Identification of 40 S Ribosomal Protein S6 Phosphorylation Sites in Swiss Mouse 3T3 Fibroblasts Stimulated with Serum (original) (raw)
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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.
The phosphorylation of eukaryotic ribosomal protein S6 by protein kinase C
European Journal of Biochemistry, 1985
Purified Ca2 +-dependent and phospholipid-dependent protein kinase (protein kinase C) from bovine brain catalysed the phosphorylation of ribosomal protein S 6 when incubated with 40 S 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 S 6 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 S 6 in certain circumstances in vivo.
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...
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...
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;
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.
FEBS Letters, 1994
We have purified a 40 kDa protein from the rat 40 S ribosomal subunit and determined its primary structure by amino acid and cDNA sequencing. The amino acid sequence of the 40 kDa protein shared 29-37% homology with prokaryotic ribosomal protein S2 of eubacteria and chloroplasts, indicating that the protein is a eukaryotic counterpart to prokaryotic S2. Moreover, the amino acid sequence shared 99% identity with those deduced from cDNAs for 68 kDa lam&n binding proteins of human, murine and bovine origins. The cDNAs are capable of encoding polypeptides with predicted molecuiar mass of 33,000 which lacked typical signal sequences, N-linked glycosylation sites and putative transmembrane domains. These results indicate that the cDNAs for 68 kDa laminin binding proteins actually code for the 40 kDa ribosomal protein.
The primary structure of rat ribosomal protein S6
Journal of Biological Chemistry, 1988
The amino acid sequence of rat ribosomal protein S6, the major phosphoprotein in the organelle, was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed from the sequence of amino acids in portions of the protein. Ribosomal protein S6 contains 249 amino acids and has a molecular weight of 28,683. The protein has 15 seryl residues; 7 are located in the carboxyl-terminal sequence of 15 amino acids and probably include most if not all of the residues that are phosphorylated. There are related repeated sequences of 10 amino acids each that occur at four separate positions in S6 and that are very basic. Rat S6 is homologous to Saccharomyces cerevisiae S 10 (the extent of the identity is 75%) and most likely also to Schizosaccharomyces pombe S6.