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Regulation of mTORC1 by lysosomal calcium and calmodulin.

ABSTRACT: Blockade of lysosomal calcium release due to lysosomal lipid accumulation has been shown to inhibit mTORC1 signaling. However, the mechanism by which lysosomal calcium regulates mTORC1 has remained undefined. Herein we report that proper lysosomal calcium release through the calcium channel TRPML1 is required for mTORC1 activation. TRPML1 depletion inhibits mTORC1 activity, while overexpression or pharmacologic activation of TRPML1 has the opposite effect. Lysosomal calcium activates mTORC1 by inducing association of calmodulin (CaM) with mTOR. Blocking the interaction between mTOR and CaM by antagonists of CaM significantly inhibits mTORC1 activity. Moreover, CaM is capable of stimulating the kinase activity of mTORC1 in a calcium-dependent manner in vitro. These results reveal that mTOR is a new type of CaM-dependent kinase, and TRPML1, lysosomal calcium and CaM play essential regulatory roles in the mTORC1 signaling pathway.

SUBMITTER: Li RJ

PROVIDER: S-EPMC5106211 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Regulation of mTORC1 by lysosomal calcium and calmodulin.

Li Ruo-Jing RJ Xu Jing J Fu Chenglai C Zhang Jing J Zheng Yujun George YG Jia Hao H Liu Jun O JO

eLife 20161027

Blockade of lysosomal calcium release due to lysosomal lipid accumulation has been shown to inhibit mTORC1 signaling. However, the mechanism by which lysosomal calcium regulates mTORC1 has remained undefined. Herein we report that proper lysosomal calcium release through the calcium channel TRPML1 is required for mTORC1 activation. TRPML1 depletion inhibits mTORC1 activity, while overexpression or pharmacologic activation of TRPML1 has the opposite effect. Lysosomal calcium activates mTORC1 by i ...[more]

PMID: 27787197

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Project description:Memory and long term potentiation require de novo protein synthesis. A key regulator of this process is mTORC1, a complex comprising the mTOR kinase. Growth factors activate mTORC1 via a pathway involving PI3-kinase, Akt, the TSC complex and the GTPase Rheb. In non-neuronal cells, translocation of mTORC1 to late endocytic compartments (LEs), where Rheb is enriched, is triggered by amino acids. However, the regulation of mTORC1 in neurons remains unclear. In mouse hippocampal neurons, we observed that BDNF and treatments activating NMDA receptors trigger a robust increase in mTORC1 activity. NMDA receptors activation induced a significant recruitment of mTOR onto lysosomes even in the absence of external amino acids, whereas mTORC1 was evenly distributed in neurons under resting conditions. NMDA receptor-induced mTOR translocation to LEs was partly dependent on the BDNF receptor TrkB, suggesting that BDNF contributes to the effect of NMDA receptors on mTORC1 translocation. In addition, the combination of Rheb overexpression and artificial mTORC1 targeting to LEs by means of a modified component of mTORC1 fused with a LE-targeting motif strongly activated mTOR. To gain spatial and temporal control over mTOR localization, we designed an optogenetic module based on light-sensitive dimerizers able to recruit mTOR on LEs. In cells expressing this optogenetic tool, mTOR was translocated to LEs upon photoactivation. In the absence of growth factor, this was not sufficient to activate mTORC1. In contrast, mTORC1 was potently activated by a combination of BDNF and photoactivation. The data demonstrate that two important triggers of synaptic plasticity, BDNF and NMDA receptors, synergistically power the two arms of the mTORC1 activation mechanism, i.e., mTORC1 translocation to LEs and Rheb activation. Moreover, they unmask a functional link between NMDA receptors and mTORC1 that could underlie the changes in the synaptic proteome associated with long-lasting changes in synaptic strength.

Project description:Screening of cDNA expression libraries with labelled calmodulin (CaM) as a probe resulted in the isolation of cDNA encoding proteins designated CAMTA (for calmodulin-binding transcription activators). The Arabidopsis genome contains 6 members of this protein family (AtCAMTA1-6) all containing in addition to a defined CaM-binding domain a DNA-binding domain and ankyrin-repeat motifs. RT-PCR analysis revealed that all 6 genes are expressed in all organ throughout plant development. Therefore functional regulation of AtCAMTA proteins is likely mediated by second messengers (e.g. calcium/calmodulin signalling) and protein levels rather than by or in addition to gene expression levels. Based on domain organisation and sequence homologies we identified putative members of this protein family in C. elegans and in human. A yeast system was used to express chimeric fusion proteins comprised of the DNA-binding domain of the bacterial LexA protein with various segments of AtCAMTA1. This analysis revealed a distinct domain of AtCAMTA1 capable of activating transcription. Similar results were obtained with two human CAMTA homologues. To identify the gene targets of CAMTA proteins in Arabidopsiswe plan to analyse the transcriptome in loss_of_function and gain_of_function AtCAMTA mutants. For this we have already isolated a T-DNA insertion mutant of AtCAMTA1 (two alleles) and have requested two other insertion mutants of AtCAMTA2 and AtCAMTA3 identified in other labs. Screening for insertion mutants in the three remaining genes is underway. In addition we have initiated a gain_of_function approach in which DL10 proteins are expressed under the control of the dexamethasone-inducible promoter. Genes whose expression will be found modulated in the mutant plants will be considered candidate targets of AtCAMTA proteins. This analysis will complement an in vitro study of DNA-protein interactions to identify target-binding sites (part of the BBSRC funded project). As a first step in the project proposed to GARNet we suggest to compare the transcriptome in WT whole plants (2 weeks old) with that of three T-DNA insertion AtCAMTA mutants and one line expressing AtCAMTA1 under the control of an inducible promoter. Keywords: strain_or_line_design

Dataset Information

Regulation of mTORC1 by lysosomal calcium and calmodulin.

Publications

Regulation of mTORC1 by lysosomal calcium and calmodulin.

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