Regulation of mTOR by RTK-PI-3K
Several reports indicate that insulin [104-106] induces a modest (~ twofold) increase in the activity of immuno-precipitated mTOR; a more robust increase in mTOR activity is observed in response to neurotrophin (CNTF, BDNF) treatment of primary neurons and cell lines [107,108]. Overexpression of active forms of PI-3K or PKB results in increased 4E-BP phosphorylation at the rapamycin-/ wortmannin-sensitive sites  and activation of p70 S6K , although PKB itself does not phosphorylate either 4E-BP or p70 S6K. Whether the effects of insulin, neurotrophins, recombinant PI-3K, and PKB reflect a direct modification and activation of TOR kinase or more indirect mechanisms (e.g., phosphorylation of and disinhibition from TSC , PDK1 and/or PKB recruitment of other 4E-BP and/or p70 S6K kinases, negative regulation of protein phosphatase, or some combination of these actions) is not known. In addition to the modest (twofold) increase in the activity of immunoprecipitated mTOR, activation of PKB in vivo is associated with increased phosphorylation of mTOR at Ser2448 [106,111], a canonical PKB site. The functional significance of PKB-catalyzed mTOR phosphorylation is unclear, as mutation of Ser2448 to Ala on the rapamycin-resistant mTOR (Ser2035 Thr) does not alter its ability to rescue 4E-BP or p70 S6K from rapamycin-induced dephosphorylation . Nevertheless, deletion of the mTOR segment surrounding Ser2448  or the binding of a polyclonal Ab [66,105] to this site each increase the in vitro mTOR kinase activity by five- to tenfold, suggesting that a regulatory input (e.g., TSC inhibition) whose mechanism remains to be elucidated may be effected through this segment of mTOR.
Microinjection of a prokaryotic recombinant FRB fragment into MG63 osteosarcoma cells, a cell line whose growth is reliably arrested in G: by rapamycin, prevents entry into S , suggesting that the function of the FRB domain in maintaining TOR activity might be regulatory rather than structural. An important insight into the function of the FRB domain was the finding that this segment binds selectively to lipid vesicles that contain phosphatidic acid (PA) . This binding can be inhibited by addition of an FKBP12/rapamycin complex or partially by mutation of mTOR Arg2109 to Ala; introduction of this mutation into a rapamycin-resistant mTOR (S2035T) mutant reduces by approximately 40% its ability to rescue coexpressed p70S6K from rapamycin inhibition. PA added exogenously can activate p70 S6K and 4E-BP phosphorylation. Mitogens, though activation of PLD, increase cellular PA levels, and butanol, which sequesters intracellular PA as an ester, inhibits the serum-induced activation of p70 S6K and the phosphorylation of 4E-BP without affecting phosphorylation of PKB or MAPK. Moreover, a mutant of p70 S6K resistant to inhibition by rapamycin (see above) is also resistant to inhibition by butanol. The noncovalent interaction of PA with the mTOR FRB domain appears to provide one component of the mitogen activation of mTOR signaling.
In summary, mTOR kinase activity is regulated in mammalian cells by tyrosine-kinase-linked receptors through a PI-3K-PDK1-Akt pathway, at least in some cell backgrounds and possibly for some, but not all, substrates. This activation persists to some degree after extraction and immunoprecipitation and is probably attributable to PI-3K/PB-induced mTOR phosphorylation. An additional important site of PKB regulation is through the phosphory-lation of, and disinhibition from, TSC2 . Moreover, as described above, an RTK-PLD-induced accumulation of phosphatidic acid  probably promotes mTOR activity through a noncovalent interaction; this activation is unlikely to survive cell extraction. An understanding of the TOR scaffold protein Raptor [70,71] as a site of regulation will be necessary for understanding the specific mechanisms that regulate the mTOR kinase activity toward each of its physiologic substrates. Finally, the role of gephyrin  in the receptor regulation of mTOR remains unclear. Gephyrin is a ubiquitously expressed tubulin-binding protein necessary for the postsynaptic clustering of glycine receptors in neurons. Gephrin binds to mTOR (AA1010-1128). Mutations in this region that abolish mTOR interactions with gephyrin abrogate the ability of a rapamycin-resistant mTOR (Ser2035Thr) to rescue p70 S6K and 4E-BP from rapamycin-induced dephosphorylation.
The evidence that TOR retains its role as a nutrient-sensor in metazoans is entirely indirect; in contrast to the generally reproducible, if modest, stimulatory effects of insulin and neurotrophins, there has been no demonstration that alterations in the nutrient environment cause stable changes in mTOR kinase activity that can be measured by mTOR kinase assay in vitro, although it is reported that nutrient deprivation promotes an inhibitory interaction between Raptor and mTOR [70,71]. Nevertheless, there is persuasive evidence that TOR kinase activity in vivo is controlled by inputs related to amino acid and overall energy sufficiency. Thus, in a wide range of cultured mammalian cells, withdrawal of medium amino acids leads to progressive deacti-vation of the p70 S6K and dephosphorylation of eIF 4E-BP over 1 to 2 hours and completely inhibits the ability of insulin to promote these phosphorylations [87,115-117]. Amino acid withdrawal, however, does not significantly affect, at least over this initial interval, the upstream elements of the insulin signaling pathway (i.e., IR/IRS tyrosine phosphorylation, PI-3K activity, PKB or MAPK activation). Readdition of amino acids restores phosphorylation of p70 S6K on 4E-BP and their responsiveness to insulin. Moreover, elevation of ambient concentration of amino acids to higher than usual levels causes a progressive increase in p70 S6K activity and 4E-BP phosphorylation to levels observed with maximal insulin stimulation; at these high amino acid concentrations, addition of insulin gives no further stimulation of p70 S6K activity . Thus, amino acid deficiency, like rapamycin, results in the selective dephosphorylation of these two well characterized TOR targets in a manner that overrides the activating input of the RTK/PI-3K pathway. Notably, the doubly deleted rapamycin-resistant p70 S6K mutant, p70A2-46/ACT104, is also highly resistant to deactivation/dephosphorylation by amino acid withdrawal . This finding establishes that amino acid withdrawal, like rapamycin, does not inhibit any of the steps necessary for the insulin/PI-3K phosphorylation of p70 S6K Thr252 (the PDK1 site) and Thr412 (unknown kinase), but rather promotes the dephosphorylation of these sites; mTOR and amino acid sufficiency thus appear to inhibit the same p70 S6K phosphatase. Although rapamycin inhibits the ability of amino acid readdition to restore p70 S6K phosphorylation, it has not been formally established whether amino acids require mTOR to inhibit this putative p70 S6K phosphatase. Wortmannin also inhibits amino-acid-induced p70 S6K phosphorylation, but concentrations higher than those sufficient to inhibit type 1a PI-3K are required  and correspond to those necessary for inhibition of mTOR itself . Subsequent work demonstrated that the rapamycin-sensitive phosphorylation sites in the nPKCs (5 and e) are also dephosphorylated in response to amino acid withdrawal [100,101].
The complete restoration of p70 S6K and 4E-BP phos-phorylation in cell culture requires the readdition of all 20 amino acids; readdition of single amino acids is without effect, except for leucine, which enables a variable extent of partial restoration in many cell lines [39,88,115,119]. Similarly, whereas removal of any single amino acid usually results in some dephosphorylation, the most substantial inhibition is observed on removal of leucine and occasionally arginine. A large body of in vivo experiments by Jefferson et al. [120-123] indicate that leucine exerts significant stimulatory action on the phosphorylation of p70 S6K and 4E-BP and is uniquely effective in promoting the synthesis of ribosomal proteins, providing strong evidence that the amino-acid-activated, mTOR-dependent pathways evident in cell culture are operative in vivo and are one component of the multiple mechanisms by which amino acids and insulin coordinately regulate protein synthesis especially in skeletal muscle.
Although evidence has been provided for the existence of a membrane-localized leucine receptor, at least in regard to amino-acid-regulation of autophagy in hepatocytes , it is likely that the majority of amino-acid-dependent responses mediated by mTOR are initiated at an intracellu-lar site . Thus, inhibition of mRNA translation, either at the level of initiation (anisomycin)  or elongation (cycloheximide) , results in the activation of p70 S6K and hyperphosphorylation of 4E-BP  in a rapamycin-sensitive manner. Reciprocally, overexpression of eIF-4E (although transforming in many cell backgrounds) is accompanied by hypophosphorylation of 4E-BP and p70 S6K . An attractive hypothesis is that the ability of protein synthesis inhibitors to stimulate the phosphorylation of p70 S6K and 4E-BP might reflect the activation of mTOR, induced by the accumulation of some intermediate in the translational process (e.g., a minor acylated tRNA) or by a byproduct of stalled translation, analogous to the synthesis of guanosine tetraphosphate during the "stringent" response in bacteria . Support for such a mechanism is provided by the observation that amino acid alcohols, which inhibit cognate tRNA synthetase activity and protein synthesis, nevertheless cause dephosphorylation of 4E-BP and p70 S6K (suggesting a decrease in mTOR activity), in contrast to anisomycin . Moreover, cycloheximide overcomes the dephosphorylation of p70 S6K/4E-BP caused by amino acid withdrawal. Similarly, CHO cells bearing a temperature-sensitive mutant histidyl tRNA synthetase, when shifted to the nonpermissive temperature, exhibit dephosphorylation of 4E-BP and p70 S6K . If mTOR is regulated by the charging of tRNAs, this is accomplished through a mechanism distinct from that regulating the GCN2 kinase , as well as that underlying the bacterial stringent response .
Although the phosphorylation of 4E-BP is suppressed by amino acid withdrawal, some degree of insulin-stimulated 4E-BP phosphorylation persists under these conditions, to a degree sufficient to displace 4E-BP from 4E and to promote an increased association of eIF-4E with eIF-4G . In CHO cells deprived of both amino acids and glucose, the basal- and insulin-stimulated phosphorylation of Thr 36/45 is severely inhibited; readdition of glucose alone, although insufficient to enable detectable phosphorylation of p70 S6K1 Thr412 or Ser444/447, allows substantial insulin-stimulated Thr36/45 phosphorylation and significant basal and insulin stimulated protein synthesis. This effect of glucose requires its metabolism and can be reproduced in part by lactate . Although the identity of the kinase responsible for the glucose-dependent, insulin-stimulated phos-phorylation of 4E-BP Thr36/45 is not known, a plausible candidate is mTOR, inasmuch as these are the primary sites of mTOR-catalyzed 4E-BP phosphorylation. The dependence of this response on glucose metabolism suggests that mTOR kinase activity is itself, to some extent, dependent on and regulated by some product of glucose metabolism, independent of amino acid sufficiency, PI-3K, and PKB. Several observations indicate that this input is related to the state of overall energy sufficiency, as reflected by the concentration of adenine nucleotides. Thus, inhibitors of glycosis such as 2-deoxyglucose (2DG) and inhibitors of mitochondr-ial oxidative phosphorylation, e.g., rotenone or CN- both cause a marked inhibition of 4E-BP1 and p70 S6K phospho-rylation, at concentrations that have little effect on PKB or MAPK activation . Notably, a rapamycin-resistant mutant of p70 S6K previously shown to be resistant to inhibition on withdrawal of amino acids is also entirely resistant to the inhibitory effects of 2DG, strongly supporting the conclusion that the inhibitory effects of energy depletion on p70 S6K and presumably 4E-BP are mediated by inhibition of mTOR. It has been suggested that mTOR is directly sensing the concentration of ATP itself, based on the apparently high ED50 for ATP (~1.0 mM) in the mTOR-catalyzed phosphorylation of 4E-BP in vitro . This estimate of Km for ATP, however, is probably compromised by the copurifica-tion with mTOR of protein phosphatases and other contaminants. A more plausible mediator of TOR inhibition in the setting of energy depletion is the AMP-activated kinase (AMPK) system [134,135]. AICAR, a precursor of the AMPK activator ZMP, can inhibit p70 S6K and 4E-BP phosphorylation in cell culture, at least in cells able to efficiently convert this precursor to ZMP, without inhibition of PKB and MAPK. In addition, mTOR directly and specifically associates with AMPK. AICAR given by subcutaneous injection to rats results in an inhibition in skeletal muscle of p70 S6K (Thr412) and 4E-BP (Thr37) phospho-rylation, accompanied by a decrease in the association of eIF-4E with eIF-4G and an inhibition of protein synthesis . The effects of AICAR in skeletal muscle may also be mediated by an inhibition of the PI-3K pathway inasmuch as AICAR injection in vivo also results in decreased phosphorylation of PKB (Ser473) and mTOR Ser2448, a canonical site of PKB-catalyzed phosphorylation in vivo; the latter responses are not seen upon glucose withdrawal from cultured cells. In summary, mTOR output is regulated by the cellular energy state, although this appears to be secondary in importance to regulation by amino acid sufficiency. Inhibition of mTOR by AMPK is likely to contribute an important component of the energy-dependent regulation of TOR.
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