• 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.

• Microbiology and Biotechnology Letters
• /
• v.37 no.2
• /
• pp.105-109
• /
• 2009

Among signal transduction systems by protein phosphorylation Akt/PKB protein kinase which is one of serine/threonine kinases, is known to regulate the survival and death of the cell and glucose metabolism. Thus, Akt/PKB protein kinase has been used as one of the target proteins to find anti-cancer agents from natural products. In this study, human Akt/PKB protein kinase was expressed in Escherichia coli expression system for the mass production. Human Akt/PKB protein kinase expressed in E. coli formed inclusion body under the general condition. However, most of the expressed protein was solubilized under the culture temperature at $27^{\circ}C$ and 0.01-0.09 mM of IPTG for induction of the protein expression. The expressed protein was purified using $Ni^{2+}$-NTA agarose column and confirmed by using anti-Akt antibody. Subsequently, the purified human Akt/PKB protein kinase was activated by in vitro phosphorylation using cellular extract containing kinases. The activated protein was confirmed to phosphorylate the specific fluorescent peptide specially designed as the artificial substrate for Akt/PKB protein kinase.

• Journal of Life Science
• /
• v.22 no.4
• /
• pp.447-455
• /
• 2012

Akt plays an important role in a variety of cellular physiologies such as growth, proliferation, and differentiation. In skeletal muscle, Akt has been implicated in regulating regeneration, hypertrophy, and atrophy. In this study, the role of Akt has been examined during skeletal muscle differentiation. Culturing C2C12 myoblasts under low serum (1% horse serum) and high density converted cell morphology from a round shape to an elongated and multi-nucleated shape. Morphological changes were initiated from day 2 of differentiation. In addition, the expression of both myogenin G and myogenin D was elevated from day 2 of differentiation. Skeletal muscle differentiation was abolished by silencing Akt1 or Akt2, but was significantly enhanced by the over-expression of either Akt1 or Akt2. The activation of Akt was observed from day 2 of differentiation and disappeared after day 7. The expression of kruppel-like factor 4 was observed from day 6 of differentiation. Moreover, this expression was blocked in cells silencing either Akt1 or Akt2. In addition, the promoter activity of kruppel-like factor 4 was significantly reduced in cells silencing Akt1 or Akt2. These results suggest that Akt regulates skeletal muscle differentiation through the regulation of kruppel-like factor 4 expression.

• Journal of Life Science
• /
• v.22 no.1
• /
• pp.55-61
• /
• 2012

Akt is known to play an important role in cell proliferation and differentiation, and is also over-expressed in several types of cancer cells. In this study, we explored the anti-proliferative effects of selenium in HT-29 colon cancer cells, mediated through effects on Akt and COX-2. Selenium treatments at different concentrations and for different durations inhibited proliferation of HT-29 colon cancer cells and increased apoptotic cell death. Selenium treatment decreased Akt phosphorylation and COX-2 expression. Treatment with LY294002 (an Akt inhibitor) decreased proliferation of HT-29 cells, while a combined treatment with LY294002 and selenium resulted in even further decreases in cell proliferation. Inactivation of Akt by Akt siRNA treatment abolished these inhibitory effects on cell growth. COX-2 expression decreased in Akt transfected cells compared to non-transfected cells. These results suggest that selenium induced both anti-proliferative and apoptotic effects by inhibiting Akt phosphorylation and COX-2 expression. Selenium treatment also appeared to induce synergistic anti-proliferative effects by inhibition of Akt in HT-29 colon cancer cells.

• Journal of Life Science
• /
• v.17 no.2 s.82
• /
• pp.185-191
• /
• 2007

Akt/PKB plays pivotal roles in many physiological responses such as proliferation, differentiation, apoptosis, and angiogenesis. Here we show that tyrosine phosphorylation of Akt/PKB is essential for the subsequent phosphorylation at $Thr^{\308}$. Tyrosine phosphorylation of Akt/PKB was induced by stimulation of COS-7 cells with epidermal growth factor receptor (EGF) and its phosphorylation was significantly enhanced by constitutive targeting of Akt/PKB to the plasma membrane by myristoylation. Interestingly, incubation of affinity purified Myc-tagged Akt/PKB with purified EGF receptor resulted in tyrosine phosphorylation as well as $Ser^{\473}$ phosphorylation of Akt/PKB. In addition, tyrosine-phosphorylated Akt/PKB could directly associate with activated EGF receptor in vitro. Finally, alanine mutation at putative tyrosine phosphorylation site $(Tyr^{\326})$ abolished EGF induced $Thr^{\308}$ phosphorylation of wild type as well as constitutively active form of Akt/PKB. Given these results we suggest here that direct tyrosine phosphorylation of Akt/PKB by EGF receptor could be another mechanism of EGF-induced control of many physiological responses.

• BMB Reports
• /
• v.45 no.9
• /
• pp.521-525
• /
• 2012

In addition to its pivotal role in neuronal survival, PI3K/Akt signaling is integral to neuronal differentiation and neurite outgrowth. However, the exact role of Akt in neuronal differentiation is still controversial. Here, we found that nuclear expression of CA-Akt resulted in unusual rapid neurite outgrowth and overexpression of KD-Akt caused multiple dendrite growth without specific axon elongation. Moreover, microarray data revealed that the expression of FOXQ1 expression was about 10-fold higher in cells with nuclear, active Akt than in control cells. Quantitative real-time PCR analysis showed that mRNA levels were upregulated in NLS-CA-Akt cells as compared to KD or EV cells. Furthermore, our FACS analysis demonstrated that overexpression of NLS-CA-Akt accumulate cells in the G1 phase within 24 h, fitting with the rapid sprouting of neuritis. Thus, our data implied that at least in this early time frame, the overexpression of nuclear, active Akt forced cells into neurite development through probably FOXQ1regulation.

• YAKHAK HOEJI
• /
• v.48 no.2
• /
• pp.105-110
• /
• 2004

Akt (or Protein Kinase B; PKB) is a serine/threonine kinase and is activated by phosphoinositide 3-kinase (PI3K) pathway. Recent evidence indicates that the abnormal activities or expression of Akt is closely associated with cancer, diabetes and neuro-degenerative diseases. These findings mean that Akt is likely to be a new therapeutic target for the treatment of disease. Here, we screened the effects of dithiolo-dithione derivatives such as SWU-20004, SWU-20009 and SWU-20025 on Akt activities. Among these compounds, only SWU-20009 (2-Thioxo-[1,3]dithiolo[4,5- $\beta$][1,4]dithiine-5,6-dicarboxylic acid dimethyl ester) inhibited the growth of KATOIII cell at micromolar range of concentration. Further investigation also revealed that SWU-20009 inhibited cellular Akt activity and induced apoptotic cell death.

• The Korean Journal of Physiology and Pharmacology
• /
• v.21 no.5
• /
• pp.547-554
• /
• 2017

Previous studies have demonstrated the role of hydroquinone (HQ), a hydroxylated benzene metabolite, in modulating various immune responses; however, its role in macrophage-mediated inflammatory responses is not fully understood. In this study, the role of HQ in inflammatory responses and the underlying molecular mechanism were explored in macrophages. HQ down-regulated the expression of interferon $(IFN)-{\beta}$ mRNA in LPS-stimulated RAW264.7 cells without any cytotoxicity and suppressed interferon regulatory factor (IRF)-3-mediated luciferase activity induced by TIR-domain-containing adapter-inducing interferon-${\beta}$ (TRIF) and TANK-binding kinase 1 (TBK1). A mechanism study revealed that HQ inhibited IRF-3 phosphorylation induced by lipopolysaccharide (LPS), TRIF, and AKT by suppressing phosphorylation of AKT, an upstream kinase of the IRF-3 signaling pathway. IRF-3 phosphorylation is highly induced by wild-type AKT and poorly induced by an AKT mutant, AKT C310A, which is mutated at an inhibitory target site of HQ. We also showed that HQ inhibited IRF-3 phosphorylation by targeting all three AKT isoforms (AKT1, AKT2, and AKT3) in RAW264.7 cells and suppressed IRF-3-mediated luciferase activities induced by AKT in HEK293 cells. Taken together, these results strongly suggest that HQ inhibits the production of a type I IFN, $IFN-{\beta}$, by targeting AKTs in the IRF-3 signaling pathway during macrophage-mediated inflammation.

• Biomedical Science Letters
• /
• v.19 no.3
• /
• pp.266-269
• /
• 2013

Hox genes encode transcription factors important for anterior-posterior body patterning at early stages of embryonic development. However, the precise mechanisms by which signal pathways are stimulated to regulate Hox gene expression are not clear. In the previous study, protein kinase B alpha (Akt1) has been identified as a putative upstream regulator of Hox genes, and Akt1 has shown to regulate Gcn5, a prototypical histone acetyltransferase (HAT), in a negative way in mouse embryonic fibroblast (MEF) cells. Since the activity of HAT such as the CBP/p300, and PCAF (a Gcn5 homolog), was down-regulated by Akt through a phosphorylation at the Akt consensus substrate motif (RXRXXS/T), the amino acid sequence of Gcn5 protein was analyzed. Mouse Gcn5 contains an Akt consensus substrate motif as RQRSQS sequence while human Gcn5 does not have it. In order to see whether Akt1 directly binds to Gcn5, immunoprecipitation with anti-Akt1 antibody was carried out in wild-type (WT) mouse embryonic fibroblast (MEF) cells, and then western blot analysis was performed with anti-Akt1 and anti-Gcn5 antibodies. Gcn5 protein was detected in the Akt1 immunoprecipitated samples of MEFs. This result demonstrates that Akt1 directly binds to Gcn5, which might have contributed the down regulation of the 5' Hoxc gene expressions in wild type MEF cells.

• Korean Journal of Head & Neck Oncology
• /
• v.21 no.2
• /
• pp.158-164
• /
• 2005

Purpose: The serine/threonine kinase Akt was described to inhibit apoptosis in cancer. This study was to examine the effect of Gleevec on head and neck squamous cell carcinoma(HNSCC) through the mechanism of Akt. Experimental Design: Gleevec was introduced into the HNSCC cell lines UMSCC10B, HN12 and HN30 in a range of concentrations. Cell viability was assessed by clonogenic survival analysis. Targets of Gleevec(PDGFR, c-Kit, and c-Abl) were evaluated by Western blot. HNSCC tissue samples were stained for PDGFR, c-Kit and phosphorylated Akt. Akt phosphorylation following Gleevec treatment was assessed using Western blot. Akt siRNA was used to as the positive control. Results: Colony forming efficiency decreased with an increase in concentration of Gleevec. Expressions of PDGFR, c-Kit, and c-Abl were observed in HNSCC cells. Immunohistochemistry confirmed high expression of PDGFR, c-Kit, and p-Akt in human HNSCC tissues. Akt kinase activity was significantly inhibited with increasing concentration of Gleevec in HNSCC cells, and near complete dephosphorylation of Akt was observed at $6{\mu}M$ of Gleevec in the UMSCC10B and HN30 cell lines. Conclusions: Gleevec at clinically comparable concentrations caused a dose dependant decrease in HNSCC survival. The decreased cell survival was related to the inhibition of Akt kinase activity and dephosphorylation of Akt. Akt signaling pathway may be a relevant target for Gleevec in treating HNSCC.