Effect of essential amino acids on the phosphorylation of a 40S ribosomal protein and protein synthesis in Acanthamoeba castellanii

Reversible and multiple phosphorylation of a 40S ribosomal protein is observed in a variety of eukaryotic cells. In the primitive eukaryote Acanthamoeba, one or three phosphorylated S3 derivatives are observed during growth phase in nondefined nutrient medium (ND cells) or in chemically defined nutrient medium (D cells), respectively. In both cases, stationary phase cells exhibit nonphosphorylated S3; however, transfer of these cells into the respective fresh nutrient media results in a transient accumulation of four phosphorylated S3 derivatives. Transfer of D cells into nutrient medium, deficient in all or any single essential amino acids, leads to reversible inhibition of S3 phosphorylation and growth arrest. The low level of phosphorylated S3 is not simply the consequence of growth arrest, since in cells where growth is arrested differently, the level of phosphorylated S3 can be high. In response to amino acid deficiency, a number of other changes can be observed. These include a 2-3-fold decrease of total protein synthesis, 13 changes in the cellular protein pattern, and specific alterations in the ribosome absorbance profiles and in the distribution of poly-A+ RNA within subribosomal and ribosomal fractions. While the rate of total protein synthesis seems to be associated with the level of phosphorylated S3, the level of the synthesis of at least 10 of the particular proteins can be dissociated from the level of S3 phosphorylation.     

TPA stimulates S6 phosphorylation but not protein synthesis in Ehrlich cells

Increased phosphorylation of ribosomal protein S6 has been extensively correlated with an increased rate of protein synthesis. We report here that under two separate conditions in Ehrlich cells an increase in the level of S6 phosphorylation does not result in any increase in the rate of protein synthesis. 1) In glutamine-deprived cells TPA stimulates S6 phosphorylation but has no effect on the rate of protein synthesis, 2) In cells deprived of serum growth factors, addition of serum stimulates both S6 phosphorylation and protein synthesis while TPA stimulates only S6 phosphorylation. These results show that increased phosphorylation of S6 is not sufficient to cause increased rates of protein synthesis, and suggest that additional factors may play a more direct role.     

In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.

From the high salt wash of the ribosomes of the yeast Saccharomyces cerevisiae, three protein kinases have been isolated and separated by DEAE-cellulose chromatography. The three kinases differ in their abilities to phosphorylate substrates such as histones (calf thymus), casein, and S. cerevisiae ribosomes; two of the kinases showed increased activity in the presence of cyclic adenosine 3',5'-monophosphate when histones and 40S ribosomal subunits were used as substrates. The protein kinases catalyzed phosphorylation of certain proteins of the 40S and 60S ribosomal subunits, and 80S ribosomes in vitro. Nine proteins of the 80S ribosome, seven proteins of the 40S subunit, and eleven of the 60S subunit were phosphorylated; different proteins were modified to various extents when different kinases were used. We have identified several proteins of 40S and 60S ribosomal subunits which are not available to the kinases in the 80S particles. Ribosomes isolated from S. cerevisiae cells growing in logarithmic phase of growth were found to contain a number of phosphorylated proteins. Studies by two-dimensional polyacrylamide gel electrophoresis indicated that the ribosomal proteins phosphorylated in vivo correspond with those phosphorylated in vitro. The relationship of in vivo phsophorylation of ribosomes to the growth and physiology of S. cerevisiae is not known.     

Methionine-Dependent Synthesis of Ribosomal Ribonucleic Acid During Sporulation and Vegetative Growth of Saccharomyces cerevisiae

Methionine limitation during growth and sporulation of a methionine-requiring diploid of Saccharomyces cerevisiae causes two significant changes in the normal synthesis of ribonucleic acid (RNA). First, whereas 18S ribosomal RNA is produced, there is no significant accumulation of either 26S ribosomal RNA or 5.8S RNA. The effect of methionine on the accumulation of these RNA species occurs after the formation of a common 35S precursor molecule which is still observed in the absence of methionine. During sporulation, diploid strains of S. cerevisiae produce a stable, virtually unmethylated 20S RNA which has previously been shown to be largely homologous to methylated 18S ribosomal RNA. The appearance of this species is not affected by the presence or absence of methionine from sporulation medium. However, when exponentially growing vegetative cells are starved for methionine, unmethylated 20S RNA is found. The 20S RNA, which had previously been observed only in cells undergoing sporulation, accumulates at the same time as a methylated 18S RNA. These effects on ribosomal RNA synthesis are specific for methionine limitation, and are not observed if protein synthesis is inhibited by cycloheximide or if cells are starved for a carbon source or for another amino acid. The phenomena are not marker specific as analogous results have been obtained for both a methionine-requiring diploid homozygous for met13 and a diploid homozygous for met2. The results demonstrate that methylation of ribosomal RNA or other methionine-dependent events plays a critical role in the recognition and processing of ribosomal precursor RNA to the final mature species.     

Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ribosomal protein rp51 is encoded by two interchangeable genes, RP51A and RP51B. We altered the RP51 gene dose by creating deletions of the RP51A or RP51B genes or both. Deletions of both genes led to spore inviability, indicating that rp51 is an essential ribosomal protein. From single deletion studies in haploid cells, we concluded that there was no intergenic dosage compensation at the level of mRNA abundance or mRNA utilization (translational efficiency), although phenotypic analysis had previously indicated a small compensation effect on growth rate. Similarly, deletions in diploid strains indicated that no strong mechanisms exist for intragenic dosage compensation; in all cases, a decreased dose of RP51 genes was characterized by a slow growth phenotype. A decreased dose of RP51 genes also led to insufficient amounts of 40S ribosomal subunits, as evidenced by a dramatic accumulation of excess 60S ribosomal subunits. We conclude that inhibition of 40S synthesis had little or no effect on the synthesis of the 60S subunit components. Addition of extra copies of rp51 genes led to extra rp51 protein synthesis. The additional rp51 protein was rapidly degraded. We propose that rp51 and perhaps many ribosomal proteins are normally oversynthesized, but the unassembled excess is degraded, and that the apparent compensation seen in haploids, i.e., the fact that the growth rate of mutant strains is less depressed than the actual reduction in mRNA, is a consequence of this excess which is spared from proteolysis under this circumstance.     

Protein synthesis, ribosomal protein S6 phosphorylation in vitro and the effects of amiloride: SDS gel electrophoresis studies in the Yoshida ascites tumor (AH 130) grown in vivo

Cell-free cytosolic extracts from the Yoshida (AH 130) rat ascites hepatoma cell line, grown in vivo, showed high ribosomal protein S6 kinase activity in vitro, as measured by transfer of 32P to exogenous 40S rat liver ribosomal subunits, in both exponential growing and stationary phase cells. A significant decrease of protein synthesis (3H-leucine incorporation into total cell protein) was found to occur in cells reaching the stationary phase of growth, suggesting that S6 phosphorylation was not tightly coupled to the rate of the intraperitoneal cell growth and of protein synthesis in these tumor cells. When the cell-free cytosolic extracts were prepared from cells exposed to amiloride, at concentrations that inhibit the Na+/H+ exchange, a decrease of S6 kinase activity was observed only in exponential growing cells, suggesting the possibility of coupling of the Na+/H+ exchange with phosphorylation of intracellular proteins in these tumor cells. Actually, stationary phase cells showed unchanged S6 kinase activity under the same conditions, possibly due to the extremely low Na+/H+ exchange activity, previously demonstrated (Cell Biol. Int. Rep., 1985, 9, 1017-1025). The present experiments support the hypothesis that the regulation of protein synthesis is not tightly coupled to phosphorylation-dephosphorylation cycles, at least of ribosomal protein S6, in cells characterized by a rather uncontrolled growth such as the Yoshida (AH 130) rat ascites hepatoma. In this connection, an elevated degree of protein phosphorylation, such as that of the ribosomal protein S6, could be a general phenomenon of neoplastic transformation.     

EGF receptor transactivation mediates ANG II-stimulated mitogenesis in intestinal epithelial cells through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway

The role of epidermal growth factor receptor (EGFR) tyrosine kinase and its downstream targets in the regulation of the transition from the G0/G1 phase into DNA synthesis in response to ANG II has not been previously investigated in intestinal epithelial IEC-18 cells. ANG II induced a rapid and striking EGFR tyrosine phosphorylation, which was prevented by selective inhibitors of EGFR tyrosine kinase activity (e.g., AG-1478) or by broad-spectrum matrix metalloproteinase (MMP) inhibitor GM-6001. Pretreatment of these cells with either AG-1478 or GM-6001 reduced ANG II-stimulated DNA synthesis by approximately 50%. To elucidate the downstream targets of EGFR, we demonstrated that ANG II stimulated phosphorylation of Akt at Ser473, mTOR at Ser2448, p70S6K1 at Thr389, and S6 ribosomal protein at Ser(235/236). Pretreatment with AG-1478 inhibited Akt, p70S6K1, and S6 ribosomal protein phosphorylation. Inhibition of phosphatidylinositol (PI)3-kinase with LY-294002 or mTOR/p70S6K1 with rapamycin reduced [3H]thymidine incorporation by 50%, i.e., to levels comparable to those achieved by addition of either AG-1478 or GM-6001. Utilizing Akt small-interfering RNA targeted to Akt1 and Akt2, Akt protein knockdown dramatically inhibited p70S6K1 and S6 ribosomal protein phosphorylation. In contrast, AG-1478 or Akt gene silencing exerted no detectable inhibitory effect on ANG II-induced extracellular signal-regulated kinase 1/2 phosphorylation in IEC-18 cells. Taken together, our results demonstrate that EGFR transactivation mediates ANG II-stimulated mitogenesis through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway in IEC-18 cells.     

Physiological control of phosphorylation ribosomal protein S6 in Mucor racemosus.

The level of phosphorylation of ribosomal protein S6 increased with accelerating rates of growth and protein synthesis in Mucor racemosus. Lowered levels of phosphorylation were seen under conditions of metabolic shift-down or the onset of stationary phase, and no phosphorylation was detected in sporangiospores. Changing metabolic states, changing intracellular levels of adenosine triphosphatase, and the level of phosphorylation of protein S6 were correlated in M. racemosus.     

Regulation of 40S ribosomal protein S6 phosphorylation in Swiss mouse 3T3 cells

Addition of serum to resting cultures of Swiss mouse 3T3 cells causes an immediate multiple phosphorylation of 40S ribosomal protein S6. After 60 min of stimulation, changing to medium containing no serum led to the net dephosphorylation of S6. During this same period, a second protein, as yet unidentified, became increasingly phosphorylated. Incubation of cells with cycloheximide prior to the addition of serum almost completely blocked the activation of protein synthesis. There was no effect on the serum-induced phosphorylation of S6. If cells were stimulated in the presence of cAMP phosphodiesterase inhibitors theophylline or SQ 20006, both S6 phosphorylation and the activation of protein synthesis were inhibited. Stimulation of cells with serum also led to an immediate drop in total intracellular cAMP levels. This was blocked by prostaglandin E1 (PGE1), which caused a 10 fold increase in total intracellular cyclic AMP. However, PGE1 had no effect on protein synthesis or S6 phosphorylation.     

The specific phosphorylation of a 40S ribosomal protein in growth-arrested tetrahymena is induced by sodium

In the past few years, in vivo phosphorylation of ribosomal proteins has been the subject of extensive studies and the results have shown that reversible phosphorylation of small subunit ribosomal protein S6, ubiquitous in eukaryotic cells, is apparently related to regulation of protein synthesis initiation. Thus the level of protein synthesis under various conditions is correlated with the level of S6 phosphorylation. In exponentially growing Tetrahymena, however, such phosphorylation does not occur, but when these cells are transferred to starvation buffers, the rate of protein synthesis is drastically reduced and a 40S ribosomal protein analogous to S6 of higher eukaryotic cells is fully and rapidly phosphorylated in all the ribosomes. We have studied the conditions which lead to this phosphorylation in growth-arrested Tetrahymena, in order to understand the physiological significance of this process. Our results show that there is no obvious correlation between this phosphorylation and starvation. Moreover, it is not a developmentally regulated process related to the conjugation cycle, but a modification induced by the presence of sodium ions or high concentration of Tris in the starvation buffer. The physiological significance of this process is discussed in terms of accumulation of negative charge density probably required for initiation of protein synthesis in the growth-arrested cells starving in Na+-containing buffers.     

The phosphorylation of ribosomal protein S6 by protein kinases from cells infected with pseudorabies virus.

We examined the ability of protein kinase activities from BHK (baby-hamster kidney) cells infected with pseudorabies virus to catalyse the phosphorylation of ribosomal protein S6 in vitro. When the cytosol from infected cells was fractionated on DEAE-cellulose, 40S ribosomal protein kinase activity was found associated with the two isoforms of the cyclic AMP-dependent protein kinase, protein kinase C and a protein kinase (ViPK, virus-induced protein kinase) only detected in infected cells. The phosphorylation of ribosomal protein by ViPK was of particular interest because the appearance of the protein kinase and the increase in the phosphorylation of protein S6 in infected cells shared a similar time course. At moderate concentrations of KCl the major ribosomal substrate for ViPK was ribosomal protein S7, a protein not found to be phosphorylated in vivo. However, at 600 mM-KCl, or in the presence of 5-10 mM-spermine at 60-150 mM-KCl, the phosphorylation of ribosomal protein S7 was suppressed and ribosomal protein S6 became the major substrate. The maximum stoichiometry of phosphorylation obtained under the latter conditions was 1-2 mol of phosphate/mol of S6, and only mono- and di-phosphorylated forms of S6 were detected on two-dimensional gel electrophoresis. As the infection of BHK cells by pseudorabies virus results in the appearance of phosphorylated species of S6 containing up to 5 mol of phosphate/mol of S6 protein, it appears unlikely that ViPK alone can be responsible for the multiple phosphorylation seen in vivo. Nevertheless, tryptic phosphopeptide analysis did indicate that in vitro ViPK catalysed the phosphorylation of at least one of the sites on ribosomal protein S6 phosphorylated in vivo, so that a contributory role for the enzyme in the phosphorylation in vivo cannot be excluded.     

Insulin-mediated phosphorylation of ribosomal protein S6 in mouse melanoma cells and melanoma x fibroblast hybrid cells in relation to cell proliferation

The possible role of insulin-mediated phosphorylation of ribosomal protein S6 in the control of cell proliferation was examined in insulin-unresponsive mouse melanoma calls (PG19) and insulin-responsive melanoma x fibroblast clone 100A. In the hybrid cells, under conditions of growth arrest in medium with low serum, ribosomal protein S6 was rapidly phosphorylated in response to insulin or serum. The phosphorylation of the S6 protein increased over a wide range of insulin concentrations, suggesting that insulin stimulated the phosphorylation by interacting with both high- and low-affinity receptors. In contrast, in growth-arrested melanoma cells, an intermediate level of S6 phosphorylation was observed. Insulin caused only a marginal increase and serum caused a small but consistent increase in the level of S6 phosphorylation in the melanoma cells. Cell cycle analysis revealed that both cell lines arrested growth to a similar degree in the G1 phase of the cell cycle; thus, the higher baseline level of S6 phosphorylation observed in the melanoma cells was not attributable to less complete growth arrest of these cells in medium with low serum. The S6 phosphorylation results correlate well with previous results suggesting that the hybrid cells, but not the parental melanoma cells, can become growth-limited for processes regulated by insulin.     

Synergistic stimulation of S6 ribosomal protein phosphorylation and DNA synthesis by epidermal growth factor and insulin in quiescent 3T3 cells

Using an improved method to quantify the level of phosphorylation of the S6 ribosomal protein, we have analyzed the effect of growth stimuli on S6 phosphorylation in quiescent murine Swiss/3T3 cells to see if it can be dissociated from the later increase in DNA synthesis. Saturating concentrations of epidermal growth factor (EGF), insulin and serum each stimulate phosphorylation of the S6 ribosomal protein to the same maximal level; this is not so for DNA synthesis. Subsaturating concentrations of EGF and insulin act synergistically to stimulate both S6 phosphorylation and DNA synthesis, but qualitatively the two synergistic interactions are expressed differently. Insulin increases the maximal response of DNA synthesis to EGF, whereas it decreases the concentration of EGF required for half-maximal stimulation of S6 phosphorylation. We conclude that S6 phosphorylation is not a principal regulator of DNA synthesis, and that insulin and EGF regulate both S6 phosphorylation and DNA synthesis through different, but interacting, pathways of action.     

Control of Protein Synthesis in Acanthamoeba castellanii

During development of Acanthamoeba castellanii in a non-nutrient medium, the pattern of synthesis of proteins changes. Comparison of in vivo and in vitro patterns of protein synthesis reveals concomitant relative increases of five proteins, indicating a control of synthesis of these proteins at the level of the RNA content. The decrease in the overall rate of protein synthesis and relative decreases in the synthesis of actin and ribosomal proteins during development, not accompanied by equivalent changes in the content of mRNA, suggest control mechanisms also at the level of translation. Patterns of ribosomal proteins do not change qualitatively during encystation, indicating that the inhibition in the overall rate of protein synthesis and the formation of inactive monosomes is not controlled by this mechanism; however, phosphorylation of one ribosomal protein, S 3, is observed occasionally during encystation. Phosphorylation of S 3 is also detected after transfer of stationary phase cells into fresh nutrient medium. It was found that only such cells having RNA of aberrant properties are able to phosphorylate S 3 after transfer into fresh nutrient medium. Since these changes in the property of RNA are never observed in cysts, in which phosphorylation of S 3 sometimes occurs, it is concluded that either other alterations in the properties of RNA than those detected or other parameters are responsible for changes in phosphorylation of S 3.     

Coordinate control of syntheses of ribosomal ribonucleic acid and ribosomal proteins during nutritional shift-up in Saccharomyces cerevisiae.

We investigated the regulation of ribosome synthesis in Saccharomyces cerevisiae growing at different rates and in response to a growth stimulus. The ribosome content and the rates of synthesis of ribosomal ribonucleic acid and of ribosomal proteins were compared in cultures growing in minimal medium with either glucose or ethanol as a carbon source. The results demonstrated that ribosome content is proportional to growth rate. Moreover, these steady-state concentrations are regulated at the level of synthesis of ribosomal precursor ribonucleic acid and of ribosomal proteins. When cultures growing on ethanol were enriched with glucose, the rate of ribosomal ribonucleic acid synthesis, measured by pulsing cells with [methyl-3H]methionine, increased by 40% within 5 min, doubled within 15 min, and reached a steady state characteristic of the new growth medium by 30 min. Labeling with [3H]leucine reveal a coordinate increase in the rate of synthesis of 30 or more ribosomal proteins as compared with that of total cellular proteins. Their synthesis was stimulated approximately 2.5-fold within 15 min and nearly 4-fold within 60 min. The data suggest that S. cerevisiae responds to a growth stimulus by preferential stimulation of the synthesis of ribosomal ribonucleic acid and ribosomal proteins.     

Depletion of yeast ribosomal proteins L16 or rp59 disrupts ribosome assembly

Two strains of Saccharomyces cerevisiae were constructed that are conditional for synthesis of the 60S ribosomal subunit protein, L16, or the 40S ribosomal subunit protein, rp59. These strains were used to determine the effects of depriving cells of either of these ribosomal proteins on ribosome assembly and on the synthesis and stability of other ribosomal proteins and ribosomal RNAs. Termination of synthesis of either protein leads to diminished accumulation of the subunit into which it normally assembles. Depletion of L16 or rp59 has no effect on synthesis of most other ribosomal proteins or ribosomal RNAs. However, most ribosomal proteins and ribosomal RNAs that are components of the same subunit as L16 or rp59 are rapidly degraded upon depletion of L16 or rp59, presumably resulting from abortive assembly of the subunit. Depletion of L16 has no effect on the stability of most components of the 40S subunit. Conversely, termination of synthesis of rp59 has no effect on the stability of most 60S subunit components. The implications of these findings for control of ribosome assembly and the order of assembly of ribosomal proteins into the ribosome are discussed.     

Nutrient-dependent multimerization of the mammalian target of rapamycin through the N-terminal HEAT repeat region

The mammalian target of rapamycin (mTOR) plays a pivotal role in the regulation of cell growth in response to a variety of signals such as nutrients and growth factors. mTOR forms two distinct complexes in vivo. mTORC1 (mTOR complex 1) is rapamycin-sensitive and regulates the rate of protein synthesis in part by phosphorylating two well established effectors, S6K1 (p70 ribosomal S6 kinase 1) and 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1). mTORC2 is rapamycin-insensitive and likely regulates actin organization and activates Akt/protein kinase B. Here, we show that mTOR forms a multimer via its N-terminal HEAT repeat region in mammalian cells. mTOR multimerization is promoted by amino acid sufficiency, although the state of multimerization does not directly correlate with the phosphorylation state of S6K1. mTOR multimerization was insensitive to rapamycin treatment but hindered by butanol treatment, which inhibits phosphatidic acid production by phospholipase D. We also found that mTOR forms a multimer in both mTORC1 and mTORC2. In addition, Saccharomyces cerevisiae TOR proteins Tor1p and Tor2p also exist as homomultimers. These results suggest that TOR multimerization is a conserved mechanism for TOR functioning.     

Further characterization of recessive suppression in yeast. Isolation of the low-temperature sensitive mutant of Saccharomyces cerevisiae defective in the assembly of 60 S ribosomal subunit

It has been shown that recessive suppressor mutations in the yeast Saccharomyces cerevisiae may cause sensitivity towards low temperatures (very slow growth or lack of growth at 10 degrees C). One of the sup 1 low temperature sensitive (Lts-) mutants, 26-125A-P-2156, was studied in detail. After a prolonged period of incubation (70 h) under restrictive conditions the protein synthesis apparatus in the mutant cells was irreversibly damaged. In addition, Lts- cells incubated under restrictive conditions synthesize unequal amounts of ribosomal subunits, the level of 60 S subunit being reduced. It has been suggested that the recessive suppression is mediated by a mutation in the gene coding for 60 S subunit component, probably a ribosomal protein. The mutation leads simultaneously to a defect in the assembly of 60 S subunit and to low-temperature sensitive growth of the mutant.