• Title/Summary/Keyword: PDK-1

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PKCθ-Mediated PDK1 Phosphorylation Enhances T Cell Activation by Increasing PDK1 Stability

  • Kang, Jung-Ah;Choi, Hyunwoo;Yang, Taewoo;Cho, Steve K.;Park, Zee-Yong;Park, Sung-Gyoo
    • Molecules and Cells
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    • v.40 no.1
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    • pp.37-44
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    • 2017
  • PDK1 is essential for T cell receptor (TCR)-mediated activation of $NF-{\kappa}B$, and PDK1-induced phosphorylation of $PKC{\theta}$ is important for TCR-induced $NF-{\kappa}B$ activation. However, inverse regulation of PDK1 by $PKC{\theta}$ during T cell activation has not been investigated. In this study, we found that $PKC{\theta}$ is involved in human PDK1 phosphorylation and that its kinase activity is crucial for human PDK1 phosphorylation. Mass spectrometry analysis of wild-type $PKC{\theta}$ or of kinase-inactive form of $PKC{\theta}$ revealed that $PKC{\theta}$ induced phosphorylation of human PDK1 at Ser-64. This $PKC{\theta}$-induced PDK1 phosphorylation positively regulated T cell activation and TCR-induced $NF-{\kappa}B$ activation. Moreover, phosphorylation of human PDK1 at Ser-64 increased the stability of human PDK1 protein. These results suggest that Ser-64 is an important phosphorylation site that is part of a positive feedback loop for human PDK1-$PKC{\theta}$-mediated T cell activation.

PI3-Kinase and PDK-1 Regulate HDAC1-mediated Transcriptional Repression of Transcription Factor NF-κB

  • Choi, Yong Seok;Jeong, Sunjoo
    • Molecules and Cells
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    • v.20 no.2
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    • pp.241-246
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    • 2005
  • PDK-1 activates PI3-kinase/Akt signaling and regulates fundamental cellular functions, such as growth and survival. NF-${\kappa}B$ is involved in the induction of a variety of cellular genes affecting immunity, inflammation and the resistance to apoptosis induced by some anti-cancer drugs. Even though the crucial involvement of the PI3-kinase/Akt pathway in the anti-apoptotic activation of NF-${\kappa}B$ is well known, the exact role of PDK-1 as well as PI3-kinase/Akt in NF-vactivation is not understood. Here we demonstrate that PDK-1 plays a pivotal role in transcriptional activation of NF-${\kappa}B$ by dissociating the transcriptional co-repressor HDAC1 from the p65 subunit of NF-${\kappa}B$. The association of CBP with p65 was not directly modulated by PDK-1 or by PI3-kinase. Etoposide activated NF-${\kappa}B$ through PI3-kinase/Akt, and the transcription activation domain (TAD) of p65 was further activated by wild-type PDK-1. Overexpression of a dominant negative PDK-1 mutant decreased etoposide-induced NF-${\kappa}B$ transcription and further down-regulated the ectopic HDAC1-mediated decrease in NF-${\kappa}B$ transcriptional activity. Thus activation of PDK-1 relieves the HDAC1-mediated repression of NF-${\kappa}B$ that may be related to basal as well as activated transcription by NF-${\kappa}B$. This effect may also explain the role of the PI3-kinase/PDK-1 pathway in the anti-apoptotic function of NF-${\kappa}B$ associated with the chemoresistance of cancer cells.

Silencing of PDK1 Gene Expression by RNA Interference Suppresses Growth of Esophageal Cancer

  • Yu, Jing;Chen, Kui-Sheng;Li, Ya-Nan;Yang, Juan;Zhao, Lu
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.8
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    • pp.4147-4151
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    • 2012
  • The current study was conducted to explore the inhibitory effects of a small interfering RNA (siRNA) on 3-phosphoinositide-dependent protein kinase 1 (PDK1) expression in esophageal cancer 9706 (EC9706) cells and the influence on their biological behavior. After transfection of a synthesized PDK1 siRNA, PDK1 mRNA and protein expression and the phosphorylation level of the downstream Akt protein were assessed using RT-PCR and Western blot analysis. Proliferation, apoptosis, cell invasion and in vivo tumor formation capacity were also investigated using MTT, flow cytometry, Transwell invasion trials, and nude mouse tumor transplantion, respectively. PDK1 siRNA effectively suppressed PDK1 mRNA and protein expression, and down-regulated the phosphorylation level of the Akt protein in the EC9706 cells (P < 0.05). It also inhibited cell proliferation and invasion, and promoted apoptosis; such effects were particularly obvious at 48 h and 72 h after transfection (P < 0.05). Growth of transplanted tumors was inhibited in nude mice, with decreased PDK1 expression in tumor tissues. PDK1 may be closely correlated with proliferation, apoptosis and invasion of esophageal cancer cells and thus may serve as an effective target for gene therapy.

Serine 389 phosphorylation of 3-phosphoinositide-dependent kinase 1 by UNC-51-like kinase 1 affects its ability to regulate Akt and p70 S6kinase

  • Kim, Kidae;Park, Sung Goo;Park, Byoung Chul;Kim, Jeong-Hoon;Kim, Sunhong
    • BMB Reports
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    • v.53 no.7
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    • pp.373-378
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    • 2020
  • Phosphorylation of the signaling component by protein kinase often leads to a kinase cascade or feedback loop. 3-Phosphoinositide-dependent kinase 1 (PDK1) signaling pathway diverges into various kinases including Akt and p70 S6 kinase (p70S6k). However, the PDK1 feedback mechanism remains elusive. Here, we demonstrated that UNC-51-like kinase (ULK1), an autophagy initiator kinase downstream of mechanistic target of rapamycin (mTOR), directly phosphorylated PDK1 on serine 389 at the linker region. Furthermore, our data showed that this phosphorylation affected the kinase activity of PDK1 toward downstream substrates. These results suggest a possible negative feedback loop between PDK1 and ULK1.

Drug evaluation based on phosphomimetic PDHA1 reveals the complexity of activity-related cell death in A549 non-small cell lung cancer cells

  • Jin, Ling;Cho, Minkyoung;Kim, Bo-Sung;Han, Jung Ho;Park, Sungmi;Lee, In-Kyu;Ryu, Dongryeol;Kim, Jae Ho;Bae, Sung-Jin;Ha, Ki-Tae
    • BMB Reports
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    • v.54 no.11
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    • pp.563-568
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    • 2021
  • Cancer cells predominantly generate energy via glycolysis, even in the presence of oxygen, to support abnormal cell proliferation. Suppression of PDHA1 by PDK1 prevents the conversion of cytoplasmic pyruvate into Acetyl-CoA. Several PDK inhibitors have been identified, but their clinical applications have not been successful for unclear reasons. In this study, endogenous PDHA1 in A549 cells was silenced by the CRISPR/Cas9 system, and PDHA1WT and PDHA13SD were transduced. Since PDHA13SD cannot be phosphorylated by PDKs, it was used to evaluate the specific activity of PDK inhibitors. This study highlights that PDHA1WT and PDHA13SD A549 cells can be used as a cell-based PDK inhibitor-distinction system to examine the relationship between PDH activity and cell death by established PDK inhibitors. Leelamine, huzhangoside A and otobaphenol induced PDH activity-dependent apoptosis, whereas AZD7545, VER-246608 and DCA effectively enhanced PDHA1 activity but little toxic to cancer cells. Furthermore, the activity of phosphomimetic PDHA1 revealed the complexity of its regulation, which requires further in-depth investigation.

Pyruvate Dehydrogenase Kinase Protects Dopaminergic Neurons from Oxidative Stress in Drosophila DJ-1 Null Mutants

  • Lee, Yoonjeong;Kim, Jaehyeon;Kim, Hyunjin;Han, Ji Eun;Kim, Sohee;Kang, Kyong-hwa;Kim, Donghoon;Kim, Jong-Min;Koh, Hyongjong
    • Molecules and Cells
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    • v.45 no.7
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    • pp.454-464
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    • 2022
  • DJ-1 is one of the causative genes of early-onset familial Parkinson's disease (PD). As a result, DJ-1 influences the pathogenesis of sporadic PD. DJ-1 has various physiological functions that converge to control the levels of intracellular reactive oxygen species (ROS). Based on genetic analyses that sought to investigate novel antioxidant DJ-1 downstream genes, pyruvate dehydrogenase (PDH) kinase (PDK) was demonstrated to increase survival rates and decrease dopaminergic (DA) neuron loss in DJ-1 mutant flies under oxidative stress. PDK phosphorylates and inhibits the PDH complex (PDC), subsequently downregulating glucose metabolism in the mitochondria, which is a major source of intracellular ROS. A loss-of-function mutation in PDK was not found to have a significant effect on fly development and reproduction, but severely ameliorated oxidative stress resistance. Thus, PDK plays a critical role in the protection against oxidative stress. Loss of PDH phosphatase (PDP), which dephosphorylates and activates PDH, was also shown to protect DJ-1 mutants from oxidative stress, ultimately supporting our findings. Further genetic analyses suggested that DJ-1 controls PDK expression through hypoxia-inducible factor 1 (HIF-1), a transcriptional regulator of the adaptive response to hypoxia and oxidative stress. Furthermore, CPI-613, an inhibitor of PDH, protected DJ-1 null flies from oxidative stress, suggesting that the genetic and pharmacological inhibition of PDH may be a novel treatment strategy for PD associated with DJ-1 dysfunction.

The effect of thiamine and endurance training of 4weeks for PDH activity in skeletal muscle (4주간의 지구성 트레이닝과 thiamine 섭취가 골격근 내 PDH 활성에 미치는 영향)

  • Hwang, Hyejung;Km, Jisoo;Jang, Jiwoong;Lim, Kiwon;Joung, Seungsam;Choi, Sungkeun
    • 한국체육학회지인문사회과학편
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    • v.55 no.3
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    • pp.649-658
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    • 2016
  • This study aimed to analyze PDH(Pyruvate dehydrogenase) and protein expression of PDK4(Pyruvate dehydrogenase kinase 4), PDP1(PDH phosphatase 1), enzymes that are involved in the activation of PDH, in skeletal muscle and to investigate the concentration of thiamine administration in liver and muscle following 4 weeks of endurance training. Methods : 6 weeks old male ICR mice were divided into two groups: sedentary group (CON, n=10; TH, n=10), and exercise group (EX, n=10, THEX, n=10). Thiamine(thiamine tetrahydrofurfuryl disulfide: TTFD) TTFD was orally administrated into TH and THEX groups in 50mg/kg body weight for 4 weeks. Treadmill training was performed in EX and THEX groups at about 70% of VO2max for 5 times a week for 4 weeks. Results : In this study, the concentration of glycogen was significantly increased following 4 weeks of endurance training, but a significant difference was not found following thiamine administration. Similarly, there was a significant effect of the training on PDH and the expression of PDK4 and PDP1 as PDH was increased by about 40% along with the increase in PDK4 and PDP1. However, there was no significant difference found between the groups following thiamine administration. Discussion : This result shows that there was no synergistic effect of thiamine administration, potentially due to adaptation of skeletal muscle from a long-term endurance training. Therefore, it will be necessary to consider the intake timing of thiamine and to analyze proteins that are related to PDH following the administration of complex carbohydrates.

Dichloroacetate Inhibits the Proliferation of a Human Anaplastic Thyroid Cancer Cell Line via a p53-independent Pathway (Dichloroacetate의 p53 비의존적 경로를 통한 인간 역분화 갑상선 암세포주의 성장억제 효과)

  • KC, Yam Bahadur;Poudel, Sunil;Jeon, Eon Ju;Shon, Ho Sang;Byun, Sung June;Jeoung, Nam Ho
    • Journal of Life Science
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    • v.28 no.12
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    • pp.1469-1476
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    • 2018
  • Occurrence of the Warburg effect in solid tumors causes resistance to cancer chemotherapy, and targeting energy metabolisms such as aerobic glycolysis is a potential strategy for alternative treatment. Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), shifts glucose metabolism from aerobic glycolysis to oxidative phosphorylation (OxPhos) in many cancers. In this study, we investigated the anticancer effect of DCA on a human anaplastic thyroid cancer (ATC) cell line, 8505C. We found that DCA selectively inhibits cell proliferation of the 8505C line but not of a normal thyroid line. In 8505C, the cell cycle was arrested at the G1/S phase with DCA treatment as a result of decreased antiapoptotic proteins such as $HIF1{\alpha}$, PDK1, and Bcl-2 and increased proapoptotic proteins such as Bax and p21. DCA treatment enhanced the production of reactive oxygen species which consequently induced cell cycle arrest and apoptosis. Interestingly, DCA treatment not only reduced lactate production but also increased the expression of sodium-iodine symporter, indicating that it restores the OxPhos of glucose metabolism and the iodine metabolism of the ATC. Taken together, our findings suggest that PDK inhibitors such as DCA could be useful anticancer drugs for the treatment of ATC and may also be helpful in combination with chemotherapy and radiotherapy.

Dimethyl Cardamonin Exhibits Anti-inflammatory Effects via Interfering with the PI3K-PDK1-PKCα Signaling Pathway

  • Yu, Wan-Guo;He, Hao;Yao, Jing-Yun;Zhu, Yi-Xiang;Lu, Yan-Hua
    • Biomolecules & Therapeutics
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    • v.23 no.6
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    • pp.549-556
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    • 2015
  • Consumption of herbal tea [flower buds of Cleistocalyx operculatus (Roxb.) Merr. et Perry (Myrtaceae)] is associated with health beneficial effects against multiple diseases including diabetes, asthma, and inflammatory bowel disease. Emerging evidences have reported that High mobility group box 1 (HMGB1) is considered as a key "late" proinflammatory factor by its unique secretion pattern in aforementioned diseases. Dimethyl cardamonin (2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone, DMC) is a major ingredient of C. operculatus flower buds. In this study, the anti-inflammatory effects of DMC and its underlying molecular mechanisms were investigated on lipopolysaccharide (LPS)-induced macrophages. DMC notably suppressed the mRNA expressions of TNF-${\alpha}$, IL-$1{\beta}$, IL-6, and HMGB1, and also markedly decreased their productions in a time- and dose-dependent manner. Intriguingly, DMC could notably reduce LPS-stimulated HMGB1 secretion and its nucleo-cytoplasmic translocation. Furthermore, DMC dose-dependently inhibited the activation of phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent kinase 1 (PDK1), and protein kinase C alpha (PKC${\alpha}$). All these data demonstrated that DMC had anti-inflammatory effects through reducing both early (TNF-${\alpha}$, IL-$1{\beta}$, and IL-6) and late (HMGB1) cytokines expressions via interfering with the PI3K-PDK1-PKC${\alpha}$ signaling pathway.

Postmortem mRNA Expression Patterns in Left Ventricular Myocardial Tissues and Their Implications for Forensic Diagnosis of Sudden Cardiac Death

  • Son, Gi Hoon;Park, Seong Hwan;Kim, Yunmi;Kim, Ji Yeon;Kim, Jin Wook;Chung, Sooyoung;Kim, Yu-Hoon;Kim, Hyun;Hwang, Juck-Joon;Seo, Joong-Seok
    • Molecules and Cells
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    • v.37 no.3
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    • pp.241-247
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    • 2014
  • Sudden cardiac death (SCD), which is primarily caused by lethal heart disorders resulting in structural and arrhythmogenic abnormalities, is one of the prevalent modes of death in most developed countries. Myocardial ischemia, mainly due to coronary artery disease, is the most common type of heart disease leading to SCD. However, postmortem diagnosis of SCD is frequently complicated by obscure histological evidence. Here, we show that certain mRNA species, namely those encoding hemoglobin A1/2 and B (Hba1/2 and Hbb, respectively) as well as pyruvate dehydrogenase kinase 4 (Pdk4), exhibit distinct postmortem expression patterns in the left ventricular free wall of SCD subjects when compared with their expression patterns in the corresponding tissues from control subjects with non-cardiac causes of death. Hba1/2 and Hbb mRNA expression levels were higher in ischemic SCD cases with acute myocardial infarction or ischemic heart disease without recent infarction, and even in cardiac death subjects without apparent pathological signs of heart injuries, than control subjects. By contrast, Pdk4 mRNA was expressed at lower levels in SCD subjects. In conclusion, we found that altered myocardial Hba1/2, Hbb, and Pdk4 mRNA expression patterns can be employed as molecular signatures of fatal cardiac dysfunction to forensically implicate SCD as the primary cause of death.