The phosphorylation of eukaryotic ribosomal protein S6 by protein kinase C

Purified Ca2+-dependent and phospholipid-dependent protein kinase (protein kinase C) from bovine brain catalysed the phosphorylation of ribosomal protein S6 when incubated with 40S ribosomal subunits from rat liver or from hamster fibroblasts. The phosphorylation was dependent on Ca2+ and phospholipid, and occurred under ionic conditions similar to those which support protein biosynthesis in vitro. Protein kinase C phosphorylated at least three sites on ribosomal protein S6 when incubated with unphosphorylated ribosomes, and increased the extent of phosphorylation of ribosomes previously phosphorylated predominantly on two sites by cyclic-AMP-dependent protein kinase, converting some molecules to the tetraphosphorylated or pentaphosphorylated form. This indicates that protein kinase C can phosphorylate sites on ribosomal protein S6 other than those phosphorylated by the cyclic-AMP-dependent protein kinase, and this conclusion was confirmed by analysis of tryptic phosphopeptides. These results strengthen the possibility that protein kinase C might be involved in catalysing the multisite phosphorylation of ribosomal protein S6 in certain circumstances in vivo.     

Ordered multisite phosphorylation of Xenopus ribosomal protein S6 by S6 kinase II.

Phosphorylated ribosomal proteins were isolated from Xenopus 40 S ribosomal subunits by reversed-phase high performance liquid chromatography (HPLC) to enable direct analysis of the phosphorylation sites in ribosomal protein S6. Xenopus S6 closely resembled mammalian S6 with respect to the following properties: (i) reversed-phase HPLC elution behavior, (ii) amino-terminal sequence (96% identity in the first 37 residues), and (iii) an identical sequence within the region of its phosphorylation sites. Whereas S6 was the only ribosomal protein phosphorylated in vitro by Xenopus S6 kinase II, ribosomes phosphorylated in vivo were found to be associated with an additional phosphoprotein having an amino-terminal sequence identical to that of the ubiquitin carboxyl-terminal extension protein CEP 80. S6 kinase II phosphorylated at least four sites (serines 1-3 and 5) in the sequence Arg-Arg-Leu-Ser(1)-Ser(2)-Leu-Arg-Ala-Ser(3)-Thr-Ser(4)-Lys-Ser(5)-, which correspond to the residues known to be phosphorylated in the carboxyl-terminal region of mammalian S6. The in vivo S6 phosphorylation sites in maturing Xenopus oocytes were shown to be located within the same cluster of serine residues, although individual sites were not identified. Kinetic analysis of S6 kinase II-catalyzed phosphorylation events indicated a simple sequential mechanism of multisite phosphorylation initiating at either serine 2 (preferred) or serine 1, with the rates of phosphorylation of individual sites occurring in the order serine 2 greater than serine 1 greater than serine 3 greater than serine 5.     

Regulation of ribosomal protein S6 phosphorylation in heat-shocked HeLa cells

Decreases in energy charge, ribosomal protein phosphorylation and rate of protein synthesis are well-documented facets of the cellular response to hyperthermia in non-vertebrates. We have tried to reproduce this response pattern in 32P-labelled HeLa cells in order to investigate the hypothetical causal relationship between these effects. In HeLa cells shifted from 36 degrees C to 42 degrees C, dephosphorylation of S6 and inhibition of protein synthesis, owing to a decreased initiation rate, were observed, but could not have been mediated by changes in the cells' general energy charge since the ATP and GTP levels were not reduced. In addition, we found that the hyperthermic translation block developed faster than the overall dephosphorylation of S6, showing that S6 dephosphorylation cannot be responsible for the translation block unless site-specific effects play a critical role.     

Ethionine and the phosphorylation of ribosomal protein S6.

Ribosome phosphorylation was studied by monitoring the phosphorylation state of small subunit protein S6 as visualized on two-dimensional electrophoretograms of ribosomal proteins isolated from rat liver. No phosphorylation of S6 was observed under conditions of ethionine-induced inhibition of protein synthesis. Moderate phosphorylation, detected as the appearance of S6 and four or five phosphorylated derivatives, was observed in saline-treated animals. Reversal of ethionine-induced inhibition of protein synthesis by treatment with adenine led to extensive phosphorylation of S6. A model for protein synthesis which includes requisite phosphorylation of ribosomes during initiation is proposed. Cyclic adenosine 3':5'-monophosphate concentration was significantly elevated in liver of both ethionine- and ethionine plus adenine-treated rats, relative to that of saline-treated animals.     

In vivo phosphorylation and activation of ribosomal protein S6 kinases during Xenopus oocyte maturation

Previous studies have shown that increased ribosomal protein S6 kinase activity in unfertilized Xenopus eggs can be resolved by DEAE-Sephacel chromatography into two peaks, designated S6 kinase I and S6 kinase II. We show here that antibody against bacterially expressed S6 kinase II cross-reacts with S6 kinase I. Both S6 kinases undergo marked phosphorylation when they are activated during oocyte maturation, and both become deactivated and dephosphorylated upon activation of eggs. Immunoblotting of extracts of oocytes reveals that all S6 kinase molecules undergo a decrease and increase in electrophoretic mobility upon activation and deactivation, respectively. The increase in electrophoretic mobility can be produced in vitro by incubation of activated S6 kinase with purified phosphatases. Phosphoamino acid analysis of S6 kinase II labeled in vivo during maturation reveals both phosphoserine and phosphothreonine, and phosphopeptide maps suggest that several kinases may phosphorylate and activate S6 kinase II in vivo. These results demonstrate that, during oocyte maturation and early development, S6 kinase activation and deactivation are regulated by phosphorylation and dephosphorylation, suggesting a probable mechanism for S6 kinase regulation in other mitogenically stimulated cells.     

Oxidants induce phosphorylation of ribosomal protein S6

We have investigated the phosphorylation of the ribosomal S6 protein which may be on the pathway of mitogenic stimulation in response to oxidants. Mouse epidermal cells JB6 (clone 41) were exposed to active oxygen generated extracellularly by glucose/glucose oxidase (producing H2O2) or xanthine oxidase (producing H2O2 plus superoxide) or active oxygen produced intracellularly by the metabolism of menadione (producing mostly superoxide). All three sources of active oxygen induced rapidly a protein kinase activity which phosphorylated S6 in cellular extracts prepared in the presence of the phosphatase inhibitor beta-glycerophosphate. Maximal activity was reached within 15 min of exposure, and phosphorylation occurred specifically at serine residues. Strong activation of the protein kinase activity was also observed by diamide which selectively oxidizes SH functions. The following observations characterize the reaction: 1) Extracellular addition of catalase but not Cu,Zn-superoxide dismutase was inhibitory, implicating H2O2 rather than superoxide as the active species. 2) Exposure of JB6 cells to reagent H2O2 or H2O2 released by glucose/glucose oxidase resulted in a measurable increase in intracellular free Ca2+. 3) The intracellular Ca2+ complexer quin 2 suppressed the reaction. 4) The calmodulin antagonist trifluoperazine prevented the activation of the protein kinase. 5) Exposure of cells to Mn2+ and La3+, which stimulate calmodulin-dependent activities, potently increased the S6 kinase activity of the cell extracts. 6) Desalted extracts strictly required the addition of Mg2+ and their activity was inhibited by Mn2+. In contrast, the phosphorylation of a 95-kDa protein was strongly stimulated by Mn2+. 7) For several agonists, i.e. active oxygen, phorbol 12-myristate 13-acetate, and serum, tryptic peptide analysis yielded the same phosphopeptides, suggesting that a common S6 kinase is involved in these reactions. From these data we propose that oxidants induce an increase in intracellular free Ca2+ which activates a Ca2+/calmodulin-dependent protein kinase and, as a consequence, an S6 kinase.     

Semliki Forest virus capsid protein associates with the 60S ribosomal subunit in infected cells.

Semlike forest virus capsid protein cosedimented with the large ribosomal subunit at 60S in sucrose gradients after treatment of cytoplasm from infected cells with Triton X-100 and EDTA. In CsCl gradients the capsid protein banded with the subunit at a density of 1.56 to 1.57 g/cm3. Most of the capsid protein could be detached from the 60S structure by treatment with 0.8 M KCl. The ribonucleoprotein of the 26S RNA had a sedimentation value of 53S and a density of 1.50 g/cm3 and could thus be separated from the 60S structure. The data suggest that the capsid protein binds to the large ribosomal subunit, but not to the viral 26S RNA.     

Kinetics of ribosomal protein S6 phosphorylation by HepG-2 cells in response to insulin

The effect of insulin on the phosphorylation of ribosomal protein S6 was studied in a human liver cell line (HepG-2), using [32P] inorganic phosphate. Increased rate of protein S6 phosphorylation was detected 8 min following the addition of insulin to serum starved cells. Maximum enhancement of phosphorylation was observed at 80 nM insulin. Minimum level of insulin required to produce measurable increase of S6 phosphorylation was 20 nM. Radioactivity of protein S6 increased most in the native subunit and polysome fractions. Significant increase in radioactivity of this protein was not observed in the monosome fraction during the first 30 min of insulin stimulation. Increase in the specific radioactivity of native 40S subunit was higher than that of polysomes. These results suggest that phosphorylation takes place in the subunit compartment and moves preferentially into the polysomes.     

Increased phosphorylation of ribosomal protein S6 in hamster fibroblasts transformed by polyoma virus and simian virus 40

The extent of phosphorylation of ribosomal protein S6 was compared in normal hamster fibroblasts and in fibroblasts transformed by polyoma virus or simian virus 40. In both strains of transformed cells the protein was more highly phosphorylated than in the normal cells.     

Role of pseudorabies virus glycoprotein gI in virus release from infected cells.

The Bartha vaccine strain of pseudorabies virus has a deletion in the short unique (Us) region of its genome which includes the genes that code for glycoproteins gI and gp63 (E. Petrovskis, J. G. Timmins, T. M. Gierman, and L. E. Post, J. Virol. 60:1166-1169, 1986). Restoration of an intact Us to the Bartha strain enhances its ability to be released from infected rabbit kidney cells and increases the size of the plaques formed on these cells (T. Ben-Porat, J. M. DeMarchi, J. Pendrys, R. A. Veach, and A. S. Kaplan, J. Virol. 57:191-196, 1986). To determine which gene function plays a role in virus release from rabbit kidney cells, deletions were introduced into the genomes of both wild-type virus and the "rescued" Bartha strain (Bartha strain to which an intact Us had been restored) that abolish the expression of either the gI gene alone or both gI and gp63 genes. The effect of these deletions on the phenotype of the viruses was studied. Deletion mutants of wild-type virus defective in either gI or gI and gp63 behave like wild-type virus with respect to virus release and plaque size on rabbit kidney cells. Deletion of gI from the rescued Bartha strain, however, strongly affects virus release and causes a decrease in plaque size. We conclude that gI affects virus release but that at least one other viral function also affects this process. This function is defective in the Bartha strain but not in wild-type virus; in its absence gI is essential to efficient release of the virus from rabbit kidney cells.     

The stimulation of the phosphorylation of ribosomal protein S6 by cycloheximide and puromycin

The administration of cycloheximide or puromycin to rats in amounts that all but completely inhibited hepatic protein synthesis caused an increase in the amount of radioactive phosphate incorporated into the liver ribosomal protein S6; there was also an increase in the prominence of the derivatives of S6 which contain increasing numbers of phosphorylated serine residues.     

Phosphorylation of ribosomal proteins in hamster fibroblasts infected with pseudorabies virus or herpes simplex virus.

In BHK cells infected with pseudorabies virus, there was a substantial increase in the phosphorylation of ribosomal protein S6. This increase occurred between 2 and 4 h after infection and persisted at least until 9 h. We estimated that in mock-infected cells S6 contained, on an average, one phosphate group per protein chain, whereas in infected cells this rose to between four and five phosphate groups per protein chain. A second ribosomal protein, either S16 or S18, was also phosphorylated after infection. No increase in cyclic AMP was found at the time of phosphorylation. We also found an increased phosphorylation of S6 in herpes simplex virus-infected BHK 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.     

The ubiquitination of ribosomal S6 kinases is independent from the mitogen-induced phosphorylation/activation of the kinase

Ribosomal protein S6 kinase plays a critical role in the regulation of cell growth and energy metabolism. S6K belongs to the AGC family of serine/threonine kinases and is a downstream effector of the mTOR and PI3K signalling pathways. The activity and subcellular localisation of S6K are tightly controlled by phosphorylation/dephosphorylation events. We have recently demonstrated that steady-state levels of S6K isoforms, S6K1 and S6K2, are regulated by ubiquitination-mediated proteasomal degradation. In this study, we report for the first time that the ubiquitination status of S6K isoforms is coordinated by signalling pathways induced by mitogenic stimuli and extracellular stresses. The induction of signal transduction by serum and growth factors significantly increases the level of S6K ubiquitination, while the treatment of cells with UV and staurosporine has the opposite effect. Furthermore, we found that the phosphorylation/activation of S6Ks does not correlate directly with the induction of their ubiquitination in response to diverse cellular stimuli. This study suggests that the ubiquitination and subsequent proteasomal degradation of S6K are controlled by signalling pathways, which could possibly facilitate their association with the components of the ubiquitination machinery.     

The phosphorylation of ribosomal protein S6 in baby hamster kidney fibroblasts

Ribosomal protein S6 was extensively phosphorylated in pre-confluent but not in post-confluent baby hamster kidney fibroblasts. This appears to be the first example of increased phosphorylation of S6 under physiological conditions where the cellular concentration of cyclic AMP is not elevated. The extent of the phosphorylation of S6 was also independent of alterations in the protein synthetic activity of the cells, suggesting that the biological role of this phosphorylation may be unrelated to the functional ability of the ribosomes.     

Insulin- and phorbol ester-stimulated phosphorylation of ribosomal protein S6

The relative abilities of insulin and the phorbol ester tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) to lead to the phosphorylation of ribosomal protein S6 in vivo were compared in a Reuber H35 hepatoma cell line shown previously to be highly responsive to these agents. In quiescent (serum-starved) cultures of H35 cells incubated with 32Pi, both insulin (10(-7) M) and TPA (1.6 X 10(-6) M) resulted in the marked phosphorylation of S6 compared to the unstimulated cultures as evidenced by an increase in radioactivity associated with S6 and by a corresponding shift in the mobility of phosphorylated S6 during two-dimensional electrophoresis. Following incubation with insulin or TPA, greater than 95% of the phosphate was in derivatives containing four to five phosphate groups. The site-specific phosphorylation of S6 in response to both optimal and suboptimal concentrations of insulin and/or TPA was examined by two-dimensional peptide mapping of the trypsin-digested ribosomal protein S6. The tryptic phosphopeptides of S6 obtained following treatment of the H35 cells with insulin and/or TPA were identical and were the same phosphopeptides as those observed previously following the phosphorylation in vitro of 40 S ribosomal subunits from reticulocytes with purified protease-activated kinase II (Perisic, O., and Traugh, J. A. (1983) J. Biol. Chem. 258, 13998-14002).     

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.     

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 phosphorylation of ribosomal protein S6 from progesterone-stimulated Xenopus laevis oocytes. Kinetic studies and phosphopeptide analysis

Xenopus laevis oocytes were prelabeled with [32P]orthophosphate overnight before maturation was induced by progesterone stimulation. The phosphorylation status of ribosomal protein S6 from control oocytes and the temporal changes in S6 phosphorylation after progesterone treatment were analyzed by two-dimensional gel electrophoresis. S6 protein was separated in up to five distinct S6 species, which differed in their degree of phosphorylation. 32P labeling of S6, as judged from the shift of radioactivity into more highly phosphorylated S6 derivatives, continuously increased in progesterone-stimulated oocytes even at later times when germinal vesicle breakdown was completed. S6 protein of unstimulated oocytes was labeled to a lower degree. Trypsin cleavage of total S6 protein, isolated from control and maturing oocytes, gave rise to different complex phosphopeptide patterns reflecting the existence of various multiply phosphorylated S6 derivatives in both samples. Two of the more highly phosphorylated S6 derivatives showed considerable differences between the phosphopeptide elution profiles of control and stimulated oocytes indicating that dissimilar sites had been modified under both physiological conditions. Only phosphoserine was detected in the phosphoamino acid analysis of individual S6 derivatives.