Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2

Feldman, Michael R.; Apsel, Beth; Uotila, Aino; Loewith, Robbie; Knight, Zachary A.; Ruggero, Davide; Shokat, Kevan M.

doi:10.1371/journal.pbio.1000038

Cited by 998 publications

(1,054 citation statements)

References 54 publications

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“…For all three ligands we obtained scores which are similar to those obtained for the crystal ligands in PDK1 and PKC-alpha. Thus, our simulation studies corroborate recent observations on pp242 (24) and also support our experimental findings with PG and OBX.…”

Section: In Silico Docking Predictionsupporting

confidence: 92%

“…is structurally similar to prodiginines (24). In silico models suggest that prodiginines, like pp242, could interact in the active-site of mTOR and provided several of the recognition motifs involved in their interaction.…”

Section: Discussionmentioning

confidence: 90%

“…For PDK1 and PKC-alpha we docked the crystallographic ligands together with PG. For mTOR we docked PG, OBX and PP242, a ligand that inhibits mTOR with an IC 50 of 8 nM (24). The induced fit was modelled by 600 iterations with PELE (Protein Energy Landscape Exploration), a stochastic method of mapping large conformational rearrangements and induced fit events in protein-ligand interactions (25).…”

Section: Theoretical Methodsmentioning

confidence: 99%

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Identification of dual mTORC1 and mTORC2 inhibitors in melanoma cells: Prodigiosin vs. obatoclax

Espona-Fiedler

Soto‐Cerrato

Hosseini

et al. 2012

Biochemical Pharmacology

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The PI3K/AKT/mTOR signaling pathway regulates cell proliferation, survival and angiogenesis. The mammalian target of rapamycin (mTOR) is a protein kinase ubiquitously expressed within cells that regulates cell growth and survival by integrating nutrient and hormonal signals. mTOR exists in two complexes, mTORC1 and mTORC2. Hyperactivation of the mTOR protein has been linked to development of cancer, raising mTOR as an attractive target for cancer therapy. Prodigiosin (PG) and Obatoclax (OBX), two members of the prodiginines family, are small molecules with anticancer properties which are currently under clinical trials. In the present paper, we demonstrate that mTOR is a molecular target of both prodiginines in melanoma, a highly drug-resistant cancer model. The inhibition of mTORC1 and mTORC2 complexes by PG or OBX resulted in a loss of AKT phosphorylation at S473, preventing its full activation, with no significant effect on T308. The strongest activity inhibition (89%) was induced by PG on mTORC2. Binding assays using Surface Plasmon Resonance (SPR) provide kinetic and affinity data of the interaction of these small molecules with mTOR. In addition, in silico modelling produced a detailed atomic description of the binding modes. These results provide new data to understand the mechanism of action of these molecules, and provide new structural data that will allow the development of more specific mTOR inhibitors for cancer treatment.3

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Section: In Silico Docking Predictionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 90%

Section: Theoretical Methodsmentioning

confidence: 99%

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Identification of dual mTORC1 and mTORC2 inhibitors in melanoma cells: Prodigiosin vs. obatoclax

Espona-Fiedler

Soto‐Cerrato

Hosseini

et al. 2012

Biochemical Pharmacology

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“…If TM phosphorylation-dependent machinery capable of suppressing A-loop phosphorylation exists in these examples, differences in A-loop phosphorylation may reflect differences in TM phosphorylation. In fact, a rational pattern of TM phosphorylation has been observed in some cases (Feldman et al, 2009). Although TM phosphorylation and its contribution to A-loop phosphorylation need to be re-evaluated in each case, these previous reports suggest that the novel regulatory mechanism revealed in this study could be applicable to mammalian cells.…”

Section: Mutual Phosphorylation Of Akt On Conserved Motifs D Hiraoka mentioning

confidence: 53%

“…Inhibition of mTORC2 by chemical inhibitors or RNA interference results in diminished HM phosphorylation and decreased A-loop phosphorylation (Sarbassov et al, 2005(Sarbassov et al, , 2006Feldman et al, 2009;Garcı´a-Martı´nez et al, 2009). However, A-loop phosphorylation was not disrupted in the absence of HM phosphorylation in mouse embryonic fibroblasts from mTORC2 component-knockout mice (Guertin et al, 2006;Jacinto et al, 2006;Facchinetti et al, 2008;Ikenoue et al, 2008;Feldman et al, 2009;Garcı´a-Martı´nez et al, 2009). These discrepancies have not been explained sufficiently and imply the existence of an unknown mechanism that controls Akt phosphorylation.…”

Section: Introductionmentioning

confidence: 99%

Turn motif phosphorylation negatively regulates activation loop phosphorylation in Akt

2011

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Akt, also known as protein kinase B, has a central role in various signaling pathways that regulate cellular processes such as metabolism, proliferation and survival. On stimulation, phosphorylation of the activation loop (Aloop) and hydrophobic motif (HM) of Akt by the kinase phosphoinositide-dependent kinase 1 (PDK1) and the mammalian target of rapamycin complex 2 (mTORC2), respectively, results in Akt activation. A well-conserved threonine in the turn motif (TM) is also constitutively phosphorylated by mTORC2 and contributes to the stability of Akt. The role of TM phosphorylation in HM and A-loop phosphorylation has not been sufficiently evaluated. Using starfish oocytes as a model system, this study provides the first evidence that TM phosphorylation has a negative role in A-loop phosphorylation. In this system, the maturation-inducing hormone, 1-methyladenine, stimulates Akt to reinitiate meiosis through activation of cyclin B-Cdc2. The phosphorylation status of Akt was monitored via introduction of exogenous human Akt (hAkt) in starfish oocytes. TM and HM phosphorylation was inhibited by microinjection of an anti-starfish TOR antibody, but not by rapamycin treatment, suggesting that both phosphorylation events depend on TORC2, as reported in mammalian cells. A single or double alanine substitution at each of three phosphorylation residues revealed that TM phosphorylation renders Akt susceptible to dephosphorylation on the A-loop. When A-loop phosphatase was inhibited by okadaic acid (OA), TM phosphorylation still reduced Aloop phosphorylation, suggesting that the effect is caused at least partially through reduction of sensitivity to PDK1. Negative regulation by TM phosphorylation was also observed in constitutively active Akt and was functionally reflected in meiosis resumption. By contrast, HM phosphorylation enhanced A-loop phosphorylation and achieved full activation of Akt via a mechanism at least partially independent of TM phosphorylation. These observations provide new insight into the mechanism controlling Akt phosphorylation in the cell.

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Cyclin‐dependent kinase 4 inhibits the translational repressor 4E‐BP1 to promote cap‐dependent translation during mitosis–G1 transition

2019

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Phosphorylation of translational repressor eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) controls the initiation of capdependent translation, a type of protein synthesis that is frequently upregulated in human diseases such as cancer. Because of its critical cellular function, it is not surprising that multiple kinases can post-translationally modify 4E-BP1 to drive aberrant cap-dependent translation. We recently reported a site-selective chemoproteomic method for uncovering kinase-substrate interactions, and using this approach, we discovered the cyclin-dependent kinase (CDK)4 as a new 4E-BP1 kinase. Herein, we describe our extension of this work and reveal the role of CDK4 in modulating 4E-BP1 activity in the transition from mitosis to G1, thereby demonstrating a novel role for this kinase in cell cycle regulation.Cap-dependent translation is an important cellular process that controls the translation of select mRNAs typically encoding for growth factors and oncogenes [1][2][3][4]. The initiation of cap-dependent mRNA translation is governed by the availability of eIF4E, the m 7 GpppX-cap-binding translation initiation factor [5,6]. This protein is highly regulated, primarily through the work of the 4E-BPs, which sequester eIF4E from eIF4G and the eIF4F translation initiation complex [7][8][9][10][11][12][13]. The activity of 4E-binding protein 1 (4E-BP1) is in turn regulated by phosphorylation, where hypophosphorylated 4E-BP1 binds strongly to eIF4E to inhibit translation, while hyperphosphorylated 4E-BP1 releases eIF4E to initiate capdependent translation [13][14][15][16]. For many years, the only validated kinase known to affect 4E-BP1 phosphorylation has been mechanistic target of rapamycin complex 1 (mTORC1), which was shown to hierarchically phosphorylate 4E-BP1 at T37 and T46 followed by T70 and S65 [14,15,17,18]. However, several findings have called into question the exclusivity of mTORC1 for each of these phosphorylation sites [19], namely reports demonstrating that other unknown kinases can also phosphorylate 4E-BP1 to stimulate cap-dependent translation [20][21][22], particularly in cases of mTOR inhibitor drug resistance [23][24][25][26].Recently, our laboratory has developed a chemoproteomic pipeline by which to identify site-specific kinase-substrate interactions, Phosphosite-Accurate kinase-substrate cross(X)linking Assay or PhAXA [27]. Using this methodology, we discovered that cyclin-dependent kinase 4 (CDK4), which is primarily responsible for controlling the cell cycle checkpoint at the G 1 /S transition through phosphorylation of the retinoblastoma tumor suppressor protein (Rb) Abbreviations 4E-BP1, 4E-binding protein 1; CDK, cyclin-dependent kinase; FDR, false discovery rate; mTORC1, mechanistic target of rapamycin complex 1; PSMs, peptide-spectrum matches; WT, wild-type.

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Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2

Cited by 998 publications

References 54 publications

Identification of dual mTORC1 and mTORC2 inhibitors in melanoma cells: Prodigiosin vs. obatoclax

Identification of dual mTORC1 and mTORC2 inhibitors in melanoma cells: Prodigiosin vs. obatoclax

Turn motif phosphorylation negatively regulates activation loop phosphorylation in Akt

Cyclin‐dependent kinase 4 inhibits the translational repressor 4E‐BP1 to promote cap‐dependent translation during mitosis–G1 transition

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