Review

TORC2 Structure and

Function
Christl Gaubitz,1 Manoel Prouteau,1 Beata Kusmider,1 and
Robbie Loewith1,2,*
The target of rapamycin (TOR) kinase functions in two multiprotein complexes,
Trends
TORC1 and TORC2. Although both complexes are evolutionarily conserved,
The target of rapamycin (TOR) kinase
only TORC1 is acutely inhibited by rapamycin. Consequently, only TORC1 assembles into two, evolutionarily con-
signaling is relatively well understood; and, at present, only mammalian TORC1 served multiprotein complexes, TORC1
and TORC2.
is a validated drug target, pursued in immunosuppression and oncology. How-
ever, the knowledge void surrounding TORC2 is dissipating. Acute inhibition of Unlike TORC1, TORC2 is not acutely
TORC2 with small molecules is now possible and structural studies of both inhibited by rapamycin and, conse-
quently, the signaling pathways in
TORC1 and TORC2 have recently been reported. Here we review these recent which this complex operates are rela-
advances as well as observations made from tissue-specific mTORC2 knockout tively less understood.
mice. Together these studies help define TORC2 structure–function relation-
Recent electron microscopy structures
ships and suggest that mammalian TORC2 may one day also become a bona provide a framework for understanding
fide clinical target. how TORC2 is regulated as well as the
specific functions of individual subunits.

The Discovery of Rapamycin and Its Target Tissue-specific ablation of mammalian

Rapamycin is a macrolide antibiotic produced by the soil bacterium Streptomyces hygro- TORC2 demonstrates a role for this
scopicus. It was discovered and purified by virtue of its antifungal activity [1,2]. Not long after it complex in the regulation of growth,
proliferation, and metabolism and sug-
was shown to be a potent immunosuppressant and, subsequently, to inhibit growth of
gests that, like mTORC1, mTORC2
transformed cells in vitro and in rodent xenograph models [3,4]. These intriguing properties inhibition may also bring clinical gain.
sparked a massive effort to try to identify the target of rapamycin. Ultimately, a simple, but
nevertheless elegant, genetic dissection of Saccharomyces cerevisiae mutants displaying
spontaneous resistance to rapamycin yielded the target of this drug–TOR–and helped define
the nonessential proline isomerase FKBP12 as a receptor for rapamycin, necessary to mediate
its toxic effects [5].

S. cerevisiae encodes two TOR paralogs, Tor1 and Tor2. Biochemical purification of these
proteins demonstrated that they assemble into two distinct multiprotein complexes named TOR
complex 1 (TORC1) and TORC2 (see Glossary) [6,7]. Both complexes are widely conserved
[6,8–11] but only TOR in TORC1 is bound and inhibited by FKBP12–rapamycin. Importantly,
mammalian TORC1 (MTORC1; for clarity, mammalian factors are written in small Caps) is a
validated drug target as rapamycin was eventually approved for clinical use in both immuno-
1
suppression and oncology. Rapamycin has additionally been a valuable tool to study TORC1 Department of Molecular Biology, and
Institute of Genetics and Genomics of
signaling; and, over the years, it has become abundantly clear that both TORC1 regulators Geneva (iGE3), University of Geneva,
and effectors are robustly conserved from yeast to human [12–15]. By contrast, in the absence 30 quai Ernest Ansermet, CH1211
of a specific inhibitor, the utility of MTORC2 inhibition as a therapeutic option remains unclear and Geneva, Switzerland
2
National Centre of Competence in
the molecular characterization of TORC2 signaling has comparatively lagged. Research “Chemical Biology”,
University of Geneva, Geneva CH-
However, new tools are starting to become available to study TORC2. For example, ATP- 1211, Switzerland
competitive MTOR inhibitors that inhibit MTORC2 (and MTORC1) have been described [3] and
reverse chemical genetic approaches enable the specific inhibition of TORC2 in yeast *Correspondence:
[16–18]. These chemical tools are complemented by tissue-specific MTORC2 knockout mouse Robbie.Loewith@unige.ch (R. Loewith).

532 Trends in Biochemical Sciences, June 2016, Vol. 41, No. 6 http://dx.doi.org/10.1016/j.tibs.2016.04.001
© 2016 Elsevier Ltd. All rights reserved.
lines. Furthermore, several exciting structural insights have recently been made. Building on Glossary
these advances, in this review we detail the current understanding of the functions of individual AGC kinase: a family of
TORC2 subunits. We then try to summarize the often controversial literature describing the approximately 60 related protein
localization and regulation of MTORC2. Lastly, we close with a discussion of what these studies kinases named after protein kinase A,
protein kinase G, and protein kinase
suggest regarding MTORC2 inhibition as a clinical strategy. C. A common prerequisite for AGC
kinase activation are phosphorylation
Common Architecture of the Two TOR Complexes events mediated by PDK, in the
TORC1 and TORC2 are similarly large, weighing in at
1.2 and
1.4 MDa, respectively. TORC1/ activation loop of the kinase domain,
and TOR, in C-terminal motifs.
mTORC1 contains TOR/MTOR, Lst8/MLST8, Kog1/RAPTOR, Tco89 (which is yeast-specific), and ATM: ataxia telangiectasia mutated
DEPTOR (which is vertebrate-specific). TORC2/MTORC2 contains TOR/MTOR, Lst8/MLST8, Avo3/ plays important roles in DNA repair
RICTOR, Avo1/hSIN1, Bit61 and Bit2/PROTOR-1 and PROTOR-2, Avo2 (which is yeast-specific), and and is recruited to double-strand
breaks (DSBs) via the MRE11–RAD50–
DEPTOR (which again is vertebrate-specific) (Figure 1A). A search of subunit paralogs in published
NBS1 (MRN) complex. Loss of kinase
genomes suggests that TOR complexes are broadly conserved in eukaryotes with notable activity results in ataxia telangiectasia
absences in some parasites, including Microsporidia and Plasmodia [12], and, curiously, a lack (A-T), an autosomal recessive
of TORC2 in Planta [19] (Figure 1A). disorder.
ATR: ataxia telangiectasia and RAD3-
related protein serves essential
In recent years there have been five major structural insights into TOR complex structure and functions in maintaining genomic
function: a low-resolution cryo-electron microscopy (cryo-EM) reconstruction of MTORC1 integrity and binds to replication
[20]; a high-resolution cryo-EM reconstruction of MTORC1 including a docked crystal structure of protein A (RPA) at sites of DNA
damage via interaction with ATRIP.
the RAPTOR subunit [21]; a low-resolution negative-stain EM reconstruction of yeast TORC2 [16]
cryo-EM (cryo-electron
(Figure 2A); an atomic resolution crystal structure of the kinase domain of MTOR in complex with microscopy): is a structural biology
MLST8 [22] (Figure 1B); and, most recently, a high-resolution cryo-EM reconstruction of full-length technique used to image single
Tor–Lst8 complex from the thermotolerant yeast Kluyveromyces marxianus [23]. A comparison particle specimens without the need
for crystals. Owing to recent
of the EM reconstructions demonstrates that the two TOR Complexes have in common some technological advances, it is possible
interesting design features, suggesting that they share fundamental structure–function proper- to determine the structure of
ties. In both MTORC1 and TORC2 there are two copies of each subunit. These subunits are macromolecular complexes and
divided into one of two protomers, which are themselves related by C2 rotational (pseudo-) proteins at near atomic resolution.
DEP: the DEP (dishevelled, egl-10,
symmetry. Together the two protomers give an overall parallelepiped (3D rhomboid) structure to pleckstrin) domain folds into a
each complex and delineate a relatively large central cavity that could potentially accommodate a globular structure of approximately
globular protein up to
60 kDa in size [16]. The function of this cavity is unknown but it has been 80 amino acid residues, which is
found in proteins that regulate G
proposed to provide an interaction surface for upstream regulators [16]. Both TOR complexes
protein signaling.
are known to interact with membranes and it is likely one of the broad faces of the parallelepiped DNA–PKcs: this DNA-dependent
that is membrane proximal [16]. protein kinase catalytic subunit forms
the DNA–PK holoenzyme with the
Ku70/80 heterodimer to mediate
Several of the TORC2 subunits can be localized within the TORC2 EM reconstruction, which
non-homologous end joining, the
helps to define their otherwise enigmatic functions (Figure 2). Furthermore, the arrangements of main pathway used in mammals to
the MTOR and MLST8 subunits, which are shared between the two complexes, were well defined in repair double-strand DNA breaks.
the high-resolution cryo-EM MTORC1 reconstruction [21] and the high-resolution cryo-EM DNA–PKcs is the largest of all PIKKs.
Eisosome: plasma membrane
KmTor–Lst8 reconstruction [23]. Using this same orientation, these two subunits can be
invaginations that may serve as
tentatively placed within the low-resolution TORC2 reconstruction (Figure 2B [21]). This place- plasma membrane reservoirs in yeast
ment questions the accuracy of the previous positioning of the Avo1 subunit but supports the and appear to act analogously to
notion that, as with MTORC1 [21], access to the deeply recessed active sites of TORC2 is gained caveolae in mammalian cells.
FAT: FRAP–ATM–TRRAP is a 600-residue
from the external periphery [16]. domain made up of /-helical bundles
located adjacent to the N terminus of
Structure–Function of TORC2 Subunits the kinase domain in PIKKs.
TOR/MTOR and Lst8/MLST8 FRB: the FKBP12–rapamycin binding
domain folds into a bundle of four
TOR and its binding partner Lst8 form the core of both TORC1 and TORC2. TOR is the founding /-helices within the kinase domain,
member of the phosphatidylinositol 3-kinase (PI3K)-related kinases (PIKK) family [24]. Included in thereby extending the N-lobe side of
this family are the mammalian PIKKs: DNA–PKcs, ATM,ATR,SMG1, and (the catalytically inactive) the kinase cleft.
TRRAP. Like TOR, all of these proteins are similarly large and possess common domain structures HEAT: HUNTINGTON, EF3A, PP2A, TOR is
a repeat motif consisting of /-helices
including a serine–threonine kinase domain that shares a curious resemblance to the catalytic that fold into flexible super helical
domains of PI3Ks (Figure 1B). Lst8 is composed entirely of WD40 repeats, which fold into a

Trends in Biochemical Sciences, June 2016, Vol. 41, No. 6 533

seven-bladed b-propeller that, in other WD40-repeat containing proteins, generally serves as a arm-like structures and often mediate
scaffold for protein interactions [25]. protein–protein interactions.
HbrB: is a conserved structural motif
found in proteins that are involved in
The TOR kinase domain has a bilobal structure and possesses catalytic residues and a catalytic hyphal growth and polarity
mechanism resembling those of canonical protein kinases [22]. The activation loop is well hyperbranching in fungi.
ordered in the crystal structure, suggesting the kinase domain is in an intrinsically active LBE: the Lst8 binding element is a
two /-helix 40-residue structural
conformation. This implies that TORC1 signaling output is regulated by the controlled access motif that forms the binding site for
of substrates. This regulation has been proposed to be facilitated by two structural features, the Lst8, thereby extending the C-lobe
FKBP12–rapamycin binding domain (FRB) domain and the Lst8 Binding Element (LBE), bound site of the kinase cleft.
MCTs: membrane compartments
to Lst8, which serve to recess and thus hinder access to the active site (Figure 1B). Building on
containing TORC2 are highly dynamic
this model, FKBP12–rapamycin binding is proposed to inhibit TORC1 signaling by further foci that appear at the plasma
restricting access of substrate to the active site [21,22]. membrane for a few seconds and are
observed by light microscopy.
MTORC2: mammalian TORC2 is
1.3
The C-terminal kinase domain occupies only approximately one-fifth of the primary sequence of
MDa and contains two copies of
TOR; the remainder is composed of two blocks of HEAT (Huntington, EF3A, ATM, TOR) repeats each of six subunits MTOR, MLST8,
followed by a block of tetratricopeptide (TPR) repeats that form the so-called FAT (Frap, ATM, RICTOR, SIN1, DEPTOR, and PRR5 or its

TRRAP) domain (Figure 1B). In the high-resolution MTORC1 reconstruction [21], the two HEAT paralog PRR5L.
PH: pleckstrin homology domains are
repeats form /-solenoid superstructures named the ‘horn’ and ‘bridge’, whereas the TPRs of approximately 100 amino acid
the FAT domain form a compact structure that cradles the kinase domain [21,22]. The ‘horn’ and residues in length and serve as
‘bridge’ pack against one another but also form part of the solvent-exposed surface of MTORC1. membrane targeting modules; they
The ‘horn’ additionally interacts with an adjacent FAT domain to form an interlocking structure bind to a diverse set of lipids
including phosphatidylinositol
sufficient to mediate the observed dimerization of two protomers in MTORC1 [21]. Given the phosphates.
structural similarities between MTORC1 and TORC2, it is reasonable to suspect that the HEAT and PIKK: phosphatidylinositol-3 kinase-
TPR repeats of TOR also mediate dimerization of TORC2. Such a structural role does not related kinases form a family of
serine–threonine kinases, which
preclude additional functions for these repeats in the recruitment of regulators, for example, nor
comprises six members in mammals:
does it preclude scaffolding roles for other subunits in the complex. SMG1, ATM, ATR, DNA–PKc, MTOR, and
TRRAP. The concerted activity of all

Avo3/RICTOR members ensures the maintenance of

metabolic homeostasis, genomic
After TOR, Avo3/RICTOR is the largest subunit in TORC2 (Figure 1C). Depletion of Avo3 results in
integrity, and transcriptional fidelity.
the disassembly of TORC2, suggesting that it performs an important scaffolding function [26], PRD: PIKK regulatory domain (PRD)
analogous to the role RAPTOR performs in the stabilization of the dimerization of mTORC1 [21]. is a domain that comprises a stretch
Consistent with this role, Avo3 localizes to the core (palm) of TORC2 [16]. Avo3 possesses a of approximately 20 amino acids,
which when deleted results in
central Armadillo-like (ARM-like) domain, which likely folds into an /-helical solenoid similar to
hyperactivation of TOR.
the HEAT repeats. ARM-containing proteins are known to bind large substrates like proteins RasGEFN: this domain is found
or nucleic acids [27]. Crosslinking suggests that the ARM-like domain of Avo3 is tightly looped N-terminal to the catalytic RasGEF
around the FAT and the kinase domain of Tor2 [16]. This is supported by biochemical and domain in guanine nucleotide exchange
factors (GEFs) of small GTPases.
genetic studies that demonstrated that the C terminus of Avo3 sterically occludes the FRB SMG1: SMG1 kinase is recruited,
domain of Tor2, which explains why TORC2 is insensitive to FKBP12–rapamycin [16]. together with SMG8 and SMG9, to
premature termination codons to
Beyond the ARM-like domain, Avo3 paralogs show limited conservation with both the N and C initiate nonsense-mediated mRNA
decay.
termini being particularly divergent (Figure 1C). Curiously, Avo3 contains a RasGEFN domain. TORC2: target of rapamycin complex
These domains are typically found N-terminal to the catalytic domain of RAS guanine nucleotide 2 is an
1.4 MDa multiprotein complex
exchange factors but their specific function is unknown [28]. The presence of this domain could that contains two copies of each of six
subunits, Tor2, Lst8, Avo1, Avo2,
suggest an interaction between TORC2 and RAS signaling. Intriguingly, Avo3 is not the only
Avo3, and Bit61 or its paralog Bit2.
TORC2 subunit with a connection to RAS; the slime mold paralog of Avo1, the next biggest TORC2 TPR: tetratricopeptide repeat is a
subunit, was originally isolated as a Ras-interacting protein 3 (RIP3) in a two-hybrid screen [29]. repeat motif of 34 amino acids that
fold into adjacent /-helices.
TRRAP: TRAF and TNF receptor-
Avo1/SIN1
associated protein acts as part of
Interestingly, SIN1, the human ortholog of Avo1, was also identified in a genetic screen for distinct histone acetyltransferase
suppressors of RAS function in yeast [30] and Avo1/SIN1 orthologs display several regions of complexes and can associate with
high conservation (Figure 1D), including a RAS binding domain (RBD) [31,32]. C-terminal of the several transcription factors. TRRAP
has no kinase activity.
RBD is an essential pleckstrin homology (PH) domain, which localizes to the tip of the ‘thumbs’ in

534 Trends in Biochemical Sciences, June 2016, Vol. 41, No. 6

TORC2 (Figure 2B) and, in yeast, is necessary to tether TORC2 to the plasma membrane [33].
The PH domains of both yeast Avo1 and mammalian SIN1 have been crystalized and have been
reported to bind various phosphatidylinositol polyphosphates and potentially also sphingoid
bases [34,35]. Preceding the RBD is the so-called conserved region in the middle (CRIM) [36].
This region can be extensively crosslinked to Lst8 [16] and has been implicated in substrate
binding [37,38]. The ability of Avo1 to bind membrane, substrate, and the kinase domain of Tor2
(via Lst8) is intriguing in that it would suggest that this subunit is ideally suited to couple correct
TORC2 (membrane) localization to signaling output of the complex. Avo1/SIN1 appears to also
have a general scaffolding role because their loss prevents assembly of TORC2/MTORC2 [26,39].
A number of different SIN1 splice variants have been described, three of which can assemble
with MTOR. Although their specific functions are unclear, these splice variants present curious
tissue-specific expression patterns and distinct subcellular localizations [40,41].

BITs/PRRs
The last remaining conserved TORC2 subunit is known as Bit61 in yeast [7] and PRR5 (PROTOR-1)
in mammals [42,43]. Each has a paralog–Bit2 and PRR5L (PROTOR-2)–that also associates with
TORC2/MTORC2. The BITs are not essential and the specific functions of neither the BITs nor the
PRRs are known. They bind TORC2 peripherally via Avo3/RICTOR and Avo1/SIN1 but are not
themselves required for complex assembly [16,26,44]. They possess a conserved domain
shared with the Aspergillus nidulans protein HbrB (Figure 1E) [45,46]. HbrB plays a role in
filamentous growth in A. nidulans but how this relates to BIT/PRR function in TORC2/MTORC2 is
unclear.

Avo2
Avo2 is a nonessential, yeast-specific TORC2 subunit. It has a peripheral localization within
TORC2 [16] and is predicted to contain ankyrin repeats (Figure 1F) [46]. Together with the BITs
[47], it may bind to the TORC2 regulators Slm1 and Slm2, which are described in the next
section. Otherwise, the molecular function of Avo2 within TORC2 is unknown.

DEPTOR
DEPTOR is a vertebrate-specific subunit of both MTORC2 and MTORC1 [48]. It possesses two tandem
DEP (dishevelled, egl-10, pleckstrin) motifs at its N terminus in addition to a PDZ (postsynaptic
density 95, discs large, zonula occludens-1) domain (Figure 1G). DEP domains provide spatial
control to diverse signaling pathways by recruiting signaling modules to cellular membranes [49].
PDZ domains are often implicated in protein binding and DEPTOR seems to bind MTOR via its PDZ
domain. DEPTOR interferes with the signaling output of the two MTOR complexes through unknown
mechanisms [48].

Regulation of TORC2
TORC2 Localizes to the Plasma Membrane and Is Regulated by Membrane Tension
TORC2 localizes to puncta at the plasma membrane that are named membrane compartments
containing TORC2 (MCTs) [50]. MCTs are adjacent to, but never overlapping with, distinct
membrane nanodomains known as eisosomes. Eisosomes contain dozens of different pro-
teins, the major ones being the Bin/Amphiphysin/Rvs (BAR) domain containing proteins Lsp1
and Pil1 [51]. Lsp1 and Pil1 contour eisosomes, and the underlying plasma membrane, into
canoe-shaped structures that invaginate into the cytoplasm. This puckering of the membrane
could potentially serve to sense membrane tension [52].

Eisosomes regulate TORC2 signaling via the Slm1 and Slm2 paralogs [50] (Figure 3A). In
rapidly growing cells, the Slm1/2 proteins are partitioned approximately equally between
eisosomes and MCTs. However, upon an increase in plasma membrane tension, triggered
by inhibition of endogenous lipid biosynthesis, hypotonic shock or direct mechanical

Trends in Biochemical Sciences, June 2016, Vol. 41, No. 6 535

 (A) (B) mLst8 FAT
Protor
Mammals mTOR mLst8 Rictor Sin1 1/2
Deptor
100%
Birds Sequence
Amphibians conservaon
0
Fish
Echinodermata mLst8 WD40 370 aa
Insects
Nematodes
Fungi Tor2 Lst8 Avo3 Avo1 Bit2/61 Avo2 LBE
Dictyostelium Lst8 WD40 303 aa FATC
Plants
0
ATP
Green algae Sequence

FRB KD
Red algae conservaon
100%
Ciliates
Trypanosomes 200 aa

100%
Sequence
conservaon
0

mTOR HEAT FAT FRB KD 2549 aa

FATC

Tor2 HEAT FAT FRB KD 2474 aa

0
Sequence
conservaon
100%

(C) 100%
(D) 100%
Sequence
Sequence
conservaon
0
conservaon 0

Rictor ARM 1708 aa Sin1 CRIM RB PH 522 aa

Avo3 ARM 1430 aa Avo1 CRIM RB PH 1176 aa

0 0
Sequence Sequence
conservaon conservaon
100% 100%

100%
Sequence
(E) conservaon (F) (G) 100%
0 Sequence
conservaon
0
Protor-1 HbrB 370 aa
Avo2 ANK 426 aa Deptor DEP DEP PDZ 409 aa

100%
Sequence
Bit2 HbrB 543 aa conservaon
0
0
Sequence
conservaon
100%

Figure 1. Conservation of TORC2 Subunits. (A) Conservation of TORC2 subunits across the eukaryotic kingdom. Names of mammalian and budding yeast proteins
are included in the table. TOR and Lst8 form the core of both TORC1 and TORC2 and are conserved in all eukaryotes with the exception of a few intracellular parasites
[12]. PROTOR-1/2 paralogs are present in most vertebrates but their yeast orthologs Bit2/61 are conserved in only a minority of fungi. (B–G) Sequence conservation
diagrams of yeast and mammalian TORC2 subunits. The plots indicate the percentage conservation across orthologs found in representative eukaryote species. The
protein sequence alignment was generated with Clustal using default parameters. Insertions/deletions between the cartooned yeast and the mammalian orthologs are
shown by light gray sectors delimited by broken lines. (B) Structure and conservation of Tor2/MTOR and Lst8/MLST8. Illustrated are the HEAT (HUNTINGTON, EF3A, PP2A, TOR),
FAT (FRAP–ATM–TRRAP), FRB (FKBP12–rapamycin binding), Kinase, and FATC (found in the C terminus of FAT-containing proteins) domains of TOR and the WD40 repeats
of Lst8. Also shown is the atomic resolution structure [22] of the C-terminal portion of MTOR in complex with MLST8 via the small LBE (Lst8 binding element). Note how
access to the active site (where ATP is bound) is restricted by MLST8 and the FRB domain. (C) The amino- and carboxy-terminal tails of Avo3/RICTOR are poorly conserved,
do not display any known domain, and show many species-specific sequence insertions/deletions. These variable tails frame a conserved central domain consisting of an
ARM (Armadillo-like) and RAS–GEFN domains. (D) The overall sequence of Avo1/SIN1 is variable in size, poorly conserved, and displays many insertions/deletions
(Figure legend continued on the bottom of the next page.)

536 Trends in Biochemical Sciences, June 2016, Vol. 41, No. 6

(A) mTORC1

Acve site

10 nm

26Å cryo-EM reconstrucon, 2010 5.9Å cryo-EM reconstrucon, 2016 Atomic model of mTOR and mLst8

(B) TORC2

mLst8
FAT Acve site
Bit61/2 FRB

90°

Avo2 HEAT
KD
Avo1 PH FATC
26Å negave stain EM reconstrucon, 2015 Atomic model of mTOR and mLst8 placed into TORC2 reconstrucon

Figure 2. Comparison of TORC2 and MTORC1 Electron Microscopy (EM) Reconstructions. (A) From left to right, EM maps, generated in Chimera, showing a
low resolution (26 Å) reconstruction of mTORC1 [20] (EMD ID: 5197), a high resolution (5.9 Å) reconstruction of mTORC1 [21] (EMD ID: 3213) and the corresponding
atomic model (PDB ID: 5FLC) placed within this reconstruction. (B) Left: Low resolution (26 Å) reconstruction of TORC2 [16] (EMD ID: 2990). The PH domain of Avo1
(which tethers TORC2 to the plasma membrane), Avo2 and Bit61/2 are indicated. Center and right: Atomic model of MTOR and MLST8 (PDB ID: 5FLC) was placed within the
TORC2 reconstruction using Chimera. The domains within MTOR are colour-coded: HEAT repeats are in gray, FAT in green, kinase domain in orange, FRB in yellow and
FATC in red. In this placement, MLST8, represented in purple, sits in the ‘thumb’ of TORC2 presumably just below the PH domain of Avo1. This simple placement does not
account for spatial constraints that TORC2-specific subunits will undoubtedly evoke.

manipulation, Slm1/2 exit eisosomes and accumulate in MCTs. In MCTs, Slm1/2 activate
TORC2 signaling output, potentially by corecruitment of the major TORC2 substrate Ypk1
[50,53].

Regulation of MTORC2
As several potential mechanisms have been proposed in the literature (Figure 3B), the regulation
of MTORC2 is currently debated. There is some evidence to support the notion that MTORC2,
similarly to TORC2 in yeast, is regulated by membrane tension. Specifically, stretching mam-
malian cells triggers MTORC2-dependent phosphorylation of AKT [54–57]. Intriguingly, this signal-
ing requires CAVEOLIN, the defining protein of caveolae, which are vertebrate-specific membrane
invaginations thought to function as membrane reservoirs and may function analogously to
eisosomes in yeast. Furthermore, as in yeast, hyperosmotic stress, which decreases membrane
tension, inhibits MTORC2 activity [58,59].

between orthologs. The CRIM (conserved region in the middle), RB (Ras binding), and PH (pleckstrin homology) domains are indicated. (E) Bit2/61–Protor1/2 contains a
conserved HbrB (hyperbranching) domain of unknown function. (F) Avo2 is a small subunit, found only in fungi, containing a domain composed of ANK (Ankyrin) repeats.
(G) DEPTOR displays two DEP (dishevelled, egl-10, pleckstrin) domains and associates with MTOR, in both MTORC1 and MTORC2, through its very well-conserved PDZ
(postsynaptic density protein, Drosophila disc large tumor suppressor, and zonula occludens-1 protein) domain.

Trends in Biochemical Sciences, June 2016, Vol. 41, No. 6 537

(A) MCT eisosome

Avo1 plasma membrane

Avo1 Lst8 SLM
Tor2
Avo2 BIT
Avo3 plasma membrane tension
t8
Lst8
Lst8
Ls

Ypk1/2
P P Ypk1/2
P P hypotonic shock
mechanical stress
YPK Pkc1 impaired lipid biosynthesis
methylglyoxal

turgor pressure
plasma membrane tension
acn polarizaon
cell wall integrity pathway
G2/M cell cycle transion
genome integrity
pentose phosphate pathway

(B) Rac1
Ribosome
Rit1
A

A acetyl coenzyme A
A

A A
PtdIns(3,4,5)P3 SIN1 RICTOR

mTOR
PROTOR
DEPTOR mLST8
Ypk1/2

Ypk1/2
P P
hyperosmoc XPLN AGC
stress kinase

methylglyoxal

cell survival and differenaon

proliferaon
metabolism

Figure 3. Upstream Regulators and Downstream Effectors of Yeast (A) and Mammalian TORC2 (B). (A)
Membrane tension, which triggers the translocation of the Slm1/2 proteins into MCT (membrane compartments containing
TORC2) is the best described regulator of TORC2 in yeast. Ypk1, and to a lesser extent Ypk2 and Pkc1, are the best
described substrates of TORC2 in yeast. (B) Many upstream regulators of MTORC2 have been proposed. The best described
substrates of MTORC2 are the AGC kinases AKT/PKB, SGK, and various PKC isoforms. See text for details and abbreviations.

Localization to the plasma membrane would support a role for MTORC2 downstream of mem-
brane tension; however, here too the literature is unclear because MTORC2 is reportedly localized
to the plasma membrane, but also mitochondria, the endoplasmic reticulum (ER), mitochondria–
ER junctions, the lysosome, and other locales [60,61]. Why the localization of MTORC2 is so

538 Trends in Biochemical Sciences, June 2016, Vol. 41, No. 6

controversial is mysterious. Potentially, technical limitations such as off-target labeling of anti-
bodies could account for some of this controversy. Alternatively, MTORC2 localization may differ
between cell types or perhaps is transient with recruitment to discrete loci occurring only upon
certain upstream cues.

Consistent with the latter proposition, MTORC2 is reportedly activated upon binding to PtdIns
(3,4,5)P3 produced at the plasma membrane downstream of growth factor signaling [62,63].
This recruitment is mediated by the PH domain in SIN1. Moreover, in this report it was suggested
that, in the absence of lipids, the PH domain of SIN1 binds to the kinase domain of MTOR and
inhibits its activity. Binding of PtdIns(3,4,5)P3 was thus proposed to not only recruit MTORC2 to the
plasma membrane where it would meet its substrates but to also reorient the PH domain of SIN1
such that substrates could gain access to the kinase pocket.

Regulation of MTORC2 by Subunit Phosphorylation

In addition to lipid messengers, growth factor signaling may also influence MTORC2 activity by the
regulated phosphorylation of different subunits. Indeed, many phosphorylation sites have been
identified in both TORC2 and MTORC2. Although their functions remain uncharacterized, at least
52 phosphorylation sites have been mapped in Avo1, Avo2, Avo3, and Bit61 in high-throughput
phosphoproteomics work [64]. RICTOR too is heavily phosphorylated with >20 phosphorylation
sites mapped [65]. The partial characterization of MTORC2 subunit phosphorylation events has
been reported in many recent studies, which are summarized in Table 1. Some of the described

Table 1. Phosphorylation of MTORC2 Subunits

Subunit Sitea Upstream Kinase Functional Consequence of Refs
Phosphorylation

MTOR S1261 in HEAT Unknown, not Phosphorylation promotes MTORC1 [112–114]

autophosphorylation activity, unknown function in MTORC2

MTOR S2159 and T2164 Unknown Phosphorylation promotes MTORC1 [113]

in kinase domain activity, not known if site
phosphorylated in MTORC2

MTOR T2173 in kinase AKT Feedback regulation of MTORC2, [114]

domain phosphorylation inhibits MTORC2
activity, phosphorylation is conserved
in S. pombe

MTOR T2446 in PRD S 6K Feedback regulation of MTORC1 [115]

MTOR S2448 in PRD S 6K Feedback regulation of MTORC1 [115]

MTOR S2481 in PRD MTORC2 Unknown, phosphorylation often [116]

autophosphorylation interpreted as a marker for active/
intact MTORC2

RICTOR T1135 S 6K Phosphorylation promotes binding of [65,117,118]

14-3-3 proteins and antagonizes
MTORC2-dependent phosphorylation
of Akt

RICTOR S1235 GSK-3b Phosphorylation interferes with [58]

substrate binding

SIN1 T86 AKT Controversial: phosphorylation [119–121]

stimulates MTORC2 activity,
phosphorylation disrupts MTORC2

SIN1 S260 in CRIM MTOR Phosphorylation prevents SIN1 [122]

degradation in the lysosome

SIN1 T398 in PH domain S 6K Phosphorylation disrupts MTORC2 [120]

a
Numbering refers to human sequences.

Trends in Biochemical Sciences, June 2016, Vol. 41, No. 6 539

phosphorylation events are likely due to autophosphorylation, while others are mediated by
distinct kinases. Beyond being generally activating or inhibitory, the functional consequences
of MTORC2 subunit phosphorylation are ill defined.

Regulation of MTORC2 by Metabolic Cues

In addition to being phosphorylated, RICTOR is also acetylated on lysines 1116, 1119, and 1125
[66,67]. Acetylation appears to stimulate MTORC2 kinase activity, although the mechanistic details
underlying this stimulation are not known. The acetylation capacity of a cell is thought to be linked
to metabolic status [68]. Specifically, the acetyl moieties used to charge coenzyme A are derived
from glycolytic and/or lipid metabolism while histone deacetylases are linked to metabolism
through use of NAD+ as a cofactor or via allosteric regulation by other metabolites. Thus, MTORC2
activity could be wired to cellular metabolic status via RICTOR acetylation.

Methylglyoxal is another potential link between metabolic status and MTORC2 activity. A reactive
intermediate produced during glycolysis, methylglyoxal was recently reported to activate TORC2
in budding yeast as well as MTORC2 [69].

Other Regulators of MTORC2

It is well established that yeast and mammalian TORC1 are regulated by small GTPases
including the RAG family GTPases [15]. Emerging evidence suggests that MTORC2 may
also be regulated by GTPases. The first hint at such regulation is that RICTOR and SIN1,
respectively, contain RasGEFN and Ras association domains as described in the
previous section. Furthermore, work in fission yeast has suggested that glucose activates
TORC2 via the Rab family GTPase Ryh1 [70,71]. Subsequently, the RAC1 GTPase was
proposed to be a regulator of both MTORC1 and MTORC2 by directly binding to MTOR and
facilitating localization of both complexes to the appropriate cellular membranes [72]. Lastly,
the Ras family GTPase RIT was suggested to bind to and activate MTORC2 in response to
oxidative stress [73]. Interestingly, oxidative stress has also been described as an activator of
yeast TORC2 [74].

In addition to GTPases, other MTORC2 protein binding partners have been described as regu-
lators of MTORC2 activity. These include XPLN, a guanine nucleotide exchange factor (GEF) for Rho
GTPases that antagonizes MTORC2 signaling [75], and the ribosome, which activates MTORC2
signaling downstream of growth factor signaling [76,77]. The mechanisms by which these
MTORC2 partners influence MTORC2 signaling output are not known.

TORC2 Effectors
The best characterized substrates of yeast and mammalian TORC2 are the AGC family kinases,
which are activated downstream of TORC2 by phosphorylation on the so-called turn and
hydrophobic motifs found in their C termini [78].

TORC2 Regulates Cell Turgor and Membrane Tension

In yeast, the major TORC2 substrate is the AGC kinase Ypk1. Expression of a hyperactive
allele of Ypk1 rescues the lethality of yeast cells lacking essential TORC2 components and
suppresses all known phenotypes associated with loss of TORC2 activity [53]. YPK2 is a
paralog of YPK1 that is essential only in the absence of YPK1 [79,80]. Ypk1 has a number
of known targets including the Orm1/2 paralogs and ceramide synthase subunits that
regulate the biosynthesis of complex sphingolipids; and, the glycerol efflux pump Fps1
and the glycerol-3-phosphate dehydrogenase Gpd1, which regulate intracellular accumula-
tion of glycerol, a major osmoprotectant. Collectively, through the phosphorylation of these
substrates, TORC2/Ypk1 coordinately regulate turgor pressure and plasma membrane
tension [81].

540 Trends in Biochemical Sciences, June 2016, Vol. 41, No. 6

Beyond membrane tension, TORC2, via Ypk1, additionally regulates the G2/M cell cycle
transition [16], actin organization and endocytosis [18], genome integrity [82], and the pentose
phosphate pathway [17]. Lastly, TORC2 also phosphorylates the sole PKC ortholog in yeast,
Pkc1, presumably to regulate the downstream cell wall integrity MAP kinase pathway [69]. The
conservation of these effectors remains to be determined but from these studies it is safe to
conclude that, like TORC1, TORC2 plays a central role in regulating multiple aspects of
eukaryotic physiology.

Definition of the Pathways Downstream of MTORC2 Is Ongoing

Like TORC2 in yeast, MTORC2 phosphorylates the turn and hydrophobic motifs in multiple AGC
family kinases including AKT, SGK1, and PKC-/ [82–86]. However, two major obstacles have
hindered further characterization of the pathways downstream of MTORC2 and these AGC
kinases.

The first obstacle in the interrogation of MTORC2 effector pathways lies in the curious fact that, in
mammalian cells, phosphorylation of the turn and hydrophobic motifs by MTORC2 seems to be
required only for maximal activity and thus influences only substrate specificity of the down-
stream AGC kinase (reviewed in [39,87]). This means that known AGC kinase substrates are
not necessarily MTORC2 distal effectors even if the AGC kinase in question is a substrate of
MTORC2. Neglecting this fact has greatly confused the study of MTORC2 effectors. For example, in
mouse embryonic fibroblasts, loss of MTORC2-mediated phosphorylation on Ser473 in the
hydrophobic motif of AKT diminishes AKT-mediated phosphorylation of the FOXO1/3A (forkhead
box O1/3a) transcription factor but not TSC2 (a GTPase activating protein that acts on the
MTORC1-activating GTPase RHEB) nor GSK3-b (glycogen synthase kinase). Thus, it is inappropri-
ate to cartoon MTORC2 ! AKT ! TSC2 ! RHEB as a mechanism of MTORC1 regulation as is often
done in MTOR reviews.

The second obstacle in the interrogation of MTORC2 effector pathways is the absence of small
molecules that specifically and acutely inhibit this complex. This problem has recently been
surmounted in yeast using reverse chemical genetics (reviewed in [81]), but analogous systems
have yet to be developed in mammalian systems. Although ATP competitive MTOR inhibitors are
available [3], these compounds inhibit both MTOR complexes which makes it difficult to assign
functions to one complex over the other.

Because of these challenges, MTORC2 functions have most commonly been ascertained through
genetic studies, typically involving deletion of the gene encoding the essential RICTOR subunit.
However, unlike acute inhibition attainable through pharmacological approaches, genetic stud-
ies, due to their chronic nature, often permit adaptive events to occur, which can confound
results. A potential example of this can be seen in two studies where RICTOR was deleted in
muscle. In one study [88], RICTOR-deficient muscle exhibited decreased insulin-stimulated glu-
cose uptake, decreased AKT Ser473 phosphorylation and the mice displayed glucose intolerance.
While in another study [89], RICTOR-deficient muscle was indistinguishable from wild-type con-
trols, although one should note that the ex vivo analyses of RICTOR-deficient muscle was relatively
less rigorous in this study. One possible mechanism of adaptation is the reacquisition of AKT
Ser473 phosphorylation in RICTOR-deficient cells as was observed in the latter study. Indeed,
DNA–PKcs [90] among other kinases have been proposed to phosphorylate AKT Ser
473
, the so-
called PDK2 site, independently of MTORC2. Other possible confounding factors can of course be
the choice of promoter driving the conditional knockout [91], or inherent differences in the mouse
genetic backgrounds.

Other, tissue-specific RICTOR knockouts have now been reported. Global deletion of RICTOR
causes lethality during embryonic development likely due to defects in the fetal vasculature

Trends in Biochemical Sciences, June 2016, Vol. 41, No. 6 541

[84,92]. In line with this, inducible deletion of RICTOR in the mouse endothelium demonstrated that Outstanding Questions
MTORC2 is essential for midgestational development but not later in development [93,94]. In the treatment of cancer, metabolic
Interestingly, RICTOR deletion prevented hyperneovascularization induced by ectopic fibroblast syndrome, and other maladies, would
MTORC2-specific inhibitors be better
growth factor treatment, or by implantation of tumor cells. Subsequent ex vivo studies sug- than MTOR inhibitors that inhibit both
gested that MTORC2 regulates vascular assembly and endothelial cell proliferation via AKT and MTOR Complexes?

PKC-/, respectively [94].

Relative to RAPTOR (MTORC1) deletions,
conditional RICTOR (MTORC2) deletions
RICTOR deletion in the central nervous system demonstrated an important role for MTORC2, via PKC appear to be well tolerated. Thus, phar-
isoforms, in regulating the size and function of neurons and in long-term memory consolidation macophores targeting MTORC2 may
[95]; and, analogous studies demonstrated a role for MTORC2, via multiple effectors, in T cell retain efficacy while presenting a better
toxicity profile compared with ATP
functions [96,97]. Conditional RICTOR knockouts also demonstrated that MTORC2 signaling from
competitive MTOR inhibitors.
adipose tissue plays an unexpected role as a negative regulator of whole-body growth [98,99]
and is indispensable for the growth of brown adipose tissue (BAT) [91]. Interestingly, the latter How can one specifically target
study raised the possibility that targeting MTORC2 in BAT could be a useful strategy to increase mTORC2?

energy expenditure in obese people. However, fat cell-specific RICTOR ablation provoked glucose
Continuing improvements on the 3D
intolerance, hyperinsulinemia, and hepatic steatosis. Beta-cell-specific RICTOR ablation also structure of mTORC2 will hopefully facil-
provoked hyperglycemia and glucose intolerance [100]. Furthermore, RICTOR deletion in the liver itate the rational design of compounds
[101–103] was found to perturb glucose metabolism leading to hyperglycemia and hyper- that (i) prevent or disrupt mTORC2
assembly, (ii) block access of substrates
insulinemia. Interestingly, however, these mice also presented reduced serum cholesterol and to the active site, (iii) prevent the interac-
reduced hepatic steatosis. In summary, these observations suggest that MTORC2 inhibitors tion of upstream activators, or (iv) pre-
would trigger both desired as well as unwanted metabolic consequences. vent membrane association (via the PH
domain of SIN1).

Concluding Remarks and Future Perspectives Might mTORC2 agonists be clinically

Despite recent advances in exploring the structure and function of MTORC2, small molecules that useful?
specifically target this kinase remain to be described (see Outstanding Questions). Thus, the
In some cases, it might be clinically
question of whether MTORC2-specific inhibitors will be clinically useful remains hypothetical.
interesting to hyperactivate mTORC2.
Furthermore, although the field is profiting from a mix of pharmacological studies in yeast, Such agonists may serve to transiently
genetic studies in metazoans, and structural biology studies, our understanding of MTORC2 stimulate glucose uptake in diabetics,
regulation and signaling outputs is still rudimentary making it very difficult to predict the or to prevent cell death upon hypoxia.
As we learn more about mTORC2
therapeutic costs versus gains of MTORC2 inhibition. It is encouraging to note that despite being
effector pathways, the relevant clinical
essential during early development, MTORC2 inhibition in adults appears to be relatively well circumstances where mTORC2 ago-
tolerated as exemplified by the fact that ATP competitive inhibitors of MTOR, which inhibit both nists could be useful will become more
MTORC1 and MTORC2, are in advanced clinical trials. In contrast, as described, mouse studies obvious.

predict that MTORC2 inhibitors may provoke unwanted metabolic issues. Indeed, some of the
How is mTORC2 regulated?
undesirable side effects of prolonged rapamycin treatment, including impaired sugar metabo-
lism, are attributed to inhibition of MTORC2, although these might be mitigated with optimized Presently, the regulation of mTORC2
treatment regimens [104,105]. Furthermore, RICTOR deletion in mouse heart results in cardiac is fairly controversial. Structural data to
date suggest that the kinase domain per
dysfunction [106] and RICTOR deletion in mouse liver or induced systemically in all mouse cells se is in a constitutively active conforma-
late in life specifically reduces male lifespan [107]. It will be interesting to see how well these tion. This would suggest that signaling
mouse studies will resonate with human clinical studies. Targeted drug delivery to affected output is regulated by recruitment of
organs may also help assuage eventual side effects. substrate to mTORC2, or, recruitment
of mTORC2 to substrate. Key to this
will be a definitive determination of
Although loss of MTORC2 appears to affect basal AKT activity only modestly, elegant genetic mTORC2 localization within the cell.
experiments in flies demonstrate that dTORC2 activity is absolutely required for pathologies Also important will be a molecular under-
standing of why the TOR complexes
stemming from dAkt hyperactivation [108]. The requirement for MTORC2 in pathologies driven by
are dimeric and why they possess such
hyperactive AKT has also been reported. For example, in mice, RICTOR deletion is sufficient to block a large central cavity.
aberrant fibroblast growth factor (FGF)2- or tumor-induced neovascularization, and tumor
development induced by loss of the PTEN tumor suppressor [93,94,109]. These observations
suggest that it would be interesting to evaluate MTORC2-specific inhibitors in the treatment of any
pathology associated with hyperactive AKT, which includes many cancers with PIK3CA mutations
[110]. Lastly, according to the COSMIC (Catalog of Somatic Mutations in Cancer) databasei,
RICTOR appears to be amplified in a large percentage of cancers, suggesting that MTORC2 itself

542 Trends in Biochemical Sciences, June 2016, Vol. 41, No. 6

could be an oncogenic driver. This hypothesis is supported by a very recent, exciting report
describing the so-far efficacious treatment of an 18-year-old never-smoker lung adenocarci-
noma patient [111]. Interestingly, this malignancy appears to be driven by RICTOR amplification
and the successful treatment has been with the MTORC1/2 inhibitor MLN0128.
Acknowledgments
The authors thank Georgia Konstantinidou and the anonymous reviewers for their critical reading of the manuscript and
apologize to authors of relevant studies that were not cited owing to space restrictions. The Loewith lab receives funding
from the Canton of Geneva, the Swiss National Science Foundation, and the European Research Council Consolidator
Grant Programme.
Resources
i
http://cancer.sanger.ac.uk/cosmic/gene/analysis?ln=RICTOR
References
1. Sehgal, S.N. et al. (1975) Rapamycin (AY-22,989), a new anti-
fungal antibiotic. II. Fermentation, isolation and characterization.
J. Antibiot. (Tokyo) 28, 727–732
2. Vezina, C. et al. (1975) Rapamycin (AY-22,989), a new antifungal
antibiotic. I. Taxonomy of the producing streptomycete and
isolation of the active principle. J. Antibiot. (Tokyo) 28, 721–726
3. Benjamin, D. et al. (2011) Rapamycin passes the torch: a
new generation of mTOR inhibitors. Nat. Rev. Drug Discov.
10, 868–880
4. Houchens, D.P. et al. (1983) Human brain tumor xenografts in
nude mice as a chemotherapy model. Eur. J. Cancer Clin. Oncol.
19, 799–805
5. Heitman, J. et al. (1991) Targets for cell cycle arrest by the
immunosuppressant rapamycin in yeast. Science 253, 905–909
6. Loewith, R. et al. (2002) Two TOR complexes, only one of which
is rapamycin sensitive, have distinct roles in cell growth control.
Mol. Cell 10, 457–468
7. Wedaman, K.P. et al. (2003) Tor kinases are in distinct mem-
brane-associated protein complexes in Saccharomyces cerevi-
siae. Mol. Biol. Cell 14, 1204–1220
8. Hara, K. et al. (2002) Raptor, a binding partner of target of
rapamycin (TOR), mediates TOR action. Cell 110, 177–189
9. Jacinto, E. et al. (2004) Mammalian TOR complex 2 controls the
actin cytoskeleton and is rapamycin insensitive. Nat. Cell Biol. 6,
1122–1128
10. Kim, D-H. et al. (2002) mTOR interacts with raptor to form a
nutrient-sensitive complex that signals to the cell growth machin-
ery. Cell 110, 163–175
11. Sarbassov, D.D. et al. (2004) Rictor, a novel binding partner of
mTOR, defines a rapamycin-insensitive and raptor-independent
pathway that regulates the cytoskeleton. Curr. Biol. 14, 1296–
1302
12. van Dam, T.J.P. et al. (2011) Evolution of the TOR Pathway. J.
Mol. Evol. 73, 209–220
13. Loewith, R. and Hall, M.N. (2011) Target of rapamycin (TOR)
in nutrient signaling and growth control. Genetics 189, 1177–
1201
14. Panchaud, N. et al. (2013) SEACing the GAP that nEGOCiates
TORC1 activation: evolutionary conservation of Rag GTPase
regulation. Cell Cycle 12, 2948–2952
15. Zoncu, R. et al. (2011) mTOR: from growth signal integration to
cancer, diabetes and ageing. Nat. Rev. Mol. Cell Biol. 12, 21–35
16. Gaubitz, C. et al. (2015) Molecular basis of the rapamycin insen-
sitivity of target of rapamycin complex 2. Mol. Cell 58, 977–988
17. Kliegman, J.I. et al. (2013) Chemical genetics of rapamycin-
insensitive TORC2 in S. cerevisiae. Cell Rep. 5, 1725–1736
18. Rispal, D. et al. (2015) Target of rapamycin complex 2 regulates
actin polarization and endocytosis via multiple pathways. J. Biol.
Chem. 290, 14963–14978
19. Rexin, D. et al. (2015) TOR signalling in plants. Biochem. J. 470,
1–14
20. Yip, C.K. et al. (2010) Structure of the human mTOR complex I and
its implications for rapamycin inhibition. Mol. Cell 38, 768–774
21. Aylett, C.H.S. et al. (2016) Architecture of human mTOR complex
1. Science 351, 48–52
22. Yang, H. et al. (2013) mTOR kinase structure, mechanism and
regulation. Nature 497, 217–223
, D. et al. (2016) Tor forms a dimer through an N-terminal
23. Baretic
helical solenoid with a complex topology. Nat. Commun. Published
online April 13, 2016. http://dx.doi.org/10.1038/ncomms11016
24. Keith, C.T. and Schreiber, S.L. (1995) PIK-related kinases: DNA
repair, recombination, and cell cycle checkpoints. Science 270,
50–51
25. Neer, E.J. et al. (1994) The ancient regulatory-protein family of
WD-repeat proteins. Nature 371, 297–300
26. Wullschleger, S. et al. (2005) Molecular organization of target of
rapamycin complex 2. J. Biol. Chem. 280, 30697–30704
27. Javadi, Y. and Itzhaki, L.S. (2013) Tandem-repeat proteins:
regularity plus modularity equals design-ability. Curr. Opin.
Struct. Biol. 23, 622–631
28. Marchler-Bauer, A. (2004) CDD: a Conserved Domain Database
for protein classification. Nucleic Acids Res. 33, D192–D196
29. Lee, S. et al. (1999) A novel Ras-interacting protein required for
chemotaxis and cyclic adenosine monophosphate signal relay in
Dictyostelium. Mol. Biol. Cell 10, 2829–2845
30. Colicelli, J. et al. (1991) Expression of three mammalian cDNAs
that interfere with RAS function in Saccharomyces cerevisiae.
Proc. Natl. Acad. Sci. U.S.A. 88, 2913–2917
31. Lee, S. et al. (2005) TOR complex 2 integrates cell movement
during chemotaxis and signal relay in Dictyostelium. Mol. Biol.
Cell 16, 4572–4583
32. Schroder, W.A. et al. (2007) Human Sin1 contains Ras-binding
and pleckstrin homology domains and suppresses Ras signal-
ling. Cell. Signal. 19, 1279–1289
33. Berchtold, D. and Walther, T.C. (2009) TORC2 plasma mem-
brane localization is essential for cell viability and restricted to a
distinct domain. Mol. Biol. Cell 20, 1565–1575
34. Gallego, O. et al. (2010) A systematic screen for protein–lipid
interactions in Saccharomyces cerevisiae. Mol. Syst. Biol. 6, 430
35. Pan, D. and Matsuura, Y. (2012) Structures of the pleckstrin
homology domain of Saccharomyces cerevisiae Avo1 and its
human orthologue Sin1, an essential subunit of TOR complex
2. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 68,
386–392
36. Schroder, W. et al. (2004) Alternative polyadenylation and splic-
ing of mRNAs transcribed from the human Sin1 gene. Gene 339,
17–23
37. Cameron, A.J.M. et al. (2011) mTORC2 targets AGC kinases
through Sin1-dependent recruitment. Biochem. J. 439, 287–297
38. Liao, H-C. and Chen, M-Y. (2012) Target of rapamycin complex 2
signals to downstream effector yeast protein kinase 2 (Ypk2)
through adheres-voraciously-to-target-of-rapamycin-2 protein 1
Trends in Biochemical Sciences, June 2016, Vol. 41, No. 6 543
 (Avo1) in Saccharomyces cerevisiae. J. Biol. Chem. 287, 6089–
6099
39. Polak, P. and Hall, M.N. (2006) mTORC2 caught in a SINful Akt.
Dev. Cell 11, 433–434
40. Frias, M.A. et al. (2006) mSin1 is necessary for Akt/PKB phos-
phorylation, and its isoforms define three distinct mTORC2s.
Curr. Biol. 16, 1865–1870
41. Yuan, Y. et al. (2015) Characterization of Sin1 isoforms reveals an
mTOR-dependent and independent function of Sin1g. PLoS
ONE 10, e0135017
42. Pearce, L.R. et al. (2011) Protor-1 is required for efficient
mTORC2-mediated activation of SGK1 in the kidney. Biochem.
J. 436, 169–179
43. Thedieck, K. et al. (2007) PRAS40 and PRR5-like protein are new
mTOR interactors that regulate apoptosis. PLoS ONE 2, e1217
44. Woo, S-Y. et al. (2007) PRR5, a novel component of mTOR
complex 2, regulates platelet-derived growth factor receptor beta
expression and signaling. J. Biol. Chem. 282, 25604–25612
45. Gatherar, I. et al. (2004) Identification of a novel gene hbrB
required for polarised growth in Aspergillus nidulans. Fungal
Genet. Biol. 41, 463–471
46. Goujon, M. et al. (2010) A new bioinformatics analysis tools
framework at EMBL-EBI. Nucleic Acids Res. 38, W695–W699
47. Fadri, M. et al. (2005) The pleckstrin homology domain proteins
Slm1 and Slm2 are required for actin cytoskeleton organization in
yeast and bind phosphatidylinositol-4,5-bisphosphate and
TORC2. Mol. Biol. Cell 16, 1883–1900
48. Peterson, T.R. et al. (2009) DEPTOR is an mTOR inhibitor fre-
quently overexpressed in multiple myeloma cells and required for
their survival. Cell 137, 873–886
49. Consonni, S.V. et al. (2014) DEP domains: structurally similar but
functionally different. Nat. Rev. Mol. Cell Biol. 15, 357–362
50. Berchtold, D. et al. (2012) Plasma membrane stress induces
relocalization of Slm proteins and activation of TORC2 to pro-
mote sphingolipid synthesis. Nat. Cell Biol. 14, 542–547
51. Douglas, L.M. and Konopka, J.B. (2014) Fungal membrane
organization: the eisosome concept. Annu. Rev. Microbiol. 68,
377–393
52. Kabeche, R. et al. (2015) Eisosomes provide membrane reser-
voirs for rapid expansion of the yeast plasma membrane. J. Cell
Sci. 128, 4057–4062
53. Niles, B.J. et al. (2012) Plasma membrane recruitment and
activation of the AGC kinase Ypk1 is mediated by target of
rapamycin complex 2 (TORC2) and its effector proteins Slm1
and Slm2. Proc. Natl. Acad. Sci. U.S.A. 109, 1536–1541
54. Kippenberger, S. et al. (2005) Mechanical stretch stimulates
protein kinase B/Akt phosphorylation in epidermal cells via angio-
tensin II type 1 receptor and epidermal growth factor receptor.
J. Biol. Chem. 280, 3060–3067
55. Sedding, D.G. (2005) Caveolin-1 facilitates mechanosensitive
protein kinase B (Akt) signaling in vitro and in vivo. Circ. Res.
96, 635–642
56. Yu, J. (2006) Direct evidence for the role of caveolin-1 and
caveolae in mechanotransduction and remodeling of blood ves-
sels. J. Clin. Invest. 116, 1284–1291
57. Zhang, B. et al. (2007) Caveolin-1 phosphorylation is required for
stretch-induced EGFR and Akt activation in mesangial cells. Cell.
Signal. 19, 1690–1700
58. Chen, C-H. et al. (2011) ER stress inhibits mTORC2 and Akt
signaling through GSK-3b-mediated phosphorylation of rictor.
Sci. Signal 4, ra10
59. Muir, A. et al. (2014) TORC2-dependent protein kinase Ypk1
phosphorylates ceramide synthase to stimulate synthesis of
complex sphingolipids. Elife 3, e03779
60. Betz, C. and Hall, M.N. (2013) Where is mTOR and what is it
doing there? J. Cell Biol. 203, 563–574
61. Arias, E. et al. (2015) Lysosomal mTORC2/PHLPP1/Akt regulate
chaperone-mediated autophagy. Mol. Cell 59, 270–284
62. Gan, X. et al. (2011) Evidence for direct activation of mTORC2
kinase activity by phosphatidylinositol 3,4,5-trisphosphate. J.
Biol. Chem. 286, 10998–11002
544 Trends in Biochemical Sciences, June 2016, Vol. 41, No. 6
63. Liu, P. et al. (2015) PtdIns(3,4,5)P3-dependent activation of the
mTORC2 kinase complex. Cancer Discov 5, 1194–1209
64. Swaney, D.L. et al. (2013) Global analysis of phosphorylation and
ubiquitylation cross-talk in protein degradation. Nat. Methods 10,
676–682
65. Dibble, C.C. et al. (2009) Characterization of rictor phosphory-
lation sites reveals direct regulation of mTOR complex 2 by S6K1.
Mol. Cell. Biol. 29, 5657–5670
66. Glidden, E.J. et al. (2012) Multiple site acetylation of Rictor
stimulates mammalian target of rapamycin complex 2
(mTORC2)-dependent phosphorylation of Akt protein. J. Biol.
Chem. 287, 581–588
67. Masui, K. et al. (2013) mTOR complex 2 controls glycolytic
metabolism in glioblastoma through FoxO acetylation and upre-
gulation of c-Myc. Cell Metab. 18, 726–739
68. Choudhary, C. et al. (2014) The growing landscape of lysine
acetylation links metabolism and cell signalling. Nat. Rev. Mol.
Cell Biol. 15, 536–550
69. Nomura, W. and Inoue, Y. (2015) Methylglyoxal activates the
target of rapamycin complex 2-protein kinase C signaling
pathway in Saccharomyces cerevisiae. Mol. Cell. Biol. 35,
1269–1280
70. Hatano, T. et al. (2015) Fission yeast Ryh1 GTPase activates TOR
complex 2 in response to glucose. Cell Cycle 14, 848–856
71. Tatebe, H. et al. (2010) Rab-family GTPase regulates TOR com-
plex 2 signaling in fission yeast. Curr. Biol. 20, 1975–1982
72. Saci, A. et al. (2011) Rac1 regulates the activity of mTORC1 and
mTORC2 and controls cellular size. Mol. Cell 42, 50–61
73. Cai, W. and Andres, D.A. (2014) mTORC2 is required for Rit-
mediated oxidative stress resistance. PLoS ONE 9, e115602
74. Niles, B.J. and Powers, T. (2014) TOR complex 2–Ypk1 signaling
regulates actin polarization via reactive oxygen species. Mol. Biol.
Cell 25, 3962–3972
75. Khanna, N. et al. (2013) XPLN is an endogenous inhibitor of
mTORC2. Proc. Natl. Acad. Sci. U.S.A. 110, 15979–15984
76. Oh, W.J. et al. (2010) mTORC2 can associate with ribosomes to
promote cotranslational phosphorylation and stability of nascent
Akt polypeptide. EMBO J. 29, 3939–3951
77. Zinzalla, V. et al. (2011) Activation of mTORC2 by association
with the ribosome. Cell 144, 757–768
78. Pearce, L.R. et al. (2010) The nuts and bolts of AGC protein
kinases. Nat. Rev. Mol. Cell Biol. 11, 9–22
79. Kamada, Y. et al. (2005) Tor2 directly phosphorylates the AGC
kinase Ypk2 to regulate actin polarization. Mol. Cell. Biol. 25,
7239–7248
80. Roelants, F.M. (2004) Differential roles of PDK1- and PDK2-
phosphorylation sites in the yeast AGC kinases Ypk1, Pkc1
and Sch9. Microbiology 150, 3289–3304
81. Eltschinger, S. and Loewith, R. (2016) TOR complexes and
the maintenance of cellular homeostasis. Trends Cell Biol 26,
148–159
82. Shimada, K. et al. (2013) TORC2 signaling pathway guarantees
genome stability in the face of DNA strand breaks. Mol. Cell 51,
829–839
83. García-Martínez, J.M. and Alessi, D.R. (2008) mTOR complex 2
(mTORC2) controls hydrophobic motif phosphorylation and acti-
vation of serum- and glucocorticoid-induced protein kinase 1
(SGK1). Biochem. J. 416, 375–385
84. Guertin, D.A. et al. (2006) Ablation in mice of the mTORC com-
ponents raptor, rictor, or mLST8 reveals that mTORC2 is
required for signaling to Akt-FOXO and PKC/, but not S6K1.
Dev. Cell 11, 859–871
85. Ikenoue, T. et al. (2008) Essential function of TORC2 in PKC and
Akt turn motif phosphorylation, maturation and signalling. EMBO
J. 27, 1919–1931
86. Sarbassov, D.D. et al. (2005) Phosphorylation and regulation
of Akt/PKB by the Rictor–mTOR complex. Science 307,
1098–1101
87. Sparks, C.A. and Guertin, D.A. (2010) Targeting mTOR: pros-
pects for mTOR complex 2 inhibitors in cancer therapy. Onco-
gene 29, 3733–3744
88. Kumar, A. et al. (2008) Muscle-specific deletion of Rictor impairs
insulin-stimulated glucose transport and enhances basal glyco-
gen synthase activity. Mol. Cell. Biol. 28, 61–70
89. Bentzinger, C.F. et al. (2008) Skeletal muscle-specific ablation of
raptor, but not of rictor, causes metabolic changes and results in
muscle dystrophy. Cell Metab. 8, 411–424
90. Bozulic, L. and Hemmings, B.A. (2009) PIKKing on PKB: regula-
tion of PKB activity by phosphorylation. Cell Regul. 21, 256–261
91. Hung, C-M. et al. (2014) Rictor/mTORC2 loss in the Myf5 lineage
reprograms brown fat metabolism and protects mice against
obesity and metabolic disease. Cell Rep. 8, 256–271
92. Shiota, C. et al. (2006) Multiallelic disruption of the rictor gene in
mice reveals that mTOR complex 2 is essential for fetal growth
and viability. Dev. Cell 11, 583–589
93. Aimi, F. et al. (2015) Endothelial Rictor is crucial for midgestational
development and sustained and extensive FGF2-induced neo-
vascularization in the adult. Sci. Rep. 5, 17705
94. Wang, S. et al. (2015) Regulation of endothelial cell proliferation
and vascular assembly through distinct mTORC2 signaling path-
ways. Mol. Cell. Biol. 35, 1299–1313
95. Thomanetz, V. et al. (2013) Ablation of the mTORC2 component
rictor in brain or Purkinje cells affects size and neuron morphol-
ogy. J. Cell Biol. 201, 293–308
96. Chou, P-C. et al. (2014) Mammalian target of rapamycin complex
2 modulates TCR processing and surface expression during
thymocyte development. J. Immunol. 193, 1162–1170
97. Lee, K. et al. (2010) Mammalian target of rapamycin protein
complex 2 regulates differentiation of Th1 and Th2 cell subsets
via distinct signaling pathways. Immunity 32, 743–753
98. Cybulski, N. et al. (2009) mTOR complex 2 in adipose tissue
negatively controls whole-body growth. Proc. Natl. Acad. Sci. U.
S.A. 106, 9902–9907
99. Kumar, A. et al. (2010) Fat cell-specific ablation of Rictor in mice
impairs insulin-regulated fat cell and whole-body glucose and
lipid metabolism. Diabetes 59, 1397–1406
100. Gu, Y. et al. (2011) Rictor/mTORC2 is essential for maintaining a
balance between b-cell proliferation and cell size. Diabetes 60,
827–837
101. Hagiwara, A. et al. (2012) Hepatic mTORC2 activates glycolysis
and lipogenesis through Akt, glucokinase, and SREBP1c. Cell
Metab. 15, 725–738
102. Lamming, D.W. et al. (2012) Rapamycin-induced insulin resis-
tance is mediated by mTORC2 loss and uncoupled from longev-
ity. Science 335, 1638–1643
103. Lamming, D.W. et al. (2014) Hepatic signaling by the mechanistic
target of rapamycin complex 2 (mTORC2). FASEB J. 28, 300–315
104. Arriola Apelo, S.I. et al. (2016) Alternative rapamycin treatment
regimens mitigate the impact of rapamycin on glucose homeo-
stasis and the immune system. Aging Cell 15, 28–38
105. Lamming, D.W. et al. (2013) Rapalogs and mTOR inhibitors as
anti-aging therapeutics. J. Clin. Invest. 123, 980–989
106. Sciarretta, S. et al. (2015) mTORC2 regulates cardiac response
to stress by inhibiting MST1. Cell Rep. 11, 125–136
107. Lamming, D.W. et al. (2014) Depletion of Rictor, an essential
protein component of mTORC2, decreases male lifespan. Aging
Cell 13, 911–917
108. Hietakangas, V. and Cohen, S.M. (2007) Re-evaluating AKT
regulation: role of TOR complex 2 in tissue growth. Genes
Dev. 21, 632–637
109. Guertin, D.A. et al. (2009) mTOR complex 2 is required for the
development of prostate cancer induced by Pten loss in mice.
Cancer Cell 15, 148–159
110. Ciriello, G. et al. (2013) Emerging landscape of oncogenic sig-
natures across human cancers. Nat. Genet. 45, 1127–1133
111. Cheng, H. et al. (2015) RICTOR amplification defines a novel
subset of patients with lung cancer who may benefit from
treatment with mTORC1/2 inhibitors. Cancer Discov. 5,
1262–1270
112. Dan, H.C. et al. (2014) Akt-dependent activation of mTORC1
complex involves phosphorylation of mTOR (mammalian tar-
get of rapamycin) by IB kinase (IKK). J. Biol. Chem. 289,
25227–25240
113. Ekim, B. et al. (2011) mTOR kinase domain phosphorylation
promotes mTORC1 signaling, cell growth, and cell cycle pro-
gression. Mol. Cell. Biol. 31, 2787–2801
114. Halova, L. et al. (2013) Phosphorylation of the TOR ATP binding
domain by AGC kinase constitutes a novel mode of TOR inhibi-
tion. J. Cell Biol. 203, 595–604
115. Holz, M.K. (2005) Identification of S6 kinase 1 as a novel mam-
malian target of rapamycin (mTOR)-phosphorylating kinase. J.
Biol. Chem. 280, 26089–26093
116. Copp, J. et al. (2009) TORC-specific phosphorylation of mam-
malian target of rapamycin (mTOR): phospho-Ser2481 is a
marker for intact mTOR signaling complex 2. Cancer Res. 69,
1821–1827
117. Julien, L-A. et al. (2010) mTORC1-activated S6K1 phosphory-
lates Rictor on threonine 1135 and regulates mTORC2 signaling.
Mol. Cell. Biol. 30, 908–921
118. Treins, C. et al. (2010) Rictor is a novel target of p70 S6 kinase-1.
Oncogene 29, 1003–1016
119. Humphrey, S.J. et al. (2013) Dynamic adipocyte phosphopro-
teome reveals that Akt directly regulates mTORC2. Cell Metab.
17, 1009–1020
120. Liu, P. et al. (2014) Dual phosphorylation of Sin1 at T86 and T398
negatively regulates mTORC2 complex integrity and activity.
Protein Cell 5, 171–177
121. Yang, G. et al. (2015) A positive feedback loop between Akt and
mTORC2 via SIN1 phosphorylation. Cell Rep. 12, 937–943
122. Chen, C-H. et al. (2013) Autoregulation of the mechanistic
target of rapamycin (mTOR) complex 2 integrity is controlled
by an ATP-dependent mechanism. J. Biol. Chem. 288,
27019–27030
Trends in Biochemical Sciences, June 2016, Vol. 41, No. 6 545