• BMB Reports
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• v.51 no.10
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• pp.484-485
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• 2018

Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is a serine-threonine kinase largely essential for necroptotic cell death; it also plays a role in some inflammatory diseases. High levels of RIP3 are likely sufficient to activate necroptotic and inflammatory pathways downstream of RIP3 in the absence of an upstream stimulus. For example, we have previously detected high levels or RIP3 in the skin of Toxic Epidermal Necrolysis patients; this correlates with increased phosphorylation of MLKL found in these patients. We have long surmised that there are molecular mechanisms to prevent anomalous activity of the RIP3 protein, and so prevent undesirable cell death and inflammatory effects when inappropriately activated. Recent discovery that Carboxyl terminus of Hsp 70-Interacting Protein (CHIP) could mediate ubiquitylation- and lysosome-dependent RIP3 degradation provides a potential protein that has this capacity. However, while screening for RIP3-binding proteins, we discovered that pellino E3 ubiquitin protein ligase 1 (PELI1) also interacts directly with RIP3 protein; further investigation in this study revealed that PELI1 also targets RIP3 for proteasome-dependent degradation. Interestingly, unlike CHIP, which targets RIP3 more generally, PELI1 preferentially targets kinase active RIP3 that has been phosphorylated on T182, subsequently leading to RIP3 degradation.

• BMB Reports
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• v.39 no.3
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• pp.311-318
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• 2006

One of the biggest group of proteins influenced by protein kinase CK2 is formed by factors engaged in gene expression. Here we have reported recently identified yeast transcription elongation factor Elf1 as a new substrate for both monomeric and tetrameric forms of CK2. Elf1 serves as a substrate for both the recombinant CK2$\alpha$' ($K_m$ 0.38 ${\mu}M$) and holoenzyme ($K_m$ $0.13\;{\mu}M$). By MALDI-MS we identified the two serine residues at positions 95 and 117 as the most probable in vitro phosphorylation sites. Co-immunoprecypitation experiments show that Elf1 interacts with catalytic ($\alpha$ and $\alpha$') as well as regulatory ($\beta$ and $\beta$') subunits of CK2. These data may help to elucidate the role of protein kinase CK2 and Elf1 in the regulation of transcription elongation.

• Journal of Microbiology
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• v.34 no.4
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• pp.374-378
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• 1996

In the aquatic fungus Allomyces macrogynus the effects of $Ca^{2+}$ and cAMP on the intracellular signal transduction of zoospore germination were studied using in vitro protein phosphorylation assay system. An endogenously phosphorylated protein (p50) having molecular weight of 50 kDa on SDS-PAGE was found in soluble fractions of both zoospore and mycelium. In zoospore extract, the endogenous phosphorylation of p50 was weak without any effectors, but was enhanced by $Ca^{2+}$ and even more by cAMP. Phosphorylation of the same protein in mycelial extract was high only in the absence of cAMP. Irrespective of the presence of $Ca^{2+}$ and cAMP, its phosphorylation was antagonistically suppressed in assay of combined zoospore and mycelial extracts. These results suggest that p50 is interconvertible in phosphorylation/dephosphorylation as a novel protein involved in germination of A. macrogynus. The antagonistic effect of cAMP to the phosphorylation of p50s from different developmental stages may be important in the regulation of cellular differentiation.

• IMMUNE NETWORK
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• v.6 no.2
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• pp.67-75
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• 2006

Background: Cytotoxic function of killer cells is inhibited by specific recognition of class I MHC molecules on target cells by inhibitory killer Ig-like receptors (KIR) expressed on NK cells and some cytotoxic T cells. The inhibitory effect of KIR is accomplished by recruitment of SH2-containing protein tyrosine phosphatase (SHP) to the phosphotyrosine residues in the cytoplasmic tail. Methods: By in vitro coprecipitation experiments and transfection analysis, we investigated the association of KIR with an adaptor protein Shc in Jurkat T cells. Results: The cytoplasmic tail of KIR appeared to associate with an adaptor protein Shc in Jurkat T celilysates. Similar in vitro experiments showed that phosphorylated KIR cytoplasmic tail bound SHP-1 and Shc in Jurkat T cell lysates. The association of KIR with Shc was further confirmed by transfection analysis in 293T cells. Interestingly, however, Shc appeared to be replaced by SHP-2 upon engagement of KIR in 293T cells. Conclusion: Our data indicate that KIR associate with an adaptor protein Shc in Jurkat T cells, and suggest that KIR might have an additional role which is mediated by this adaptor protein.

• Proceedings of the Microbiological Society of Korea Conference
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• 1998.04a
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• pp.49.1-49.1
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• 1998

• Journal of Plant Biology
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• v.33 no.1
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• pp.59-64
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• 1990

In order to study the effect of GA3 on the phosphorylation of ribosomal proteins during germination in Zea mays, ribosomal proteins were labelled with 32P, extracted, electrophoresed and autoradiographed. There are five phosphorylated ribosomal proteins. One of these is in 40S subunit and has molecular weight of 33,000 daltons. Others are in 60S subunit and have molecular weights of 37,000, 16,000, 15,200 and 13,500, respectively. Phosphorylation of ribosomal proteins was increased maximum 47.7% in shoots of Zea mays treated with GA3.

• Microbiology and Biotechnology Letters
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• v.33 no.3
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• pp.189-193
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• 2005

IgE-dependent histamine-releasing factor(HRF) was initially described as a secretagogue for secretion of histamine from IgE+ basophils from a subset of allergic donors. Previously, we identified that S98 residue of HRF was phosphorylated using anti-HRFpS98 antibody which specifically recognizes the phosphorylated serine residue of HRF and HRFS98A mutant construct. In vitro kinase assay, only wild type HRF was phosphorylated by PKC, and S98A HRF was not affected by PKC. In this study, we attempted to characterize the phosphatase which specifically dephosphorylates HRF by immunoprecipitation and pull-down assay. In RBL-2H3 cells, HRF interacted only with calcineurin (also called as PP2B, calcium/calmodulin-dependent phosphatase) but not with PP1 or PP2A. The results suggest that HRF is most likely dephosphory-lated by calcineurin.

• Molecules and Cells
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• v.26 no.1
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• pp.41-47
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• 2008

Mitogen-activated protein kinase (MAPK) signaling is a crucial component of eukaryotic cells; it plays an important role in responses to extracelluar stimuli and in the regulation of various cellular activities. The signaling cascade is evolutionarily conserved in the eukaryotic kingdom from yeast to human. In response to a variety of extracellular signals, MAPK activity is known to be regulated via phosphorylation of a conserved $T{\times}Y$ motif at the activation loop in which both threonine and tyrosine residues are phosphorylated by the upstream kinase. However, the mechanism by which both residues are phosphorylated continues to remain elusive. In the budding yeast, Saccharomyces cerevisiae, Fus3 MAPK is involved in the mating signaling pathway. In order to elucidate the functional mechanism of MAPK activation, we quantitatively profiled phosphorylation of the $T{\times}Y$ motif in Fus3 using mass spectrometry (MS). We used synthetic heavy stable isotope-labeled phosphopeptides and nonphosphopeptides corresponding to the proteolytic $T{\times}Y$ motif of Fus3 and accompanying data-dependent tandem MS to quantitatively monitor dynamic changes in the phosphorylation events of MAPK. Phosphospecific immunoblotting and the MS data suggested that the tyrosine residue is dynamically phosphorylated upon stimulation and that this leads to dual phosphorylation. In contrast, the magnitude of threonine phosphorylation did not change significantly. However, the absence of a threonine residue leads to hyperphosphorylation of the tyrosine residue in the unstimulated condition, suggesting that the threonine residue contributes to the control of signaling noise.

• Journal of Korean Ophthalmic Optics Society
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• v.21 no.1
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• pp.69-76
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• 2016

Purpose: The study was conducted to determine that photoreceptors of mouse having pigment in RPE(retinal pigment epithelium) can be damaged by blue-light and apoptosis of specific cells among photoreceptors are induced by blue-light, and to assist the investigation of AMD(Age-related macular degeneration) mechanisms and development of AMD drugs. Methods: C57Black mice were injured by irradiating $2800{\pm}10lux$ of 463 nm LED for 6 hours after 24 hours dark adaptation and eyes were enucleated 1, 3, 7 days. Damage of retina induced by blue-light was determined by western blotting GFAP(Glial fibrillary acidic protein) expression. In the light-injured retina, cell death of photoreceptors was determined by TUNEL(Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. ERK(Extracellular signal-regulated kinases), JNK, and SRC(sarcoma) expression were assessed by western blotting to determine regulated pathway. Blue light-injured retina were immunostained with antibodies against Opsin and Rhodopsin as markers of photoreceptors to compared the damage cone cells with rod cells. Results: After 1, 3 and 7 days from exposure to blue-light, thickness of retina was more decreased than control, and more decreased at nuclear layer than at outer plexiform layer and GFAP expression was increased day 1 after blue-light injured. While phosphorylated ERK and SRC protein expressions at day 1 were increased after blue-light injured, phosphorylated c-JUN was decreased. Fluorescence intensity analysis showed that markers of cone and rod cells were decreased after blue-light injured and Opsin was more decreased than Rhodopsin. Conclusions: The study suggests possibilities that the blue-light promotes retinal damage and causes apoptotic cell death via ERK and SRC pathway in mouse retina, and blue-light retinal damage is more induced cone cells apoptosis than rod cells directly.

• Journal of Ginseng Research
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• v.46 no.5
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• pp.666-674
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• 2022

Background: Ginsenosides and their metabolites have antidepressant-like effects, but the underlying mechanisms remain unclear. We previously identified 14-3-3 ζ as one of the target proteins of 20 (S)-protopanaxadiol (PPD), a fully deglycosylated ginsenoside metabolite. Methods: Corticosterone (CORT) was administered repeatedly to induce the depression model, and PPD was given concurrently. The tail suspension test (TST) and the forced swimming test (FST) were used for behavioral evaluation. All mice were sacrificed. Golgi-cox staining, GSK 3β activity assay, and Western blot analysis were performed. In vitro, the kinetic binding analysis with the Biolayer Interferometry (BLI) was used to determine the molecular interactions. Results: TST and FST both revealed that PPD reversed CORT-induced behavioral deficits. PPD also ameliorated the CORT-induced expression alterations of hippocampal Ser9 phosphorylated glycogen synthase kinase 3β (p-Ser9 GSK 3β), Ser133 phosphorylated cAMP response element-binding protein (p-Ser133 CREB), and brain-derived neurotrophic factor (BDNF). Moreover, PPD attenuated the CORT-induced increase in GSK 3β activity and decrease in dendritic spine density in the hippocampus. In vitro, 14-3-3 ζ protein specifically bound to p-Ser9 GSK 3β polypeptide. PPD promoted the binding and subsequently decreased GSK 3β activity. Conclusion: These findings demonstrated the antidepressant-like effects of PPD on the CORT-induced mouse depression model and indicated a possible target-based mechanism. The combination of PPD with the 14-3-3 ζ protein may promote the binding of 14-3-3 ζ to p-GSK 3β (Ser9) and enhance the inhibition of Ser9 phosphorylation on GSK 3β kinase activity, thereby activating the plasticity-related CREBeBDNF signaling pathway.