• Molecules and Cells
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• v.44 no.7
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• pp.500-516
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• 2021

Cardiac hypertrophic signaling cascades resulting in heart failure diseases are mediated by protein phosphorylation. Recent developments in mass spectrometry-based phosphoproteomics have led to the identification of thousands of differentially phosphorylated proteins and their phosphorylation sites. However, functional studies of these differentially phosphorylated proteins have not been conducted in a large-scale or high-throughput manner due to a lack of methods capable of revealing the functional relevance of each phosphorylation site. In this study, an integrated approach combining quantitative phosphoproteomics and cell-based functional screening using phosphorylation competition peptides was developed. A pathological cardiac hypertrophy model, junctate-1 transgenic mice and control mice, were analyzed using label-free quantitative phosphoproteomics to identify differentially phosphorylated proteins and sites. A cell-based functional assay system measuring hypertrophic cell growth of neonatal rat ventricle cardiomyocytes (NRVMs) following phenylephrine treatment was applied, and changes in phosphorylation of individual differentially phosphorylated sites were induced by incorporation of phosphorylation competition peptides conjugated with cell-penetrating peptides. Cell-based functional screening against 18 selected phosphorylation sites identified three phosphorylation sites (Ser-98, Ser-179 of Ldb3, and Ser-1146 of palladin) displaying near-complete inhibition of cardiac hypertrophic growth of NRVMs. Changes in phosphorylation levels of Ser-98 and Ser-179 in Ldb3 were further confirmed in NRVMs and other pathological/physiological hypertrophy models, including transverse aortic constriction and swimming models, using site-specific phospho-antibodies. Our integrated approach can be used to identify functionally important phosphorylation sites among differentially phosphorylated sites, and unlike conventional approaches, it is easily applicable for large-scale and/or high-throughput analyses.

• The Korean Journal of Zoology
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• v.37 no.1
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• pp.130-136
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• 1994

Although alteration in protein phosphorylation by specific protein kinases is of importance in transducing cellular signals in a variety of neural/endocrine systems, little is known about protein phosphorylation in the hvpothalamus. The present study aims to explore whether activation of the second messenger-dependent protein kinases affects phosphorylation of specific proteins using a cell free phosphorylation system followed by SDS-polvacrylamide gel electrophoresis. Cytoplasmic fractions derived from hvpothalami of immature rats were used as substrates and several activators and/or inhibitors of CAMP-, phosphatidylinositol- and Ca2+-calmodulin-dependent protein kinases were assessed. Many endogenous proteins were extensively phosphorylated and depending on the signal transduction pathways, phosphorvlation profiles were markedly different. The present data indicate that extracellular signals may affect cellular events through protein phosphorylation by second messengers-protein kinases in the rat hypothalamus.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.6
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• pp.3725-3728
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• 2013

Background: The mammalian target of rapamycin (mTOR) /RPS6KB1 activation has recently been implicated in tumour development, but its role in lung cancer remains unclear. The aim of this study was to explore the role of mTOR/RPS6KB1 signaling pathway in non-small-cell lung cancer (NSCLC). Methods: Immunohistochemistry was performed to assess the expression of phosphorylated mammalian target of rapamycin (p-mTOR) and its downstream ribosomal phosphorylated RPS6KB1 (p-RPS6KB1) in NSCLC patients. We also analyzed p-mTOR/p-RPS6KB1 protein expression in 45 fresh NSCLC tissues using Western blotting. Results: The expression level of p-mTOR and p-RPS6KB1 was significantly higher in NSCLC tumor specimens than that in adjacent noncancerous normal lung tissues (P<0.01). p-mTOR expression correlated with p-RPS6KB1. Furthermore, high expression level of p-mTOR or p-RPS6KB1 in NSCLC was associated with a shorter overall survival (both P<0.01). Multivariate analysis indicated high level of p-mTOR expression was an independent prognostic factor (HR=2.642, 95%CI 1.157-4.904, p=0.002). Conclusions: p-mTOR and p-RPS6KB1 could be useful prognostic markers for NSCLC.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.27 no.2
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• pp.110-122
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• 2005

Adenoid cystic carcinoma (ACC) of salivary glands has a protracted clinical course with perineural invasion, delayed onset of hematogenous metastasis, and poor responses to classical cytotoxic chemotherapic agents. Most deaths from salivary ACC are caused by lung metastases that are resistant to conventional therapy. Therefore, knowledge of cellular properties and tumor-host interactions that influence the dissemination of metastatic cells is important for the design of more effective therapy of salivary cancer. I determined in vitro expression of epidermal growth factor receptor (EGFR) and its downstream effectors and vascular endothelial growth factor receptor (VEGFR)-2 on a human salivary ACC cell line (ACC2). I also evaluated the expression of EGF and VEGF signaling molecules and metastasis-related proteins on human salivary ACC cells orthotopically growing in nude mice. In Western blot and immunohistochemical analyses, EGFR and VEGFR-2 were presented and phosphorylated in ACC2 cells. In human parotid cancer xenografts in nude mice, EGF and VEGF signaling molecules, IL-8, and MMP-9 were expressed at markedly higher levels than in normal parotid tissues. Moreover, tumor-associated endothelial cells of this orthotopic parotid tumor expressed phosphorylated VEGFR-2 and phosphorylated Akt, which is a cell-survival protein. These data show that those biomarkers can be molecular targets for therapy of salivary ACC, which has a propensity for delayed lung metastasis.

• BMB Reports
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• v.44 no.8
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• pp.541-546
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• 2011

It is generally accepted that spermatozoa capacitation is associated with protein kinase A-mediated tyrosine phosphorylation. In our previous study, we identified the fibrous sheath CABYR binding protein (FSCB), which was phosphorylated by PKA. However, the phosphorylation status of FSCB protein during spermatozoa capacitation should be further investigated. To this aim, in this study, we found that phosphorylation of this 270-kDa protein occurred as early as 1 min after mouse spermatozoa capacitation, which increased over time and remained stable after 60 min. Immunoprecipitation assays demonstrated that the tyrosine and Ser/Thr phosphorylation of FSCB occurred during spermatozoa capacitation. The extent of phosphorylation and was closely associated with the PKA activity and spermatozoa motility characteristics. FSCB phosphorylation could be induced by PKA agonist DB-cAMP, but was blocked by PKA antagonist H-89.Therefore, FSCB contributes to spermatozoa capacitation in a tyrosine-phosphorylated format, which may help in further elucidating the molecular mechanism of spermatozoa capacitation.

• BMB Reports
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• v.50 no.11
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• pp.566-571
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• 2017

Weight loss ${\geq}5$ percent is sufficient to significantly reduce health risks for obese people; therefore, development of novel weight loss compounds with reduced toxicity is urgently required. After screening of natural compounds with anti-adipogenesis properties in 3T3-L1 cells, we determined that kahweol, a coffee-specific diterpene, inhibited adipogenesis. Kahweol reduced lipid accumulation and expression levels of adipogenesis and lipid accumulation-related factors. Levels of phosphorylated AKT and phosphorylated JAK2, that induce lipid accumulation, decreased in kahweol-treated cells. Particularly, kahweol treatment significantly increased AMP-activated protein kinase (AMPK) activation. We revealed that depletion of AMPK alleviated reduction in lipid accumulation from kahweol treatment, suggesting that inhibition of lipid accumulation by kahweol is dependent on AMPK activation. We detected more rapid reduction in blood glucose levels in mice administrated kahweol than in control mice. We suggest that kahweol has anti-obesity effects and should be studied further for possible therapeutic applications.

• Animal cells and systems
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• v.10 no.1
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• pp.1-6
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• 2006

Previous studies showed that Cdc7 kinase of Schizosaccharomyces pombe phosphorylated the minichromosome maintenance (Mcm) complex efficiently in the presence of spMcm10 protein. The biochemical properties of the phosphorylated Mcm complexes were examined to understand the activation mechanism of the Mcm complex by Cdc7 kinase. The phosphorylation of Mcm complex in the presence of spMcm10 by Cdc7 kinase did not affect the stability of the Mcm complex containing all six subunits, and the changes in the sedimentation properties were not observed after the phosphorylation. The reconstitution of the Mcm complex using the purified proteins showed that the phosphorylation of Mcm2 proteins did not affect the interactions between Mcm proteins. The phosphorylation of the Mcm2-7 complex at the same condition also did not activate the other biochemical activities such as DNA helicase and single stranded (ss) DNA binding activities. On the other hand, spMcm10 protein that was used for the stimulation of Mcm phosphorylation showed single stranded DNA binding activity, and inhibited the DNA helicase activity of the Mcm4/6/7 complex. These inhibitory effects were reduced by the addition of Cdc7 kinase, suggesting that the phosphorylation by Cdc7 kinase decreased the interactions between spMcm10 and the Mcm complex. Taken together, these results suggested that the phosphorylation by Cdc7 kinase alone is not sufficient for the remodeling and the activation of the Mcm complex, and the additional factors or the phosphorylations might be required for the activation of the Mcm complex.

• Molecules and Cells
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• v.44 no.7
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• pp.493-499
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• 2021

Parkinson's disease (PD) is characterized by a progressive loss of dopamine-producing neurons in the midbrain, which results in decreased dopamine levels accompanied by movement symptoms. Oral administration of l-3,4-dihydroxyphenylalanine (L-dopa), the precursor of dopamine, provides initial symptomatic relief, but abnormal involuntary movements develop later. A deeper understanding of the regulatory mechanisms underlying dopamine homeostasis is thus critically needed for the development of a successful treatment. Here, we show that p21-activated kinase 4 (PAK4) controls dopamine levels. Constitutively active PAK4 (caPAK4) stimulated transcription of tyrosine hydroxylase (TH) via the cAMP response element-binding protein (CREB) transcription factor. Moreover, caPAK4 increased the catalytic activity of TH through its phosphorylation of S⁴⁰, which is essential for TH activation. Consistent with this result, in human midbrain tissues, we observed a strong correlation between phosphorylated PAK4^S474, which represents PAK4 activity, and phosphorylated TH^S40, which reflects their enzymatic activity. Our findings suggest that targeting the PAK4 signaling pathways to restore dopamine levels may provide a new therapeutic approach in PD.

• BMB Reports
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• v.31 no.3
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• pp.281-286
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• 1998

Hepatitis C virus (HCV) core proteins from two different isolates (HCV-1 and HCV-RH) were expressed in Spotioptera Jrugiperda (Sf9) insect cells. The RH core consisted of two major species of proteins (21 kDa and 19 kDa). On the other hand, the HCV-1 core was approximately 16 kDa in a SDS-PAGE gel. Both core proteins were phosphorylated in vivo on serine residues. Furthermore, the RH core but not HCV-1 core formed dimers, indicating that the protein conformation of the core in these two isolates is dfferent from one another. Immunofluorescence studies showed that the RH core was present in the cytoplasm, whereas the HCV-1 core was localized predominantly to the nucleus in recombinant baculovirus-infected insect cells. Since the major difference between the two isolates is the codon 9 of the core protein, a single amino acid substitution appears to play a major role in the protein conformation and these properties may reflect the different biological functions of core proteins in HCV-infected cells.

• Bulletin of the Korean Chemical Society
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• v.34 no.5
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• pp.1503-1507
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• 2013

The two major symptoms characterizing Alzheimer's disease are the formation of amyloid-${\beta}$ extracellular deposits in the form of senile plaques and intracellular neurofibrillary tangles (NFTs) that consist of pathological hyperphosphorylated tau protein aggregated into insoluble paired helical filaments (PHFs). Neurons of the central nervous system have appreciable amounts of tau protein, a microtubule-associated protein. To maintain an optimal operation of nerves, the microtubules are stabilized, which is necessary to support cell structure and cellular processes. When the modified tau protein becomes dysfunctional, the cells containing misfolded tau cannot maintain cell structure. One of the pathological hallmarks of Alzheimer's disease is hyperphosphorylated tau protein. This paper shows that the small heat shock protein from humans (Hsp27) reduces hyperphosphorylated tau and prevents hyperphosphorylated tau-induced cell death of the human neuroblastoma cell line SH-SY5Y.