Mol. Cells 2020; 43(8): 705-717
Published online August 7, 2020
https://doi.org/10.14348/molcells.2020.0089
© The Korean Society for Molecular and Cellular Biology
Correspondence to : jkc@snu.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
While the growth factors like insulin initiate a signaling cascade to induce conformational changes in the mechanistic target of rapamycin complex 1 (mTORC1), amino acids cause the complex to localize to the site of activation, the lysosome. The precise mechanism of how mTORC1 moves in and out of the lysosome is yet to be elucidated in detail. Here we report that microtubules and the motor protein KIF11 are required for the proper dissociation of mTORC1 from the lysosome upon amino acid scarcity. When microtubules are disrupted or KIF11 is knocked down, we observe that mTORC1 localizes to the lysosome even in the amino acid-starved situation where it should be dispersed in the cytosol, causing an elevated mTORC1 activity. Moreover, in the mechanistic perspective, we discover that mTORC1 interacts with KIF11 on the motor domain of KIF11, enabling the complex to move out of the lysosome along microtubules. Our results suggest not only a novel way of the regulation regarding amino acid availability for mTORC1, but also a new role of KIF11 and microtubules in mTOR signaling.
Keywords Drosophila, KIF11, lysosome, microtubule, mTORC1
A cell must decide whether it is the right time to run anabolic pathways for growth and proliferation according to its internal and external status. The mechanistic target of rapamycin complex 1 (mTORC1) lies at the center of the decision (Kim et al., 2013; 2019), and a myriad of signals including hormones and nutrients are converged to mTORC1, which promotes cell growth and division. When activated, mTORC1 directly phosphorylates ribosomal protein S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (Burnett et al., 1998; Chung et al., 1992). The phosphorylated and activated S6K phosphorylates ribosomal protein S6 (RpS6), whereas 4E-BP1 phosphorylation by mTORC1 frees the eukaryotic initiation factor 4E from its negative regulator, 4E-BP1.
Numerous signals that are responsible for regulating the activity of mTORC1 are relayed to a small G protein Ras homolog-enriched in the brain (RHEB) and its negative regulator TSC complex, which functions as a GTPase-activating protein of RHEB (Inoki et al., 2003). RHEB directly binds to mTORC1 to induce a global conformational change that increases its catalytic activity (Yang et al., 2017). Insulin growth factor signaling is probably one of the most-studied upstream cues, which upon activation frees RHEB from the TSC complex by Akt-dependent phosphorylation (Inoki et al., 2002; Menon et al., 2014).
Apart from the growth factor signaling, amino acids are required to fully activate mTORC1 (Hara et al., 1998; Wang et al., 1998). Unlike hormones that affect mTORC1 by inducing protein phosphorylation, the presence of amino acids triggers the movement of the complex from the cytosol to the lysosome, the subcellular organelle where RHEB is constantly located. The main players that recruit mTORC1 to the lysosome are Rag GTPases (Nguyen et al., 2017; Sancak et al., 2010). RagA or B form a heterodimer with RagC or D, and the Rag complex is localized to the lysosome by the Ragulator complex. The Ragulator complex is a protein complex consisting of five subunits, which is attached to the cytosolic surface of the lysosome and binds to the C-terminal domains of RagA/B and RagC/D. When amino acids are present, RagA/B in the Rag complex is in GTP-bound form whereas RagC/D is GDP-bound. The Rag complex composed of GTP-bound RagA/B and GDP-bound RagC/D possesses a higher affinity with mTORC1 compared to the oppositely composed complex. Through extensive biochemical studies, some protein complexes lying upstream of the Rag complex are discovered including GTPase-activating proteins toward Rags complex (GATOR) 1 and 2 (Bar-Peled et al., 2013;Panchaud et al., 2013; Peng et al., 2017; Wolfson et al., 2017). Notably, not all amino acids are of equal importance, and the sensors for key signaling amino acids for activating mTORC1 have been revealed recently (Chantranupong et al., 2016; Saxton et al., 2016; Wolfson et al., 2016).
Microtubules and motor proteins are crucial for a variety of cellular events from maintaining cell structures and shape to cargo trafficking and cell division (Lee et al., 2019). Two members of the tubulin superfamily, α- and β-tubulins are the building blocks for microtubules, which are polymerized and depolymerized dynamically. Moving along microtubules are motor proteins, which walk through microtubules with their motor domains, for trafficking cargos or appropriately positioning microtubules during cell division. The motor proteins walking on microtubules fall into two kinds, kinesin motors which move from the minus-end to the plus-end (Porter et al., 1987; Vale et al., 1985) and dynein motors which move in the opposite direction (Paschal and Vallee, 1987) with several exceptional kinesins which move toward the minus-end (McDonald et al., 1990; Middleton and Carbon, 1994; Noda et al., 2001; Tseng et al., 2018; Walker et al., 1990).
Motor proteins are required for the movement of large protein complexes and intracellular components such as subcellular organelles. Both microtubules and actin filaments act as a track for the cargos to move along for long-distance and local transport, respectively (Gross, 2004). With each motor protein having its partner cargos, 45 kinesin-coding genes are classified into 14 families in mammals, and there are over 30 genes encoding dynein proteins reported to the current date (Hirokawa et al., 2009).
Motor proteins are also critical for cell division, as they participate in mitotic spindle aligning. One of the well-known mitotic motor proteins is KIF11, a member of the kinesin 5 families (Lawrence et al., 2004). KIF11 mainly regulates the assembly of the mitotic spindle (Cole et al., 1994; Enos and Morris, 1990; Slangy et al., 1995). KIF11 forms a bipolar homo-tetramer, which allows it to help two anti-parallel microtubules to be cross-linked, aligned, and able to glide appropriately with respect to each other (Kapitein et al., 2005; Kashlna et al., 1996; Sharp et al., 1999). Another study, however, suggested a different role of KIF11 in intracellular trafficking. In the study, KIF11 was proved to be involved in delivering CARTS (carriers of the trans-Golgi network [TGN] to the cell surface) from the TGN to the cell surface in non-mitotic cells (Wakana et al., 2013).
In a genetic screen for searching a previously unknown regulator of mTORC1, we have found that destabilizing microtubules increases TORC1 activity in
Species:
For immunostaining
Clones of homozygous mutant cells were generated using
A single guide RNA (sgRNA) was designed to target the early region of the coding sequence of
For immunostaining mammalian cells, cells were seeded on coverslips. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, and then permeabilized with 0.1% Triton X-100. After blocking in goat serum for 1 h, slides were incubated with primary antibody for 1 h at room temperature or at 4°C overnight, washed 3 times with phosphate-buffered saline (PBS), and then incubated with FITC- or TRITC-conjugated secondary antibodies (1:1,000; Invitrogen, USA) and Hoechst 33342 (blue) for 1 h at room temperature. The primary antibodies against LAMP2 (1:100, sc-18822; Santa Cruz Biotechnology, USA), mTOR (1:100, #2983; Cell Signaling Technology, USA), and β-tubulin (1:100, ab6046; Abcam, UK) were used for immunofluorescence. The slides were then washed 3 times with PBS and mounted. Cell images were captured with a confocal microscope (Zeiss, Germany).
For immunostaining
HEK293E cells were cultured in DMEM (Welgene, Korea) supplemented with 10% fetal bovine serum (Invitrogen) at 37°C in a humidified atmosphere of 5% CO2. For amino acid-starvation or stimulation, cells were seeded in 9.6 cm2 6-well plates and incubated in media without or with amino acids, respectively. Cells were treated with 10 nM colchicine, 100 nM insulin, 200 nM rapamycin, 250 nM torin1 or U0126 for indicated hour(s). For immunostaining cells, cells were seeded in 3.5 cm2 12-well plates and deprived of serum for 16 h before amino acid-starvation or stimulation. Cells were seeded in 9.6 cm2 6-well plates and incubated in serum starved media for 16 h to identify serum-dependency. siRNAs for control (1003; Bioneer, Korea),
Cells were lysed in a lysis buffer (150 mM NaCl, 20 mM Tris, pH 7.4, 1 mM EGTA, 1 mM EDTA, 1% NP-40, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 10% glycerol, protease inhibitors pepstatin A, PMSF, and leupeptin). Equivalent protein quantities were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% bovine serum albumin-containing PBS for 1 h at room temperature and then probed with the indicated primary antibodies, followed by the appropriate horseradish peroxidase (HRP)-conjugated anti-mouse/rabbit secondary antibodies. Immuno-reactive bands were visualized with enhanced chemiluminescence (ECL) reagent. For immunoprecipitation assay, cells were collected and lysed in 0.5 ml lysis buffer plus protease inhibitors for 30 min at 4°C. After 12,000
All experiments were repeated at least three times, and all results were expressed as mean ± SD. Student’s two-tailed
As a previous work revealed a patterned activation of TORC1 in
As microtubules are appreciated for the movement of intracellular cargos including protein complexes, we hypothesized that disrupting microtubules would increase mTORC1 activity by having it mislocalized in the cell. As the Rag complex is responsible for the lysosomal localization of mTORC1, we expressed a dominant-negative form of RagA (RagADN) with RNAi targeted for
We moved on to another question of whether there is a motor protein responsible for the movement of mTORC1 along microtubules in the cell. Given the data indicating the lysosome-to-cytosol movement of mTORC1 through microtubules, we hypothesized that knockdown of the motor protein would increase RpS6 phosphorylation and render resistance of mTORC1 activity during amino acid starvation. We also predicted that the motor protein would have a direct physical interaction with mTORC1. To find out the motor protein involved, we have done a genetic screen using
We then tested whether the phenotype could be observed in mammalian cells. When HEK293E cells were treated with siRNA-targeting
Subsequently, mTORC1 activity was measured in mammalian cells starved by administering an amino acid-free medium with or without
We then immunostained mTOR and LAMP2, markers of mTORC1 and lysosomes, respectively, in various conditions in order to confirm that microtubules regulate the subcellular localization of mTORC1 according to amino acid availability. In the control group, as expected, the two marker proteins only co-localized when cells were stimulated by amino acids (Figs. 3A and 3B, 3F and 3G). In experimental groups, however, both
As
As we have done in the case of microtubule destabilization, the subcellular localization of mTORC1 was monitored by labeling mTOR and LAMP2 in
To further speculate how KIF11 interacts with mTORC1, we performed co-immunoprecipitation assays. In an open database BioGRID (https://thebiogrid.org/), KIF11 was reported to bind mLST8, a member of mTORC1, which led us to test the interaction between KIF11 and mLST8. Indeed, Myc-tagged mLST8 was co-immunoprecipitated with Flag-tagged KIF11 (Fig. 6A). Moreover, when we tested whether KIF11 interacts with other components of mTORC1, we found that, like mLST8, RAPTOR and mTOR were co-immunoprecipitated with KIF11 (Supplementary Fig. S5). We then sought for the region of the interaction using truncated forms of KIF11 and full-length mLST8. Interestingly, the N-terminal motor domain of KIF11 was found to be important for the interaction between KIF11 and mLST8 (Fig. 6B). These results suggest that KIF11 binds to mTORC1 through its N-terminal motor domain.
To investigate which one of the two mTOR-containing complexes is responsible for the increased phosphorylation of RpS6 and S6K upon
This study highlights the role of microtubules and a specific motor protein KIF11 on the subcellular movement of mTORC1 from the lysosome to the cytosol. The inhibition of microtubule polymerization, either done genetically or pharmacologically, results in aberrant mTORC1 activity
In the dorsal region of Drosophila eye disc, ectopic expression of RNAi targeting TBCC or Klp61F was applied for disrupting microtubules or depleting Klp61F, respectively. Interestingly, the higher TORC1 activity caused by such genetic manipulations was mainly restricted to cells in the anterior region (see Figs. 1A and 1B, 1D-1F). This different responsiveness is possible because of the different repertoire of regulation on TORC1 in the eye disc regarding the location. This is not the only case in which cells with different differentiation status responded differently to genetic manipulations intended to alter TORC1 activity. For example, when a constitutively active form of insulin receptor or myristoylated Akt was expressed clonally in the eye disc, only cells near the second mitotic wave (the cells positive for phosphorylated RpS6 in the wild-type eye disc) showed higher RpS6 phosphorylation (Supplementary Fig. S7).
As the microtubule/KIF11-dependent movement of mTORC1 enhances its activity in both normal and amino acid-starved conditions, we assume this movement occurs specifically when mTORC1 is moving out of the lysosome. This is further ensured by the fact that stimulating the starved cells by amino acids increases mTORC1 activity regardless of microtubule stability or KIF11 availability. This means that the complex coming to the lysosome was not affected by those factors. In short, the movement of mTORC1 through microtubules via KIF11 is in a one-way fashion, allowing the complex to move out of the lysosome. The microtubule- and motor protein-independent way of mTORC1 translocating from the cytosol to the lysosome would be an interesting topic to pursue in the future. In contrast to our expectation that mTORC1 would bind to the C-terminus of KIF11 like a typical cargo trafficking using a kinesin motor dimer, mLST8 was found to bind KIF11 through the motor domain (Fig. 6B). As KIF11 is known to work in a homo-tetrameric manner for the mitosis (Mann and Wadsworth, 2019), we suspect that when trafficking mTORC1, one end of tetramer would bind to the microtubule and the other to mLST8. Especially, electron microscopy explaining the binding of the two would provide valuable information. Indeed, a research indicated that the bipolar assembly (BASS) domain of KLP61F is critical in the formation of homo-tetramer (Scholey et al., 2014). This research also noticed that multiple KLP61F mutants in the BASS domain form monomer or dimer, but not tetramer. Therefore, a further research is needed to verify whether mTORC1 translocation is affected by KIF11/KLP61F mutants which are unable to form tetramers.
The physiological meaning of the movement of mTORC1 by the motor protein KIF11 is yet elucidated. Since mTROC1 activity control involves a change in mTORC1 localization between the lysosome and the cytosol according to amino acid availability, the simplest answer for why mTORC1 is transported between these spaces through microtubules would be by detaching the complex from its activation base camp. There are, however, other possibilities as well. According to recent studies, the subcellular localization of mTORC1 does not perfectly correspond to its regulation by amino acids (Manifava et al., 2016). In the article, the authors showed that when amino acid stimulation induces lysosomal localization of mTORC1, the complex gets out of the lysosome quickly, and mTORC1 activity did not fall immediately after the detachment. This suggests the localization of the complex has more to do than just activity regulation. We, therefore, cannot rule out the possibility that mTORC1 is transported to a place other than lysosomes for a purpose that is not activity regulation.
Collectively, this study consistently indicates that microtubules and KIF11 are required for a proper movement of mTORC1 from the lysosome to the cytosol in response to amino acid starvation and mTROC1 hyperactivation upon the blockage of the transport system. As there is some room for further investigation of this phenomenon, further studies will provide valuable information on how this critical signaling pathway is delicately regulated by changes in subcellular localization of mTORC1.
We would like to thank Dr. Hongyan Wang for kindly providing the
J.C. funding acquisition; J.C. and Y.G.J. conceptualization; J.C. supervision; Y.G.J. and Y.C. investigation; Y.G.J. and Y.C. visualization; Y.G.J. and Y.C. writing - original draft; J.C., Y.G.J., Y.C., and K.J. writing - review & editing.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2020; 43(8): 705-717
Published online August 31, 2020 https://doi.org/10.14348/molcells.2020.0089
Copyright © The Korean Society for Molecular and Cellular Biology.
Yoon-Gu Jang1,2,3 , Yujin Choi1,2,3
, Kyoungho Jun1,2
, and Jongkyeong Chung1,2,*
1Institute of Molecular Biology and Genetics, Seoul National University, Seoul 08826, Korea, 2School of Biological Sciences, Seoul National University, Seoul 08826, Korea, 3These authors contributed equally to this work.
Correspondence to:jkc@snu.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
While the growth factors like insulin initiate a signaling cascade to induce conformational changes in the mechanistic target of rapamycin complex 1 (mTORC1), amino acids cause the complex to localize to the site of activation, the lysosome. The precise mechanism of how mTORC1 moves in and out of the lysosome is yet to be elucidated in detail. Here we report that microtubules and the motor protein KIF11 are required for the proper dissociation of mTORC1 from the lysosome upon amino acid scarcity. When microtubules are disrupted or KIF11 is knocked down, we observe that mTORC1 localizes to the lysosome even in the amino acid-starved situation where it should be dispersed in the cytosol, causing an elevated mTORC1 activity. Moreover, in the mechanistic perspective, we discover that mTORC1 interacts with KIF11 on the motor domain of KIF11, enabling the complex to move out of the lysosome along microtubules. Our results suggest not only a novel way of the regulation regarding amino acid availability for mTORC1, but also a new role of KIF11 and microtubules in mTOR signaling.
Keywords: Drosophila, KIF11, lysosome, microtubule, mTORC1
A cell must decide whether it is the right time to run anabolic pathways for growth and proliferation according to its internal and external status. The mechanistic target of rapamycin complex 1 (mTORC1) lies at the center of the decision (Kim et al., 2013; 2019), and a myriad of signals including hormones and nutrients are converged to mTORC1, which promotes cell growth and division. When activated, mTORC1 directly phosphorylates ribosomal protein S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (Burnett et al., 1998; Chung et al., 1992). The phosphorylated and activated S6K phosphorylates ribosomal protein S6 (RpS6), whereas 4E-BP1 phosphorylation by mTORC1 frees the eukaryotic initiation factor 4E from its negative regulator, 4E-BP1.
Numerous signals that are responsible for regulating the activity of mTORC1 are relayed to a small G protein Ras homolog-enriched in the brain (RHEB) and its negative regulator TSC complex, which functions as a GTPase-activating protein of RHEB (Inoki et al., 2003). RHEB directly binds to mTORC1 to induce a global conformational change that increases its catalytic activity (Yang et al., 2017). Insulin growth factor signaling is probably one of the most-studied upstream cues, which upon activation frees RHEB from the TSC complex by Akt-dependent phosphorylation (Inoki et al., 2002; Menon et al., 2014).
Apart from the growth factor signaling, amino acids are required to fully activate mTORC1 (Hara et al., 1998; Wang et al., 1998). Unlike hormones that affect mTORC1 by inducing protein phosphorylation, the presence of amino acids triggers the movement of the complex from the cytosol to the lysosome, the subcellular organelle where RHEB is constantly located. The main players that recruit mTORC1 to the lysosome are Rag GTPases (Nguyen et al., 2017; Sancak et al., 2010). RagA or B form a heterodimer with RagC or D, and the Rag complex is localized to the lysosome by the Ragulator complex. The Ragulator complex is a protein complex consisting of five subunits, which is attached to the cytosolic surface of the lysosome and binds to the C-terminal domains of RagA/B and RagC/D. When amino acids are present, RagA/B in the Rag complex is in GTP-bound form whereas RagC/D is GDP-bound. The Rag complex composed of GTP-bound RagA/B and GDP-bound RagC/D possesses a higher affinity with mTORC1 compared to the oppositely composed complex. Through extensive biochemical studies, some protein complexes lying upstream of the Rag complex are discovered including GTPase-activating proteins toward Rags complex (GATOR) 1 and 2 (Bar-Peled et al., 2013;Panchaud et al., 2013; Peng et al., 2017; Wolfson et al., 2017). Notably, not all amino acids are of equal importance, and the sensors for key signaling amino acids for activating mTORC1 have been revealed recently (Chantranupong et al., 2016; Saxton et al., 2016; Wolfson et al., 2016).
Microtubules and motor proteins are crucial for a variety of cellular events from maintaining cell structures and shape to cargo trafficking and cell division (Lee et al., 2019). Two members of the tubulin superfamily, α- and β-tubulins are the building blocks for microtubules, which are polymerized and depolymerized dynamically. Moving along microtubules are motor proteins, which walk through microtubules with their motor domains, for trafficking cargos or appropriately positioning microtubules during cell division. The motor proteins walking on microtubules fall into two kinds, kinesin motors which move from the minus-end to the plus-end (Porter et al., 1987; Vale et al., 1985) and dynein motors which move in the opposite direction (Paschal and Vallee, 1987) with several exceptional kinesins which move toward the minus-end (McDonald et al., 1990; Middleton and Carbon, 1994; Noda et al., 2001; Tseng et al., 2018; Walker et al., 1990).
Motor proteins are required for the movement of large protein complexes and intracellular components such as subcellular organelles. Both microtubules and actin filaments act as a track for the cargos to move along for long-distance and local transport, respectively (Gross, 2004). With each motor protein having its partner cargos, 45 kinesin-coding genes are classified into 14 families in mammals, and there are over 30 genes encoding dynein proteins reported to the current date (Hirokawa et al., 2009).
Motor proteins are also critical for cell division, as they participate in mitotic spindle aligning. One of the well-known mitotic motor proteins is KIF11, a member of the kinesin 5 families (Lawrence et al., 2004). KIF11 mainly regulates the assembly of the mitotic spindle (Cole et al., 1994; Enos and Morris, 1990; Slangy et al., 1995). KIF11 forms a bipolar homo-tetramer, which allows it to help two anti-parallel microtubules to be cross-linked, aligned, and able to glide appropriately with respect to each other (Kapitein et al., 2005; Kashlna et al., 1996; Sharp et al., 1999). Another study, however, suggested a different role of KIF11 in intracellular trafficking. In the study, KIF11 was proved to be involved in delivering CARTS (carriers of the trans-Golgi network [TGN] to the cell surface) from the TGN to the cell surface in non-mitotic cells (Wakana et al., 2013).
In a genetic screen for searching a previously unknown regulator of mTORC1, we have found that destabilizing microtubules increases TORC1 activity in
Species:
For immunostaining
Clones of homozygous mutant cells were generated using
A single guide RNA (sgRNA) was designed to target the early region of the coding sequence of
For immunostaining mammalian cells, cells were seeded on coverslips. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, and then permeabilized with 0.1% Triton X-100. After blocking in goat serum for 1 h, slides were incubated with primary antibody for 1 h at room temperature or at 4°C overnight, washed 3 times with phosphate-buffered saline (PBS), and then incubated with FITC- or TRITC-conjugated secondary antibodies (1:1,000; Invitrogen, USA) and Hoechst 33342 (blue) for 1 h at room temperature. The primary antibodies against LAMP2 (1:100, sc-18822; Santa Cruz Biotechnology, USA), mTOR (1:100, #2983; Cell Signaling Technology, USA), and β-tubulin (1:100, ab6046; Abcam, UK) were used for immunofluorescence. The slides were then washed 3 times with PBS and mounted. Cell images were captured with a confocal microscope (Zeiss, Germany).
For immunostaining
HEK293E cells were cultured in DMEM (Welgene, Korea) supplemented with 10% fetal bovine serum (Invitrogen) at 37°C in a humidified atmosphere of 5% CO2. For amino acid-starvation or stimulation, cells were seeded in 9.6 cm2 6-well plates and incubated in media without or with amino acids, respectively. Cells were treated with 10 nM colchicine, 100 nM insulin, 200 nM rapamycin, 250 nM torin1 or U0126 for indicated hour(s). For immunostaining cells, cells were seeded in 3.5 cm2 12-well plates and deprived of serum for 16 h before amino acid-starvation or stimulation. Cells were seeded in 9.6 cm2 6-well plates and incubated in serum starved media for 16 h to identify serum-dependency. siRNAs for control (1003; Bioneer, Korea),
Cells were lysed in a lysis buffer (150 mM NaCl, 20 mM Tris, pH 7.4, 1 mM EGTA, 1 mM EDTA, 1% NP-40, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 10% glycerol, protease inhibitors pepstatin A, PMSF, and leupeptin). Equivalent protein quantities were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% bovine serum albumin-containing PBS for 1 h at room temperature and then probed with the indicated primary antibodies, followed by the appropriate horseradish peroxidase (HRP)-conjugated anti-mouse/rabbit secondary antibodies. Immuno-reactive bands were visualized with enhanced chemiluminescence (ECL) reagent. For immunoprecipitation assay, cells were collected and lysed in 0.5 ml lysis buffer plus protease inhibitors for 30 min at 4°C. After 12,000
All experiments were repeated at least three times, and all results were expressed as mean ± SD. Student’s two-tailed
As a previous work revealed a patterned activation of TORC1 in
As microtubules are appreciated for the movement of intracellular cargos including protein complexes, we hypothesized that disrupting microtubules would increase mTORC1 activity by having it mislocalized in the cell. As the Rag complex is responsible for the lysosomal localization of mTORC1, we expressed a dominant-negative form of RagA (RagADN) with RNAi targeted for
We moved on to another question of whether there is a motor protein responsible for the movement of mTORC1 along microtubules in the cell. Given the data indicating the lysosome-to-cytosol movement of mTORC1 through microtubules, we hypothesized that knockdown of the motor protein would increase RpS6 phosphorylation and render resistance of mTORC1 activity during amino acid starvation. We also predicted that the motor protein would have a direct physical interaction with mTORC1. To find out the motor protein involved, we have done a genetic screen using
We then tested whether the phenotype could be observed in mammalian cells. When HEK293E cells were treated with siRNA-targeting
Subsequently, mTORC1 activity was measured in mammalian cells starved by administering an amino acid-free medium with or without
We then immunostained mTOR and LAMP2, markers of mTORC1 and lysosomes, respectively, in various conditions in order to confirm that microtubules regulate the subcellular localization of mTORC1 according to amino acid availability. In the control group, as expected, the two marker proteins only co-localized when cells were stimulated by amino acids (Figs. 3A and 3B, 3F and 3G). In experimental groups, however, both
As
As we have done in the case of microtubule destabilization, the subcellular localization of mTORC1 was monitored by labeling mTOR and LAMP2 in
To further speculate how KIF11 interacts with mTORC1, we performed co-immunoprecipitation assays. In an open database BioGRID (https://thebiogrid.org/), KIF11 was reported to bind mLST8, a member of mTORC1, which led us to test the interaction between KIF11 and mLST8. Indeed, Myc-tagged mLST8 was co-immunoprecipitated with Flag-tagged KIF11 (Fig. 6A). Moreover, when we tested whether KIF11 interacts with other components of mTORC1, we found that, like mLST8, RAPTOR and mTOR were co-immunoprecipitated with KIF11 (Supplementary Fig. S5). We then sought for the region of the interaction using truncated forms of KIF11 and full-length mLST8. Interestingly, the N-terminal motor domain of KIF11 was found to be important for the interaction between KIF11 and mLST8 (Fig. 6B). These results suggest that KIF11 binds to mTORC1 through its N-terminal motor domain.
To investigate which one of the two mTOR-containing complexes is responsible for the increased phosphorylation of RpS6 and S6K upon
This study highlights the role of microtubules and a specific motor protein KIF11 on the subcellular movement of mTORC1 from the lysosome to the cytosol. The inhibition of microtubule polymerization, either done genetically or pharmacologically, results in aberrant mTORC1 activity
In the dorsal region of Drosophila eye disc, ectopic expression of RNAi targeting TBCC or Klp61F was applied for disrupting microtubules or depleting Klp61F, respectively. Interestingly, the higher TORC1 activity caused by such genetic manipulations was mainly restricted to cells in the anterior region (see Figs. 1A and 1B, 1D-1F). This different responsiveness is possible because of the different repertoire of regulation on TORC1 in the eye disc regarding the location. This is not the only case in which cells with different differentiation status responded differently to genetic manipulations intended to alter TORC1 activity. For example, when a constitutively active form of insulin receptor or myristoylated Akt was expressed clonally in the eye disc, only cells near the second mitotic wave (the cells positive for phosphorylated RpS6 in the wild-type eye disc) showed higher RpS6 phosphorylation (Supplementary Fig. S7).
As the microtubule/KIF11-dependent movement of mTORC1 enhances its activity in both normal and amino acid-starved conditions, we assume this movement occurs specifically when mTORC1 is moving out of the lysosome. This is further ensured by the fact that stimulating the starved cells by amino acids increases mTORC1 activity regardless of microtubule stability or KIF11 availability. This means that the complex coming to the lysosome was not affected by those factors. In short, the movement of mTORC1 through microtubules via KIF11 is in a one-way fashion, allowing the complex to move out of the lysosome. The microtubule- and motor protein-independent way of mTORC1 translocating from the cytosol to the lysosome would be an interesting topic to pursue in the future. In contrast to our expectation that mTORC1 would bind to the C-terminus of KIF11 like a typical cargo trafficking using a kinesin motor dimer, mLST8 was found to bind KIF11 through the motor domain (Fig. 6B). As KIF11 is known to work in a homo-tetrameric manner for the mitosis (Mann and Wadsworth, 2019), we suspect that when trafficking mTORC1, one end of tetramer would bind to the microtubule and the other to mLST8. Especially, electron microscopy explaining the binding of the two would provide valuable information. Indeed, a research indicated that the bipolar assembly (BASS) domain of KLP61F is critical in the formation of homo-tetramer (Scholey et al., 2014). This research also noticed that multiple KLP61F mutants in the BASS domain form monomer or dimer, but not tetramer. Therefore, a further research is needed to verify whether mTORC1 translocation is affected by KIF11/KLP61F mutants which are unable to form tetramers.
The physiological meaning of the movement of mTORC1 by the motor protein KIF11 is yet elucidated. Since mTROC1 activity control involves a change in mTORC1 localization between the lysosome and the cytosol according to amino acid availability, the simplest answer for why mTORC1 is transported between these spaces through microtubules would be by detaching the complex from its activation base camp. There are, however, other possibilities as well. According to recent studies, the subcellular localization of mTORC1 does not perfectly correspond to its regulation by amino acids (Manifava et al., 2016). In the article, the authors showed that when amino acid stimulation induces lysosomal localization of mTORC1, the complex gets out of the lysosome quickly, and mTORC1 activity did not fall immediately after the detachment. This suggests the localization of the complex has more to do than just activity regulation. We, therefore, cannot rule out the possibility that mTORC1 is transported to a place other than lysosomes for a purpose that is not activity regulation.
Collectively, this study consistently indicates that microtubules and KIF11 are required for a proper movement of mTORC1 from the lysosome to the cytosol in response to amino acid starvation and mTROC1 hyperactivation upon the blockage of the transport system. As there is some room for further investigation of this phenomenon, further studies will provide valuable information on how this critical signaling pathway is delicately regulated by changes in subcellular localization of mTORC1.
We would like to thank Dr. Hongyan Wang for kindly providing the
J.C. funding acquisition; J.C. and Y.G.J. conceptualization; J.C. supervision; Y.G.J. and Y.C. investigation; Y.G.J. and Y.C. visualization; Y.G.J. and Y.C. writing - original draft; J.C., Y.G.J., Y.C., and K.J. writing - review & editing.
The authors have no potential conflicts of interest to disclose.
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