-$ and ${\beta}-tubulin$, and tubulin transport plays an important role in defining the rate of microtubule growth inside cellular appendages, such as the cilia and flagella. Heterotrimeric kinesin 2 is a molecular motor member of the kinesin superfamily (KIF) that moves along the microtubules to transport multiple cargoes. It consists of two motor subunits (KIF3A and KIF3B) and a kinesin-associated protein 3 (KAP3), forming a heterotrimeric complex. Heterotrimeric kinesin 2 interacts with many different binding proteins through the cargo-binding domains of the KIF3s, but these binding proteins have not yet been specified. To identify these proteins for KIF3A, we performed yeast two-hybrid (Y2H) screening and found a specific interaction with ${\beta}2-tubulin$ (Tubb2), a microtubule component. Tubb2 was found to bind to the cargo-binding domain of KIF3A but did not interact with KIF3B, KIF5B, or kinesin light chain 1 in the Y2H assay. The carboxyl-terminal region of Tubb2 is essential for interaction with KIF3A. Other Tubb isoforms, including Tubb1, Tubb3, Tubb4, and Tubb5, also interacted with KIF3A in the Y2H screening. However, ${\alpha}1-tubulin$ (Tuba1) did not interact with KIF3A. In addition, an antibody to KIF3A specifically co-immunoprecipitated the KIF3B and KAP3 associated with Tubb2 from mouse brain extracts. In combination, these results suggest that a heterotrimeric kinesin 2 motor protein is capable of binding to tubulin and may transport it in cells.

• BMB Reports
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• v.40 no.2
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• pp.218-225
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• 2007

In Arabidopsis thaliana, the microtubule-associated protein AtMAP65-1 shows various functions on microtubule dynamics and organizations. However, it is still an open question about whether AtMAP65-1 binds to tubulin dimers and how it regulates microtubule dynamics. In present study, the tubulin-binding activity of AtMAP65-1 was investigated. Pull-down and co-sedimentation exp eriments demonstrated that AtMAP65-1 bound to tubulin dimers,at a molar ratio of 1 : 1. Cross-linking experiments showed that AtMAP65-1 bound to tubulin dimers by interacting with $\alpha$-tubulin of the tubulin heterodimer. Interfering the bundling effect of AtMAP65-1 by addition of salt and monitoring the tubulin assembly, the experiment results indicated that AtMAP65-1 promoted tubulin assembly by interacting with tubulin dimers. In addition, five truncated versions of AtMAP65-1, namely AtMAP65-1 $\Delta$N339 (amino acids 340-587); AtMAP65-1 $\Delta$N494 (amino acids 495-587); AtMAP65-1 340-494 (amino acids 340-494); AtMAP65-1 $\Delta$C495 (amino acids 1-494) and AtMAP65-1 $\Delta$C340 (amino acids 1-339), were tested for their binding activities and roles in tubulin polymerization in vitro. Four (AtMAP65-1 $\Delta$N339, $\Delta$N494, AtMAP65-1 340-494 and $\Delta$C495) from the five truncated proteins were able to co-sediment with microtubules, and three (AtMAP65-1 $\Delta$N339, $\Delta$N494 and AtMAP65-1 340-494) of them could bind to tubulin dimers in vitro. Among the three truncated proteins, AtMAP65-1 $\Delta$N339 showed the greatest activity to promote tubulin polymerization, AtMAP65-1 $\Delta$N494 exhibited almost the same activity as the full length protein in promoting tubulin assembly, and AtMAP65-1 340-494 had minor activity to promote tubulin assembly. On the contrast, AtMAP65-1 $\Delta$C495, which bound to microtubules but not to tubulin dimers, did not affect tubulin assembly. Our study suggested that AtMAP65-1 might promote tubulin assembly by binding to tubulin dimers in vivo.

• Journal of Life Science
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• v.27 no.10
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• pp.1191-1198
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• 2017

Vesicles and organelles are transported along microtubule and delivered to appropriate compartments in cells. The intracellular transport process is mediated by molecular motor proteins, kinesin, and dynein. Kinesin is a plus-end-directed molecular motor protein that moves the various cargoes along microtubule tracks. Kinesin 1 is first isolated from squid axoplasm is a dimer of two heavy chains (KHCs, also called KIF5s), each of which is associated with the light chain (KLC). KIF5s interact with many different binding proteins through their carboxyl (C)-terminal tail region, but their binding proteins have yet to be specified. To identify the interacting proteins for KIF5A, we performed the yeast two-hybrid screening and found a specific interaction with Ras-GTPase-activating protein (GAP) Src homology3 (SH3)-domain-binding protein 2 (G3BP2), which is involved in stress granule formation and mRNA-protein (mRNP) localization. G3BP2 bound to the C-terminal 73 amino acids of KIF5A but did not interact with the KIF5B, nor the KIF5C in the yeast two-hybrid assay. The arginine-glycine-glycine (RGG)/Gly-rich region domain of G3BP2 is a minimal binding domain for interaction with KIF5A. However, G3BP1 did not interact with KIF5A. When co-expressed in HEK-293T cells, G3BP2 co-localized with KIF5A and was co-immunoprecipitated with KIF5A. These results indicate that G3BP2, which was originally identified as a Ras-GAP SH3 domain-binding protein, is a protein that interacts with KIF5A.

• Journal of Korean Neurosurgical Society
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• v.49 no.1
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• pp.13-19
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• 2011

Objective: The purpose of this study is to investigate the combined effects of ginkgo biloba extract, ginkgolide A and B and aspirin on SK-N-MC, human neuroblastoma cell viability and mRNA expression of growth associated protein43 (GAP43), Microtubule-associated protein 2 (MAP2), B-cell lymphoma2 (Bcl2) and protein53 (p53) gene in hypoxia and reperfusion condition. Methods: SK-N-MC cells were cultured with Dulbecco's Modified Eagle's Medium (DMEM) media in $37^{\circ}C$, 5% $CO_2$ incubator. The cells were cultured for 8 hours in non-glucose media and hypoxic condition and for 12 hours in normal media and $O_2$ concentration. Cell survival rate was measured with Cell Counting Kit-8 (CCK-8) reagent assay. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to estimate mRNA levels of GAP43, MAP2, Bcl2, and p53 genes. Results: The ginkgolide A and B increased viable cell number decreased in hypoxic and reperfused condition. The co-treatment of ginkgolide B with aspirin also increased the number of viable cells, however, there was no additive effect. Although there was no increase of mRNA expression of GAP43, MAP2, and Bcl2 in SK-N-MC cells with individual treatment of ginkgolide A, B or aspirin in hypoxic and reperfused condition, the co-treatment of ginkgolide A or B with aspirin significantly increased GAP43 and Bcl2 mRNA levels. In MAP2, only the co-treatment of ginkgolide A and aspirin showed increasing effect. The mRNA expression of p53 had no change in all treating conditions. Conclusion: This study suggests that the combined treatments of Ginkgo biloba extracts and aspirin increase the regeneration of neuroblastoma cells injured by hypoxia and reperfusion.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.10a
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• pp.101-101
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• 2018

Resveratrol is a polyphenolic compound, which is a naturally occurring phytochemical and is found in a variety of plants, including food such as grapes, berries and peanuts. Although several studies have demonstrated that resveratrol possesses anti-cancer activity against various types of human cancer, the molecular mechanisms of resveratrol-mediated overcome drug resistance potential are unclear. In this study, we determined whether resveratrol attenuates drug resistance responses in 5-fluorouracil-resistant colon cancer (SNUC5/5-FUR) cells. Treatment with resveratrol significantly enhanced apoptosis in a concentration-dependent manner, which was associated with the modulation of anti- and/or pro-apoptotic protein expression, activation of caspases and activation of AMP-activated protein kinase. Resveratrol treatment also increased the induction of autophagy through up-regulation of autophagy-related genes such as Microtubule-associated protein 1A/1B-light chain 3, P62 and beclin-1. However, blocking of autophagy by bafilomycin A1 reduced apoptotic cell death, suggesting that resveratrol-induced autophagy functions as a cell death mechanism in SNUC5/5-FU cells. Although the further studies are needed, these findings suggest that resveratrol may have therapeutic potential to overcome drug resistance in colon cancer patients.

• BMB Reports
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• v.51 no.10
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• pp.508-513
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• 2018

Fibronectin fragments found in the synovial fluid of patients with osteoarthritis (OA) induce the catabolic responses in cartilage. Nuclear high-mobility group protein Box 1 (HMGB1), a damage-associated molecular pattern, is responsible for the regulation of signaling pathways related to cell death and survival in response to various stimuli. In this study, we investigated whether changes induced by 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) in HMGB1 expression influences the pathogenesis of OA via an HMGB1-modulated autophagy signaling pathway. Human articular chondrocytes were enzymatically isolated from articular cartilage. The level of mRNA was measured by quantitative real-time PCR. The expression of proteins was examined by western blot analysis, immnunofluorescence assay, and enzyme-linked immunosorbent assay. Interaction of proteins was evaluated by immunoprecipitation. The HMGB1 level was significantly lower in human OA cartilage than in normal cartilage. Although 29-kDa FN-f significantly reduced the HMGB1 expression at the mRNA and protein levels 6 h after treatment, the cytoplasmic level of HMGB1 was increased in chondrocytes treated with 29-kDa FN-f, which significantly inhibited the interaction of HMGB1 with Beclin-1, increased the interaction of Bcl-2 with Beclin-1, and decreased the levels of Beclin-1 and phosphorylated Bcl-2. In addition, the level of microtubule-associated protein 1 light chain 3-II, an autophagy marker, was down-regulated in chondrocytes treated with 29-kDa FN-f, whereas the effect was antagonized by mTOR knockdown. Furthermore, prolonged treatment with 29-kDa FN-f significantly increased the release of HMGB1 into the culture medium. These results demonstrated that 29-kDa FN-f inhibits chondrocyte autophagy by modulating the HMGB1 signaling pathway.

• Biomolecules & Therapeutics
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• v.25 no.6
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• pp.609-617
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• 2017

Pancreatic cancer is one of the most lethal and aggressive cancers in the world. However, no effective treatment is currently available for pancreatic cancer. The objective of this study was to determine the anti-pancreatic cancer effect of ${\alpha}$-mangostin (${\alpha}M$) and ${\gamma}$-mangostin (${\gamma}M$) extracted from the pericarp of Garcinia mangostana L.. Both ${\alpha}$M and ${\gamma}M$ reduced the viability of pancreatic cancer cells MIA PaCa-2 and PANC-1 in a dose-dependent manner. These compounds induced apoptosis by increasing c-PARP and c-Caspase 3 levels. They also induced autophagy by increasing levels of microtubule-associated protein 1A/1B light chain 3B (LC3II) in both cell lines while decreasing sequestosome 1 (p62) in MIA PaCa-2. Both ${\alpha}$M and ${\gamma}M$ induced autophagy through increasing phosphorylation levels of AMP-activated protein kinase (p-AMPK) and p38-mitogen activated protein kinase (p-p38) while decreasing phosphorylation level of mammalian target of rapamycin complex 1 (p-mTOR). Of various microRNAs (miRNA), miR-18a was found to be a putative regulatory miRNA for autophagy induced by ${\alpha}$M or ${\gamma}M$. In combination with gemcitabine, a compound frequently used in pancreatic cancer treatment, ${\alpha}$M and ${\gamma}M$ showed synergistic anti-cancer effects in MIA PaCa-2. Collectively, these results suggest that ${\alpha}$M and ${\gamma}M$ can induce apoptosis and autophagy in pancreatic cancer cells and that their anti-cancer effect is likely to be associated with miR-18a. In conclusion, ${\alpha}$M and ${\gamma}M$ might be used as a potential new therapy for pancreatic cancer.