Amino acid deprivation inhibits global translational activity through phosphorylation of eukaryotic initiation factor 2α (eIF2α) and dephosphorylation of eIF4E-BP (reviewed in reference 27
). The decreased polysomal association of non-TOP mRNAs, like that of mRNAs encoding actin (Fig. and c) and Act(28)-GFP (Fig. ), in amino acid-starved cells reflects this global effect on the translational machinery. Other non-TOP mRNAs, like those encoding SOD (Fig. and b), Act(28)-GH, and L32(−1C→A
)-GH (Fig. ), seem to be essentially refractory to this repression. Conceivably, this diverse sensitivity primarily reflects different affinities of the individual transcripts for the translational initiation factors. However, the data presented here clearly demonstrate that translation of TOP mRNAs is much more sensitive to this nutritional control and that its response is the same as that observed in nonproliferating cells (reference 39
and references therein).
Growth is characterized by an elevated production of the translational apparatus needed to cope with the increasing demand for protein synthesis. Indeed, according to one estimate most of the energy consumed during cellular growth is utilized for generating components of protein synthesis machinery (56
). The apparent advantages in regulating the synthesis of the translational apparatus at the translational level are the rate and the readily reversible nature of the response to altering physiological conditions. These two features enable cells to rapidly repress the biosynthesis of the translational machinery upon shortage of amino acids or growth arrest, thus rapidly blocking energy wastage.
Two experimental approaches have been used to examine the causal relationship between rpS6 phosphorylation and translational control of TOP mRNAs upon mitogenic stimulation by serum refeeding. First, rapamycin indirectly blocked the mitogenic activation of S6K1 and prevented rpS6 phosphorylation (10
) by a poorly understood mechanism. Indeed, rapamycin treatment of some cell lines selectively abolished translational activation of TOP mRNAs upon mitogenic stimulation (26
). However, in other cell lines rapamycin exhibited only a minor, if any, repressive effect, even though S6K1 activity was completely inhibited (22
). Second, transfection of cells with expression vectors encoding a mutant version of S6K1, p70S6K
A229, which functioned as a dominant-negative mutant, completely inhibited S6K1 activity. Nevertheless, in a single reported experiment the overexpression of this mutant exerted a modest inhibitory effect on the translational activation of a chimeric TOP mRNA following mitogenic stimulation (22
). It should be noted that the phosphorylation of S10, the Saccharomyces cerevisiae
homolog of mammalian S6, has been shown to be dispensable for optimal yeast growth (25
). Nonetheless, the lack of TOP mRNAs in yeast and the fact that synthesis of yeast rp is primarily regulated at the transcriptional level (70
) renders this observation irrelevant to the present discussion.
The data presented herein provide very strong support for the conclusion that the phosphorylation of rpS6 is neither necessary nor sufficient to enable the polysomal recruitment and translational initiation of TOP mRNAs in response to amino acid sufficiency. In addition, the complete inhibition or loss of S6K1 activity, whether caused by rapamycin, recombinant polypeptide inhibitors (i.e., p70S6K,K123 M or p70Δ2-46/ΔCT104,K123 M/T412E), or deletion of the S6K1 gene, does not impair the ability of amino acids to restore TOP mRNA polysomal recruitment after prior amino acid withdrawal. Nevertheless, S6K1−/−
cells express a second S6K, S6K2 (32
), which is less strongly inhibited by rapamycin than is S6K1 (K. Yonezawa and K. Hara, personal communication), so that its residual activity might account for the rapamycin resistance of amino acid-regulated TOP mRNA recruitment. Although this possibility cannot be eliminated by the present results, our data do establish that such an action of S6K2 cannot be attributed to the phosphorylation of S6. In fact, rapamycin, despite its inability to inhibit fully S6K2, completely inhibits S6 phosphorylation, suggesting that S6 may not be a major substrate of S6K2. Moreover, other observations argue against a role for S6K2 in the amino acid regulation of TOP mRNA recruitment. Thus, S6K2 is resistant not only to inhibition by rapamycin but also to inhibition by amino acid withdrawal (N. Terada, personal communication, and K. Yonezawa and K. Hara, personal communication); however the S6K2 activity persisting in the face of amino acid withdrawal (at least 50% of basal) is not sufficient to prevent the inhibition of TOP mRNA translation upon amino acid withdrawal. Taken together, these observations have reopened the intriguing question concerning the physiological function of S6K1 and -2 and rpS6 phosphorylation in translational regulation; the solution awaits the establishment of S6K1 and -2 double knockout.
The involvement of mTOR in the amino acid regulation of S6K1 has been demonstrated through studies using rapamycin, which blocks the activation of S6K1 by amino acid refeeding. On the other hand, amino acid reintroduction could induce S6K1 activation in the presence of rapamycin in cells expressing a rapamycin-resistant mTOR mutant (21
). Likewise, rapamycin-resistant S6K1 mutant p70Δ2-46/ΔCT104 is resistant to amino acid deficiency, indicating that both amino acid sufficiency and an mTOR signal to S6K1 through a common effector, which could be mTOR itself or an mTOR-regulated downstream mediator (18
). Nonetheless, experiments presented here clearly show that rapamycin has only a moderate effect on the translational activation of TOP mRNAs upon amino acid activation of 293 cells. Likewise, 3 h of rapamycin treatment of p70S6K−/−
ES cells inhibited the translation of rpL32 mRNA considerably less efficiently than 2 h of amino acid starvation (from 82% to 61 or 31% in polysomes, respectively) (Fig. ) (E. Hornstein, unpublished data). This discrepancy between the effects of rapamycin and amino acids suggest that the latter exert their effect on TOP mRNA primarily in an mTOR-independent fashion. Furthermore, the delayed effect on TOP mRNAs might suggest that rapamycin elicits its repression indirectly by inhibiting one or more of the many growth-related mTOR readouts, such as transcription of specific genes (reviewed in reference 55
). Indeed, rapamycin has been shown to block cell cycle progression and to inhibit the proliferation of a variety of lymphocyte and nonlymphocyte cell types (reviewed in reference 57
The ability of rapamycin to prevent the translational activation of TOP mRNAs only partially and in a delayed manner is underscored by the apparent ability of LY294002 to completely block within 30 min this activation in amino acid-refed cells (Fig. ). Previous reports, with the exception of one case (47
), have demonstrated that amino acid withdrawal and readdition had a minimal effect on the activity of PI3-kinase and PKB, yet inhibitors of PI3-kinase completely block the amino acid-induced activation of S6K1 (16
). Two possible explanations for these seemingly conflicting results can be proposed. (i) PI3-kinase inhibitors are not as specific as initially claimed, and they also inhibit other kinases (7
). Indeed, the complete inhibition of rpS6 phosphorylation by 50 μM LY294002 can be attributed to the inhibitory effect of this concentration on mTOR activity (7
). (ii) Signaling of amino acids to TOP mRNAs requires an active PI3-kinase for continuous supply of phosphoinositides phosphorylated at position 3 on the inositol ring. Conceivably, these phosphoinositides are necessary for anchoring to the membrane (for review see reference 51
) of one or more kinases, whose activity is regulated by amino acid sufficiency. The latter explanation seems particularly applicable to the amino acid-induced translational activation of TOP mRNAs, as this activation is blocked by overexpression of either the phosphatase PTEN or the dominant-negative regulatory subunit of PI3-kinase, p85 (Fig. ). Overexpression of both these constructs has previously been shown to block the accumulation of PI-3,4,5-triphosphate (34
). Candidate kinases, other than S6K1 and -2, whose activity is regulated by amino acid sufficiency, are the novel protein kinase Cδ (PKCδ) and PKC
. Thus, the activation loop of these kinases is phosphorylated by PI-dependent kinase 1, and their hydrophobic regulatory site is dephosphorylated by amino acid deprivation (reference 43
and references therein).
It is noteworthy that, when enhanced S6K1 variants [p70Δ2-46/ΔCT104 and p70Δ2-46/ΔCT104, T412E), but not the wild-type enzyme, were overexpressed in 293 cells, they were able to relieve the translational repression of L32-GFP exerted by 1 h of amino acid starvation (G. Levy and O. Meyuhas, unpublished data). In light of all other data presented in this report, it is conceivable that this relief simply reflects artifactual consequences of the nonphysiologically high activity of S6K1 obtained by overexpression. Thus, it is possible that a substrate other than rpS6, which is only fortuitously phosphorylated by endogenous S6K1, is now significantly phosphorylated to an extent that might affect the translational efficiency of TOP mRNAs. Similarly, we have shown here that overexpression of constitutively active RSK2 mutant K3H.RSK2(Y707A) led to efficient phosphorylation of rpS6 (Fig. ), even though this substrate is primarily phosphorylated by S6K1 rather than RSK2 (10
). It should be mentioned that previously reported results derived from overexpression of various active and dominant-negative kinases have been a subject for controversial interpretations (2
A tentative model depicting the signaling pathways leading to the translational activation of TOP mRNAs by amino acid sufficiency is presented in Fig. . According to this model, amino acids signal into TOP mRNAs through an unknown target (denoted X) in a PI3-kinase-dependent fashion. However, the signaling from amino acids bifurcates upstream of mTOR, as inferred from the ability of rapamycin to discriminate between the activity of mTOR and S6K on the one hand and the translational efficiency of TOP mRNAs on the other hand.
FIG. 11 Schematic representation of signal transduction pathways involved in activation of rpS6 phosphorylation and translational control of TOP mRNAs in amino acid-stimulated cells. Arrows delineate the flow of information. Open arrowhead, partial and delayed (more ...)
It might be argued that the apparent lack of inhibitory effect of rapamycin or of dominant-negative S6K1 mutants on the early response of TOP mRNA translation in amino acid-refed cells reflects the involvement of a signaling pathway which differs from that transducing mitogenic signals. However, our recent experiments with serum-starved and serum-refed cells (including S6K1−/− cells) clearly show that the minor role of the mTOR-mediated pathway in the translational control of TOP mRNAs is not confined to nutritional signals but is also applicable to mitogenic signals (M. Stolovich, H. Tang, E. Hornstein, and O. Meyuhas, unpublished data).