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

강정아,최현우,양태우,CHO STEVE KYUNGRAE,박지용,박성규 한국분자세포생물학회 2017 Molecules and cells Vol.40 No.1

PDK1 is essential for T cell receptor (TCR)-mediated activation of NF-κB, and PDK1-induced phosphorylation of PKCθ is important for TCR-induced NF-κB activation. However, inverse regulation of PDK1 by PKCθ during T cell activation has not been investigated. In this study, we found that PKCθ is involved in human PDK1 phosphorylation and that its kinase activity is crucial for human PDK1 phosphorylation. Mass spectrometry analysis of wild-type PKCθ or of kinase-inactive form of PKCθ revealed that PKCθ induced phosphorylation of human PDK1 at Ser-64. This PKCθ-induced PDK1 phosphorylation positively regulated T cell activation and TCR-induced NF-κB 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-PKCθ-mediated T cell activation.

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 Korean Society for Molecular and Cellular Biology 2017 Molecules and cells Vol.40 No.1

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

Sunjoo Jeong,Yong Seok Choi 한국분자세포생물학회 2005 Molecules and cells Vol.20 No.2

PDK-1 activates PI3-kinase/Akt signaling and regulates fundamental cellular functions, such as growth and survival. NF-κB 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-κB is well known, the exact role of PDK-1 as well as PI3-kinase/Akt in NF-κB activation is not understood. Here we demonstrate that PDK-1 plays a pivotal role in transcriptional activation of NF-κB by dissociating the transcriptional co-repressor HDAC1 from the p65 subunit of NF-κB. The association of CBP with p65 was not directly modulated by PDK-1 or by PI3-kinase. Etoposide activated NF-κB 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-κB transcription and further downregulated the ectopic HDAC1-mediated decrease in NF-κB transcriptional activity. Thus activation of PDK-1 relieves the HDAC1-mediated repression of NF-κB that may be related to basal as well as activated transcription by NF-κB. This effect may also explain the role of the PI3-kinase/PDK-1 pathway in the anti-apoptotic function of NF-κB associated with the chemoresistance of cancer cells.

Loss of PTEN expression does not contribute to PDK-1 activity and PKC activation-loop phosphorylation in Jurkat leukaemic T cells

Freeley, Michael,Park, Jongsun,Yang, Keum-Jin,L.Wange, Ronald,Volkov, Yuri,Kelleher, Dermot,Long, Aideen 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10

Unopposed PI3-kinase activity and 3´-phosphoinositide production in Jurkat T cells, due to a mutation in the PTEN tumour suppressor protein, results in deregulation of PH domain-containing proteins including the serine/threonine kinase PKB/Akt. In Jurkat cells, PKB/Akt is constitutively active and phosphorylated at the activation-loop residue (Thr308) 3´-phosphoinositide-dependent protein kinase-1(PDK-1), an enzyme that also contains a PH domain, is thought to catalyse Thr308 phosphorylation of PKB/Akt in addition to other kinase-1 families such as PKC isoforms. It is unknown however if the loss ofPTEN in Jurkat cells also results in unregulated PDK-l activity and whether such loss impacts on activation-loop phosphorylation of other putative PDK-1 substrates such as PKC. In this study we have addressed if loss ofPTFN in Jurkat T cells affects PDK-1 catalytic activity and intracellular localisation. We demonstrate that reducing the level of3´-phosphoinositidcs in Jurkat cells with pharmacological inhibitors of PI3-kinase or expression of PTEN does not affect PDK-1 activity, Ser241 phosphorylation or intracellular localisation. In support of this finding, we show that the levels of PKC activation-loop phosphorylation are unaffected by reductions in the levels of 3´-phosphoinositides Instead, the dephosphorylation that occurs on PKB/Akt at Thr308 following reductions in 3´-phosphoinositides is dependent on PP2A-like phosphatase activity. Our finding that PDK-1 functions independently of 3´-phosphoinositides in T cells is also confirmed by studies in HuT-78 T cells, a PTEN-expressing cell line with undetectable levels of 3´-phosphoinosilides. We conclude therefore that loss of PTEN expression in Jurkat T cells docs not impact on the PDK-1 /PKC pathway and that only a subset of kinases, such as PKB/ Akt are perturbed as a consequence PTEN loss.

Metabolic Connection of Inflammatory Pain: Pivotal Role of a Pyruvate Dehydrogenase Kinase-Pyruvate Dehydrogenase-Lactic Acid Axis

Jha, Mithilesh Kumar,Song, Gyun Jee,Lee, Maan Gee,Jeoung, Nam Ho,Go, Younghoon,Harris, Robert A.,Park, Dong Ho,Kook, Hyun,Lee, In-Kyu,Suk, Kyoungho Society for Neuroscience 2015 The Journal of neuroscience Vol.35 No.42

<P>Pyruvate dehydrogenase kinases (PDK1–4) are mitochondrial metabolic regulators that serve as decision makers via modulation of pyruvate dehydrogenase (PDH) activity to convert pyruvate either aerobically to acetyl-CoA or anaerobically to lactate. Metabolic dysregulation and inflammatory processes are two sides of the same coin in several pathophysiological conditions. The lactic acid surge associated with the metabolic shift has been implicated in diverse painful states. In this study, we investigated the role of PDK-PDH-lactic acid axis in the pathogenesis of chronic inflammatory pain. Deficiency of <I>Pdk2</I> and/or <I>Pdk4</I> in mice attenuated complete Freund's adjuvant (CFA)-induced pain hypersensitivities. Likewise, <I>Pdk2/4</I> deficiency attenuated the localized lactic acid surge along with hallmarks of peripheral and central inflammation following intraplantar administration of CFA. <I>In vitro</I> studies supported the role of PDK2/4 as promoters of classical proinflammatory activation of macrophages. Moreover, the pharmacological inhibition of PDKs or lactic acid production diminished CFA-induced inflammation and pain hypersensitivities. Thus, a PDK-PDH-lactic acid axis seems to mediate inflammation-driven chronic pain, establishing a connection between metabolism and inflammatory pain.</P><P><B>SIGNIFICANCE STATEMENT</B> The mitochondrial pyruvate dehydrogenase (PDH) kinases (PDKs) and their substrate PDH orchestrate the conversion of pyruvate either aerobically to acetyl-CoA or anaerobically to lactate. Lactate, the predominant end product of glycolysis, has recently been identified as a signaling molecule for neuron-glia interactions and neuronal plasticity. Pathological metabolic shift and subsequent lactic acid production are thought to play an important role in diverse painful states; however, their contribution to inflammation-driven pain is still to be comprehended. Here, we report that the PDK-PDH-lactic acid axis constitutes a key component of inflammatory pain pathogenesis. Our findings establish an unanticipated link between metabolism and inflammatory pain. This study unlocks a previously ill-explored research avenue for the metabolic control of inflammatory pain pathogenesis.</P>

Mitochondrial Akt Regulation of Hypoxic Tumor Reprogramming

Chae, Young Chan,Vaira, Valentina,Caino, M. Cecilia,Tang, Hsin-Yao,Seo, Jae Ho,Kossenkov, Andrew V.,Ottobrini, Luisa,Martelli, Cristina,Lucignani, Giovanni,Bertolini, Irene,Locatelli, Marco,Bryant, Ke Elsevier 2016 CANCER CELL Vol.30 No.2

<P><B>Summary</B></P> <P>Hypoxia is a universal driver of aggressive tumor behavior, but the underlying mechanisms are not completely understood. Using a phosphoproteomics screen, we now show that active Akt accumulates in the mitochondria during hypoxia and phosphorylates pyruvate dehydrogenase kinase 1 (PDK1) on Thr346 to inactivate the pyruvate dehydrogenase complex. In turn, this pathway switches tumor metabolism toward glycolysis, antagonizes apoptosis and autophagy, dampens oxidative stress, and maintains tumor cell proliferation in the face of severe hypoxia. Mitochondrial Akt-PDK1 signaling correlates with unfavorable prognostic markers and shorter survival in glioma patients and may provide an “actionable” therapeutic target in cancer.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A pool of active Akt is recruited to tumor mitochondria during hypoxia </LI> <LI> Mitochondrial Akt phosphorylates PDK-1 in hypoxia on a T346 site </LI> <LI> Akt-PDK1 activation maintains tumor cell proliferation in hypoxia </LI> <LI> PDK1 phosphorylation by Akt is a negative prognostic factor in gliomas </LI> </UL> </P>

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

( Ling Jin ),( Minkyoung Cho ),( Bo-sung Kim ),( Jung Ho Han ),( Sungmi Park ),( In-kyu Lee ),( Dongryeol Ryu ),( Jae Ho Kim ),( Sung-jin Bae ),( Ki-tae Ha ) 생화학분자생물학회 2021 BMB Reports Vol.54 No.11

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 PDHA1^WT and PDHA1^3SD were transduced. Since PDHA1^3SD cannot be phosphorylated by PDKs, it was used to evaluate the specific activity of PDK inhibitors. This study highlights that PDHA1^WT and PDHA1^3SD 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. [BMB Reports 2021; 54(11): 563-568]

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

김기대,Sung Goo Park,박병철,김정훈,김선홍 생화학분자생물학회 2020 BMB Reports Vol.53 No.7

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.

4주간의 지구성 트레이닝과 thiamine 섭취가 골격근 내 PDH활성에 미치는 영향

황혜정(HyeJungHwang),김지수(JiSooKm),장지웅(JiWoongJang),임기원(KiWonLim),정승삼(SeungSamJoung),최성근(SungKeunChoi) 한국체육학회 2016 한국체육학회지 Vol.55 No.3

본 연구는 지구성 트레이닝과 thiamine(thiamine tetrahydrofurfuryl disulfide: TTFD)의 투여가 골격근 내 글리코겐과 PDH(Pyruvate dehydrogenase), 그리고 PDH 활성에 관여하는 효소의 단백질인 PDK4(Pyruvate dehydrogenase kinase 4)와 PDP1(PDH phosphatase 1)의 발현에 어떠한 영향을 미치는지 알아보는 것을 목적으로 하였다. 6주령의 ICR 마우스를 대상으로 비운동집단(Sedentary; CON, TH), 운동집단(Exercise; EX, THEX)으로 나누어 4주간의 지구성 트레이닝과 체중kg 당 50 mg의 thiamine을 경구투여 하였다. 4주간의 지구성 트레이닝은 간과 근육 내 glycogen의 저장량에 유의한 증가가 나타났지만 thiamine 투여에 따르는 차이는 나타나지 않았다. 마찬가지로 골격근 내 PDH와 PDH 조절에 관련한 PDK4, PDP1의 단백질 발현을 측정한 결과 4주간의 지구성 트레이닝에 따르는 효과는 관찰되었지만, thiamine 투여에 집단간 유의한 효과는 나타나지 않았다. 이러한 결과는 장기간 지구성 트레이닝에 따른 골격근의 적응으로 인하여 thiamine투여에 따른 시너지 효과가 나타나지 않은 것으로 보인다. 따라서 추후 연구에서는 지구성 트레이닝의 기간을 고려한 thiamine의 섭취 타이밍 그리고 탄수화물의 복합투여에 따른 PDH와 관련 단백질의 분석이 필요할 것으로 보인다. 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.

Extracellular vesicles derived from Lactobacillus plantarum restore chemosensitivity through the PDK2-mediated glucose metabolic pathway in 5-FU-resistant colorectal cancer cells

JaeJin An,Eun-Mi Ha 한국미생물학회 2022 The journal of microbiology Vol.60 No.7

Metabolic abnormalities are one of the main hallmarks of cancer and are associated with chemoresistance. Therefore, targeting the metabolic reprogramming of cancer cells has the potential to overcome chemoresistance. Probiotic-derived extracellular vesicles (EVs) play important roles in biological function and intracellular communication. However, the inhibitory effect of Lactobacillus plantarum-derived EVs (LpEVs) on colorectal cancer (CRC) cells has not yet been elucidated. This study clearly revealed that increased glycolysis in 5-fluorouracil (5-FU)-resistant CRC cells (CRC/5FUR) is directly related to chemoresistance and that the metabolic shift reversed by LpEVs inhibits cancer cell proliferation and eventually leads to apoptosis. Pyruvate dehydrogenase kinase 2 (PDK2), one of the crucial enzymes for enhancing glycolysis, was upregulated in CRC/5FUR cells. In our study, LpEVs sensitized CRC/5FUR cells to 5-FU by attenuating PDK2 expression in p53-p21-dependent metabolic signaling, thereby circumventing 5-FU resistance. We demonstrated the effect of cellular responses to 5-FU by modifying the PDK2 expression level in both 5-FU-sensitive parental CRC and 5- FU resistant CRC cell lines. Finally, we revealed that the PDK2 signaling pathway can potentially be targeted using LpEVs treatment to overcome chemoresistant CRC, thereby providing a potential strategy for CRC treatment by intervening in tumor metabolism.