Mammalian Target of Rapamycin Complex 1 (mTORC1) Activity Is Associated
with Phosphorylation of Raptor by
mTOR |
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Authors: | Lifu Wang John C Lawrence Jr Thomas W Sturgill and Thurl E Harris |
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Institution: | Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908 |
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Abstract: | mTORC1 contains multiple proteins and plays a central role in cell growth
and metabolism. Raptor (regulatory-associated protein of mammalian target of
rapamycin (mTOR)), a constitutively binding protein of mTORC1, is essential
for mTORC1 activity and critical for the regulation of mTORC1 activity in
response to insulin signaling and nutrient and energy sufficiency. Herein we
demonstrate that mTOR phosphorylates raptor in vitro and in
vivo. The phosphorylated residues were identified by using phosphopeptide
mapping and mutagenesis. The phosphorylation of raptor is stimulated by
insulin and inhibited by rapamycin. Importantly, the site-directed mutation of
raptor at one phosphorylation site, Ser863, reduced mTORC1 activity
both in vitro and in vivo. Moreover, the Ser863
mutant prevented small GTP-binding protein Rheb from enhancing the
phosphorylation of S6 kinase (S6K) in cells. Therefore, our findings indicate
that mTOR-mediated raptor phosphorylation plays an important role on
activation of mTORC1.Mammalian target of rapamycin
(mTOR)2 has been shown
to function as a critical controller in cellular growth, survival, metabolism,
and development (1). mTOR, a
highly conserved Ser-Thr phosphatidylinositol 3-kinase-related protein kinase,
structurally forms two distinct complexes, mTOR complex 1 (mTORC1) and mTOR
complex 2 (mTORC2), each of which catalyzes the phosphorylation of different
substrates (1). The best
characterized substrates for mTORC1 are eIF4E-binding protein (4E-BP, also
known as PHAS) and p70 S6 kinase (S6K)
(1), whereas mTORC2
phosphorylates the hydrophobic and turn motifs of protein kinase B
(Akt/protein kinase B) (2) and
protein kinase C (3,
4). mTORC1 constitutively
consists of mTOR, raptor, and mLst8/GβL
(1), whereas the proline-rich
Akt substrate of 40 kDa (PRAS40) is a regulatory component of mTORC1 that
disassociates after growth factor stimulation
(5,
6). Raptor is essential for
mTORC1 activity by providing a substrate binding function
(7) but also plays a regulatory
role on mTORC1 with stimuli of growth factors and nutrients
(8). In response to insulin,
raptor binding to substrates is elevated through the release of the
competitive inhibitor PRAS40 from mTORC1
(9,
10) because PRAS40 and the
substrates of mTORC1 (4E-BP and S6K) appear to bind raptor through a consensus
sequence, the TOR signaling (TOS) motif
(10–14).
In response to amino acid sufficiency, raptor directly interacts with a
heterodimer of Rag GTPases and promotes mTORC1 localization to the
Rheb-containing vesicular compartment
(15).mTORC1 integrates signaling pathways from growth factors, nutrients,
energy, and stress, all of which generally converge on the tuberous sclerosis
complex (TSC1-TSC2) through the phosphorylation of TSC2
(1). Growth factors inhibit the
GTPase-activating protein activity of TSC2 toward the small GTPase Rheb via
the PI3K/Akt pathway (16,
17), whereas energy depletion
activates TSC2 GTPase-activating protein activity by stimulating AMP-activated
protein kinase (AMPK) (18).
Rheb binds directly to mTOR, albeit with very low affinity
(19), and upon charging with
GTP, Rheb functions as an mTORC1 activator
(6). mTORC1 complexes isolated
from growth factor-stimulated cells show increased kinase activity yet do not
contain detectable levels of associated Rheb. Therefore, how Rheb-GTP binding
to mTOR leads to an increase in mTORC1 activity toward substrates, and what
the role of raptor is in this activation is currently unknown. More recently,
the AMPK and p90 ribosomal S6 kinase (RSK) have been reported to directly
phosphorylate raptor and regulate mTORC1 activity. The phosphorylation of
raptor directly by AMPK reduced mTORC1 activity, suggesting an alternative
regulation mechanism independent of TSC2 in response to energy supply
(20). RSK-mediated raptor
phosphorylation enhances mTORC1 activity and provides a mechanism whereby
stress may activate mTORC1 independent of the PI3K/Akt pathway
(21). Therefore, the
phosphorylation status of raptor can be critical for the regulation of mTORC1
activity.In this study, we investigated phosphorylation sites in raptor catalyzed by
mTOR. Using two-dimensional phosphopeptide mapping, we found that
Ser863 and Ser859 in raptor were phosphorylated by mTOR
both in vivo and in vitro. mTORC1 activity in vitro
and in vivo is associated with the phosphorylation of
Ser863 in raptor. |
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