region and rapamycin treatment should elicit identical phenotypes. Thus, whereas it is clear that rapamycin functions through an inhibition of downstream signaling from the TOR proteins, this repression may involve mechanisms other than a direct suppression of TOR kinase activity.
What Signals to TOR? The TOR proteins do not appear to function as components of a conventional linear signaling pathway. Rather, several lines of evidence suggest that the TOR proteins function in a nutrient-sensing checkpoint control capacity. As discussed further below, both TOR and phosphoinositide 3-kinase (PI3K) signaling are required for the activation (or inactivation) of several downstream effector proteins. However, whether TOR activity is regulated by PI3K, or whether the two signaling pathways function independently, is unknown. Over-expression of a membrane-targeted Akt/PKB protein (a downstream effector of PI3K) in mammalian cells leads only to a modest increase (or no change) in mTOR kinase activity (as assayed in vitro), and moderately increases mTOR autophosphorylation in vivo, as assessed with the S2481 phosphospecific antibody (32–34).
A putative Akt consensus phosphorylation site, S2448, was observed to be phosphorylated on mTOR in vivo, as determined with a phosphospecific antibody. Addition of insulin or IL-3 engenders an increase in S2448 phosphorylation in a PI3K- and Akt-dependent manner (34, 35). However, an mTOR mutant protein possessing an alanine substitution at this site retains the ability to activate S6K1 (a downstream effector of mTOR, see below) after growth factor stimulation (34). Thus, the role of this phosphorylation event in the regulation of mTOR activity is not clear.
Inactivation of the TOR proteins, or rapamycin treatment, mimics nutrient deprivation in yeast, Drosophila, and mammalian cells (21, 36–38). Thus, a current working model for TOR signaling proposes that these kinases relay a permissive signal to downstream targets only in the presence of sufficient nutrients to fuel protein synthesis (Fig. 1). In some cases, the TOR proteins appear to function in a coregulatory capacity with other conventional, linear signaling pathways (such as the PI3K pathway; see below). In this way, a passive nutrient sufficiency signal may be combined with stimulatory signaling from a second pathway to coordinate cellular processes that require the uptake of nutrients. The absence of either signal is predicted to prohibit activation of downstream targets.
A Model for TOR Signaling. How does TOR signal to downstream effectors? TOR signaling is thought to be effected through a combination of direct phosphorylation of downstream targets, and repression of phosphatase activity (Fig. 2). Genetic screening in S. cerevisiae has identified the PP2A-like phosphatase Sit4p, two PP2A regulatory subunits (CDC55 and TPD3), and a phosphatase-associated protein (Tap42p), as components of a rapamycin-sensitive signaling pathway (38, 39). Tap42p interacts directly with the catalytic subunits of PP2A and Sit4p. S. cerevisiae expressing a temperature-sensitive Tap42 mutant protein exhibit a dramatic defect in translation initiation at the nonpermissive temperature (39). Thus, Tap42p is thought to repress PP2A (or Sit4p) activity (also see refs. 40 and 41).
Phosphorylation of Tap42p regulates its interaction with phosphatases. Whereas phosphorylated Tap42p competes with the phosphatase adapter (A) subunit for binding to the catalytic subunit, dephosphorylated Tap42p does not efficiently compete for binding (42). Tap42p phosphorylation is modulated by Tor signaling. The Tap42p-PP2A association in vivo is disrupted by nutrient deprivation or rapamycin treatment (39, 42). Further, a yeast Tor2p immunoprecipitate can phosphorylate Tap42p in vitro (42), and Tap42p phosphorylation is rendered rapamycin resistant in yeast strains expressing a rapamycin-resistant Tor1 protein (42).
Tap42 orthologs are found in Arabidopsis (43), Drosophila, (GenBank accession number AAF53289), and mammalian cells (44, 45). The B cell receptor binding protein a4 (a.k.a Ig binding protein 1, IGBP1) is the mammalian ortholog of Tap42p (44, 45). The ability of this protein to interact with PP2A-like phosphatases is conserved in mammals, as a4 binds directly to the catalytic subunits of PP2A (46, 47), PP4, and PP6 (48, 49). Like Tap42p, a4 is also a phosphoprotein, and the a4-PP2A interaction was reported to be abrogated by rapamycin treatment (although this finding remains somewhat controversial; refs. 46 and 47). These observations suggest that Tap42p/a4 phosphorylation, and PP2A binding, are regulated by TOR signaling, and that an inhibition in TOR signaling leads to Tap42p/a4 dephosphorylation, dissociation of the Tap42p/a4-phosphatase complex, and phosphatase derepression.
Interestingly, mTOR was reported to undergo nucleocytoplasmic shuttling (50). Abrogation of shuttling (by treatment with leptomycin B, a specific inhibitor of the nuclear export receptor Crml, or by transfection of mTOR tagged with exogenous nuclear export or import signals) was demonstrated to inhibit signaling to S6K1 and 4E-BP1 (50). Why mTOR shuttling may be important for 4E-BP1 and S6K1 activity is unknown (50).
Tor and Translation in S. cerevisiae. Inhibition of Tor activity in yeast potently represses translation initiation, concomitant with polysome disaggregation and cell cycle arrest in G1 (36). The mechanism for this translational repression is not understood, but could be due, at least in some strains, to the degradation of the initiation factor eIF4G (51, 52). A putative regulator of yeast eIF4E function, termed Eap1p (eIF4E-associated protein 1), may also be involved in this process, as disruption of the EAP1 gene results in partial rapamycin resistance (53). The G1 arrest in response to Tor inactivation was suggested to be due to the inhibition of translation of an mRNA coding for a cyclin involved in G1 to S progression, CLN3, because the cell cycle block can be overcome by forced expression of CLN3 (54–56).