scholarly journals Localization of mTORC2 activity inside cells

2017 ◽  
Vol 216 (2) ◽  
pp. 343-353 ◽  
Author(s):  
Michael Ebner ◽  
Benjamin Sinkovics ◽  
Magdalena Szczygieł ◽  
Daniela Wolfschoon Ribeiro ◽  
Ivan Yudushkin
Keyword(s):  
Plasma Membrane ◽  
Growth Factors ◽  
Intracellular Localization ◽  
Mammalian Target Of Rapamycin ◽  
Pleckstrin Homology Domain ◽  
Growth And Survival ◽  
Membrane Recruitment ◽  
Akt Phosphorylation ◽  
Protein Kinase Akt ◽  
Cell Growth And Proliferation

Activation of protein kinase Akt via its direct phosphorylation by mammalian target of rapamycin (mTOR) complex 2 (mTORC2) couples extracellular growth and survival cues with pathways controlling cell growth and proliferation, yet how growth factors target the activity of mTORC2 toward Akt is unknown. In this study, we examine the localization of the obligate mTORC2 component, mSin1, inside cells and report the development of a reporter to examine intracellular localization and regulation by growth factors of the endogenous mTORC2 activity. Using a combination of imaging and biochemical approaches, we demonstrate that inside cells, mTORC2 activity localizes to the plasma membrane, mitochondria, and a subpopulation of endosomal vesicles. We show that unlike the endosomal pool, the activity and localization of mTORC2 via the Sin1 pleckstrin homology domain at the plasma membrane is PI3K and growth factor independent. Furthermore, we show that membrane recruitment is sufficient for Akt phosphorylation in response to growth factors. Our results indicate the existence of spatially separated mTORC2 populations with distinct sensitivity to PI3K inside cells and suggest that intracellular localization could contribute to regulation of mTORC2 activity toward Akt.

scholarly journals Real Time Fluorescence Imaging of Plcγ Translocation and Its Interaction with the Epidermal Growth Factor Receptor

2001 ◽  
Vol 153 (3) ◽  
pp. 599-612 ◽  
Author(s):  
Miho Matsuda ◽  
Hugh F. Paterson ◽  
Rosie Rodriguez ◽  
Amanda C. Fensome ◽  
Moira V. Ellis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Real Time ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Pleckstrin Homology Domain ◽  
Membrane Structures ◽  
Main Site ◽  
Membrane Recruitment ◽  
Src Homology

The translocation of fluorescently tagged PLCγ and requirements for this process in cells stimulated with EGF were analyzed using real time fluorescence microscopy applied for the first time to monitor growth factor receptor–effector interactions. The translocation of PLCγ to the plasma membrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidylinositol (PI) 3-kinase. An array of domains specific for PLCγ isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLCγ, relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-P2), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane ruffles where PI 4,5-P2 substrate could also be visualized. At later times, internalization of PLCγ, which could lead to separation from the substrate, was observed. The data support a direct binding of PLCγ to the receptor as the main site of the plasma membrane recruitment. The presence of PLCγ in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intracellular vesicles, leading to downregulation of the PLC activity.

scholarly journals Estradiol-induced regulation of GLUT4 in 3T3-L1 cells: involvement of ESR1 and AKT activation

2017 ◽  
Vol 59 (3) ◽  
pp. 257-268 ◽  
Author(s):  
Raquel S Campello ◽  
Luciana A Fátima ◽  
João Nilton Barreto-Andrade ◽  
Thais F Lucas ◽  
Rosana C Mori ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Subcellular Distribution ◽  
Glucose Transporter ◽  
Biological Effects ◽  
Glut4 Translocation ◽  
Akt Phosphorylation ◽  
Akt Activation ◽  
Glut4 Expression ◽  
Protein Kinase Akt

Impaired insulin-stimulated glucose uptake involves reduced expression of the GLUT4 (solute carrier family 2 facilitated glucose transporter member 4, SLC2A4 gene). 17β-estradiol (E2) modulates SLC2A4/GLUT4 expression, but the involved mechanisms are unclear. Although E2 exerts biological effects by binding to estrogen receptors 1/2 (ESR1/2), which are nuclear transcriptional factors; extranuclear effects have also been proposed. We hypothesize that E2 regulates GLUT4 through an extranuclear ESR1 mechanism. Thus, we investigated the effects of E2 upon (1) subcellular distribution of ESRs and the proto-oncogene tyrosine-protein kinases (SRC) involvement; (2) serine/threonine-protein kinase (AKT) activation; (3) Slc2a4/GLUT4 expression and (4) GLUT4 subcellular distribution and glucose uptake in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were cultivated or not with E2 for 24 h, and additionally treated or not with ESR1-selective agonist (PPT), ESR1-selective antagonist (MPP) or selective SRC inhibitor (PP2). Subcellular distribution of ESR1, ESR2 and GLUT4 was analyzed by immunocytochemistry; Slc2a4 mRNA and GLUT4 were quantified by qPCR and Western blotting, respectively; plasma membrane GLUT4 translocation and glucose uptake were analyzed under insulin stimulus for 20 min or not. E2 induced (1) translocation of ESR1, but not of ESR2, from nucleus to plasma membrane and AKT phosphorylation, effects mimicked by PPT and blocked by MPP and PP2; (2) increased Slc2a4/GLUT4 expression and (3) increased insulin-stimulated GLUT4 translocation and glucose uptake. In conclusion, E2 treatment promoted a SRC-mediated nucleus-plasma membrane shuttle of ESR1, and increased AKT phosphorylation, Slc2a4/GLUT4 expression and plasma membrane GLUT4 translocation; consequently, improving insulin-stimulated glucose uptake. These results unravel mechanisms through which estrogen improves insulin sensitivity.

mTORC1 regulates the efficiency and cellular capacity for protein synthesis

2013 ◽  
Vol 41 (4) ◽  
pp. 923-926 ◽  
Author(s):  
Christopher G. Proud
Keyword(s):  
Protein Synthesis ◽  
Growth Factors ◽  
Cell Growth ◽  
Translation Initiation ◽  
Ribosomal Proteins ◽  
Mammalian Target Of Rapamycin ◽  
Rna Polymerases ◽  
Short Term ◽  
Cellular Capacity ◽  
Cell Growth And Proliferation

mTORC1 (mammalian target of rapamycin complex 1) is activated by nutrients, growth factors and certain hormones. Signalling downstream of mTORC1 promotes protein synthesis by both activating the processes of translation initiation and elongation, in the short term, and the production of new ribosomes, in the longer term. mTORC1 signalling stimulates the translation of the mRNAs encoding the ribosomal proteins, activates RNA polymerases I and III, which make the rRNAs, and promotes the processing of the precursor for the main rRNAs. Taken together, these effects allow mTORC1 signalling to drive cell growth and proliferation.

scholarly journals Regulation of mTORC1 and mTORC2 Complex Assembly by Phosphatidic Acid: Competition with Rapamycin

2008 ◽  
Vol 29 (6) ◽  
pp. 1411-1420 ◽  
Author(s):  
Alfredo Toschi ◽  
Evan Lee ◽  
Limei Xu ◽  
Avalon Garcia ◽  
Noga Gadir ◽  
...  
Keyword(s):  
Cell Growth ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Cancer Survival ◽  
Mammalian Target Of Rapamycin ◽  
Inhibitory Effect ◽  
S6 Kinase ◽  
Growth And Survival ◽  
Akt Phosphorylation ◽  
Mtorc2 Complex

ABSTRACT mTOR, the mammalian target of rapamycin, is a critical node for control of cell growth and survival and has widely been implicated in cancer survival signals. mTOR exists in two complexes: mTORC1 and mTORC2. Phospholipase D (PLD) and its metabolite phosphatidic acid (PA) have been implicated in the regulation of mTOR; however, their role has been controversial. We report here that suppression of PLD prevents phosphorylation of the mTORC1 substrate S6 kinase (S6K) at Thr389 and the mTORC2 substrate Akt at Ser473. Suppression of PLD also blocked insulin-stimulated Akt phosphorylation at Ser473 and the mTORC2-dependent phosphorylation of PRAS40. Importantly, PA was required for the association of mTOR with Raptor to form mTORC1 and that of mTOR with Rictor to form mTORC2. The effect of PA was competitive with rapamycin—with much higher concentrations of rapamycin needed to compete with the PA-mTORC2 interaction than with PA-mTORC1. Suppressing PA production substantially increased the sensitivity of mTORC2 to rapamycin. Data provided here demonstrate a PA requirement for the stabilization of both mTORC1 and mTORC2 complexes and reveal a mechanism for the inhibitory effect of rapamycin on mTOR. This study also suggests that by suppressing PLD activity, mTORC2 could be targeted therapeutically with rapamycin.

scholarly journals Characterization of Galpha13-dependent plasma membrane recruitment of p115RhoGEF

2003 ◽  
Vol 371 (3) ◽  
pp. 709-720 ◽  
Author(s):  
Raja BHATTACHARYYA ◽  
Philip B. WEDEGAERTNER
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Small Gtpase ◽  
Pleckstrin Homology Domain ◽  
Nucleotide Exchange ◽  
Pleckstrin Homology ◽  
Α Subunit ◽  
Membrane Recruitment ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The Ras homology (Rho) guanine nucleotide exchange factor (GEF), p115RhoGEF, provides a direct link between the G-protein α subunit, α13, and the small GTPase Rho. In the present study, we demonstrate that activated mutants of α13 or α12, but not αq, promote the redistribution of p115RhoGEF from the cytoplasm to the plasma membrane (PM). We also show that the PM translocation of p115RhoGEF is promoted by stimulation of thromboxane A2 receptors. Furthermore, we define domains of p115RhoGEF required for its regulated PM recruitment. The RhoGEF RGS (regulators of G-protein signalling) domain of p115RhoGEF is required for PM recruitment, but it is not sufficient for strong α13-promoted PM recruitment, even though it strongly interacts with activated α13. We also identify the pleckstrin homology domain as essential for α13-mediated PM recruitment. An amino acid substitution of lysine to proline at position 677 in the pleckstrin homology domain of p115RhoGEF inhibits Rho-mediated gene transcription, but this mutation does not affect α13-mediated PM translocation of p115RhoGEF. The results suggest a mechanism whereby multiple signals contribute to regulated PM localization of p115RhoGEF.

scholarly journals A Rictor-Myo1c Complex Participates in Dynamic Cortical Actin Events in 3T3-L1 Adipocytes

2008 ◽  
Vol 28 (13) ◽  
pp. 4215-4226 ◽  
Author(s):  
G. Nana Hagan ◽  
Yenshou Lin ◽  
Mark A. Magnuson ◽  
Joseph Avruch ◽  
Michael P. Czech
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Molecular Motor ◽  
Regulatory Protein ◽  
Mammalian Target Of Rapamycin ◽  
Cortical Actin ◽  
Actin Remodeling ◽  
Membrane Ruffling ◽  
Akt Phosphorylation ◽  
Protein Kinase Akt

ABSTRACT Insulin signaling through phosphatidylinositol 3-kinase (PI 3-kinase) activates the protein kinase Akt through phosphorylation of its threonine 308 and serine 473 residues by the PDK1 protein kinase and the Rictor-mammalian target of rapamycin complex (mTORC2), respectively. Remarkably, we show here that the Rictor protein is also present in cultured adipocytes in complexes containing Myo1c, a molecular motor that promotes cortical actin remodeling. Interestingly, the Rictor-Myo1c complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Furthermore, while RNA interference-directed silencing of Rictor results in the expected attenuation of Akt phosphorylation at serine 473, depletion of Myo1c is without effect. In contrast, loss of either Rictor or Myo1c inhibits phosphorylation of the actin filament regulatory protein paxillin at tyrosine 118. Furthermore, Myo1c-induced membrane ruffling of 3T3-L1 adipocytes is also compromised following Rictor knockdown. Interestingly, neither the mTORC2 inhibitor rapamycin nor the PI 3-kinase inhibitor wortmannin affects paxillin tyrosine 118 phosphorylation. Taken together, our findings suggest that the Rictor-Myo1c complex is distinct from mTORC2 and that Myo1c, in conjunction with Rictor, participates in cortical actin remodeling events.

scholarly journals Phosphatidylinositol-3,4,5-Trisphosphate Is Required for Insulin-Like Growth Factor 1-Mediated Survival of 3T3-L1 Preadipocytes**This work was supported by a grant (to A.S.) from the Medical Research Council of Canada.

Endocrinology ◽  
2001 ◽  
Vol 142 (1) ◽  
pp. 205-212 ◽  
Author(s):  
AnneMarie Gagnon ◽  
Patti Dods ◽  
Nicolas Roustan-Delatour ◽  
Ching-Shih Chen ◽  
Alexander Sorisky
Keyword(s):  
Cell Death ◽  
Growth Factor ◽  
Mammalian Target Of Rapamycin ◽  
Inhibitory Effect ◽  
Tunel Staining ◽  
Insulin Like Growth Factor ◽  
Tissue Mass ◽  
Akt Phosphorylation ◽  
Dependent Cell ◽  
Phosphoinositide 3 Kinase

Abstract Adipocyte number, a determinant of adipose tissue mass, reflects the balance between the rates of proliferation/differentiation vs. apoptosis of preadipocytes. The percentage of 3T3-L1 preadipocytes undergoing cell death following serum deprivation was reduced by 10 nm insulin-like growth factor (IGF)-1 (from 50.0 ± 0.7% for control starved cells to 27.5 ± 3.1%). TUNEL staining confirmed the apoptotic nature of the cell death. The protective effect of IGF-1 was blocked by phosphoinositide 3-kinase (PI3K) inhibitors, wortmannin, and LY294002, but was unaffected by rapamycin, PD98059, or SB203580, which inhibit mammalian target of rapamycin (mTOR), ERK kinase (MEK1), and p38 MAPK respectively. Exogenous PI(3,4,5)P3 (10 μm), the principal product of IGF-1-stimulated PI3K in 3T3-L1 preadipocytes, had a modest survival effect on its own, reducing cell death from 47.9± 3.4% to 35.6 ± 3.5%. When added to the combination of IGF-1 and LY294002, PI(3,4,5)P3 reversed most of the inhibitory effect of LY294002 on IGF-1-dependent cell survival, protein kinase B/Akt phosphorylation, and caspase-3 activity. Taken together, these results implicate PI(3,4,5)P3 as a necessary signal for the anti-apoptotic action of IGF-1 on 3T3-L1 preadipocytes.

scholarly journals Akt-Dependent Phosphorylation of p21Cip1 Regulates PCNA Binding and Proliferation of Endothelial Cells

2001 ◽  
Vol 21 (16) ◽  
pp. 5644-5657 ◽  
Author(s):  
Lothar Rössig ◽  
Amir S. Jadidi ◽  
Carmen Urbich ◽  
Cornel Badorff ◽  
Andreas M. Zeiher ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Intracellular Localization ◽  
Site Directed Mutagenesis ◽  
Endothelial Cell Proliferation ◽  
Inhibitory Protein ◽  
Intact Cells ◽  
Biochemical Fractionation ◽  
Protein Kinase Akt

ABSTRACT The protein kinase Akt is activated by growth factors and promotes cell survival and cell cycle progression. Here, we demonstrate that Akt phosphorylates the cell cycle inhibitory protein p21Cip1 at Thr 145 in vitro and in intact cells as shown by in vitro kinase assays, site-directed mutagenesis, and phospho-peptide analysis. Akt-dependent phosphorylation of p21Cip1 at Thr 145 prevents the complex formation of p21Cip1 with PCNA, which inhibits DNA replication. In addition, phosphorylation of p21Cip1 at Thr 145 decreases the binding of the cyclin-dependent kinases Cdk2 and Cdk4 to p21Cip1 and attenuates the Cdk2 inhibitory activity of p21Cip1. Immunohistochemistry and biochemical fractionation reveal that the decrease of PCNA binding and regulation of Cdk activity by p21Cip1 phosphorylation is not caused by altered intracellular localization of p21Cip1. As a functional consequence, phospho-mimetic mutagenesis of Thr 145 reverses the cell cycle-inhibitory properties of p21Cip1, whereas the nonphosphorylatable p21Cip1 T145A construct arrests cells in G0 phase. These data suggest that the modulation of p21Cip1 cell cycle functions by Akt-mediated phosphorylation regulates endothelial cell proliferation in response to stimuli that activate Akt.

Insulin modulates PC-1 processing and recruitment in cultured human cells

2003 ◽  
Vol 284 (3) ◽  
pp. E514-E520 ◽  
Author(s):  
C. Menzaghi ◽  
R. Di Paola ◽  
G. Baj ◽  
A. Funaro ◽  
A. Arnulfo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Plasma Membrane ◽  
Intracellular Trafficking ◽  
Posttranslational Processing ◽  
Phosphatidylinositol 3 Kinase ◽  
S6 Kinase ◽  
Fourfold Increase ◽  
Membrane Recruitment ◽  
Insulin Receptor Signaling ◽  
Cultured Human Cells

We evaluated whether insulin signaling modulates plasma cell glycoprotein (PC-1) plasma membrane recruitment, posttranslational processing, and gene expression in human cultured cell lines. Insulin induced a fourfold increase ( P < 0.01) of membrane PC-1 expression by rapid and sensitive mechanism(s). This effect was reduced ( P < 0.05–0.01) by inhibition of phosphatidylinositol 3-kinase (200 nmol/l wortmannin) and S6 kinase (50 nmol/l rapamycin) activities and intracellular trafficking (50 μmol/l monensin) and was not accompanied by PC-1 gene expression changes. Moreover, at Western blot, insulin elicited the appearance, in both plasma membrane and cytosol, of a PC-1-related 146-kDa band (in addition to bands of 163, 117, 106, and 97 kDa observed also in absence of insulin) that was sensitive to endoglycosidase H. Finally, inhibition of PC-1 translocation to plasma membrane, by wortmannin pretreatment, increases insulin-stimulated receptor autophosphorylation. Our data indicate that insulin stimulates PC-1 posttranslational processing and translocation to the plasma membrane, which in turn impairs insulin receptor signaling. Bidirectional cross talk between insulin and PC-1, therefore, takes place, which may be part of the hormone self-desensitization mechanism.

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