• BMB Reports
• /
• v.44 no.9
• /
• pp.572-577
• /
• 2011

Elevated phospholipase D (PLD) expression prevents cell cycle arrest and apoptosis. However, the roles of PLD isoforms in cell proliferation and apoptosis are incompletely understood. Here, we investigated the physiological significance of the interaction between PLD2 and protein kinase CKII (CKII) in HCT116 human colorectal carcinoma cells. PLD2 interacted with the CKII${\beta}$ subunit in HCT116 cells. The C-terminal domain (residues 578-933) of PLD2 and the N-terminal domain of CKII${\beta}$ were necessary for interaction between the two proteins. PLD2 relocalized CKII${\beta}$ to the plasma membrane area. Overexpression of PLD2 reduced CKII${\beta}$ protein level, whereas knockdown of PLD2 led to an increase in CKII${\beta}$ expression. PLD2-induced CKII${\beta}$ reduction was mediated by ubiquitin-dependent degradation. The C-terminal domain of PLD2 was sufficient for CKII${\beta}$ degradation as the catalytic activity of PLD2 was not required. Taken together, the results indicate that the C-terminal domain of PLD2 can regulate CKII by accelerating CKII${\beta}$ degradation in HCT116 cells.

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

• Bulletin of the Korean Chemical Society
• /
• v.34 no.4
• /
• pp.1165-1169
• /
• 2013

To elucidate the role of PKG isoforms in transcriptional control of cyclin D1, we employed a series of expression vectors of PKG $1{\alpha}$ and PKG $1{\beta}$ which encode HA-tagged wild type and constitutively active (SD and ${\Delta}N$) mutants. Our present study demonstrates that both the constitutively active mutants of PKG $1{\beta}$ downregulate the transcription of cyclin D1 when transiently transfected in NIH3T3 cells, whereas PKG $1{\alpha}$ mutants show weak inhibition. We further studied the transcriptional regulators of cyclin D1, such as, c-fos, NF-${\kappa}B$, and CRE by using the luciferase reporter assay. Constitutively active mutants of PKG $1{\beta}$ showed marked transcriptional downregulation of c-fos in NIH3T3 cells, whereas PKG $1{\alpha}$ downregulated c-fos to a lesser extent. We also found that the constitutively active mutants of PKG negatively regulated the activation of NF-${\kappa}B$ and CRE, suggesting their involvement in the regulation of cyclin D1.

• Journal of Veterinary Science
• /
• v.24 no.2
• /
• pp.26.1-26.11
• /
• 2023

Background: Angiotensin-converting enzyme inhibitor (ACEi) inhibits the catalysis of angiotensin I to angiotensin II and the degradation of substance P (SP) and bradykinin (BK). While the possible relationship between ACEi and SP in nociceptive mice was recently suggested, the effect of ACEi on signal transduction in astrocytes remains unclear. Objectives: This study examined whether ACE inhibition with captopril or enalapril modulates the levels of SP and BK in primary cultured astrocytes and whether this change modulates PKC isoforms (PKCα, PKCβI, and PKCε) expression in cultured astrocytes. Methods: Immunocytochemistry and Western blot analysis were performed to examine the changes in the levels of SP and BK and the expression of the PKC isoforms in primary cultured astrocytes, respectively. Results: The treatment of captopril or enalapril increased the immunoreactivity of SP and BK significantly in glial fibrillary acidic protein-positive cultured astrocytes. These increases were suppressed by a pretreatment with an angiotensin-converting enzyme. In addition, treatment with captopril increased the expression of the PKCβI isoform in cultured astrocytes, while there were no changes in the expression of the PKCα and PKCε isoforms after the captopril treatment. The captopril-induced increased expression of the PKCβI isoform was inhibited by a pretreatment with the neurokinin-1 receptor antagonist, L-733,060, the BK B1 receptor antagonist, R 715, or the BK B₂ receptor antagonist, HOE 140. Conclusions: These results suggest that ACE inhibition with captopril or enalapril increases the levels of SP and BK in cultured astrocytes and that the activation of SP and BK receptors mediates the captopril-induced increase in the expression of the PKCβI isoform.

• Biomedical Science Letters
• /
• v.16 no.2
• /
• pp.119-122
• /
• 2010

Interleukin-1${\beta}$ $(IL-1{\beta})$ is one of the key proinflammatory cytokines and it plays an important role for the antimycobacterial host defense mechanisms. In this study, we examined Mycobacterium tuberculosis (MTB)-stimulated induction of IL-1${\beta}$ and evaluated the associated signal transduction pathways. In PMA-differentiated THP-1 cells, MTB infection increased mRNA expression of IL-$1{\beta}$ in a dose-dependent manner. The expression of IL-1${\beta}$ mRNA began to be induced at 1.5 h after infection, and induced expression of IL-1${\beta}$ was retained for 48 h after MTB infection. The increase in expression of IL-1${\beta}$ caused by MTB was reduced in cells treated with Ro-31-8425 (an inhibitor of PK$C{\alpha}$, ${\beta}I$, ${\beta}II$, ${\gamma}$, ${\varepsilon}$) or PD98059 (an inhibitor of MEK1), meanwhile, pre-treatment with $G\ddot{o}6976$ (an inhibitor of $Ca^{2+}$ dependent PK$C{\alpha}$ and PK$C{\beta}I$) or Rottlerin (an inhibitor of PK$C{\delta}$) has no effect on MTB-induced expression of $IL-1{\beta}$ mRNA. These results show that the expression of $IL-1{\beta}$ mRNA caused by MTB may be mediated via MEK1 and PKC isoforms including PK$C{\beta}II$, $PKC{\gamma}$, or $PKC{\varepsilon}$. Further studies are required to determine whether other PKC isoforms $(PKC {\eta},\;{\theta},\;{\varepsilon},\;and\;{\lambda}/{\iota})$, except $PKC{\delta}$, $PKC{\alpha}$, and $PKC{\beta}I$, are also involved in $IL-1{\beta}$ mRNA expression after mycobacterial infection.

• Journal of Yeungnam Medical Science
• /
• v.18 no.1
• /
• pp.13-29
• /
• 2001

The enzyme activities of creatine kinase (CK), its isoenzyme MB (CK-MB) and of lactate dehydrogenase isoenzyme 1 (LD-1) have been used for years in diagnosing patients with chest pain in order to differentiate patients with acute myocardial infarction (AMI) from non-AMI patients. These methods are easy to perform as automated analyses, but they are not specific for cardiac muscle damage. During the early 90's the situation changed. First, creatine kinase ME mass (CK-MB mass) replaced the measurement of CK-MB activity. Subsequently cardiac-specific proteins, troponin T (cTnT) and troponin I (cTnI) appeared and displacing LD-1 analysis. However, troponin concentrations in blood increase only from four to six hours after onset of chest pain. Therefore a rapid marker such as myoglobin, fatty acid binding protein or glycogen phosphorylase BB could be used in early diagnosis of AMI. On the other hand, CK-MB isoforms alone may also be useful in rapid diagnosis of cardiac muscle damage. Myoglobin, CK-MB mass, cTnT and cTnI are nowadays widely used in diagnosing patients with acute chest pain. Myoglobin is not cardiac-specific and therefore requires supplementation with some other analyses such as troponins to support the myoglobin value. Troponins are very highly cardiac-specific. Only the sera of some patients with severe renal failure, which requires hemodialysis, have elevated cTnT and/or cTnI without there being any evidence of cardiac damage. The latest studies have shown that elevated troponin levels in sera of hemodialysis patients point to an increased risk of future cardiac events in a similar manner to the elevated troponin values in sera of patients with unstable angina pectoris. In addition, the bedside tests for cTnT and cTnI alone- or together with myoglobin and CK-ME mass can be used instead of quantitative analyses in the diagnosis of patients with chest pain. These rapid tests are easy to perform and they do not require expensive instrumentation. For the diagnosis of patient with chest pain, routinely myoglobin and CK-ME mass measurements should be performed whenever they are requested (24 h/day) and cTnT or cTnI on admission to the hospital and then 4-6 and 12 hours later and maintained less than 10% in imprecision.

• The Journal of Korean Physical Therapy
• /
• v.20 no.1
• /
• pp.57-65
• /
• 2008

Purpose: Heat shock proteins (HSPs) are a group of proteins that are activated when cells are exposed to a variety of environmental stresses, such as infection, inflammation, exposure to toxins, starvation, hypoxia, brain injury, or water deprivation. The activation of HSPs by environmental stress plays a key role in signal transduction, including cytoprotection, molecular chaperone, anti-apoptotic effect, and anti-aging effects. However, the precise mechanism for the action of small HSPs, such as HSP27 and mitogen-activated protein kinases (MAPKs: extracellular-regulated protein kinase 1/2 (ERK1/2), p38MAPK, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), is not completely understood, particularly in application of cell stimulators including platelet-derived growth factor (PDGF), angiotensin II (AngII), tumor necrosis factor $\alpha$ (TNF$\alpha$), and $H_2O_2$. This study examined the relationship between stimulators-induced enzymatic activity of HSP27 and MAPKs from rat smooth and skeletal muscles. Methods: 2-dimensional electrophoresis (2DE) and matrix assisted laser desorption ionizationtime-of-flight/time-of-flight (MALDI-TOF/TOF) analysis were used to identify HSP27 from the intact vascular smooth and skeletal muscles. Three isoforms of HSP27 were detected on silver-stained gels of the whole protein extracts from the rat aortic smooth and skeletal muscle strips. Results: The expression of PDGF, AngII, TNF$\alpha$, and $H_2O_2$-induced activation of HSP27, p38MAPK, ERK1/2, and SAPK/JNK was higher in the smooth muscle cells than the control. SB203580 (30${\mu}$M), a p38MAPK inhibitor, increased the level of HSP27 phosphorylation induced by stimulators in smooth muscle cells. Furthermore, the age-related and starvation-induced activation of HSP27 was higher in skeletal muscle cells (L6 myoblast cell lines) and muscle strips than the control. Conclusion: These results suggest, in part, that the activity of HSP27 and MAPKs affect stressors, such as PDGF, AngII, TNF$\alpha$, $H_2O_2$, and starvation in rat smooth and skeletal muscles. However, more systemic research will be needed into physical therapy, including thermotherapy, electrotherapy, radiotherapy and others.

• Genomics & Informatics
• /
• v.10 no.3
• /
• pp.167-174
• /
• 2012

AKT is a signal transduction protein that plays a central role in the tumorigenesis. There are 3 mammalian isoforms of this serine/threonine protein kinase-AKT1, AKT2, and AKT3-showing a broad tissue distribution. We first discovered 2 novel polymorphisms (AKT2 -9826 C/G and AKT3 -811 A/G), and we confirmed 6 known polymorphisms (AKT2 -9473 C/T, AKT2 -9151 C/T, AKT2 -9025 C/T, AKT2 -8618G/A, AKT3 -675 A/-, and AKT3 -244 C/T) of the AKT2 and AKT3 promoter region in 24 blood samples of Korean lung cancer patients using direct sequencing. To evaluate the role of AKT2 and AKT3 polymorphisms in the risk of Korean lung cancer, genotypes of the AKT2 and AKT3 polymorphisms (AKT2 -9826 C/G, AKT2 -9473 C/T, AKT2 -9151 C/T, AKT2 -9025 C/T, AKT2 -8618G/A, and AKT3 -675 A/-) were determined in 360 lung cancer patients and 360 normal controls. Statistical analyses revealed that the genotypes and haplotypes in the AKT2 and AKT3 promoter regions were not significantly associated with the risk of lung cancer in the Korean population. These results suggest that polymorphisms of the AKT2 and AKT3 promoter regions do not contribute to the genetic susceptibility to lung cancer in the Korean population.

• Clinical and Experimental Reproductive Medicine
• /
• v.38 no.2
• /
• pp.68-74
• /
• 2011

Objective: Previously, we found that oocyte specific homeobox (Obox) 4 plays significant role in completion of meiosis specifically at meiosis I-meiosis II (MI-MII) transition. The purpose of this study was to determine the mechanism of action of $Obox4$ in oocyte maturation by evaluating downstream signal networking. Methods: The $Obox4$ dsRNA was prepared by $in$ $vitro$ transcription and microinjected into the cytoplasm of germinal vesicle oocytes followed by $in$ $vitro$ maturation in the presence or absence of 0.2 mM 3-isobutyl-1-metyl-xanthine. Total RNA was extracted from 200 oocytes of each group using a PicoPure RNA isolation kit then amplified two-rounds. The probe hybridization and data analysis were used by Affymetrix Gene-Chip$^{(R)}$ Mouse Genome 430 2.0 array and GenPlex 3.0 (ISTECH, Korea) software, respectively. Results: Total 424 genes were up (n=80) and down (n=344) regulated after $Obox4$ RNA interference (RNAi). Genes mainly related to metabolic pathways and mitogen-activated protein kinase (MAPK) signaling pathway was changed. Among the protein kinase C (PKC) isoforms, PKC-alpha, beta, gamma were down-regulated and especially the MAPK signaling pathway PKC-gamma was dramatically decreased by $Obox4$ RNAi. In the cell cycle pathway, we evaluated the expression of genes involved in regulation of chromosome separation, and found that these genes were down-regulated. It may cause the aberrant chromosome segregation during MI-MII transition. Conclusion: From the results of this study, it is concluded that $Obox4$ is important upstream regulator of the PKC and anaphase-promoting complex action for maintaining intact germinal vesicle.

• BMB Reports
• /
• v.43 no.11
• /
• pp.738-743
• /
• 2010

The c-Jun $NH_2$-terminal kinase (JNK) signaling pathway participates in many physiological functions. In the current study we reported the cloning and characterization of five novel JNK2 transcript variants, which were designated as $JNK2\alpha3$, $JNK2\alpha4$, $JNK2\beta3$, $JNK2\gamma1$ and $JNK2\gamma2$, respectively. Among them, $JNK2\alpha4$ and $JNK2\gamma2$ are potential non-coding RNA because they contain pre-mature stop codons. Both $JNK2\alpha3$ and $JNK2\beta3$ contain an intact kinase domain, and both encode a protein product of 46 kDa, the same as those of $JNK2\alpha1$ and $JNK2\beta1$. $JNK2\gamma1$ contains a disrupted kinase domain and it showed a disable function. When over-expressed in mammalian cells, $JNK2\alpha3$ showed higher activity on AP-1 than that of $JNK2\beta3$ and $JNK2\gamma1$. Furthermore, $JNK2\alpha3$ and $JNK2\beta3$ showed different levels of substrate phosphorylation, although they both could promote the proliferation of 293T cells. Our results further demonstrate that JNK2 isoforms preferentially target different substrates and may regulate the expression of various target genes.