Characteristic of mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture

mTOR kinase is one of the basic links at the crossroad of several signal transduction pathways. De re gulated mTOR kinase signaling accompanies the progress of cancer, diabetes, neurodegenerative disorders and aging. Implication of mTOR inhibitor rapamycin decreases migration and invasion of malignant cells, and metastasis formation. However, a precise mechanism of the regulation of cellular locomotion by mTOR kinase is not fully understood. This article focuses on the recent fi ndings that demonstrated a possible role of mTOR kinase in the regulation of cytoskeleton remodeling and cell migration properties. Detailed studies on this non-canonical mTOR function will extend our knowledge about cell migration and metastasis formation and might improve anti-cancer therapeutic approaches.


Introduction
The mTOR (mammalian target of rapamycin) kinase is a central link of several signaling pathways that integrates the signals from growth factors, hor mones, stress, energy status, and amino acids to con trol the organism growth, homeostasis and aging.mTOR acts through two functionally and struc turally distinct complexes, named mTORC1 and mTORC2 (mTOR complex 1 and 2).
Taken together, active mTOR complexes stimulate the cellular growth and proliferation by positive regulation of transcription, translation, riboso me biogenesis, cell survival, inhibition of autopha gy and apoptosis [1].Overactivation of the mTOR kinase was found in a range of human diseases, such as different types of cancer, type 2 diabetes, obesity, and neurodegenerative disorders.The re fore, mTOR is considered as a perspective target of anti-cancer and anti-aging therapies [2].
One of the most dangerous stages of oncogenesis is the metastasis formation.At this stage the tumor is considered malignant.It is known, that the primary tumor causes death only in 10 % of the patients, whereas 90 % of deaths are caused by metastases [3].The metastasis formation is directly dependent on the cell motility and invasion, which allow the cells to change a position within the tissues.It was shown, that known mTOR inhibitor rapamycin and its synthetic analogs can demonstrate not only cytostatic effects, but a decrease in the motility of cancer cells as well [4,5].However, the mechanism of the regulation of cell migration and invasion by mTOR kinase is not fully understood.
The cytoskeleton, a cytoplasmic system of fi bers, is critical to sustain cell motility.Intermediate fi laments, actin-containing microfi laments and microtubules are the three main cytoskeletal systems of vertebrate and many invertebrate cells.The rearrangements of the cytoskeleton architecture are the main reason of the cell locomotion [6].Early studies revealed that mTORC2 regulates the actin cytoskeleton polarization in yeasts.Moreover, further research showed that inactivation of both mTOR complexes impairs the movement of normal and cancer cells [7].This article focuses on the recent studies revealing the role of mTOR kinase in the cytoskeleton remodeling and cell locomotion.

Structure and functions of mTOR kinase complexes
TOR (Target of Rapamycin) is a serine/threonine protein kinase, the activity of which is inhibited specifi cally by macrolide rapamycin (the other name sirolimus).Rapamycin, produced by fi lamentous bacterium Streptomyces hygroscopicus, was initially found in the soil sample of the Easter Island (Rapa Nui) in 1970s and subsequently discovered to have antifungal, immunosuppressive and cytostatic effects.Biochemical studies and genetic scree ning in the yeast mutants, resistant to the growth-inhibitory properties of rapamycin, led to the identifi cation of TOR kinase [8].Interestingly, rapamycin does not directly inhibit TOR, but it forms a complex with cytosolic protein FKBP12 (FK506 binding protein 12 kDa, the other name FKBP1A).The rapamycin-FKBP12 complex binds to the C terminal part of TOR molecule, termed FRB (FKBP12rapamycin binding domain), thereby in hibiting TOR kinase activity and functions [9].Further studies revealed the homologues of yeast TOR in the fl ies (Drosophila me lanogaster), worms (Caenorhabditis elegans), fun gus Cryptococcus neoformans, plants (Arabi dop sis tha liana) and mammals.That indicates a high evolutionary conservatism of the kinase, and hence its im por tant role in the regulation of intracellular processes.
It should be noted that although mTOR originally stood for 'mammalian TOR', it is now also used offi cially as an abbreviation for 'mechanistic TOR'.Unfortunately, sometimes the expression 'me cha nistic TOR' is used to indicate TOR kinase from any species that brings some confusion in the fi eld [10].To prevent further confusion we will use the term mTOR when discussing kinase in mammalian organisms.
In mammalian cells mTOR is a catalytic compo und of two different complexes mTORC1 and mTORC2, which coordinates anabolic and catabolic processes in response to growth factors and nutrients.The mTORcontaining complexes have different sensitivities to rapamycin as well as upstream regulators and downstream targets.

Components and substrates of mTOR complex 1
The most studied complex is mTORC1.It consists of mTOR, Raptor (regulatory-associated protein of mTOR), mLST8 (mammalian lethal Sec13 protein 8), PRAS40 (proline-rich kt substrate 40 kDa), Deptor (DEP-domain-containing mTOR-interacting protein), and the Tti1/Tel2 complex.Raptor and PRAS40 are unique components of mTORC1.The known functions of mTOR partner proteins are listed in the Table 1 [11].Cryo-electron studies revealed that mTORC1 is an obligate dimer with an overall rhomboid shape and a central cavity.It was shown that the dimeric interfaces were formed by interlocking interactions between the mTOR and Raptor subunits.It was also proposed that some mTORC1 substrates with multiple phosphorylation sites could shuttle between the two mTOR active sites within the dimer [12].mTORC1 acts as a signal integrator for four major regulatory inputs: nutrients, growth factors, energy and stress.Growth factors and hormones regulate mTORC1 through several different signaling pathways, such as PI3K/Akt network, Ras-Raf-MAPK/ Erk signaling and Wnt pathway.The implication of multiple growth factor-initiated pathways in mTORC1 regulation is likely to allow mTOR to participate in many developmental and physiological processes [1].
The most studied upstream regulators of mTORC1 are the elements of PI3K/Akt/mTOR signaling network: PI3K (phosphatidylinositol-3-kinase), Akt (the other name PKB, protein kinase B), TSC1-TSC2 (tu berous sclerosis complex 1 and 2) and small GTPase RHEB (Ras homolog enriched in brain).Binding growth factors to the receptor tyrosine kinases initiates the production of the second messenger PIP3 (phosphatidylinositol 3,4,5 triphosphate) by PI3K.This lipid serves as plasma membrane docking site for Akt.Recruiting Akt to the membrane induces its phosphorylation and activation.In turn, activated Akt phosphorylates TSC2 (also known as tuberin), a large protein that, together with TSC1 (also known as hamartin), forms the TSC1-TSC2 complex.TSC1-TSC2 acts as a GTPase activating protein (GAP) for RHEB and promotes its loading with GDP.Aktmediated phosphorylation of TSC2 inhibits GAP activity of the TSC1-TSC2 complex and induces RHEB to bind GTP.The GTP-bound form of Rheb directly interacts with mTORC1 and strongly stimulates its kinase activity [13,14].
Nutrients activate mTORC1 through amino acids availability.Import of the amino acids causes small Rag GTPases to switch to the active conformation.The active Rag heterodimer physically interacts with Raptor, causing mTORC1 to cluster onto the surface of late endosomes and lysosomes, where the Rag GTPases reside.This relocalization enables mTORC1 to interact with its activator RHEB [15,16].
In mammalian cells the most extensively studied substrates of mTORC1 are S6Ks (ribosomal protein S6 kinases) and 4E-BP1 (eIF4E-binding protein 1).The main function of these proteins is the regulation of mRNA translation initiation and progression, thus controlling the rate of protein synthesis.Previous studies have shown that both proteins contain TOS (TOR signaling) motifs.Mu tations in the amino acid sequence of the TOS motif signifi cantly reduces the level of phosphorylation of S6K and 4E-BP1 under in vitro conditions, due to the impaired ability of these proteins to interact with Raptor.Besides the canonical function of the mentioned mTOR targets, they are involved in the regulation of cell viability, migration, cytoskeleton remodeling etc. [17][18][19].

Components and substrates of mTOR complex 2
Com pared to mTORC1, less is known about mTORC2.It is insensitive to amino acids, but responds to growth factors through a poorly defi ned mechanism.
mTORC2 is formed by mTOR, Rictor (rapa mycin-insensitive companion of mTOR), mLST8, Deptor, mSIN1 (mammalian stress-activated pro tein kinase interacting protein) and Protor-1 (pro tein observed with Rictor-1, also known as PRR5).Ric tor, mSin1 and Protor-1 are unique components of mTORC2.The known functions of mTORC2 proteins are listed in the Table 1.In yeasts, TORC2 is Unknown function.The loss of mLST8 does not affect mTORC1 activity towards its substrates Tti1/Tel2 complex Scaffold proteins, which regulate mTORC1 assembly and stability mTORC2 mTOR Serine/threonine kinase, catalytic subunit of the complex Rictor Scaffold protein, regulates the assembly and substrates binding of mTORC2.Unique component of mTOR complex 2. mSin1 Scaffold protein regulating the assembly, stability of mTORC2 and its interaction with SGK1.Unique component of mTOR complex 2. Protor-1 Increases mTORC2-mediated activation of SGK1.Unique component of mTOR complex 2.

Deptor mTOR inhibitor mLST8
Unknown function.The loss of mLST8 does not affect mTORC1 activity towards its substrates Tti1/Tel2 complex Scaffold proteins, which regulate mTORC1 assembly and stability oligomeric and forms homodimers, but whether mTORC2 can form dimers/multimers in ma m ma lian cells is unknown.mTORC2 is insensitive to acute rapamycin treatment.However, chro nic treat ment with rapamycin inhibits mTORC2 functions in many cell lines, possibly, by sequestration of all mTOR molecules and, therefore, prevention of the de novo mTORC2 assembly [20].
It was revealed, that mTORC2 substrates are the AGC kinase family members, such as: Akt, cPKCs (conventional protein kinases C) and SGK (serumand glucocorticoid-regulated kinase).Thro ugh these kinases mTORC2 takes part in the regulation of cell survival, cell cycle progression and anabolism [1,20].

Participation of mTOR kinase in the regulation of cytoskeleton reorganization
The initial characterization of mTORC2 led to the discovery of the participation of mTOR signaling in actin cytoskeleton polarization and cell movements [21].Recent studies revealed that both complexes: mTORC1 and mTORC2 play a crucial role in the processes of cell motility and invasion through regulation of cytoskeleton remodeling [22].

Interplay between mTOR kinase and intermediate fi laments
Intermediate fi laments (IFs) form an extensive cytoskeletal network within the cell (Fig. 1, A).
The subunits composing intermediate fi laments constitute a superfamily of α-helical proteins that are found in the cytoplasm of different tissues and on the nuclear membrane.In humans, there are at least 67 genes that encode IF proteins, which ma kes this gene family one of the largest in the human genome.The various members of the intermediate fi lament protein family are expressed differentially in complex patterns during embryonic development and in the terminally differentiated tissues.So, this superfamily has been divided into fi ve distinct types on the basis of similarities in sequence and their patterns of expression in cells (Table 2) [6,23,24].
Phosphorylation rate plays an important role in the assembly and disassembly of the intermediate fi laments.Hyperphosphorylation of multiple sites of the IFs during mitosis causes rapid disassembly of the fi laments and their separation to the daughter cells.Recent studies also showed high level of fl exibility of the IFs even in stationary interphase cells.These fi ndings suggest that dynamic of IF cytoskeleton remodeling is under the control of kinases and phosphatases [23,25].
For a long time, IFs have been considered as components of the cell that maintain the cellular shape and provide resistance to mechanical stress.However, a lot of recent studies revealed novel non-canonical functions of intermediate fi lament proteins.For example, it was shown that keratins mediate localization of the hemidesmosomes and desmosomes in the human keratinocytes.Dep le tion of all keratins by genome engineering caused altered distribution of the hemidesmosomal proteins, which resulted in a faster adhesion and migration of keratin-free cells [26].Moreover, analyses of vimentin −/− mice have revealed that loss of vimentin leads to impaired wound healing due to defects in the capacity of fibroblasts to migrate [27].These fi ndings support a hypothesis that intermediate fi laments play important role in cell motility and that altered regulation of Characteristic of mTOR signaling and its involvement in the regulation of cell movements IFs assembly could be involved in cancer cell spreading.Also, intermediate fi laments take part in the apoptosis regulation and cell signaling.
Studies on the keratin 17 (K17)-null mouse skin keratinocytes revealed that K17 regulates cell growth and size through mTOR signaling.Keratin 17 is an intermediate filament protein rapidly induced in wounded stratified epithelia that alters cellular viscoelastic properties and optimizes tissue repair.Mouse skin keratinocytes lacking K17 show dep ressed protein translation and are of smaller size, correlating with decreased Akt/mTOR signaling activity.It was discovered that K17 regulates mTOR activation through binding to the adaptor protein 14-3-3σ.Two amino acid residues located in the amino-terminal head domain of keratin 17 are required for the serum-dependent relocalization of 14-3-3σ from the nucleus to the cytoplasm, and for the sti mulation of mTOR activity and cell growth [28].
Another evidence of the cooperation between IFs and mTOR kinase comes from the research of the transgenic mice lacking the entire keratin multiprotein family.All keratin-null embryos die from severe growth retardation at embryonic day 9.5.Em bryonic epithelia suffer no cytolysis but display mislocalized desmo-somes and glucose transporters GLUT1 and GLUT3.An altered localization of glucose transporters subsequently activates the energy sensor adenosine monophosphate kinase (AMPK).AMPK is a negative mTORC1 regulator, it inactivates mTOR signaling, thereby represses protein biosynthesis in keratin-null embryos [29].
Treatment of a human HaCaT keratinocyte cell line with mTOR inhibitors (rapamycin, temsirolimus or everolimus) resulted in selective keratin 6a (K6a) repression.Furthermore, treatment of the HaCaT cell line with the siRNAs targeting components of the mTOR pathway altered the levels of K6a expression.Oral rapamycin administration also improves the symptoms in pachyonychia congenita patients, suggesting mTOR inhibitors may be a therapeutic option for people with mutations that disrupt the intermediate fi laments formation.The se results show a possible bidirectional interplay between mTOR kinase and intermediate fi lament proteins [30].
It is known that the site-specifi c phosphorylation of IF proteins induces the disassembly of the fi lament structures.During mitosis, the hyperphos phorylation of intermediate fi laments by Cdk1 (Cyc lindependent kinase 1), Plk1 (Polo-like kinase 1), Rhoand Aurora-B kinases is essential for the effi cient segregation of IF networks into daughter cells [31].However, it was revealed that IF network is also a highly mobile structure in the interphase cells and its remodeling is under the control of protein kinases and phosphatases, such as protein kinase C (PKC) and protein kinase A (PKA) [25].

Regulation of actin cytoskeleton reorganization by mTOR kinase
A globular protein actin forms microfi laments, which are 7 nm in diameter polar fi brils that organize an extensive network in the cytoplasm of all eukaryotic cells (Fig. 1, B). Actin can be present as either a free monomer G-actin (globular) or a part of a linear polymer microfi lament called F-actin (fi lamentous).All actin subunits in the microfi lament point toward the same end of the fi lament.Actin fi lament exhibits polarity: the end that possesses an actin subunit that has its ATP binding site exposed is called the «(-) end», whereas the opposite end where the cleft is directed at a different adjacent monomer is called the «(+) end».The assembly of G-actin into F-actin is accompanied by the hydrolysis of ATP.Actin participates in many important cellular processes, including muscle contraction, cell motility, cell division, vesicle and organelle traffi c, and the establishment and maintenance of cell junctions [6,32].
Early studies on TOR kinase revealed that deletion of TOR2 disrupted the polarized organization of the actin cytoskeleton in yeasts.In mammalian cells mTORC2 also seems to regulate the remodulation of actin cytoskeleton [21].Knockdown of mTOR, Ric tor or mLST8 in the serum-starved NIH 3T3 fi broblast cells resulted in the defective F-actin fi bres formation in response to serum, whereas knockdown of raptor did not affect actin polymerization and cell spreading.Additionally, disruption of mTORC2 reduced phosphorylation of the focal adhesion proteins, as well as F-actin reorganization and cell motility [33,34] The actin cytoskeletal rearrangements are regulated by intracellular signaling pathways directed by Rac (Ras-related C3 botulinum toxin substrate), Rho (Ras homolog gene family), and Cdc42 (Cell division control protein 42 homolog), all Ras-like molecules belonging to the GTPase superfamily of switch proteins [6].So, it was interesting whether mTOR could infl uence actin cytoskeleton architecture through these proteins.Indeed, further research showed that in yeasts TOR2 activated Rho1 and Rho2 via their exchange factor ROM2 (Rho1 guanine nucleotide exchange factor 1).However, the actual mechanism by which TORC2 regulates the Rho1 GTPase pathway is not well studied [35,36].
Depletion of mTOR and Rictor, but not Raptor, impairs actin polymerization, leading edge establishment, and directional migration in neutrophils stimulated with chemoattractants.It was shown that depletion of Rictor inhibits Rac and Cdc42 activities, supposing that they are the target of mTORC2.Interestingly, depletion of mSin1, an integral component of mTORC2, caused no detectable changes in neutrophil polarity and chemotaxis [37,38].
Several recent studies pointed to the mTORC2 involvement in the formation of long-term memory by regulating and stabilizing the actin cytoskeleton in the dendritic spines of neurons.Rictor-defi cient mice showed a reduction in the ratio of fi brilar actin (F-actin) to actin monomers, as well as a reduction in the expression of a number of upstream positive regulators of actin polymerization.These data suggested that mTORC2 is required for the long-term memory formation by increasing the F-actin important for dendritic spine growth and remodeling [39,40].Characteristic of mTOR signaling and its involvement in the regulation of cell movements Current research revealed that mTORC1 also could be implicated in the actin cytoskeleton reassembly in different cells.It was shown that rapamycin treatment induced S6K inactivation, inhibited actin stress fi ber formation and cell migration in a wide range of mammalian cell lines.Further studies discovered that S6K, Akt, PDK1, and activated mTOR were localized to the actin arc of the Swiss 3T3 fi broblasts [10].Rapamycin treatment blocked the epidermal growth factor (EGF)-induced actin arc formation in these cells, supporting a hypothesis, that mTORC1/S6K axis is also important for the cytoskeleton regulation [41].It was observed that rapamycin inhibited IGF-I-induced F-actin reorganization and phosphorylation of the focal adhesion proteins, such as FAK (Focal adhesion kinase), paxillin and p130Cas, by inhibition of the S6K1 activity [7,33].Knockdown of mTORC1 and mTORC2 induced a mesenchymal-epithelial transition in the colorectal cancer cells, due to increased cell-cell contacts as well as decreased actin cytoskeletal remodeling and decreased activation of the small GTPases, RhoA and Rac1 [36].It supports the idea that mTOR could regulate cytoskeleton rearrangement through phosphorylation of the actin-remodulating proteins.
The present study showed that activated PI3K-Akt-mTOR signaling pathway promotes invasion and metastasis in hepatocellular carcinoma though up-regulation of MMP-9 (Matrix metalloproteinase 9), though, indicating that mTORC1 could infl uence cellular locomotion by several distinct directions [42,43].

The crosstalk between mTOR kinase and microtubules
A microtubule is a polymer of globular tubulin subunits, which are arranged in a cylindrical tube measuring 25 nm in diameter -the thickest fi brils of the cytoskeleton (Fig. 1, C).Similar to F-actin a microtubule is polarized and has (+) end and (-) end.Po lymerization of the tubulin subunits requires the hydrolysis of the GTP molecules.In addition to regulation of the cell motility, microtubules play a major role in organization of the cell polarity through a special structure called the microtubule-organizing center (MTOC).Loca ted near the nucleus, the MTOC directs the assembly and orientation of microtubules, the ro u te of vesicle tra ffi cking, and the orientation of organelles.Mic ro tubules play a crucial role during mitosis by the formation of mitotic spindle, which is used to separate eukaryotic chromoso mes.A large number of proteins infl uences the assembly and stability of microtubules and their association with other cell structures.These proteins are collectively called microtubule-associated prote ins (MAPs) [6,32].
Involvement of TOR1 and TOR2 in the control of various aspects of microtubule dynamics was reported in yeasts [44].However, the role of TORs in the regulation of microtubule dynamics has not been fully elucidated yet.
In mammalian cells mTOR was found to bind directly to and phosphorylate cytoplasmic linker protein of 170 kDa (CLIP-170), which is a MAP that binds to the (+) end of the microtubule and stabilizes it [45].However, the exact function of this phosphorylation is not fully understood.It was revealed that TSC2 knockout resulted in a greater abundance of stabilized microtubules underneath the cellular cortex.Time-lapse imaging of dynamic microtubules al so revealed disorganized movements of the growing microtubule plus-ends in the cellular cortex region, including growth in a direction that is parallel to the cortex.The authors suggested that the functional mTOR-CLIP-170 interaction helps microtubules grow to the cellular cortex [46].
It was shown that mTOR kinase is also involved in the regulation of intracellular transport associated with microtubules.Inhibition of the expression of TSC2, which is involved in the activation of mTOR, leads to disruption of the caveolin (scaffold protein, involved in the endocytosis) transport to the plasma membrane.Instead, it was detected in the vesicles, randomly located in the cytoplasm.Incubation of rat fi broblasts in the media that contain high concentrations of mTOR inhibitor rapamycin led to the same result, and chaotic arrangement of microtubules in the cortical zone was observed in the cells [47].
Interestingly, mTORC1-tubulin relations were observed to be bidirectional: the mTORC1 activation requires dynein-dependent transport to a position in the cell where it can be activated [48].The association between dynein and mTOR was shown by coim-munoprecipitation.Inhibition of dynein fun ction using RNAi hinders the mTORC1 activity in the human fi broblasts and the human glioblastoma-astrocytoma cell line U373-MG [48].
Moreover, the phosphorylated form of mTOR kinase (phospho-mTOR Ser2481) was observed to localize at the cleavage furrow of different cell lines during cytokinesis.Inhibition of the polymerization of microtubules by nocodazole leads to the loss of phospho-mTOR (Ser2481) ability to target the spindle midzone and the cleavage furrow during cytokinesis.At these conditions phospho-mTOR was randomly dispersed across the entire mitotic cytoplasm, indicating that mitotic traveling of phospho-mTOR (Ser2481) requires dynamic microtubules [49].
Using anti-phospho-mTOR (Ser2481) antibodies (Merck Millipore) we revealed the colocalization of phospho-mTOR (Ser2481) and tubulin β at the cleavage furrow that has not been demonstrated earlier (Fig. 2).Immunofl uorescent analysis was performed as described [50].Our fi ndings support a hypothesis that mTOR phosphorilated at Ser 2481 interacts with microtubules during cytokinesis.However, further studies are needed to understand the mechanism of this process.

Conclusion
Novel fi ndings in the mTOR signaling fi eld shed light on the non-canonical functions of the mTOR kinase.The bidirectional crosstalk bet ween mTOR and all three types of the cytoskeleton points to the important role of mTOR signaling pathway in the normal cell locomotion during embryonic development, wound healing, and che mo taxis as well as in the cancer cells spreading.Further detailed investigation of this new aspect of the mTOR activity might lead to the optimization of the current anti-cancer therapeutic approaches.К л юч е в ы е с л ов а: mTOR сигналинг, рапамицин, перестройка цитоскелета, промежуточные филаменты, микротрубочки, метастазирование.

Table 1 . The known functions of mTORC1 and mTORC2 proteins
mTORSerine/threonine kinase, catalytic subunit of the complex mTORC1 Raptor Scaffold protein, regulates the assembly, substrates binding and localization of mTORC1.Unique component of mTOR complex 1. PRAS40 mTOR inhibitor.Unique component of mTORC 1.