• Molecules and Cells
• /
• v.25 no.4
• /
• pp.566-571
• /
• 2008

Calcineurin (Cn) is a calcium/calmodulin-dependent serine/threonine protein phosphatase that has diverse functions in different cell types and organisms. We screened proteins interacting with the C. elegans CnA homolog, TAX-6, by the yeast two-hybrid system. CNP-3 (Calcineurin interacting protein-3) is a novel protein that physically interacts with the catalytic domain of TAX-6. It is strongly expressed in the nuclei of intestine, hypodermis, dorsal uterine regions and spermatheca. Expression begins around the 60-cell stage and proceeds during all larval stages and the adult. To elucidate the biological function of cnp-3 we isolated a cnp-3 deletion mutant. Since CNP-3 binds CnA, we looked at factors associated with calcineurin loss-of-function mutants, such as brood size, body size, serotonin- and levamisole-mediated egg-laying behavior. The cnp-3(jh145) single mutant had no gross defects compared to wild-type animal. However, the phenotypes of the double mutants, tax-6(p675);cnp-3(jh145) and cnb-1(jh103);cnp-3(jh145), were more severe in terms of brood size, body size and serotonin-mediated egg-laying defects than tax-6(p675) and cnb-1(jh103), respectively. These results suggest that dysfunction of cnp-3 enhances certain calcineurin loss-of-function phenotypes in C. elegans.

• The Plant Pathology Journal
• /
• v.35 no.6
• /
• pp.684-691
• /
• 2019

Evolution of adaptive mechanisms to abiotic stress is essential for plant growth and development. Plants adapt to stress conditions by activating the abscisic acid (ABA) signaling pathway. It has been suggested that the ABA receptor, clade A protein phosphatase, SnRK2 type kinase, and SLAC1 anion channel are important components of the ABA signaling pathway. In this study, we report that the shaker type potassium (K⁺) channel, GORK, modulates plant responses to ABA and abiotic stresses. Our results indicate that the full length of PP2CA is needed to interact with the GORK C-terminal region. We identified a loss of function allele in gork that displayed ABA-hyposensitive phenotype. gork and pp2ca mutants showed opposite responses to ABA in seed germination and seedling growth. Additionally, gork mutant was tolerant to the NaCl and mannitol treatments, whereas pp2ca mutant was sensitive to the NaCl and mannitol treatments. Thus, our results indicate that GORK enhances the sensitivity to ABA and negatively regulates the mechanisms involved in high salinity and osmotic stresses via PP2CA-mediated signals.

• Journal of Life Science
• /
• v.33 no.11
• /
• pp.859-867
• /
• 2023

Lupeol is a type of pentacyclic triterpene that has been reported to have therapeutic effects for treating many diseases; however, its effect on insulin resistance is unclear clear. This study examined the inhibitory effect of lupeol on the serine phosphorylation of insulin receptor substrate-1 in insulin resistance-induced 3T3-L1 adipocytes. 3T3-L1 cells were cultured and treated with tumor necrosis factor-α (TNF-α) for 24 hours to induce insulin resistance. Cells treated with different concentrations of lupeol (15 μM or 30 μM) or 100 nM of rosiglitazone were incubated. Then, lysed cells underwent western blotting. Lupeol exhibited a positive effect on the negative regulator of insulin signaling and inflammation-activated protein kinase caused by TNF-α in adipocytes. Lupeol inhibited the activation of protein tyrosine phosphatase-1B (PTP-1B)-a negative regulator of insulin signaling-and c-Jun N-terminal kinase (JNK); it was also an inhibitor of nuclear factor kappa-B kinase (IKK) and inflammation-activated protein kinases. In addition, Lupeol downregulated serine phosphorylation and upregulated tyrosine phosphorylation in insulin receptor substrate-1. Then, the downregulated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway was activated, the translocation of glucose transporter type 4 was stimulated to the cell membrane, and intracellular glucose uptake increased in the insulin resistance-induced 3T3-L1 adipocytes. Lupeol may improve TNF-α-induced insulin resistance by downregulating the serine phosphorylation of insulin receptor substrate 1 by inhibiting negative regulators of insulin signaling and inflammation-activated protein kinases in 3T3-L1 adipocytes.

• Molecules and Cells
• /
• v.39 no.4
• /
• pp.322-329
• /
• 2016

Voltage-gated $Ca^{2+}$ ($Ca_V$) channels are dynamically modulated by Gprotein-coupled receptors (GPCR). The $M_1$ muscarinic receptor stimulation is known to enhance $Ca_V2.3$ channel gating through the activation of protein kinase C (PKC). Here, we found that $M_1$ receptors also inhibit $Ca_V2.3$ currents when the channels are fully activated by PKC. In whole-cell configuration, the application of phorbol 12-myristate 13-acetate (PMA), a PKC activator, potentiated $Ca_V2.3$ currents by ~two-fold. After the PMA-induced potentiation, stimulation of $M_1$ receptors decreased the $Ca_V2.3$ currents by $52{\pm}8%$. We examined whether the depletion of phosphatidylinositol 4,5-bisphosphate ($PI(4,5)P_2$) is responsible for the muscarinic suppression of $Ca_V2.3$ currents by using two methods: the Danio rerio voltage-sensing phosphatase (Dr-VSP) system and the rapamycin-induced translocatable pseudojanin (PJ) system. First, dephosphorylation of $PI(4,5)P_2$ to phosphatidylinositol 4-phosphate (PI(4)P) by Dr-VSP significantly suppressed $Ca_V2.3$ currents, by $53{\pm}3%$. Next, dephosphorylation of both PI(4)P and $PI(4,5)P_2$ to PI by PJ translocation further decreased the current by up to $66{\pm}3%$. The results suggest that $Ca_V2.3$ currents are modulated by the $M_1$ receptor in a dual mode-that is, potentiation through the activation of PKC and suppression by the depletion of membrane $PI(4,5)P_2$. Our results also suggest that there is rapid turnover between PI(4)P and $PI(4,5)P_2$ in the plasma membrane.

• Journal of the korean academy of Pediatric Dentistry
• /
• v.27 no.2
• /
• pp.300-308
• /
• 2000

The Fibroblast growth factors(FGFs) plays an important role in the control of osteogenesis during skeletal development. Especially, FGF-2 is a potent mesodermal inducer during embryogenesis and FGF receptors (FGFRs) messages are strongly expressed in developing bones. In this study, we investigated the effect of bFGF on osteopontin(OPN) gene expression in ST-2 cells and tried to elucidate the mechanism of its stimulatory effects. The obtain results were as follows; The treatment of bFGF(1ng/ml) upregulates OPN, fibronectin mRNA levels and downregulates type I collagen mRNA levels. But, there was no remarkable difference in alkaline phosphatase mRNA levels between two groups. The OPN gene expression increased in a dose-dependent manner up to 10ng/ml and OPN gene began to occur at around 3h with continuous increase up to 24h then decreased to basal level at 48h. 30 minutues pretreatment with cycloheximide (500ng/ml), a protein synthesis inhibitor, prior to addition bFGF resulted in blocking bFGF induced OPN expression. These results suggest that bFGF increased the level of OPN mRNA in a dose and time-dependent manner via the synthesis of certain transcriptional regulatory proteins.

• The korean journal of orthodontics
• /
• v.23 no.2 s.41
• /
• pp.229-248
• /
• 1993

[ $Interferon-\gamma$ ] has been suggested as a cytokine of connective tissue stabilizer. In addition, it has also been demonstrated that this cytokine inhibited bone remodeling activities of the bone derived cells. In order to illuminate the effects of this cytokine in orthodontic force induced bone remodeling, it was administered to primary cultured periodontal ligament cells which have been known to have some osteoblast like characteristics. $Interferon-\gamma$ slightly decreased $[^3H]thymidine$ incorporation rate without a significant change in the total cellular DNA content up to 1000 U/ml, which meant these doses were not cytotoxic to the cell. Total protein synthesis was not influenced by various concentration of interferon-y whether it was determined by the $[^3H]proline$ incorporation rate or by the Lowry smethod. The effect of $interferon-\gamma$ on the individual protein was, however, differential, ie, it increased $[^3H]proline$ incorporation into the noncollagenous protein marginally, while it decreased $[^3H]proline$ incorporation into the collagen, so that it caused dose-dependent suppression of the relative collagen synthesis. On the contrary, the fibronectin synthesis determined by the ELISA was increased by 1000 U/ml of $interferon-\gamma$. The differential effects of the interferon-y on the collagen and fibronectin synthesis exhibited not only their protein level but also the steady state mRNA level. $Interferon-\gamma$ decreased steady state level of ${\alpha}1(I)$ procollagen mRNA significantly, while showing no significant changes in the fibronectin mRNA level. In addition to this, it was also found that indomethacin did not affect on the $interferon-\gamma$ induced collagen decrease in this cell, which meant prostaglandins were not involed in the process of $interferon-\gamma$ induced collagen decrease. So it can be concluded that the incubation of periodontal ligament cells with 1000 U/ml of $interferon-\gamma$ for 24 hr showed differential effects on the type I collagen and fibronectin gene expression. The decrease in relative collagen synthesis in the protein level was related with decrease in the steady state level of mRNA, while the increase in the fibronectin synthesis in the protein level was not correlated with the mRNA level.

• The Korean Journal of Physiology and Pharmacology
• /
• v.27 no.4
• /
• pp.417-426
• /
• 2023

The TRPM4 gene encodes a Ca²⁺-activated monovalent cation channel called transient receptor potential melastatin 4 (TRPM4) that is expressed in various tissues. Dysregulation or abnormal expression of TRPM4 has been linked to a range of diseases. We introduced the hemagglutinin (HA) tag into the extracellular S6 loop of TRPM4, resulting in an HA-tagged version called TRPM4-HA. This TRPM4-HA was developed to investigate the purification, localization, and function of TRPM4 in different physiological and pathological conditions. TRPM4-HA was successfully expressed in the intact cell membrane and exhibited similar electrophysiological properties, such as the current-voltage relationship, rapid desensitization, and current size, compared to the wild-type TRPM4. The presence of the TRPM4 inhibitor 9-phenanthrol did not affect these properties. Furthermore, a wound-healing assay showed that TRPM4-HA induced cell proliferation and migration, similar to the native TRPM4. Co-expression of protein tyrosine phosphatase, non-receptor type 6 (PTPN6 or SHP1) with TRPM4-HA led to the translocation of TRPM4-HA to the cytosol. To investigate the interaction between PTPN6 and tyrosine residues of TRPM4 in enhancing channel activity, we generated four mutants in which tyrosine (Y) residues were substituted with phenylalanine (F) at the N-terminus of TRPM4. The YF mutants displayed properties and functions similar to TRPM4-HA, except for the Y256F mutant, which showed resistance to 9-phenanthrol, suggesting that Y256 may be involved in the binding site for 9-phenanthrol. Overall, the creation of HA-tagged TRPM4 provides researchers with a valuable tool to study the role of TRPM4 in different conditions and its potential interactions with other proteins, such as PTPN6.

• Anatomy and Cell Biology
• /
• v.56 no.4
• /
• pp.552-561
• /
• 2023

The endocrinology of type 2 diabetes (T2D) and its predisposing factors have been studied extensively while its skeletal effects have received negligible research despite this being a global disease. The cellular and molecular association between proximal humeral fractures and T2D has not been fully elucidated. We aimed to study bone cell quantities and immunolabel osteogenic and antiosteogenic cytokines. The study used 12-week-old rats (23 males) consisting of 8 Sprague Dawley (SD) and 15 Zucker Diabetic Sprague Dawley (ZDSD). Weekly mass measurements were taken while fasting blood glucose levels were recorded every 2 weeks with oral glucose tolerance tests conducted once every 4 weeks. Upon termination at the age of 28 weeks, humeri were fixed in 10% buffered formalin, prior to decalcification in ethylenediaminetetraacetic acid. The bone samples were then processed in ascending grades of alcohol using an automatic processor before embedding in paraffin wax. Sections were cut at 5 ㎛ thickness in a series for Haematoxylin and Eosin stain, and immunohistochemistry was performed with the anti-tartrate-resistant acid phosphatase (TRAP), anti-alkaline phosphatase (ALP), anti-bone morphogenetic protein 3 (BMP3), anti-transforming growth factor beta 1 (TGFβ1), anti-aged glycation end product (AGE) antibodies in the sequence. ZDSD rats had more adipocytes, BMP3 and AGEs expression with higher numbers of TRAP positive osteocytes and fewer ALP cells although no differences were found in TGFβ1 immunopositivity. We also found that T2D increases the number of AGEs immuno-positive cells, as well as its extracellular expression, thus providing a conducive environment for the interaction of the osteogenic cytokine and its antagonist to suppress osteoblastogenesis. ZDSD groups had higher adipocyte numbers therefore increased marrow adiposity in T2D.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.9
• /
• pp.1206-1211
• /
• 2013

The selective inhibition of PTP1B has been widely recognized as a potential drug target for the treatment of type 2 diabetes and obesity. In the course of screening for PTP1B inhibitory fungal metabolites, the organic extracts of several fungal species isolated from marine environments were found to exhibit significant inhibitory effects, and the bioassay-guided investigation of these extracts resulted in the isolation of fructigenine A (1), cyclopenol (2), echinulin (3), flavoglaucin (4), and viridicatol (5). The structures of these compounds were determined mainly by analysis of NMR and MS data. These compounds inhibited PTP1B activity with 50% inhibitory concentration values of 10.7, 30.0, 29.4, 13.4, and 64.0 ${\mu}M$, respectively. Furthermore, the kinetic analysis of PTP1B inhibition by compounds 1 and 5 suggested that compound 1 inhibited PTP1B activity in a noncompetitive manner, whereas compound 5 inhibited PTP1B activity in a competitive manner.

• BMB Reports
• /
• v.40 no.3
• /
• pp.302-314
• /
• 2007

N type calcium channels (CaV2.2) play a key role in the gating of transmitter release at presynaptic nerve terminals. These channels are generally regarded as parts of a multimolecular complex that can modulate their open probability and ensure their location near the vesicle docking and fusion sites. However, the proteins that comprise this component remain poorly characterized. We have carried out the first open screen of presynaptic CaV2.2 complex members by an antibody-mediated capture of the channel from purified rat brain synaptosome lysate followed by mass spectroscopy. 589 unique peptides resulted in a high confidence match of 104 total proteins and 40 synaptosome proteome proteins. This screen identified several known CaV2.2 interacting proteins including syntaxin 1, VAMP, protein phosphatase 2A, $G_{o\alpha}$, G$\beta$ and spectrin and also a number of novel proteins, including clathrin, adaptin, dynamin, dynein, NSF and actin. The unexpected proteins were classified within a number of functional classes that include exocytosis, endocytosis, cytoplasmic matrix, modulators, chaperones, and cell-signaling molecules and this list was contrasted to previous reports that catalogue the synaptosome proteome. The failure to detect any postsynaptic density proteins suggests that the channel itself does not exhibit stable trans-synaptic attachments. Our results suggest that the channel is anchored to a cytoplasmic matrix related to the previously described particle web.