The phosphorylation of eukaryotic ribosomal protein S6 by protein kinase C (original) (raw)
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All of the phosphorylation sites in 40 S ribosomal protein S6 derived from serum-stimulated Swiss mouse 3T3 cells are found within a small cyanogen bromide (CNBr) peptide derived from the carboxyl terminus, Ly~'~~-Lys'~'. Further cleavage of the CNBr peptide or the intact protein with endoproteinase Lys-C (endo Lys-C) generated a single phosphorylated peptide, implying that all the sites of phosphorylation resided either between Arg'" and L Y S '~~ or between Arg'" and LysZ4' if cleavage at L Y S '~~ was blocked by phosphorylation at a nearby residue. To discern between these possibilities and to identify the phosphorylation sites, the protein was purified from serumstimulated cells and cleaved with endo Lys-C, and the single endo Lys-C phosphorylated peptide was isolated and sequenced following conversion of all the phosphorylated serines to S-ethylcysteine. The results show that the phosphorylated peptide extends from Arg'" to LysZ4' and that the sites of phosphorylation in vivo are Ser236, Ser'", SerZ4O, Ser244, and SerZ4'.
Recently we purified and cloned the mitogen/oncogene-activated M, 70,000 (70K) S6 kinase from the livers of rats treated with cycloheximide (Kozma, S. C., Lane, H. A., Ferrari, S., Luther, H., Siegmann, M., and Thomas, G. (1989) EMBO J. 8, 4125-4132;
Ordered phosphorylation of 40S ribosomal protein S6 after serum stimulation of quiescent 3T3 cells
Proceedings of the National Academy of Sciences, 1983
The amino acids and tryptic peptides that become phosphorylated in 40S ribosomal protein S6 after serum stimulation of quiescent 3T3 cells were examined by two-dimensional thin-layer electrophoresis. In the maximally phosphorylated form of the protein, most of the phosphate was incorporated into serine and a small amount, into threonine. Digestion of this form of the protein with trypsin revealed 10 major phosphopeptides. All 10 contained phosphoserine and 2 ofthe 10 also contained phosphothreonine. Next, the five forms ofincreasingly phosphorylated S6 were individually separated on two-dimensional polyacrylamide gels or total S6 was isolated from cells that were stimulated for only a short time and their phosphotryptic maps were analyzed. The results showed that, as larger amounts of phosphate were added to S6, the phosphopeptides appeared in a specific order.
Molecular Biology Reports, 1980
Ribosomes were isolated from baby hamster kidney fibroblasts, either 20 min or 2 days after labelling with radioactive amino acids, and their proteins subjected to two-dimensional polyacrylamide gel electrophoresis. No significant differences were observed between the amounts of radioactivity associated with the position of the phosphorylated derivatives of protein $6. This suggests that the phosphorylation is. unlikely to be important in ribosomal biogenesis or extranuclear transport.
The phosphorylation of ribosomal proteins in baby hamster kidney fibroblasts
1982
This thesis describes studies directed towards understanding the function of the phosphorylation of a particular ribosomal protein - S6 - in eukaryotic cells. Two experimental approaches to determine the extent of phosphorylation of ribosomal protein S6 were adopted. One involved labelling the cells with radioactive orthophosphate and quantitatively estimating the extent of phosphorylation of the ribosomes by measuring the radioactivity incorporated into the protein and the specific radioactivity of the presumed precursor, ATP, The other involved qualitative estimation of the phosphorylation by analysing the ribosomal proteins in a system of gel electrophoresis in which the phosphorylated derivatives of the protein are resolved from the unphosphorylated protein. This latter method had the advantage that it could be applied when it was not possible to label certain subcellular fractions of ribosomes with (32P)-orthophosphate. It had previously been found that the phosphorylation of r...
Ribosomal Protein S6 Phosphorylation: Four Decades of Research
International Review of Cell and Molecular Biology, 2015
The phosphorylation of ribosomal protein S6 (rpS6) has been described for the first time about four decades ago. Since then, numerous studies have shown that this modification occurs in response to a wide variety of stimuli on five evolutionarily conserved serine residues. However, despite a large body of information on the respective kinases and the signal transduction pathways, the physiological role of rpS6 phosphorylation remained obscure until genetic manipulations were applied in both yeast and mammals in an attempt to block this modification. Thus, studies based on both mice and cultured cells subjected to disruption of the genes encoding rpS6 and the respective kinases, as well as the substitution of the phosphorylatable serine residues in rpS6, have laid the ground for the elucidation of the multiple roles of this protein and its posttranslational modification. This review focuses primarily on newly identified kinases that phosphorylate rpS6, pathways that transduce various signals into rpS6 phosphorylation, and the recently established physiological functions of this modification. It should be noted, however, that despite the significant progress made in the last decade, the molecular mechanism(s) underlying the diverse effects of rpS6 phosphorylation on cellular and organismal physiology are still poorly understood.
Physiological roles of ribosomal protein S6: one of its kind
International review of cell and molecular biology, 2008
The phosphorylation of ribosomal protein S6 (rpS6), which occurs in response to a wide variety of stimuli on five evolutionarily conserved serine residues, has attracted much attention since its discovery more than three decades ago. However, despite a large body of information on the respective kinases and the signal transduction pathways, the role of this phosphorylation remained obscure. It is only recent that targeting the genes encoding rpS6, the phosphorylatable serine residues or the respective kinases that the unique role of rpS6 and its posttranslational modification have started to be elucidated. This review focuses primarily on the critical role of rpS6 for mouse development, the pathways that transduce various signals into rpS6 phosphorylation, and the physiological functions of this modification. The mechanism(s) underlying the diverse effects of rpS6 phosphorylation on cellular and organismal physiology has yet to be determined. However, a model emerging from the curre...
Experimental Cell Research, 2000
The p70 ribosomal S6 kinase (S6K1) is rapidly activated following growth factor stimulation of quiescent fibroblasts and inhibition of this enzyme results in a G 1 arrest. Phosphorylation of the ribosomal S6 protein by S6K1 regulates the translation of both ribosomal proteins and initiation factors, leading to an increase in protein synthesis. We have examined the activation of S6K1 in human fibroblasts following mitogen stimulation. In early passage fibroblasts S6K1 is activated following serum stimulation as evidenced by increased kinase activity and site-specific phosphorylation. In contrast, site-specific phosphorylation of S6K1 at Thr421/Ser424 is diminished in senescent fibroblast cultures. A second phosphorylation site within S6K1 (Ser411) is phosphorylated even in the absence of serum stimulation and the enzyme shows increased phosphorylation as judged by decreased electrophoretic mobility. Inhibitor studies indicate that this phosphorylation is dependent upon the mammalian target of rapamycin, PI 3-kinase, and the MAPK pathway. In order to understand the consequences of the altered phosphorylation of the S6K1, we examined the phosphorylation state of the ribosomal S6 protein. In early passage fibroblasts the ribosomal S6 protein is phosphorylated upon serum stimulation while the phosphorylation of the ribosomal S6 protein is drastically reduced in senescent fibroblasts. These results suggest that the intracellular regulators of S6K1 are altered during replicative senescence leading to a deregulation of the enzyme and a loss of ribosomal S6 phosphorylation.