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. Deregulated mTOR kinase signaling accompanies the progress of cancer, diabetes, neurodegenerative disorders and aging. Implication of mTOR inhibitor rapamycin decreases migration and invasion of maligna...
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irk-123456789-1524282019-06-12T01:25:25Z Characteristic of mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture Kosach, V.R. Cherednyk, O.V. Khoruzhenko, A.I. Reviews mTOR kinase is one of the basic links at the crossroad of several signal transduction pathways. Deregulated 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 findings 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. mTOR кіназa є однією з основних ланок, розташованих на перетині кількох шляхів внутрішньоклітинної передачі сигналу. Дерегуляція сигналінгу mTOR кінази супроводжує розвиток онкологічних захворювань, діабету, нейродегенеративних розладів і старіння. Застосування інгібітора mTOR рапаміцина знижує рівень міграції та інвазії злоякісних клітин і утворення метастазів. Однак, точний механізм регуляції рухливості клітин mTOR кіназою повністю не зрозумілий. Дана стаття присвячена останнім дослідженням, які демонструють можливу роль mTOR кінази в регуляції ре моделювання цитоскелету та міграції клітин. Докладні дослідження цієї неканонічної функції mTOR кінази дозволить розширити наші знання про міграцію клітин і утворення метастазів і може привести до поліпшення протиракових терапевтичних підходів. mTOR киназa является одним из основных звеньев, расположенных на пересечении нескольких путей внутриклеточной передачи сигнала. Дерегуляция сигналинга mTOR киназы сопровождает развитие онкологических заболеваний, диабета, нейродегенеративных расстройств и старения. Применение ингибитора mTOR рапамицина снижает уровень миграции и инвазии злокачественных клеток и образование метастазов. Однако, точный механизм регуляции подвижности клеток mTOR киназы полностью не изучен. Эта статья посвященна последним исследованиям, которые демонстрируют возможную роль mTOR киназы в регуляции ремоделирования цитоскелета и миграции клеток. Подробные исследования этой неканонической функции mTOR киназы позволят расширить наши знания о миграции клеток и образование метастазов и может привести к улучшению противораковых терапевтических подходов. 2015 Article Characteristic of mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture / V.R. Kosach, O.V. Cherednyk, A.I. Khoruzhenko // Biopolymers and Cell. — 2015. — Т. 31, № 1. — С. 5-14. — Бібліогр.: 50 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008C7 http://dspace.nbuv.gov.ua/handle/123456789/152428 576.322 577.22 en Інститут молекулярної біології і генетики НАН України |
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Reviews Reviews Kosach, V.R. Cherednyk, O.V. Khoruzhenko, A.I. Characteristic of mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture |
| description |
mTOR kinase is one of the basic links at the crossroad of several signal transduction pathways. Deregulated 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 findings 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. |
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Kosach, V.R. Cherednyk, O.V. Khoruzhenko, A.I. |
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Kosach, V.R. Cherednyk, O.V. Khoruzhenko, A.I. |
| author_sort |
Kosach, V.R. |
| title |
Characteristic of mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture |
| title_short |
Characteristic of mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture |
| title_full |
Characteristic of mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture |
| title_fullStr |
Characteristic of mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture |
| title_full_unstemmed |
Characteristic of mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture |
| title_sort |
characteristic of mtor signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture |
| publisher |
Інститут молекулярної біології і генетики НАН України |
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| citation_txt |
Characteristic of mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture / V.R. Kosach, O.V. Cherednyk, A.I. Khoruzhenko // Biopolymers and Cell. — 2015. — Т. 31, № 1. — С. 5-14. — Бібліогр.: 50 назв. — англ. |
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2025-07-13T03:05:37Z |
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5
ISSN 0233-7657
Biopolymers and Cell. 2015. Vol. 31. N 1. P. 5–14
doi: http://dx.doi.org/10.7124/bc.0008C7
Reviews
UDC 576.322 577.22
Characteristic of mTOR signaling and its involvement in the regulation
of cell movements through remodeling the cytoskeleton architecture
V. R. Kosach, O. V. Cherednyk, A. I. Khoruzhenko
Institute of Molecular Biology and Genetics, NAS of Ukraine,
150, Zabolotnoho Str., Kyiv, Ukraine, 03680
a.i.khoruzhenko@imbg.org.ua
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 ag-
ing. 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 for-
mation and might improve anti-cancer therapeutic approaches.
K e y w o r d s: mTOR signaling, rapamycin, cytoskeleton remodeling, intermediate fi laments, microtu-
bules, cancer metastasis.
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 stimu-
late the cellular growth and proliferation by positive
regulation of transcription, translation, riboso me bio-
genesis, cell survival, inhibition of autopha gy and
apoptosis [1]. Overactivation of the mTOR kinase
was found in a range of human diseases, such as dif-
ferent types of cancer, type 2 diabetes, obesity, and
neurodegenerative disorders. The re fore, mTOR is con-
sidered as a perspective target of anti-cancer and an-
ti-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, whe-
reas 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 syn-
thetic 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 regu-
lation of cell migration and invasion by mTOR ki-
nase is not fully understood.
The cytoskeleton, a cytoplasmic system of fi bers,
is critical to sustain cell motility. Intermediate fi la-
ments, actin-containing microfi laments and microtu-
bules are the three main cytoskeletal systems of ver-
tebrate and many invertebrate cells. The rearrange-
ments of the cytoskeleton architecture are the main
© 2015 V. R. Kosach et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
6
V. R. Kosach, O. V. Cherednyk, A. I. Khoruzhenko
reason of the cell locomotion [6]. Early studies re-
vealed that mTORC2 regulates the actin cytoskele-
ton 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 reveal-
ing the role of mTOR kinase in the cytoskeleton re-
modeling 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 na-
me sirolimus). Rapamycin, produced by fi lamentous
bacterium Streptomyces hygroscopicus, was ini-
tially 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-inhibi-
tory properties of rapamycin, led to the identifi ca-
tion of TOR kinase [8]. Interestingly, rapamycin
does not directly inhibit TOR, but it forms a com-
plex with cytosolic protein FKBP12 (FK506 bind-
ing protein 12 kDa, the other name FKBP1A). The
rapamycin-FKBP12 complex binds to the C termi-
nal part of TOR molecule, termed FRB (FKBP12-
rapamycin binding domain), thereby in hibiting TOR
kinase activity and functions [9]. Further studies re-
vealed 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 indi-
cates a high evolutionary conservatism of the ki-
nase, 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 of-
fi cially as an abbreviation for ‘mechanistic TOR’. Un-
fortunately, sometimes the expression ‘me cha nistic
TOR’ is used to indicate TOR kinase from any spe-
cies 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 mTOR-
containing complexes have different sensitivities to
rapamycin as well as upstream regulators and down-
stream 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), Dep-
tor (DEP-domain-containing mTOR-interacting pro-
tein), and the Tti1/Tel2 complex. Raptor and PRAS40
are unique components of mTORC1. The known fun-
ctions of mTOR partner proteins are listed in the Tab-
le 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 multi-
ple 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 path-
ways, 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 net-
work: 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 initi-
ates 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
7
Characteristic of mTOR signaling and its involvement in the regulation of cell movements
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. Akt-
mediated phosphorylation of TSC2 inhibits GAP ac-
tivity of the TSC1–TSC2 complex and induces
RHEB to bind GTP. The GTP-bound form of Rheb
directly interacts with mTORC1 and strongly stimu-
lates 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. [17–19].
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 my-
cin-insensitive companion of mTOR), mLST8, Dep-
tor, mSIN1 (mammalian stress-activated pro tein ki-
nase interacting protein) and Protor-1 (pro tein ob-
served with Rictor-1, also known as PRR5). Ric tor,
mSin1 and Protor-1 are unique components of
mTORC2. The known functions of mTORC2 pro-
teins are listed in the Table 1. In yeasts, TORC2 is
Table 1. The known functions of mTORC1 and mTORC2 proteins
mTOR Serine/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.
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
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
8
V. R. Kosach, O. V. Cherednyk, A. I. Khoruzhenko
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 (serum-
and 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 regu-
lation of cytoskeleton remodeling [22].
Interplay between mTOR kinase
and intermediate fi laments
Intermediate fi laments (IFs) form an extensive cy-
toskeletal 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 pro-
tein 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 ex-
pression 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 ex-
ibility of the IFs even in stationary interphase cells.
These fi ndings suggest that dynamic of IF cytoskel-
eton remodeling is under the control of kinases and
phosphatases [23, 25].
For a long time, IFs have been considered as com-
ponents 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 ex-
ample, it was shown that keratins mediate localiza-
tion 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 fi -
broblasts to migrate [27]. These fi ndings support a
hypothesis that intermediate fi laments play impor-
tant role in cell motility and that altered regulation of
Table 2. Classifi cation of the intermediate fi laments
Type
of IFs IFprotein Tissue distribution
Type I Acidic keratins All types of the epithe-
liaType II Basic keratins
Type III Vimentin
Desmin
Glial fi brillary acidic
protein
Peripherin
Mesenchymal cells
Muscle
Glial cells, astrocytes
Neurons
Type IV NF(Neurofi laments)-L
NF-M
NF-H
Nestin
Internexin
Syncolin
Neurons
Neurons
Neurons
Neuroepithelial stem
cells
Neurons
Muscle cells
Type V Lamins A, C, B1, B2 Nuclear lamina of all
types of the cells
Beaded
fi laments
Phakinin
Filensin
Intermediate fi laments
of the ocular lens
9
Characteristic of mTOR signaling and its involvement in the regulation of cell movements
IFs assembly could be involved in cancer cell spread-
ing. 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 vis-
coelastic properties and optimizes tissue repair. Mo-
use skin keratinocytes lacking K17 show dep ressed
protein translation and are of smaller size, correlat-
ing 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 se-
rum-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 multipro-
tein 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 trans-
porters subsequently activates the energy sensor ad-
enosine 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 expres-
sion. 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 in-
termediate fi laments formation. The se results show a
possible bidirectional interplay between mTOR ki-
nase and intermediate fi lament proteins [30].
It is known that the site-specifi c phosphorylation
of IF proteins induces the disassembly of the fi la-
ment structures. During mitosis, the hyperphos pho-
rylation of intermediate fi laments by Cdk1 (Cyc lin-
dependent kinase 1), Plk1 (Polo-like kinase 1), Rho-
and 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 di-
rected 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 par-
ticipates in many important cellular processes, in-
cluding muscle contraction, cell motility, cell divi-
sion, vesicle and organelle traffi c, and the establish-
ment 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 phos-
phorylation of the focal adhesion proteins, as well as
F-actin reorganization and cell motility [33, 34]
10
V. R. Kosach, O. V. Cherednyk, A. I. Khoruzhenko
The actin cytoskeletal rearrangements are regu-
lated by intracellular signaling pathways directed by
Rac (Ras-related C3 botulinum toxin substrate), Rho
(Ras homolog gene family), and Cdc42 (Cell divi-
sion control protein 42 homolog), all Ras-like mol-
ecules belonging to the GTPase superfamily of
switch proteins [6]. So, it was interesting whether
mTOR could infl uence actin cytoskeleton architec-
ture through these proteins. Indeed, further research
showed that in yeasts TOR2 activated Rho1 and
Rho2 via their exchange factor ROM2 (Rho1 gua-
nine 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 estab-
lishment, and directional migration in neutrophils
stimulated with chemoattractants. It was shown that
depletion of Rictor inhibits Rac and Cdc42 activi-
ties, supposing that they are the target of mTORC2.
Interestingly, depletion of mSin1, an integral com-
ponent of mTORC2, caused no detectable changes
in neutrophil polarity and chemotaxis [37, 38].
Several recent studies pointed to the mTORC2 in-
volvement 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 reg-
ulators 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].
Fig. 1. The architecture of different types of the cytoskeleton fi bers in the human breast adenocarcinoma MCF-7 cell line: intermedi-
ate fi laments (A), microfi laments (B), microtubules (C)
Fig. 2. Immunofl uorescent analysis of the phospho-mTOR Ser2481 (B) colocalization with tubulin β (A) during cytokinesis in the
human breast adenocarcinoma MCF-7 cell line. Nuclei were counterstained with Hoechst 33342 (C)
A B C
10 m
5 m 5 m 5 m 5 m
Merge
10 m20 m
A B C
11
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 treat-
ment 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 ki-
nase), paxillin and p130Cas, by inhibition of the S6K1
activity [7, 33]. Knockdown of mTORC1 and mTORC2
induced a mesenchymal-epithelial transition in the col-
orectal 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 regu-
late cytoskeleton rearrangement through phosphoryla-
tion of the actin-remodulating proteins.
The present study showed that activated PI3K-Akt-
mTOR signaling pathway promotes invasion and me-
tastasis in hepatocellular carcinoma though up-regu-
lation of MMP-9 (Matrix metalloproteinase 9), tho-
ugh, 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 sub-
units, which are arranged in a cylindrical tube mea-
suring 25 nm in diameter – the thickest fi brils of the
cytoskeleton (Fig. 1, C). Similar to F-actin a micro-
tubule is polarized and has (+) end and (-) end. Po ly-
merization of the tubulin subunits requires the hy-
drolysis of the GTP molecules. In addition to regula-
tion of the cell motility, microtubules play a major
role in organization of the cell polarity through a spe-
cial structure called the microtubule-organizing cen-
ter (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 sta-
bility of microtubules and their association with oth-
er cell structures. These proteins are collectively cal-
led microtubule-associated prote ins (MAPs) [6, 32].
Involvement of TOR1 and TOR2 in the control of
various aspects of microtubule dynamics was report-
ed 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 di-
rectly to and phosphorylate cytoplasmic linker pro-
tein 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 phospho-
rylation is not fully understood. It was revealed that
TSC2 knockout resulted in a greater abundance of
stabilized microtubules underneath the cellular cor-
tex. Time-lapse imaging of dynamic microtubules
al so revealed disorganized movements of the grow-
ing microtubule plus-ends in the cellular cortex re-
gion, including growth in a direction that is parallel
to the cortex. The authors suggested that the func-
tional mTOR-CLIP-170 interaction helps microtu-
bules 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 concentra-
tions 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 ob-
served to be bidirectional: the mTORC1 activation
requires dynein-dependent transport to a position in
the cell where it can be activated [48]. The associa-
tion between dynein and mTOR was shown by coim-
12
V. R. Kosach, O. V. Cherednyk, A. I. Khoruzhenko
munoprecipitation. Inhibition of dynein fun ction us-
ing RNAi hinders the mTORC1 activity in the hu-
man fi broblasts and the human glioblastoma–astro-
cytoma cell line U373-MG [48].
Moreover, the phosphorylated form of mTOR ki-
nase (phospho-mTOR Ser2481) was observed to lo-
calize 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 spin-
dle midzone and the cleavage furrow during cytoki-
nesis. At these conditions phospho-mTOR was ran-
domly 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 de-
scribed [50]. Our fi ndings support a hypothesis that
mTOR phosphorilated at Ser 2481 interacts with micro-
tubules 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 lo-
comotion during embryonic development, wound heal-
ing, and che mo taxis as well as in the cancer cells
spreading. Further detailed investigation of this new as-
pect of the mTOR activity might lead to the optimiza-
tion of the current anti-cancer therapeutic approaches.
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14
V. R. Kosach, O. V. Cherednyk, A. I. Khoruzhenko
В. Р. Косач, О. В. Чередник, А. І. Хоруженко
Характеристика mTOR
сигнального шляху та його залучення
до регуляції клітинної рухливості шляхом
реконструкції архітектури цитоскелету
mTOR кіназa є однією з основних ланок, розташованих на пе-
ретині кількох шляхів внутрішньоклітинної передачі сигналу.
Дерегуляція сигналінгу mTOR кінази супроводжує розвиток
онкологічних захворювань, діабету, нейродегенеративних роз-
ладів і старіння. Застосування інгібітора mTOR рапаміцина
знижує рівень міграції та інвазії злоякісних клітин і утворення
метастазів. Однак, точний механізм регуляції рухливості клі-
тин mTOR кіназою повністю не зрозумілий. Дана стаття при-
свячена останнім дослідженням, які демонструють можливу
роль mTOR кінази в регуляції ре моделювання цитоскелету та
міграції клітин. Докладні дослідження цієї неканонічної функ-
ції mTOR кінази дозволить розширити наші знання про мігра-
цію клітин і утворення метастазів і може привести до поліп-
шення протиракових терапевтичних підходів.
Ключов і слова: mTOR сигналінг, рапаміцин, перебудова
цитоскелету, проміжні філаменти, мікротрубочки, метаста-
зування.
В. Р. Косач, О. В. Чередник, А. И. Хоруженко
Характеристика mTOR сигнального пути
и его привлечения к регуляции клеточной подвижности
путем реконструкции архитектуры цитоскелета
mTOR киназa является одним из основных звеньев, располо-
женных на пересечении нескольких путей внутриклеточной пе-
редачи сигнала. Дерегуляция сигналинга mTOR киназы сопро-
вождает развитие онкологических заболеваний, диабета, нейро-
дегенеративных расстройств и старения. При менение ингиби-
тора mTOR рапамицина снижает уровень миграции и инвазии
злокачественных клеток и образование метастазов. Однако, точ-
ный механизм регуляции подвижности клеток mTOR киназы
полностью не изучен. Эта статья посвященна последним иссле-
дованиям, которые демонстрируют возможную роль mTOR ки-
назы в регуляции ремоделирования цитоскелета и миграции
клеток. Под роб ные исследования этой неканонической функ-
ции mTOR киназы позволят расширить наши знания о мигра-
ции клеток и образование метастазов и может привести к улуч-
шению противораковых терапевтических подходов.
Ключевые слова: mTOR сигналинг, рапамицин, пере-
стройка цитоскелета, промежуточные филаменты, микро-
трубочки, метастазирование.
Received 25.12.2014
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