Vertebrate TOP mRNAs contain an oligopyrimidine tract at their 5 termini (5TOP) and encode components of the translational machinery. Previously it has been shown that they are subject to selective translational repression upon growth arrest and that their translational behavior correlates with the activity of S6K1. We now show that the translation of TOP mRNAs is rapidly repressed by amino acid withdrawal and that this nutritional control depends strictly on the integrity of the 5TOP motif. However, neither phosphorylation of ribosomal protein ( The synthesis of many mammalian proteins associated with the translational apparatus has been shown in recent years to be selectively regulated in a growth-dependent manner at the translational level. The corresponding mRNAs are characterized by the presence of a 5Ј-terminal oligopyrimidine tract (5ЈTOP) and therefore are referred to as TOP mRNAs. This structural motif comprises the core of the translational cisregulatory element of these mRNAs (reviewed in references 35 and 39). The proportion of TOP mRNAs actively engaged in protein synthesis, i.e., the proportion associated with polysomes in a wide variety of growing mammalian cells, is significantly lower than that characteristic of other ubiquitous mRNAs (1,3,20,37,41,58). On average, only about 70% of TOP mRNAs are engaged with ribosomes compared with about 90% for the other housekeeping mRNAs. This selective repressed translation of TOP mRNAs becomes even more pronounced in cells that cease to divide, where only ϳ30% of the TOP mRNAs remain in polysomes compared with Ͼ80% for non-TOP mRNAs (36).Phosphorylation of ribosomal protein S6 (rpS6) is one of the earliest events detected following mitogenic stimuli. This phosphorylation is carried out by two closely related kinases, S6K1 (also known as p70 S6 kinase or p70 S6K ) and S6K2 (reviewed in reference 17). Several studies have shown that mitogenic stimulation of quiescent cells induces activation of S6K1 and consequently phosphorylation of rpS6. The concomitant translational activation of TOP mRNAs under these circumstances led Thomas and his colleagues to propose that rpS6 phosphorylation following S6K1 activation increases the affinity of ribosomes for TOP mRNAs and thus facilitates translation initiation (23,24,66). It should be noted, however, that this model, although supported by several correlative studies (39), has remained purely speculative. Thus, neither the involvement of rpS6 in the translational control of TOP mRNAs nor its being the only physiological substrate of S6K1 has been experimentally proven.Study of rpS6 phosphorylation in rat liver revealed that this protein is phosphorylated not only following mitogenic stimulation but also upon the refeeding of starved animals (30). The importance of amino acids in this nutritional stimulation has been demonstrated in vitro with hepatocytes isolated from starved rats. Supplementing these cells with a complete mixture of amino acids led to phosphorylation of rpS6 which could be abolished by the mTOR-spec...

Translation of terminal oligopyrimidine tract (TOP) mRNAs, which encode multiple components of the protein synthesis machinery, is known to be controlled by mitogenic stimuli. We now show that the ability of cells to progress through the cell cycle is not a prerequisite for this mode of regulation. TOP mRNAs can be translationally activated when PC12 or embryonic stem (ES) cells are induced to grow (increase their size) by nerve growth factor and retinoic acid, respectively, while remaining mitotically arrested. However, both growth and mitogenic signals converge via the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway and are transduced to efficiently translate TOP mRNAs. Translational activation of TOP mRNAs can be abolished by LY294002, a PI3-kinase inhibitor, or by overexpression of PTEN as well as by dominant-negative mutants of PI3-kinase or its effectors, PDK1 and protein kinase B␣ (PKB␣). Likewise, overexpression of constitutively active PI3-kinase or PKB␣ can relieve the translational repression of TOP mRNAs in quiescent cells. Both mitogenic and growth signals lead to phosphorylation of ribosomal protein S6 (rpS6), which precedes the translational activation of TOP mRNAs. Nevertheless, neither rpS6 phosphorylation nor its kinase, S6K1, is essential for the translational response of these mRNAs. Thus, TOP mRNAs can be translationally activated by growth or mitogenic stimuli of ES cells, whose rpS6 is constitutively unphosphorylated due to the disruption of both alleles of S6K1. Similarly, complete inhibition of mammalian target of rapamycin (mTOR) and its effector S6K by rapamycin in various cell lines has only a mild repressive effect on the translation of TOP mRNAs. It therefore appears that translation of TOP mRNAs is primarily regulated by growth and mitogenic cues through the PI3-kinase pathway, with a minor role, if any, for the mTOR pathway.Cell proliferation involves two processes: cell growth (increase in cell size) and cell division, which are normally intermingled, to the extent that cells must attain a minimal size to progress in the cell cycle. The dependence of DNA replication and cell division on cellular growth appears to enable accumulation of cellular resources to ensure daughter cell survival. Growth is characterized by elevated production of the translational apparatus needed to cope with the increasing demand for protein synthesis (42). Indeed, according to one estimate, most of the energy consumed during cellular growth is utilized for generating the components of the protein synthesis machinery (53).TOP mRNAs, which encode many components of the translational apparatus [ribosomal proteins, elongation factors eEF1A and eEF2, and poly(A)-binding protein], are translationally regulated by mitogenic signals through their 5Ј terminal oligopyrimidine tract (5ЈTOP) (35). Translational repression of TOP mRNAs is apparent when proliferation of vertebrate cells is blocked by a wide variety of physiological signals (terminal differentiation, contact inhibition, and serum starvatio...

TOP mRNAs are translationally controlled by mitogenic, growth, and nutritional stimuli through a 5-terminal oligopyrimidine tract. Here we show that LiCl can alleviate the translational repression of these mRNAs when progression through the cell cycle is blocked at G 0 , G 1 /S, or G 2 /M phases in different cell lines and by various physiological and chemical means. This derepressive effect of LiCl does not involve resumption of cell division. Unlike its efficient derepressive effect in mitotically arrested cells, LiCl alleviates inefficiently the repression of TOP mRNAs in amino acid-deprived cells and has no effect in lymphoblastoids whose TOP mRNAs are constitutively repressed even when they are proliferating. LiCl is widely used as a relatively selective inhibitor of glycogen synthase kinase-3. However, inhibition per se of this enzyme by more specific drugs failed to derepress the translation of TOP mRNAs, implying that relief of the translational repression of TOP mRNAs by LiCl is carried out in a glycogen synthase kinase-3-independent manner. Moreover, this effect is apparent, at least in some cell lines, in the absence of S6-kinase 1 activation and ribosomal protein S6 phosphorylation, thus further supporting the notion that translational control of TOP mRNAs does not rely on either of these variables.