scholarly journals Lipid-dependent Akt-ivity: where, when, and how

2019 ◽  
Vol 47 (3) ◽  
pp. 897-908 ◽  
Author(s):  
Katharina M. Siess ◽  
Thomas A. Leonard
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Kinase Domain ◽  
Target Of Rapamycin ◽  
Endomembrane System ◽  
Essential Protein ◽  
Mechanistic Target Of Rapamycin ◽  
Subcellular Compartments ◽  
Membrane Bound ◽  
Phosphoinositide 3 Kinase

Abstract Akt is an essential protein kinase activated downstream of phosphoinositide 3-kinase and frequently hyperactivated in cancer. Canonically, Akt is activated by phosphoinositide-dependent kinase 1 and mechanistic target of rapamycin complex 2, which phosphorylate it on two regulatory residues in its kinase domain upon targeting of Akt to the plasma membrane by PI(3,4,5)P3. Recent evidence, however, has shown that, in addition to phosphorylation, Akt activity is allosterically coupled to the engagement of PI(3,4,5)P3 or PI(3,4)P2 in cellular membranes. Furthermore, the active membrane-bound conformation of Akt is protected from dephosphorylation, and Akt inactivation by phosphatases is rate-limited by its dissociation. Thus, Akt activity is restricted to membranes containing either PI(3,4,5)P3 or PI(3,4)P2. While PI(3,4,5)P3 has long been associated with signaling at the plasma membrane, PI(3,4)P2 is gaining increasing traction as a signaling lipid and has been implicated in controlling Akt activity throughout the endomembrane system. This has clear implications for the phosphorylation of both freely diffusible substrates and those localized to discrete subcellular compartments.

Lupeol Inhibits Proliferation and Promotes Apoptosis of Ovarian Cancer Cells by Inactivating Phosphoinositide-3-Kinase/Protein Kinase B/ Mammalian Target of Rapamycin Pathway

2020 ◽  
Vol 19 (2) ◽  
pp. 206-210
Author(s):  
Feng Chen ◽  
Bei Zhang
Keyword(s):  
Cell Cycle ◽  
Ovarian Cancer ◽  
Protein Kinase ◽  
Cell Viability ◽  
Cancer Cells ◽  
Protein Kinase B ◽  
Mammalian Target Of Rapamycin ◽  
Target Of Rapamycin ◽  
Ovarian Cancer Cells ◽  
Phosphoinositide 3 Kinase

Lupeol exhibits multiple pharmacological activities including, anticancerous, anti-inflammatory, and antioxidant. The aim of this study was to explore the anticancerous activity of lupeol on ovarian cancer cells and examine its mechanism of action. To this end, increasing concentrations of lupeol on cell viability, cell cycle, and apoptosis in Caov-3 cells were evaluated. Lupeol inhibited cell viability, induced G1 phase arrest in cell cycle, increased cell apoptosis, and inhibited the ratio of phospho-Akt/protein kinase B and phospho-mammalian target of rapamycin/mammalian target of rapamycin. In conclusion, these data suggest that lupeol may play a therapeutic role in ovarian cancer.

scholarly journals Oligomerization-driven MLKL ubiquitylation antagonises necroptosis

2021 ◽  
Author(s):  
Zikou Liu ◽  
Laura Francesca Dagley ◽  
Kristy Lynn Shield-Artin ◽  
Samuel Nicholas Young ◽  
Aleksandra Bankovacki ◽  
...  
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Protein Kinase ◽  
Kinase Domain ◽  
Membrane Disruption ◽  
Fusion Strategy ◽  
Mixed Lineage Kinase ◽  
Independent Form ◽  
Lysine Residues ◽  
Kinetic Regulation

Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor Interacting serine/threonine Protein Kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor significance of this event have been demonstrated. Here we show that necroptosis-specific, multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin insoluble cell fraction comprising plasma/organellar membranes and protein aggregates. This ubiquitylated form is diminished by a plasma membrane located deubiquitylating enzyme. MLKL is ubiquitylated on at least 4 separate lysine residues once oligomerized, and this correlates with proteasome- and lysosome- dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licenses MLKL auto-activity independent of necroptosis signalling in mouse and human cells. Therefore, besides its role in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, ubiquitylation also contributes to the restraint of basal levels of activated MLKL to avoid errant cell death.

scholarly journals On the Structure and Mechanism of Two-Pore Channels

2017 ◽  
Author(s):  
Alexander F. Kintzer ◽  
Robert M. Stroud
Keyword(s):  
Arabidopsis Thaliana ◽  
Protein Kinase ◽  
Channel Structure ◽  
Target Of Rapamycin ◽  
Nutrient Concentrations ◽  
Pore Channel ◽  
Voltage Gated ◽  
Mechanistic Target Of Rapamycin ◽  
Terminal Domain ◽  
Pore Domain

AbstractIn eukaryotes, two-pore channels (TPC1-3) comprise a family of ion channels that regulate the conductance of Na+ and Ca2+ ions across cellular membranes. TPC1-3 form endolysosomal channels, but TPC3 can also function in the plasma membrane. TPC1/3 are voltage-gated channels, but TPC2 opens in response to binding endolysosome-specific lipid phosphatidylinositol-3,5-diphosphate (PI(3,5)P2). Filoviruses, such as Ebola, exploit TPC-mediated ion release as a means of escape from the endolysosome during infection. Antagonists that block TPC1/2 channel conductance abrogate filoviral infections. TPC1/2 form complexes with the mechanistic target of rapamycin complex 1 (mTORC1) at the endolysosomal surface that couple cellular metabolic state and cytosolic nutrient concentrations to the control of membrane potential and pH. We determined the X-ray structure of TPC1 from Arabidopsis thaliana (AtTPC1) to 2.87Å resolution–one of the two first reports of a TPC channel structure. Here we summarize these findings and the implications that the structure may have for understanding endolysosomal control mechanisms and their role in human health.AbbreviationsmTORC1Mechanistic target of rapamycin complex 1TPCTwo-pore channelPI(3,5)P2Phosphatidylinositol-3,5-diphosphateAtTPC1Arabidopsis thaliana TPC1NED19Trans-Ned-19VSDVoltage-sensing domainP1Pore domain in S5-S6P2Pore domain in S11-S12CavVoltage-gated calcium channelNavVoltage-gated sodium channelKvVoltage-gated potassium channelNTDN-terminal domainCTDC-terminal domainEFEF-hand domainNAADPNicotinic acid adenine dinucleotide phosphatePI(4,5)P2Phosphatidylinositol-4,5-diphosphateDHPDihydropyridinePAAPhenylalkylamineBTZBenzothiazepineCaa2+Activating Ca2+-ionCai2+Inhibitory Ca2+-ionfou2Fatty acid oxygenation up-regulated 2SLC38a9Sodium-coupled neutral amino acid transporter 9NPC1Niemann-Pick C1PKAProtein kinase APKCProtein kinase CPKGProtein kinase GH+ATPase - Proton Pump32P– Phosphorus-32

scholarly journals Regulation of protein kinase Cδ Nuclear Import and Apoptosis by Mechanistic Target of Rapamycin Complex-1

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Antonio Layoun ◽  
Alexander A. Goldberg ◽  
Ayesha Baig ◽  
Mikaela Eng ◽  
Ortal Attias ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Import ◽  
Target Of Rapamycin ◽  
Interferon Β ◽  
Structural Mechanism ◽  
Mechanistic Target Of Rapamycin ◽  
Kinase C ◽  
Nuclear Content ◽  
In Cells

AbstractInactivation of the protein complex ‘mechanistic target of rapamycin complex 1’ (mTORC1) can increase the nuclear content of transcriptional regulators of metabolism and apoptosis. Previous studies established that nuclear import of signal transducer and activator of transcription-1 (STAT1) requires the mTORC1-associated adaptor karyopherin-α1 (KPNA1) when mTORC1 activity is reduced. However, the role of other mTORC1-interacting proteins in the complex, including ‘protein kinase C delta’ (PKCδ), have not been well characterized. In this study, we demonstrate that PKCδ, a STAT1 kinase, contains a functional ‘target of rapamycin signaling’ (TOS) motif that directs its interaction with mTORC1. Depletion of KPNA1 by RNAi prevented the nuclear import of PKCδ in cells exposed to the mTORC1 inhibitor rapamycin or amino acid restriction. Mutation of the TOS motif in PKCδ led to its loss of regulation by mTORC1 or karyopherin-α1, resulting in increased constitutive nuclear content. In cells expressing wild-type PKCδ, STAT1 activity and apoptosis were increased by rapamycin or interferon-β. Those expressing the PKCδ TOS mutant exhibited increased STAT1 activity and apoptosis; further enhancement by rapamycin or interferon-β, however, was lost. Therefore, the TOS motif in PKCδ is a novel structural mechanism by which mTORC1 prevents PKCδ and STAT1 nuclear import, and apoptosis.

scholarly journals Activation of Mechanistic Target of Rapamycin (mTOR) in Human Endothelial Cells Infected with Pathogenic Spotted Fever Group Rickettsiae

2020 ◽  
Vol 21 (19) ◽  
pp. 7179
Author(s):  
Abha Sahni ◽  
Hema P. Narra ◽  
Sanjeev K. Sahni
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Spotted Fever ◽  
Target Of Rapamycin ◽  
Spotted Fever Group ◽  
Microvascular Endothelium ◽  
Rickettsia Species ◽  
Mechanistic Target Of Rapamycin ◽  
P70 S6k

Attributed to the tropism for host microvascular endothelium lining the blood vessels, vascular inflammation and dysfunction represent salient features of rickettsial pathogenesis, yet the details of fundamentally important pathogen interactions with host endothelial cells (ECs) as the primary targets of infection remain poorly appreciated. Mechanistic target of rapamycin (mTOR), a serine/threonine protein kinase of the phosphatidylinositol kinase-related kinase family, assembles into two functionally distinct complexes, namely mTORC1 (Raptor) and mTORC2 (Rictor), implicated in the determination of innate immune responses to intracellular pathogens via transcriptional regulation. In the present study, we investigated activation status of mTOR and its potential contributions to host EC responses during Rickettsia rickettsii and R. conorii infection. Protein lysates from infected ECs were analyzed for threonine 421/serine 424 phosphorylation of p70 S6 kinase (p70 S6K) and that of serine 2448 on mTOR itself as established markers of mTORC1 activation. For mTORC2, we assessed phosphorylation of protein kinase B (PKB or Akt) and protein kinase C (PKC), respectively, on serine 473 and serine 657. The results suggest increased phosphorylation of p70 S6K and mTOR during Rickettsia infection of ECs as early as 3 h and persisting for up to 24 h post-infection. The steady-state levels of phospho-Akt and phospho-PKC were also increased. Infection with pathogenic rickettsiae also resulted in the formation of microtubule-associated protein 1A/1B-light chain 3 (LC3-II) puncta and increased lipidation of LC3-II, a response significantly inhibited by introduction of siRNA targeting mTORC1 into ECs. These findings thus yield first evidence for the activation of both mTORC1 and mTORC2 during EC infection in vitro with Rickettsia species and suggest that early induction of autophagy in response to intracellular infection might be regulated by this important pathway known to function as a central integrator of cellular immunity and inflammation.

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