Transition from Heterotypic to Homotypic PDK1 Homodimerization Is Essential for TCR-Mediated NF-κB Activation

Kang, Jung‐Ah; Jeong, Sang Phil; Park, Daeho; Hayden, Matthew S.; Ghosh, Sankar; Park, Sung‐Gyoo

doi:10.4049/jimmunol.1202923

Cited by 17 publications

(17 citation statements)

References 22 publications

(39 reference statements)

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“…Recent studies show that phosphorylation of the PDK-1 PH domain by TCR/CD28 signaling induces formation of PDK-1 dimers able to bind and phosphorylate its substrates, PKCθ and AKT. 46 We found that DGKζ limits DAG-dependent PDK-1 activation by stabilizing PKCθ, which complements this model and helps to clarify the close interrelation of these kinases. PKCθ interacts physically and functionally with AKT to activate the NFκB pathway, 47 which correlates with enhanced NFκB activation and PDK-1-dependent AKT phosphorylation in DGKζ-null T cells.…”

Section: Discussionsupporting

confidence: 75%

“…Recent studies show that phosphorylation of the PDK‐1 PH domain by TCR/CD28 signaling induces formation of PDK‐1 dimers able to bind and phosphorylate its substrates, PKCθ and AKT 46 . We found that DGKζ limits DAG‐dependent PDK‐1 activation by stabilizing PKCθ, which complements this model and helps to clarify the close interrelation of these kinases.…”

Section: Discussionsupporting

confidence: 75%

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Predominant contribution of DGKζ over DGKα in the control of PKC/PDK‐1‐regulated functions in T cells

et al. 2017

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Diacylglycerol kinase (DGK)-mediated consumption of the diacylglycerol (DAG) generated in response to antigen recognition is an important mechanism to limit T-cell function. Targeting DGK activity presents new opportunities for therapeutic manipulation of the immune response, but assessment of individual DGK functions is complex. T cells express two DGK isoforms, DGKα and DGKζ, and there are no isoform-specific inhibitors. Here we used short interfering RNA-mediated gene silencing in human T cells and DGKα- and DGKζ-deficient mice to define DGK isoform-specific regulation of key signaling pathways during T-cell activation. Our results identify DGKζ as the predominant brake on basal/tonic conditions as well as on downstream T-cell receptor/co-stimulatory signals. DGKζ silencing triggers basal RasGTP activation and facilitates enhanced membrane stability of protein kinase C alpha as well as increased activity of AGC kinases. Downstream of T-cell receptor/co-stimulation, DGKζ silencing results in enhanced and maintained recruitment of PKC theta to the membrane, as well as phosphoinositide-dependent protein kinase-1 activation and scaffolding functions. Our studies identify a previously unrecognized DGKζ contribution as a negative regulator of the crosstalk between phospholipase C-gamma- and phosphoinositide 3-kinase-regulated pathways. This DGKζ input helps to explain previous observations in DGK-deficient mice and suggests that the development of isoform-specific DGK inhibitors is of great interest for the manipulation of distinct aspects of T-cell responses.

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Section: Discussionsupporting

confidence: 75%

Section: Discussionsupporting

confidence: 75%

Predominant contribution of DGKζ over DGKα in the control of PKC/PDK‐1‐regulated functions in T cells

et al. 2017

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“…They showed that a basal homodimer population (of ∼20 %) existed mainly in the cytoplasm of resting cells that was increased (to 40 %) upon growth factor stimulation. Even though the destabilization of the PDK1 inhibitory homodimer upon translocation and autophosphorylation would be expected to trigger an active monomeric form of PDK1, a recent article by Kang et al [19] suggested that the phosphorylation of Thr 513 would probably trigger the formation of a different PDK1 homodimer conformer (possible 'active' homodimer) [19]. Moreover, they determined that the presence of a complete PH domain was strictly necessary for the maintenance of PDK1 homodimers at steady state, but did not require a functional lipid-binding domain [18].…”

Section: Conformational Regulations Of Pdk1 and Homodimerizationmentioning

confidence: 99%

“…1 the plasma membrane by binding to PtdIns(3,4,5)P 3 via their respective PH domains [2][3][4] and involves the formation of a PKB-PDK1 heterodimer complex [5]. This consists of phosphorylation events [13,14], subcellular localization [15,16] and conformational rearrangements including PDK1 homodimerization [17][18][19]. Owing to the profuse number of signalling pathways controlled by PDK1 that are involved in cancer [7] and other diseases, great efforts have been dedicated to understanding how PDK1 itself is regulated and how it in turn regulates its different substrates spatially and temporally.…”

Section: Introductionmentioning

confidence: 99%

Acute regulation of PDK1 by a complex interplay of molecular switches

Calleja

Laguerre

Heras-Martínez

et al. 2014

Biochemical Society Transactions

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Phosphoinositide-dependent kinase 1 (PDK1) is the master regulator of at least 23 other AGC kinases whose downstream signalling has often been implicated in various diseases and in particular in cancer. Therefore there has been great interest in determining how PDK1 is controlled and how it regulates its substrates spatially and temporally. The understanding of these mechanisms could offer new possibilities for therapeutic intervention. Over the years, a more comprehensive view of the mechanisms involved in the regulation of PDK1 has emerged and these comprise serine/threonine as well as tyrosine phosphorylation, subcellular localization, regulator binding and conformation status. In the present review, we discuss how various molecular mechanisms are together responsible for the conformational regulation behind the activation of PDK1 in cells.

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“…More recently, PtdIns(3,4,5)P 3 binding to PDK1 in live cells was shown to elicit the formation of PDK1 homodimers [13, 19] and to trigger the autophosphorylation of the PDK1 PH domain residue 513. This, was suggested to be due to the disruption of an autoinhibitory PDK1 homodimer conformer [13, 21, 22]. This mechanism still remains to be defined.…”

Section: Introductionmentioning

confidence: 99%

A Complex Interplay of Anionic Phospholipid Binding Regulates 3’-Phosphoinositide-Dependent-Kinase-1 Homodimer Activation

Heras

Calleja

Bailly

et al. 2019

Preprint

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1 2 3'-Phosphoinositide-dependent-Kinase-1 is a master regulator whereby its PI3-3 kinase-dependent dysregulation in human pathologies is well documented. 4 Understanding the direct role for PtdIns(3,4,5)P 3 and other anionic 5 phospholipids in the regulation of PDK1 conformational dynamics and its 6 downstream activation remains incomplete. 7 Using advanced quantitative-time-resolved imaging, FCS and molecular 8 modelling, we show an interplay of antagonistic binding effects of 9 PtdIns(3,4,5)P 3 and other anionic phospholipids, regulating activated PDK1 10 homodimers. We demonstrate that phosphatidylserine maintains PDK1 in an 11 inactive conformation. The dysregulation of the PI3K pathway affects the spatio-12 temporal and conformational dynamics of PDK1 and the activation of its 13 downstream substrates. 14 We establish an anionic-phospholipid-dependent model for PDK1 regulation, 15 depicting the conformational dynamics of multiple homodimer states. The 16 dysregulation of the PI3K pathway perturbs equilibrium between the PDK1 17 homodimer conformations. Our findings indicate that the alteration of specific 18 basic residues of PDK1-PH domain leads to its constitutive activation, a 19 potential significance in different types of carcinomas. 20 21lipids such as PtdIns(3,4,5)P 3 , PtdIns(4,5)P 2 and PtdSer, autophosphorylation 1 of T513 and homodimerisation. However, our understanding of the interplay 2 between these events and the associated regulation that enables the 3 phosphorylation of PDK1 substrates remains largely unknown. Addressing 4 these issues has been hampered by the lack of suitable in situ quantitative 5 methods for interrogating the spatio-temporal sequence of protein-lipid 6 interactions. We have recently reported the development of a precise and well-7 resolved quantitative imaging method based on fluorescence lifetime imaging 8 (FLIM) that overcomes these technical obstacles [23]. Here, we were able to 9 monitor in situ the anionic phospholipid-mediated regulation of PDK1 10 homodimerisation and localisation leading to PDK1 activation and how this was 11 linked to T513 autophosphorylation in cells with a normal or a dysregulated 12 PI3K pathway. Using this methodology, we unravelled three major mechanisms 13 for PDK1 regulation. Firstly, that PDK1 homodimerises in an activated 14 conformation phosphorylated on T513 and capable of activating downstream 15 substrates like PKB/Akt and SGK1. Secondly, that this homodimer formation 16 was triggered by two opposite mechanisms: (i) The binding to PtdIns(3,4,5)P 3 17 upon growth factor stimulation (ii) the loss of plasma membrane binding to other 18 anionic phospholipids. This suggested a competitive regulatory mechanism 19involving PtdIns(3,4,5)P 3 and other anionic phospholipids having opposite 20 effects on PDK1 activation. Thirdly, we demonstrated that PDK1 activation 21 through recruitment to the PM was not only dependent on its PH domain but its 22 domains of PDK1 (PH PDK1 ). PH PDK1 wild type and mutants were tested using a 1 protein-li...

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Transition from Heterotypic to Homotypic PDK1 Homodimerization Is Essential for TCR-Mediated NF-κB Activation

Cited by 17 publications

References 22 publications

Predominant contribution of DGKζ over DGKα in the control of PKC/PDK‐1‐regulated functions in T cells

Predominant contribution of DGKζ over DGKα in the control of PKC/PDK‐1‐regulated functions in T cells

Acute regulation of PDK1 by a complex interplay of molecular switches

A Complex Interplay of Anionic Phospholipid Binding Regulates 3’-Phosphoinositide-Dependent-Kinase-1 Homodimer Activation

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