Identification of TOR-interacting Proteins
- Kazuyoshi Yonezawa
- Biosignal Research Center, Kobe University, Kobe 657-8501, Japan, and CREST, Japan Science and Technology Corporation
The target-of-rapamycin (TOR) proteins are protein kinases that were first identified in Saccharomyces cerevisiae through mutants that conferred resistance to growth inhibition induced by the immunosuppressive macrolide rapamycin (1 , 2). In yeast, wild-type TOR1 and TOR2 control a variety of processes contributing to cell growth—in response to nitrogen availability—including
translational initiation and early G1 progression (3) , as well as the regulation of transcription, amino-acid uptake, cytoskeletal organization, and protein degradation through
autophagy (4). In mammalian cells, rapamycin blocks the phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (5 , 6) and p70 S6 kinase (p70S6K) (7 , 8) by interfering with the function of mTOR [also known as FK506-binding protein (FKBP) 12-rapamycin–associated protein (FRAP),
or rapamycin- and FKBP-target 1 (RAFT1)] (9 , 10). Although mTOR can phosphorylate both these targets directly in vitro (11 –13) , the regulation of the kinase activity of mTOR in vivo remains incompletely understood. In addition, nutrients, especially
amino acids, which can regulate the phosphorylation of p70S6K and 4E-BP1, are necessary for insulin or mitogen regulation
(14 –19). Despite extensive efforts, how nutrients regulate the mTOR signaling pathway remains poorly understood.
The recent publications of several reports, that unveil a series of TOR-interacting proteins in yeast and mammalian cells,
have given new insights into the TOR signaling pathway. One of TOR-interacting protein is Raptor (regulatory associated protein
of mTOR) or its S. cerevisiae ortholog Kog1p, a highly conserved 150-kDa TOR-binding protein (20 –22). All Raptor orthologs contain a unique conserved region in their N-terminal half (raptor N-terminal conserved, also called
the RNC domain) followed by three HEAT (huntingtin, elongation factor 3, A subunit of protein phosphatase 2A and TOR1) repeats
and seven WD-40 repeats near the C terminus. Research on mammalian Raptor suggests that its association with mTOR promotes
the phosphorylation of downstream effectors in nutrient-stimulated cells (20 , 21). In concordance with these observations, the binding of TOR to Raptor or to Kog1p (22) is necessary for TOR signaling in vivo in Caenorhabditis elegans and S. cerevisiae (21 , 22).
Another characterized mTOR-interacting protein from S. cerevisiae Lst8p—its mammalian ortholog is mLST8/Gβ L (G protein β -subunit-like protein, pronounced “gable”)—a highly conserved 36-kDa
protein that consists almost entirely of seven WD-40 repeats with high sequence similarity to those found in the β subunits
of heterotrimeric G proteins (22 , 23). Loewith et al. reported that Lst8p interacts with Tor1p and Tor2p and showed that transiently expressed recombinant mTOR and mLST8 can
interact (22). Kim et al. (23) independently identified the same interacting protein and adopted the Gβ L name based on a previous report (24).
Regarding Raptor function, Sabatini and colleagues have also reported that the stability of the mTOR–Raptor complex is increased
when cells are amino acid– or energy-starved (20). The transition to this avid mTOR–Raptor complex correlates with the inhibition of mTOR-dependent signaling in cells and
with the repression of mTOR’s kinase activity in vitro, leading Sabatini’s group to suggest that Raptor negatively regulates
the kinase activity of mTOR. Very recently, Kim et al. observed that mLST8/Gβ L interacts constitutively with and activates the kinase domain of mTOR, and that mLST8/Gβ L is necessary
for mTOR to form a nutrient-sensitive interaction with Raptor (23). These authors favor a model in which the binding of Raptor to the complex of LST8/Gβ L and mTOR inhibits mLST8/Gβ L-mediated
activation of mTOR, and propose that the opposing effects on mTOR activity of the interactions mediated by mLST8/Gβ L and
Raptor provide a mechanism by which cellular conditions, such as nutrient levels, can positively and negatively regulate mTOR
signaling to the cell growth machinery (23).
Figure 1.
A working model for the mTOR signaling pathways in mammals. Nutrients regulate mTOR signaling pathway and Raptor and mLST8/GβL are components of the mTOR signaling complex. Raptor serves
as a scaffolding protein that binds p70S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) through
their TOR-signaling (TOS) motifs and Raptor facilitates their phosphorylation by mTOR. mLST8/GβL is another component of the
mTOR signaling complex. mLST8/GβL interacts constitutively with the kinase domain of mTOR. The right side of the figure outlines
a tentative pathway that links growth factor–dependent Akt/PKB activation through phosphatidylinositol-3’ kinase (PI3K) and
phosphoinositide-dependent kinase 1 (PDK1) to the stimulation of mTOR-dependent responses. The tuberous sclerosis complex
(TSC) proteins TSC1 and TSC2 serve as negative modulators of the mTOR pathway. The small GTPase Rheb (Ras homolog enriched
in brain) is a direct target of TSC2’s intrinsic GTPase-activating function. Rheb•GTP appears to be a positive modulator of
mTOR signaling. PKB, protein kinase B.
However, we have studied the same protein–protein interaction and have found no evidence for changes in Raptor–mTOR complex
stability when cells were shifted between nutrient-rich and nutrient-poor media (21). In addition, we observed that coprecipitation of Raptor with mTOR is essential for the phosphorylation of 4E-BP1 and p70S6K
(in immune-complex kinase assays) and that Raptor also binds to p70S6K and to 4E-BP1. Thus, we propose that Raptor participates
as an essential scaffolding protein for mTOR-catalyzed phosphorylation of 4E-BP1 and p70S6K (21). Obviously, important technical differences between our and Sabatini’s group—such as cell lysis conditions and studying transfected
or endogenous proteins—need to be addressed before firm conclusions regarding the impact of nutrient status on the stability
of the mTOR–Raptor complex in mammalian cells can be drawn. However, the following reports appear to make the scaffolding
model for Raptor function more attractive. Schalm and Blenis have identified a five amino-acid TOR-signaling (TOS) motif in
4E-BP1 and p70S6K that is required for mTOR-dependent phosphorylation of both proteins following the addition of nutrients
to starved cells (25). Whereas the TOS motif appears necessary for the binding of p70S6K or 4E-BP1 to Raptor (26 –28) , mutation of the TOS motif abolishes mTOR-catalyzed phosphorylation of 4E-BP1 in vitro in the presence of Raptor, and eliminates
the Raptor-dependent stimulation of mTOR-catalyzed p70S6K phosphorylation (26 , 27). Thus, the inhibitory effect of TOS deletion or mutation on the phosphorylation of 4E-BP1 or p70S6K in vivo can be attributed
to the inability of these mutants to bind Raptor. These results indicate one possible mechanism by which the Raptor couples
mTOR to cellular substrates.
New insights into bridging the phosphatidylinositol-3’ kinase (PI3K)–Akt signaling pathway and mTOR have emerged from recent
studies in both Drosophila melanogaster and mammalian cells involving the tumor suppressor proteins Tuberous Sclerosis Complex 1 and 2 (TSC1 and TSC2). The TSC1–TSC2
complex represses mTOR-dependent activation of p70S6K (29 –32) , and the putative GTPase-activating protein (GAP) domain of TSC2 inactivates the small GTPase Rheb (Ras homolog enriched
in brain) in vitro and in vivo (33 –36) (Figure 1⇑). TSC syndrome is an autosomal-dominant genetic disorder, characterized by mutations in either TSC1 or TSC2 that result in the widespread development of benign tumors termed hamartomas (37). In response to growth factor stimulation, the repression of mTOR signaling mediated by the TSC complex appears to be relieved
by Akt-mediated phosphorylation of TSC2, which requires the mitogen-induced activation of PI3K (30 , 31) (Figure 1⇑). Point mutations in the GAP domain of TSC2 disrupt its ability to regulate Rheb without affecting the ability of TSC2 to
form a complex with TSC1. Whether TSC1, TSC2, and/or Rheb bind to the mTOR complex containing Raptor–mLST8/Gβ L and modulate
the signaling function and kinase activity of mTOR remains an important but unanswered question. The further investigation
of Raptor, mLST8/Gβ L, and as-yet uncharacterized TOR-binding proteins will offer new targets for therapeutic invention in
human diseases, such as cancer and diabetes, in which mTOR signaling may be perturbed.
- © American Society for Pharmacology and Experimental Theraputics 2003
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Kazuyoshi Yonezawa, MD, PhD, is a Professor at the Biosignal Research Center, Kobe University. Address correspondence to KY. E-mail yonezawa{at}kobe-u.ac.jp; fax +81-78-803-5970.
