mTOR complex 2 - akt signaling is physically and functionally at mam

Betz, Charles. mTOR complex 2 - akt signaling is physically and functionally at mam. 2012, PhD Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_10188


The target of rapamycin (TOR) is a conserved protein kinase and a
central controller of growth. TOR can be part of two structurally and
functionally distinct complexes, termed TOR complex 1 and TOR
complex 2. Mammalian TOR complex 2 (mTORC2) is composed of
mTOR, Rictor, Sin1 and mLST8. Both mTORC1 and mTORC2 are activated
by growth factors. The mechanism via which growth factors
regulate mTORC2 has been elusive until recently. mTORC2 binds ribosomes
in a growth factor stimulated manner and this association is
required for mTORC2 activity.
mTOR complex 2 functions include control of spatial cell growth
and metabolism and thus, mTORC2 deregulation has been linked
to various disorders including cancer and diabetes. mTORC2 phosphorylates
and thereby activates the AGC kinase family member Akt
(PKB). Akt has many different targets and functions, not all of which
depend on mTORC2 mediated Akt phosphorylation.
In order to gain a better understanding of mTORC2 function, we
asked where mTORC2 signaling is localized. A number of studies
localized mTORC2, functionally or physically, either to the endoplasmic
reticulum (ER) or to mitochondria.We investigated whether these
seemingly unrelated observations concerning mTORC2 localization,
might be the consequence of mTORC2 signaling at MAM. MAM
or mitochondria-associated ER membrane is a quasi-synaptic subdomain
between the ER and mitochondria. MAM plays a crucial role in
the regulation of mitochondrial metabolism and cell survival by gating
both the calcium flux and phospholipid trafficking between the
ER and mitochondria.
First, we analyzed mTORC2 subcellular localization. mTORC2 is
localized to the ER adjacent to mitochondria and mTORC2 can be
biochemically isolated from MAM structures. mTOR complex 2 interacts
with the IP3R-Grp75-VDAC1 complex, a tether that connects ER
and mitochondria at MAM. Insulin stimulates mTORC2 localization
to MAM and mTORC2 interaction with the IP3R-Grp75-VDAC1 complex.
MAM localization of mTORC2 depends on mTORC2-ribosome
Next we investigated the function of mTORC2 at MAM. Rictor
(mTORC2) knockout causes a decrease in MAM formation. Growth
factors stimulate MAM formation via mTORC2 and the Akt substrate
PACS2, a MAM resident protein. As expected for MAM deficient
cells, mTORC2 disruption changes the calcium flux from the ER to
mitochondria at MAM. Furthermore, we observe a reduction of Akt
mediated phosphorylation of the MAM calcium channel IP3R upon
Rictor knockout. Thus, mTORC2 signaling at MAM controls MAM mediated
calcium release via the Akt targets PACS2 and IP3R.
Since MAM disruption and calcium signaling both affect mitochondrial
metabolism, we proceeded by analyzing the mitochondrial physiology
of mTORC2 deficient cells. Rictor knockout cells exhibit a disruption
of VDAC1-HK2 binding, caused by a lack of Akt mediated
phosphorylation of HK2 at MAM. This, together with the defect in
MAM, induces an increase in basal respiration, mitochondrial inner
membrane potential, and ATP production in the mTORC2 deficient
cells, culminating in apoptosis. Thus, mTORC2 at MAM appears to
control several aspects of mitochondrial physiology.
These findings emphasize the role of MAM as a signaling hub that
controls cell physiology. By identifying the integral role of mTORC2
at the core of this platform, our results provide new insights on
the mechanisms that regulate growth and metabolism. These observations
may offer new therapeutic strategies against mTORC2 and
MAM driven diseases such as diabetes, Alzheimer’s and cancer.
Advisors:Hall, Michael N.
Committee Members:Rüegg, Markus A.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Biochemistry (Hall)
Item Type:Thesis
Thesis no:10188
Bibsysno:Link to catalogue
Number of Pages:140 S.
Identification Number:
Last Modified:30 Jun 2016 10:51
Deposited On:27 Nov 2012 15:27

Repository Staff Only: item control page