• Animal cells and systems
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• 제9권3호
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• pp.173-179
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• 2005

Cyclic-AMP-dependent protein kinase (PKA) seems to function as a negative regulator of the c-Jun $NH_2-terminal$ kinase (JNK) signaling pathway. We demonstrate here that the activity of the PKA catalytic subunit (PKAc) is reduced in apoptotic PC12 pheochromocytoma cells. Apoptotic progress was inhibited by dibutyryl cyclic AMP (dbcAMP), an analog of cAMP. The rescue by dbcAMP was attributable to inhibition of the JNK but not of the p38 signaling pathway, due to the induction of PKA activity. JNK was present in immunocomplexes of PKAc, and PKAc phosphorylated JNK in vitro. Presence of p38 kinase, however, was not prominent in immunocomplexes of PKAc. Our data suggest that JNK is a target point of negative regulation by PKAc in the JNK signaling pathway.

• 대한의생명과학회지
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• 제13권4호
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• pp.305-311
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• 2007

cAMP-dependent protein kinase A (PKA) is the best-characterized protein kinases and has served as a model of the structure and regulation of cAMP-binding protein as well as of protein kinases. To determine the function of PKA in development, we employed the yeast two-hybrid system to screen for catalytic subunit of PKA $(C\alpha)$ interacting partners in a cDNA library from mouse embryo. A Testis-brain RNA-binding protein (TB-RBP), specifically bound to $C\alpha$. This interaction was verified by several biochemical analysis. Our findings indicate that $C\alpha$ can modulate nucleic acid binding proteins of TB-RBP and provide insights into the diverse role of PKA.

• Journal of Applied Biological Chemistry
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• 제48권3호
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• pp.120-126
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• 2005

Endothelial nitric oxide synthase (eNOS) plays a critical role in vascular biology and pathophysiology. Its activity is regulated by multiple mechanisms such as calcium/calmodulin, protein-protein interactions, sub-cellular locations and phosphorylation at various sites. Phosphorylation of eNOS-Ser1177 (based on mouse sequence) has been identified as an important mechanism of eNOS activation. However, signaling pathway leading to it phosphorylation remains controversial. The regulation of eNOS-Ser1177 phosphorylation by Src and protein kinase A (PKA) was investigated in the present study using cultured mouse aorta endothelial cells. Expression of a constitutively active Src mutant in the cells enhanced phosphorylation of eNOS and protein kinase B (Akt). The Src-stimulated phosphorylation was not attenuated by the expression of a dominant negative PKA regulatory subunit. Neither activation nor inhibition of PKA activity had any significant effect on tyrosine phosphorylation of activation or inactivation site in Src. Based on the results of this study, it is suggested that Src/Akt pathway and PKA signaling may regulate eNOS phosphorylation independently. The existence of multiple mechanisms for eNOS phosphorylation may guarantee endothelial nitric oxide production in various cellular contexts which is essential for maintenance of vascular health.

• Journal of Microbiology and Biotechnology
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• 제26권5호
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• pp.967-974
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• 2016

Zearalenone (ZEA) is an estrogenic mycotoxin that is produced by several Fusarium species, including Fusarium graminearum. One of the ZEA biosynthetic genes, ZEB2, encodes two isoforms of Zeb2 by alternative transcription, forming an activator (Zeb2L-Zeb2L homooligomer) and an inhibitor (Zeb2L-Zeb2S heterodimer) that directly regulate the ZEA biosynthetic genes in F. graminearum. Cyclic AMP-dependent protein kinase A (PKA) signaling regulates secondary metabolic processes in several filamentous fungi. In this study, we investigated the effects of the PKA signaling pathway on ZEA biosynthesis. Through functional analyses of PKA catalytic and regulatory subunits (CPKs and PKR), we found that the PKA pathway negatively regulates ZEA production. Genetic and biochemical evidence further demonstrated that the PKA pathway specifically represses ZEB2L transcription and also takes part in posttranscriptional regulation of ZEB2L during ZEA production. Our findings reveal the intriguing mechanism that the PKA pathway regulates secondary metabolite production by reprograming alternative transcription.

• Journal of Applied Biological Chemistry
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• 제64권4호
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• pp.323-331
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• 2021

Lincomycin is a lincosamide antibiotic isolated from the actinomycete Streptomyces lincolnensis. Moreover, it has been found to be effective against infections caused by Staphylococcus, Streptococcus, and Bacteroides fragillis. To identify the melanin-inducing properties of lincomycin, we used B16F10 melanoma cells in this study. The melanin content and intracellular tyrosinase activity in the cells were increased by lincomycin, without any cytotoxicity. Western blot analysis indicated that the protein expressions of tyrosinase, tyrosinase related protein 1 (TRP1) and TRP2 increased after lincomycin treatment. In addition, lincomycin enhanced the expression of master transcription regulator of melanogenesis, a microphthalmia-associated transcription factor (MITF). Lincomycin also increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and decreased the AKT phosphorylation. Moreover, the activation of tyrosinase activity by lincomycin was inhibited by the treatment with SB203580, which is p38 inhibitor. Furthermore, we also found that lincomycin-induced tyrosinase expression was reduced by H-89, a specific protein kinase A (PKA) inhibitor. These results indicate that lincomycin stimulate melanogenesis via MITF activation via p38 MAPK, AKT, and PKA signal pathways. Thus, lincomycin can potentially be used for treatment of hypopigmentation disorders.

• 대한의생명과학회지
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• 제14권2호
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• pp.77-81
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• 2008

Testis brain RNA-binding protein (TB-RBP) is a DNA/RNA binding protein. TB-RBP is mainly expressed in testis and brain and highly conserved protein with several functions, including chromosomal translocations, DNA repair, mitotic cell division, and mRNA transport, stabilization, and storage. In our previous study, we identified TB-RBP as an interacting partner for the catalytic subunit $(C{\alpha})$ of protein kinase A(PKA) and verified their interaction with several biochemical analyses. Here, we confirmed interaction between $C{\alpha}$. and TB-RBP in mammalian cells and determined the effect of $C{\alpha}$. on the function of TB-RBP. The activation of $C{\alpha}$. increased the TB-RBP function as a DNA-binding protein. These results suggest that the function of TB-RBP can be modulated by PKA and provide insights into the diverse role of PKA.

• BMB Reports
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• 제51권5호
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• pp.249-254
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• 2018

Natural pterocarpan Medicarpin (Med) has been shown to have various beneficial biological roles, including inhibition of osteoclastogenesis, stimulation of bone regeneration and induction of apoptosis. However, the effect of the Med on lipolysis in adipocytes has not been reported. Here, we show the effect of Med on lipolysis in different mouse adipocytes and elucidate the underlying mechanism. We observed that Med treatment promoted release of glycerol in the media. Differentiated mouse brown adipose tissue cells were treated with Med. RNA-Seq analysis was performed to elucidate the effect of med and subsequently was confirmed by qRT-PCR and western blotting analyses. Med treatment increased both protein and gene expression levels of hormone-sensitive lipase (Hsl) and adipose triglyceride lipase (Atgl), which are two critical enzymes necessary for lipolysis. Mechanistic study showed that Med activates Protein Kinase A (PKA) and phosphorylates Hsl at PKA target position at $Serine^{660}$. Silencing of PKA gene by short interfering RNA attenuated the Med-induced increase in glycerol release and Hsl phosphorylation. The results unveil that Med boosts lipolysis via a PKA-dependent pathway in adipocytes and may provide a possible avenue of further research of Med mediated reduction of body fat.

• Molecules and Cells
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• 제28권1호
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• pp.67-71
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• 2009

Estrogen receptor ${\alpha}$ ($ER{\alpha}$) mediates the mitogenic effects of estrogen. $ER{\alpha}$ signaling regulates the normal growth and differentiation of mammary tissue, but uncontrolled $ER{\alpha}$ activation increases the risk to breast cancer. Estrogen binding induces ligand-dependent $ER{\alpha}$ activation, thereby facilitating $ER{\alpha}$ dimerization, promoter binding and coactivator recruitment. $ER{\alpha}$ can also be activated in a ligand-independent manner by many signaling pathways, including protein kinase A (PKA) signaling. However, in several $ER{\alpha}$-positive breast cancer cells, PKA inhibits estrogen-dependent cell growth. FoxH1 represses the transcriptional activities of estrogen receptors and androgen receptors (AR). Interestingly, FoxH1 has been found to inhibit the PKA-induced and ligand-induced activation of AR. In the present study, we examined the effects of PKA activation on the ability of FoxH1 to represses $ER{\alpha}$ transcriptional activity. We found that PKA increases the protein stability of FoxH1, and that FoxH1 inhibits PKA-induced and estradiol-induced activation of an estrogen response element (ERE). Furthermore, in MCF7 cells, FoxH1 knockdown increased the PKA-induced and estradiol-induced activation of the ERE. These results suggest that PKA can negatively regulate $ER{\alpha}$, at least in part, through FoxH1.

• BMB Reports
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• 제33권2호
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• pp.103-111
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• 2000

Phosphorylation of the eukaryotic elongation factor 2 (eEF-2) blocks the elongation step of translation and stops overall protein synthesis. Although the overall rate of protein synthesis in mitosis reduces to 20% of that in S phase, it is unclear how the protein translation procedure is regulated during the cell cycle, especially in the stage of peptide elongation. To delineate the regulation of the elongation step through eEF-2 function, the changes in phosphorylation of eEF-2, and in activity of corresponding $Ca^{2+}$/calmodulin (CaM)-dependent protein kinase III (CaMK-III) during the cell cycle of NIH 3T3 cells, were determined. The in vivo level of phosphorylated eEF-2 showed an 80% and 40% increase in the cells arrested at G1 and M, respectively. The activity of CaMK-III also changed in a similar pattern, more than a 2-fold increase when arrested at G1 and M. The activity change of the kinase during one turn of the cell cycle also demonstrated the activation at G1 and M phases. The activity change of cAMP-dependent protein kinase (PKA) was reciprocal to that of CaMK-III. These results indicated: (1) the activity of CaMK-III was cell cycle-dependent and (2) the level of eEF-2 phosphorylation followed the kinase activity change. Therefore, the elongation step of protein synthesis might be cell cycle dependently regulated.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.29-29
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• 2003

Compartmentation of intracellular signaling pathways serves as an important mechanism conferring the specificity of G protein-coupled receptor (GPCR) signaling. In the heart, stimulation of $\beta$$_2$-adrenoceptor ($\beta$$_2$-AR), a prototypical GPCR, activates a tightly localized protein kinase A (PKA) signaling, which regulates substrates at cell surface membranes, bypassing cytosolic target proteins (eg, phospholamban). Although a concurrent activation of $\beta$$_2$-AR-coupled $G_{i}$ proteins has been implicated in the functional compartmentation of PKA signaling, the exact mechanism underlying the restriction of the $\beta$$_2$-AR-PKA pathway remains unclear. In the present study, we demonstrate that phosphatidylinositol 3-kinase (PI3K) plays an essential role in confining the $\beta$$_2$-AR-PKA signaling. Inhibition of PI3K with LY294002 or wortmannin enables $\beta$$_2$-AR-PKA signaling to reach intracellular substrates, as manifested by a robust increase in phosphorylation of phospholamban, and markedly enhances the receptor-mediated positive contractile and relaxant responses in cardiac myocytes. These potentiating effects of PI3K inhibitors are not accompanied by an increase in $\beta$$_2$-AR-induced cAMP formation. Blocking $G_{i}$ or $G_{$\square$$\square$}$ signaling with pertussis toxin or $\beta$ARK-ct, a peptide inhibitor of $G_{$\square$$\square$}$, completely prevents the potentiating effects induced by PI3K inhibition, indicating that the pathway responsible for the functional compartmentation of $\beta$$_2$-AR-PKA siglaling sequentially involves $G_{i}$, $G_{$\square$$\square$}$, and PI3K. Thus, PI3K constitutes a key downstream event of $\beta$$_2$-AR- $G_{i}$ signaling, which confines and negates the concurrent $\beta$$_2$-AR/Gs-mediated PKA signaling.gnaling.